Broadly reactive antibodies to influenza b viruses

Abstract

The present disclosure is directed to antibodies binding to and neutralizing influenza B virus and methods for use thereof.

Description

[0001] DESCRIPTION BROADLY REACTIVE ANTIBODIES TO INFLUENZA B VIRUSES PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 515,279, filed July 24, 2023, the entire contents of which are hereby incorporated by reference. REFERENCE TO A SEQUENCE LISITNG This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on July 23, 2024, is named VBLTP0321WO.xml and is 718,732 bytes in size. BACKGROUND 1. Field of the Disclosure The present disclosure relates generally to the fields of medicine, infectious disease, and immunology. More particular, the disclosure relates to human antibodies binding to influenza B viruses. 2. Background Influenza B viruses (IBV) are members of the order Orthomyxoviridae. These viruses contain a negative sense, single-stranded segmented RNA genome that comprises eight gene segments encoding ten viral proteins. Of these viral proteins, the surface includes the neuraminidase (NA), hemagglutinin (HA), NB, and BM2 proteins. HA is the most abundant and immunodominant surface glycoprotein on influenza viruses and enables virus binding to sialic acid on host cells (Wagner et al., 2002; Gaymard et al., 2016). The second most abundant protein, NA, is a tetrameric protein that enzymatically cleaves sialic acid. There are several proposed potential mechanisms for inhibiting NA function to protect the host, and antibodies have been used to study these mechanisms (Schneider & Coughlan, 2023). First, some anti- NA antibodies impede NA-mediated enzymatic cleavage of the virus from mucins in the airway (Matrosovich et al., 2004; Cohen et al., 2013). This activity prevents the virus from efficiently burrowing through the mucus lining the airway to achieve infection of underlying airway epithelial cells and also inhibits virus spread by preventing detachment of virion particles from

[0002] 1 4890-9150-3059, v.1 the mucins covering infected cells. Secondly, anti-NA antibodies bound to NA on virion particles can inhibit HA binding to sialic acid, impeding viral attachment and entry into host cells. Anti-NA antibodies also can mediate Fc-dependent mechanisms of protection (DiLillo et al., 2016) that eliminate virus-infected cells, including by contributing to synergistic effects for this activity with HA stalk antibodies (Momont et al., 2023). Lastly, by blocking NA enzymatic activity during viral egress, NA antibodies can prevent the cleavage of viral aggregates and hinder the release and subsequent dissemination of the virus within the host (McAuley et al., 2019; Chen et al., 2018). Historically, emphasis on the vaccine-induced immune response to HA has helped inform current understanding of the human humoral response to influenza viruses and provided a correlate of protection for regulatory purposes in the hemagglutination inhibition (HAI) assay. Recent literature, however, indicates that natural influenza virus infection induces a diverse immune response with antibody recognition of viral proteins distributed across several antigenic targets, including NA. The content of NA in most influenza subunit protein vaccines is not controlled however, and thus many current vaccines may contain suboptimal concentrations of critical protective antigens of the immune response to influenza, especially NA (Chen et al., 2018). Of the circulating influenza viruses, IBV causes a 23% median case burden rate (Zaraket et al., 2021). IBV was first described in 1940, and in subsequent sequence analyses has been shown to circulate in two distinct genetic lineages designated Victoria and Yamagata (Palese & Young, 1982). The dominance of each of these two lineages varies between influenza seasons, however, the lack of detection of Yamagata lineage strains in clinical specimens in recent years suggests that this lineage may have gone extinct in the wake of the COVID-19 pandemic (Vajo & Torzsa, 2022; Koutsakos et al., 2021). In contrast, Victoria lineage strains continue to pose a significant public health concern as they circulate each year and disproportionally affect pediatric and geriatric populations (Sharma et al., 2019), which include immunocompromised individuals. There are limited prophylactic or therapeutic options available for IBV. Clinically, patients with IBV infection cannot be distinguished from those with influenza A virus (IAV) infection (Liu et al., 2019). The presenting syndrome often includes cough, fever, congestion, vomiting, and body aches. In severe IBV cases, the clinical syndrome may progress to bronchopneumonia with relatively frequent secondary bacterial lung infections (Taubenberger & Morens, 2008). During the COVID-19 pandemic, IBV co-infections with SARS-CoV-2 contributed to causing severe disease in a setting with limited therapeutic options. Thus, there is unmet medical need for better prevention and treatment of IBV infections.

[0003] 2 4890-9150-3059, v.1 Most current approved influenza vaccines contain HA proteins from both IBV lineages yet they may fail to completely protect vaccinated individuals from disease. Currently, there are FDA-approved therapeutics to treat influenza, including M2 inhibitors that work only against the IAVs and a class of small-molecule NA inhibitors (including zanamivir, oseltamivir, laninamivir, and peramivir) that work against IAVs and IBVs (Heneghan et al., 2016). While these treatments are helpful for early-stage administration and mild infections, they provide limited benefit at later stages of infection. Furthermore, single amino acid mutations in NA can reduce efficacy or generate resistance to these drugs. Lastly, NA small molecule inhibitors are generally less effective in the treatment of IBV infection than IAV infection. Altogether, this scenario creates a need for development of additional therapeutic options for IBV infection (Farrukee et al., 2016; Yen et al., 2006; Lee & Hurt, 2018; Hurt et al., 2006; Bloom et al., 2010; Oakley et al., 2010). As such, improved methods of treating influenza B, particularly in these high-risk categories, are still needed.

[0004] 3 4890-9150-3059, v.1 SUMMARY Thus, in accordance with the present disclosure, there is provided a method of detecting an influenza B virus infection in a subject comprising (a) contacting a sample from said subject with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) detecting influenza B virus in said sample by binding of said antibody or antibody fragment to an influenza B virus antigen in said sample. The sample may be a body fluid, including blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces. Detection may comprise ELISA, RIA, lateral flow assay or western blot. The method may further comprise performing steps (a) and (b) a second time and determining a change in influenza B virus antigen levels as compared to the first assay. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone- paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. In another embodiment, there is provided a method of treating a subject infected with influenza B virus or reducing the likelihood of infection of a subject at risk of contracting influenza B virus, comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-

[0005] 4 4890-9150-3059, v.1 paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may be administered prior to infection or after infection. The subject may be a pregnant female, a sexually active female, or a female undergoing fertility treatments. Delivering may comprise antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In yet another embodiment, there is provided a monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. The

[0006] 5 4890-9150-3059, v.1 antibody or antibody fragment may further comprise a cell penetrating peptide and / or is an intrabody. In still yet another embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone- paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone- paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment mat further comprise a cell penetrating peptide and / or is an intrabody. In still yet another embodiment, there is provided a vaccine formulation comprising one or more antibodies or antibody fragments characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The one or more antibodies or antibody fragments may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The one or more antibodies or antibody fragments may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences

[0007] 6 4890-9150-3059, v.1 from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The one or more antibody fragments may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The one or more antibodies may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA- PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. At least one of said antibodies or antibody fragments further may comprise a cell penetrating peptide and / or is an intrabody. In a further embodiment, there is provided a vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment as defined herein. The expression vector(s) may be Sindbis virus or VEE vector(s). The vaccine formulation of claims may be formulated for delivery by needle injection, jet injection, or electroporation. The vaccine formulation may further comprise one or more expression vectors encoding for a second distinct antibody or antibody fragment. In yet a further embodiment, there is provided a method of reducing the risk of serious illness, hospitalization or death in a “high risk” subject infected with or at risk of infection with influenza B virus comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone- paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to

[0008] 7 4890-9150-3059, v.1 increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may be administered prior to infection or after infection. The subject may be a under the age of 12, over the age of 60, may be immunocompromised, or may suffer from an underlying medical condition increasing risk of serious illness, hospitalization or death resulting from viral infection. Delivering may comprise antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment. The antibody or antibody may reduce severity of disease and / or hospitalization as compared to an untreated control and / or may reduce risk of death as compared to an untreated control. In still yet a further embodiment, there is provided a method of determining the antigenic integrity, correct conformation and / or correct sequence of an influenza B virus antigen comprising (a) contacting a sample comprising said antigen with a first antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) determining antigenic integrity, correct conformation and / or correct sequence of said antigen by detectable binding of said first antibody or antibody fragment to said antigen. The sample may comprise recombinantly produced antigen or a vaccine formulation or vaccine production batch. Detection may comprise ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining. The first antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone- paired sequences as set forth in Table 1. The first antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The first antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment. The method may further comprise performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time.

[0009] 8 4890-9150-3059, v.1 The method may also further comprise (c) contacting a sample comprising said antigen with a second antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (d) determining antigenic integrity of said antigen by detectable binding of said second antibody or antibody fragment to said antigen. The second antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The second antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The second antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment. The method may further comprise performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0010] 9 4890-9150-3059, v.1 BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGS.1A-B. Vaccinated donor mounts influenza B NA response. (FIG.1A) Study design. A healthy adult individual was studied following intramuscular administration of the quadrivalent 2018-19 seasonal influenza vaccine Flucelvax™. Blood and bone marrow aspirates were collected after vaccination for cells secreting antibodies to IBV and the resulting mAb from single cells were expressed in microscale. The circle plot shows 17 of the total 64 clones secreted antibodies that reacted with IBV NA protein. (FIG.1B) Phylogenetic trees of the antibody variable genes encoding the mAbs, and variable gene usage table. Phylogenetic tree clonal families were constructed on heavy chain V-D-J-REGION by a neighbor-joining method using the VHgermline gene as an outgroup. Alignments were performed in MUSCLE. Trees were inferred using PHYLIP and visualized using FigTree. Circles correspond with the tissue sample from which the sequence was isolated: plasmablast (PB) or long-lived plasma cells (LLPC) in the bone marrow, or both. FIGS. 2A-B. MAbs in two clonal families bind NAs from both IBV lineages and recognize distinct binding sites. (FIG.2A) Binding of 16 NA-reactive mAbs, positive control mAb (+)r1G05 or isotype-matched negative control (-)r2D22 to recombinant NA from indicated IBV strains in ELISA. The mAb (+)r1G05 was competed for binding as a reference mAb recognizing the NA active site. The binding of reference mAbs to B / Iowa / 06 / 2017 (V) was measured in the presence of a saturating concentration of competitor mAb in a competition ELISA and normalized to the binding observed in the presence of the dengue virus mAb (-)r2D22. Black denotes full competition (<70% binding of reference mAb), and white denotes no competition (>71% binding of reference mAb). The orange box represents the clonally expanded family of mAbs that binds to the active site as indicated by competition with (+)r1G05. The pale pink box represents a family of clonally expanded mAbs that recognizes a different antigenic site. Data are representative of two independent experiments. (FIG. 2B) Binding to influenza B virus NA protein (half maximal effective concentration for binding [EC50] value; ng / mL) FIGS. 3A-C. Anti-NA human mAbs broadly inhibit viral activity. (FIG. 3A) Heatmap indicating potency of mAbs: enzyme-linked lectin assays (ELLA; IC50 values); NA-

[0011] 10 4890-9150-3059, v.1 Fluor assay (IC50values); egress assay (IC100values); real-time cell analysis (RTCA) neutralization (IC50values). Data are representative of two experiments. (FIG.3B). Air-liquid interface culture of primary human tracheal respiratory epithelial cells inoculated at an MOI of 0.1 and treated with 10 µg / mL of anti-NA mAb, isotype-matched negative control mAb, or virus-only condition. Incubation time was restricted to 8 hpi to allow for attachment but not egress. Cells were fixed and stained with anti-NP mAb (red) and anti-e-cadherin antibody (green). (FIG. 3C). Images acquired as indicated in FIG. 3B were captured on a Zeiss 710 confocal microscope. Data are representative of 9 fields of view, and two experiments were performed. FIGS. 4A-D. Anti-NA mAbs mediate protection in vivo against lethal IBV challenge in mice. (FIG. 4A) Study design. Groups of BALB / c mice were inoculated intraperitoneally (IP) 12 hours before virus challenge with 10 mg / kg of anti-NA mAb, (-)r2D22 control mAb reactive to an irrelevant antigen (dengue virus) or a control recombinant mAb recognizing IBV NA (+)r1G05. Mice were challenged intranasally (IN) with a lethal dose of indicated IBV virus and monitored daily for protection. The weights are represented as the group mean ± SEM (FIGS. 4B-C). The lower dotted line indicates the no-recovery threshold (>30% weight loss) and endpoint for euthanasia. Survival curves were estimated using the Kaplan-Meier method (FIGS. 4B-C). Survival of each group treated with an anti-NA or (+)r1G05 mAb was compared with the dengue virus mAb control treatment group using the log-rank (Mantel-Cox) test. n=5 mice per group. (FIG. 4D) Histophathology and RNAScope show efficacy against disease and virus replication in tissues mediated by FluB antibodies. FIGS.5A-D. Airway delivery of anti-influenza active site mAbs is effective. (FIG. 5A) Study design. Groups of BALB / c mice were inoculated by the intranasal (IN) route with a lethal dose of influenza B / New York / PV01181 / 2018 virus, and one day later (d1) inoculated by the IN or intraperitoneal (IP) route with 5 mg / kg of indicated anti-NA mAbs, with (-)r2D22 control mAb, or with (+)r1G05 positive control mAb, and monitored for protection (FIG. 5C and FIG.5E). The weights (FIG.5B and FIG.5D) represent the group mean ± SEM. The lower dotted line indicates the no-recovery threshold (> 30% weight loss) and endpoint for euthanasia. Survival curves were estimated using the Kaplan-Meier method and compared as indicated using log-rank (Mantel-Cox) test (FIG.5B and FIG.5C). Data represent one experiment, n= 5 mice per group. (FIG.5D) Histopathology of lungs shows reduction of virus mediated by FluB- 400. FIG.6. Cryo-EM reconstruction of NA-FluB-400. Cryo-EM reconstructions of Fab FluB-400 (top left) in complex with NA. One NA tetramer (gray) bound to four Fabs. Overall

[0012] 11 4890-9150-3059, v.1 architecture of FluB-400 binding to NA (top right). Specific interactions of FluB-400 with NA (bottom. FIGS. 7A-G. Cryo-EM reconstruction of NA-FluB-393 and NA-2D10. (FIG. 7A) IGHV and IGLV gene usage, and amino acid sequence of the HCDR3 for mAbs FluB-393 (SEQ ID NO: 811) and 2D10 (SEQ ID NO: 812). Cryo-EM reconstructions of Fab FluB-393 and 2D10 (FIGS. 7B-C) in complex with NA. One NA tetramer (gray) bound to four Fabs. (FIG.7D) Common loop being targeted by three mAbs: orange FluB-393, blue 2D10, red NA- 63. (FIGS. 7E-F) Specific interactions of FluB-393 with NA. (FIG.7G) Specific interactions of 2D10 with NA.

[0013] 12 4890-9150-3059, v.1 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS As discussed above, influenza B virus is less dangerous than the closely related influenza A virus, but still presents a significant health risk to certain individuals. Here, the inventors describe isolation of human mAbs recognizing IBV NA from the circulating blood plasmablasts or long-lived plasma cells in the bone marrow of an individual following vaccination with the 2018-2019 quadrivalent influenza vaccine. Antibody variable gene sequences from the isolated B cell clones were obtained, and selected mAbs were expressed to yield a panel of 17 antibodies reactive with IBV NA proteins. The sequences of these mAbs fell into lineages of two clonally expanded families with representative clones designated FluB- 393 or FluB-400. The mAb FluB-400 recognized contact residues within the active site of NA, exhibited potent inhibition of NA enzymatic activity, and prevented IBV attachment to primary human respiratory epithelial cells in culture. FluB-400 protected mice from IBV authentic virus airway challenge the when the mAb was administered by systemic (intraperitoneal) or topical (intranasal) routes. These and other aspects of the disclosure are described in detail below. I. Influenza B Virus Influenza B virus is the only species in the genus Betainfluenzavirus in the virus family Orthomyxoviridae. Influenza B virus is known only to infect humans and seals. This limited host range is apparently responsible for the lack of associated influenza pandemics in contrast with those caused by the morphologically similar influenza A virus as both mutate by both antigenic drift and reassortment. There are two known circulating lineages of Influenza B virus based on the antigenic properties of the surface glycoprotein hemagglutinin. The lineages are termed B / Yamagata / 16 / 88-like and B / Victoria / 2 / 87-like viruses. The quadrivalent influenza vaccine licensed by the CDC is currently designed to protect against both co- circulating lineages and has been shown to have greater effectiveness in prevention of influenza caused by Influenza B virus than the previous trivalent vaccine. Further diminishing the impact of this virus, in humans, influenza B viruses evolve slower than A viruses and faster than C viruses. Influenza B virus mutates at a rate 2 to 3 times slower than type A. Nevertheless, it is accepted that Influenza B virus could cause significant morbidity and mortality worldwide, and significantly impacts adolescents and schoolchildren. The B / Yamagata lineage might have become extinct in 2020 / 2021 due to COVID-19 pandemic measures. The influenza B virus capsid is enveloped while its virion consists of an envelope, a matrix protein, a nucleoprotein complex, a nucleocapsid, and a polymerase complex. Influenza

[0014] 13 4890-9150-3059, v.1 B virus virions can have spherical, irregular or filamentous appearances and are surrounded by a membrane. The membrane contains viral HA, NA, NB and BM2 which are membrane integrated. Type A and B influenza viruses cannot be distinguished based upon their morphology or size as determined by electron microscopy. The average size of spherical influenza B virions of the prototypic B / Lee / 40 strain grown in embryonated chicken eggs is 137 ± 27 nm. Eight viral negative-strand RNA gene segments with different configuration, together with the viral nucleoprotein and polymerase, exist as viral RNPs inside influenza B virus virions. Purified virions appear to contain 400–500 spike-like projections per spherical virion corresponding to the viral glycoproteins. M1 is the most abundant virion component followed by NP, HA and NA, and the level of NB and P proteins were the lowest. The subtypes of influenza A virus are estimated to have diverged 2,000 years ago. Influenza viruses A and B are estimated to have diverged from a single ancestor around 4,000 years ago, while the ancestor of influenza viruses A and B and the ancestor of influenza virus C are estimated to have diverged from a common ancestor around 8,000 years ago. Metatranscriptomics studies have also identified closely related “influenza B-like viruses such as the Wuhan spiny eel influenza virus and “influenza-B like” viruses in a number of vertebrate species such as salamanders and fish. II. Monoclonal Antibodies and Production Thereof An "isolated antibody" is one that has been separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In particular embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most particularly more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore

[0015] 14 4890-9150-3059, v.1 contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable region (VH) followed by three constant domains (CH) for each of the alpha and gamma chains and four CHdomains for mu and isotypes. Each L chain has at the N-terminus, a variable region (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CLis aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. They gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The term "variable" refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to

[0016] 15 4890-9150-3059, v.1 the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD). The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31- 35 (H1), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or those residues from a "hypervariable loop" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and / or those residues from a "hypervariable loop" / CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VHwhen numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63, 74- 75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VsubH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)). By "germline nucleic acid residue" is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. "Germline gene" is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A "germline mutation" refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.

[0017] 16 4890-9150-3059, v.1 The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent 4,816,567) after single cell sorting of an antigen specific B cell, an antigen specific plasmablast responding to an infection or immunization, or capture of linked heavy and light chains from single cells in a bulk sorted antigen specific collection. The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. A. General Methods It will be understood that monoclonal antibodies binding to influenza B virus will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing influenza B virus infection, as well as for treating the same. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265). The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection or vaccination with a licensed or experimental vaccine. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often

[0018] 17 4890-9150-3059, v.1 necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis- biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants in animals include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants and aluminum hydroxide adjuvant and in humans include alum, CpG, MFP59 and combinations of immunostimulatory molecules (“Adjuvant Systems”, such as AS01 or AS03). Additional experimental forms of inoculation to induce influenza B virus-specific B cells is possible, including nanoparticle vaccines, or gene-encoded antigens delivered as DNA or RNA genes in a physical delivery system (such as lipid nanoparticle or on a gold biolistic bead), and delivered with needle, gene gun, transcutaneous electroporation device. The antigen gene also can be carried as encoded by a replication competent or defective viral vector such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively a virus like particle. In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects that have been exposed to the pathogens, such as those who have been diagnosed as having contracted the disease, or those who have been vaccinated to generate protective immunity against the pathogen or to test the safety or efficacy of an experimental vaccine. Circulating anti-pathogen antibodies can be detected, and antibody encoding or producing B cells from the antibody-positive subject may then be obtained. The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate MAbs.

[0019] 18 4890-9150-3059, v.1 Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs like lung or GI tract, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal or immune human are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human / mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells. Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. In some cases, transformation of human B cells with Epstein Barr virus (EBV) as an initial step increases the size of the B cells, enhancing fusion with the relatively large-sized myeloma cells. Transformation efficiency by EBV is enhanced by using CpG and a Chk2 inhibitor drug in the transforming medium. Alternatively, human B cells can be activated by co-culture with transfected cell lines expressing CD40 Ligand (CD154) in medium containing additional soluble factors, such as IL-21 and human B cell Activating Factor (BAFF), a Type II member of the TNF superfamily. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v / v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp.71-74, 1986) and there are processes for better efficiency (Yu et al., 2008). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10-6to 1 x 10-8, but with optimized procedures one can achieve fusion efficiencies close to 1 in 200 (Yu et al., 2008). However, relatively low efficiency of fusion does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred agents are

[0020] 19 4890-9150-3059, v.1 aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the medium is supplemented with hypoxanthine. Ouabain is added if the B cell source is an EBV- transformed human B cell line, in order to eliminate EBV-transformed lines that have not fused to the myeloma. The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain may also be used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant. Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in

[0021] 20 4890-9150-3059, v.1 vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer. It also is contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. Single B cells labelled with the antigen of interest can be sorted physically using paramagnetic bead selection or flow cytometric sorting, then RNA can be isolated from the single cells and antibody genes amplified by RT-PCR. Alternatively, antigen- specific bulk sorted populations of cells can be segregated into microvesicles and the matched heavy and light chain variable genes recovered from single cells using physical linkage of heavy and light chain amplicons, or common barcoding of heavy and light chain genes from a vesicle. Matched heavy and light chain genes form single cells also can be obtained from populations of antigen specific B cells by treating cells with cell-penetrating nanoparticles bearing RT-PCR primers and barcodes for marking transcripts with one barcode per cell. The antibody variable genes also can be isolated by RNA extraction of a hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 104times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies. Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates.

[0022] 21 4890-9150-3059, v.1 B. Antibodies of the Present Disclosure Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity. Those of skill in the art, by assessing the binding specificity / affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. For example, the epitope to which a given antibody bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within the antigen molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope). Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody “interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Cross-blocking can be measured in various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke, 2004, Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, high-resolution electron microscopy techniques using single particle reconstruction, cryoEM, or tomography, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring, 1999, Analytical Biochemistry 267: 252-259; Engen and Smith, 2001, Anal. Chem. 73: 256A-265A. When the antibody

[0023] 22 4890-9150-3059, v.1 neutralizes influenza B virus, antibody escape mutant variant organisms can be isolated by propagating influenza B virus in vitro or in animal models in the presence of high concentrations of the antibody. Sequence analysis of the influenza B virus gene encoding the antigen targeted by the antibody reveals the mutation(s) conferring antibody escape, indicating residues in the epitope or that affect the structure of the epitope allosterically. The term “epitope” refers to a site on an antigen to which B and / or T cells respond. B- cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes. The present disclosure includes antibodies that may bind to the same epitope, or a portion of the epitope. Likewise, the present disclosure also includes antibodies that compete for binding to a target or a fragment thereof with any of the specific exemplary antibodies described herein. One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference, the reference antibody is allowed to bind to target under saturating conditions. Next, the ability of a test antibody to bind to the target molecule is assessed. If the test antibody is able to bind to the target molecule following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to the target molecule following saturation

[0024] 23 4890-9150-3059, v.1 binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody. To determine if an antibody competes for binding with a reference anti- influenza B virus antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to the influenza B virus antigen under saturating conditions followed by assessment of binding of the test antibody to the influenza B virus molecule. In a second orientation, the test antibody is allowed to bind to the influenza B virus antigen molecule under saturating conditions followed by assessment of binding of the reference antibody to the influenza B virus molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the influenza B virus, then it is concluded that the test antibody and the reference antibody compete for binding to the influenza B virus. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. Structural studies with EM or crystallography also can demonstrate whether or not two antibodies that compete for binding recognize the same epitope. In another aspect, there are provided monoclonal antibodies having clone-paired CDRs from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such antibodies

[0025] 24 4890-9150-3059, v.1 may be produced by the clones discussed below in the Examples section using methods described herein. In another aspect, the antibodies may be defined by their variable sequence, which include additional “framework” regions. These are provided in Tables 1 and 2 that encode or represent full variable regions. Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C, (e) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids may vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing applies to the nucleic acid sequences set forth as Table 1 and the amino acid sequences of Table 2. When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins-- Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645

[0026] 25 4890-9150-3059, v.1 Methods in Enzymology vol.183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11- 17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730. Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. One particular example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of an antibody sequence and the variable length of each gene requires multiple rounds of BLAST searches for a single antibody sequence. Also, manual assembly of different genes is difficult and error-prone. The sequence analysis tool IgBLAST (world-wide-web at ncbi.nlm.nih.gov / igblast / ) identifies matches to the germline V, D and J genes, details at rearrangement junctions, the delineation of Ig V domain framework regions and complementarity determining regions. IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches and allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation

[0027] 26 4890-9150-3059, v.1 of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. In one approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity. Yet another way of defining an antibody is as a “derivative” of any of the below- described antibodies and their antigen-binding fragments. The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non- naturally occurring amino acid residues. The term “derivative” encompasses, for example, as variants having altered CH1, hinge, CH2, CH3 or CH4 regions, so as to form, for example, antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics. The term “derivative” additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N- acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-

[0028] 27 4890-9150-3059, v.1 glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment, the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R. L. et al. (2002), J. Biol. Chem.277(30): 26733-26740; Davies J. et al. (2001), Biotechnology & Bioengineering 74(4): 288-294). Methods of altering carbohydrate contents are known to those skilled in the art, see, e.g., Wallick, S. C. et al. (1988), J. Exp. Med.168(3): 1099-1109; Tao, M. H. et al. (1989), J. Immunol. 143(8): 2595-2601; Routledge, E. G. et al. (1995), Transplantation 60(8):847-53; Elliott, S. et al. (2003), Nature Biotechnol.21:414-21; Shields, R. L. et al. (2002), J. Biol. Chem.277(30): 26733-26740). A derivative antibody or antibody fragment can be generated with an engineered sequence or glycosylation state to confer preferred levels of activity in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody- dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions as measured by bead-based or cell-based assays or in vivo studies in animal models. A derivative antibody or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative will possess a similar or identical function as the parental antibody. In another embodiment, an antibody derivative will exhibit an altered activity relative to the parental antibody. For example, a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody. C. Engineering of Antibody Sequences In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques,

[0029] 28 4890-9150-3059, v.1 or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. The following is a general discussion of relevant goals techniques for antibody engineering. Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns. Recombinant full-length IgG antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant. Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows. Expression of nucleic acids encoding antibodies, both for the purpose of subsequent antibody purification, and for immunization of a host, is also contemplated. Antibody coding sequences can be RNA, such as native RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In addition to turning off the immune / eIF2α phosphorylation-dependent inhibition of translation, incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment. Such modifications could be used to enhance antibody expression in vivo following inoculation with RNA. The RNA, whether native or modified, may be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle. Alternatively, DNA encoding the antibody may be employed for the same purposes. The DNA is included in an expression cassette comprising a promoter active in the host cell for which it is designed. The expression cassette is advantageously included in a replicable

[0030] 29 4890-9150-3059, v.1 vector, such as a conventional plasmid or minivector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses are contemplated. Replicons encoding antibody genes such as alphavirus replicons based on VEE virus or Sindbis virus are also contemplated. Delivery of such vectors can be performed by needle through intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation when in vivo expression is desired. The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g / L was achieved within 9 weeks of transfection. Antibody molecules will comprise fragments (such as F(ab′), F(ab′)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. F(ab′) antibody derivatives are monovalent, while F(ab′)2antibody derivatives are bivalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule. In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0031] 30 4890-9150-3059, v.1 It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3). It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency. Alternatively or additionally, it may be useful to combine amino acid modifications with one or more further amino acid modifications that alter C1q binding and / or the complement dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19 binding molecule. The binding polypeptide of particular interest may be one that binds to C1q and displays complement dependent cytotoxicity. Polypeptides with pre-existing C1q binding activity, optionally further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced. Amino acid modifications that alter C1q and / or modify

[0032] 31 4890-9150-3059, v.1 its complement dependent cytotoxicity function are described, for example, in WO / 0042072, which is hereby incorporated by reference. One can design an Fc region of an antibody with altered effector function, e.g., by modifying C1q binding and / or FcγR binding and thereby changing CDC activity and / or ADCC activity. “Effector functions” are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.). For example, one can generate a variant Fc region of an antibody with improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or ablated, a variant Fc region can be engineered with reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa). FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to the FcRn have been described (Shields et al., (2001). High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR, (J. Biol. Chem.276:6591-6604). A number of methods are known that can result in increased half- life (Kuo and Aveson, (2011)), including amino acid modifications may be generated through techniques including alanine scanning mutagenesis, random mutagenesis and screening to assess the binding to the neonatal Fc receptor (FcRn) and / or the in vivo behavior. Computational strategies followed by mutagenesis may also be used to select one of amino acid mutations to mutate. The present disclosure therefore provides a variant of an antigen binding protein with optimized binding to FcRn. In a particular embodiment, the said variant of an antigen binding protein comprises at least one amino acid modification in the Fc region of said antigen binding protein, wherein said modification is selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285,

[0033] 32 4890-9150-3059, v.1 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fc region as compared to said parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat. In a further aspect of the disclosure the modifications are M252Y / S254T / T256E. Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified, see for example Kontermann (2009) either by introducing an FcRn-binding polypeptide into the molecules or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fc receptors have been greatly reduced or fusing with FcRn binding domains of antibodies. Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, particularly a human. Such alterations may result in a half- life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half- lives of the antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus reduces the frequency of the administration of said antibodies or antibody fragments and / or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor. Beltramello et al. (2010) previously reported the modification of neutralizing mAbs, due to their tendency to enhance dengue virus infection, by generating in which leucine residues at positions 1.3 and 1.2 of CH2 domain (according to the IMGT unique numbering for C-domain) were substituted with alanine residues. This modification, also known as “LALA” mutation, abolishes antibody binding to FcγRI, FcγRII and FcγRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their capacity to neutralize and enhance infection by the four dengue virus serotypes. LALA variants retained

[0034] 33 4890-9150-3059, v.1 the same neutralizing activity as unmodified mAb but were completely devoid of enhancing activity. LALA mutations of this nature are therefore contemplated in the context of the presently disclosed antibodies. Altered Glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody, or antigen binding fragment thereof, containing a substantially homogeneous glycan without sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be attached to heavy chain or light chain constant regions respectively. The aforementioned substantially homogeneous glycan may be covalently attached to the heavy chain constant region. Another embodiment of the present disclosure comprises a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody, or antigen binding fragment thereof, is present in a substantially homogenous composition represented by the GNGN or G1 / G2 glycoform. Fc glycosylation plays a significant role in anti-viral and anti-cancer properties of therapeutic mAbs. The disclosure is in line with a recent study that shows increased anti- lentivirus cell-mediated viral inhibition of a fucose free anti-HIV mAb in vitro. This embodiment of the present disclosure with homogenous glycans lacking a core fucose, showed increased protection against specific viruses by a factor greater than two-fold. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs). The isolated monoclonal antibody, or antigen binding fragment thereof, comprising a substantially homogenous composition represented by the GNGN or G1 / G2 glycoform exhibits increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibody without the substantially homogeneous GNGN glycoform and with G0, G1F, G2F, GNF, GNGNF or GNGNFX containing glycoforms. In one embodiment of the present disclosure, the antibody dissociates from Fc gamma RI with a Kd of 1 x 10-8M or less and from Fc gamma RIII with a Kd of 1 x 10-7M or less. Glycosylation of an Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O- linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are

[0035] 34 4890-9150-3059, v.1 asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site. The glycosylation pattern may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and / or adding one or more glycosylation site(s) that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites. In certain embodiments, the antibody is expressed in cells that express beta (1,4)-N- acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing antibodies in such a fashion are provided in WO / 9954342, WO / 03011878, patent publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be altered to enhance or reduce or eliminate certain post-translational modifications, such as glycosylation, using genome editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). For example, CRISPR technology can be used to eliminate genes encoding glycosylating enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies. Elimination of monoclonal antibody protein sequence liabilities. It is possible to engineer the antibody variable gene sequences obtained from human B cells to enhance their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs associated with sites containing: 1) Unpaired Cys residues, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) SYE truncation, 6) Met oxidation, 7) Trp oxidation, 8) N-terminal glutamate, 9) Integrin binding,

[0036] 35 4890-9150-3059, v.1 10) CD11c / CD18 binding, or 11) Fragmentation Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding recombinant antibodies. Protein engineering efforts in the field of development of therapeutic antibodies clearly reveal that certain sequences or residues are associated with solubility differences (Fernandez- Escamilla et al., Nature Biotech., 22 (10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106 (29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21 (2), 385-392, 2010) Evidence from solubility-altering mutations in the literature indicate that some hydrophilic residues such as aspartic acid, glutamic acid, and serine contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine. Stability. Antibodies can be engineered for enhanced biophysical properties. One can use elevated temperature to unfold antibodies to determine relative stability, using average apparent melting temperatures. Differential Scanning Calorimetry (DSC) measures the heat capacity, Cp, of a molecule (the heat required to warm it, per degree) as a function of temperature. One can use DSC to study the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAb structure, producing up to three peaks in the thermogram (from unfolding of the Fab, CH2, and CH3 domains). Typically unfolding of the Fab domain produces the strongest peak. The DSC profiles and relative stability of the Fc portion show characteristic differences for the human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun.355, 751-757, 2007). One also can determine average apparent melting temperature using circular dichroism (CD), performed with a CD spectrometer. Far-UV CD spectra will be measured for antibodies in the range of 200 to 260 nm at increments of 0.5 nm. The final spectra can be determined as averages of 20 accumulations. Residue ellipticity values can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg / mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C / min. One can use dynamic light scattering (DLS) to assess for propensity for aggregation. DLS is used to characterize size of various particles including proteins. If the system is not disperse in size, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, and particle concentration. Since DLS essentially measures fluctuations in scattered light intensity due to particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays the

[0037] 36 4890-9150-3059, v.1 particle population at different diameters. Stability studies can be done conveniently using DLS. DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pI of the protein. Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pIs). This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step. The technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 µg / mL. Solubility. One can determine the intrinsic solubility score of antibody sequences. The intrinsic solubility scores can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequences for residues 95-102 (Kabat numbering) in HCDR3 of each antibody fragment such as a scFv can be evaluated via the online program to calculate the solubility scores. One also can determine solubility using laboratory techniques. Various techniques exist, including addition of lyophilized protein to a solution until the solution becomes saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most straightforward method is induction of amorphous precipitation, which measures protein solubility using a method involving protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation gives quick and accurate information on relative solubility values. Ammonium sulfate precipitation produces precipitated solutions with well-defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using induction of amorphous precipitation by ammonium sulfate also can be done easily at different pH values. Protein

[0038] 37 4890-9150-3059, v.1 solubility is highly pH dependent, and pH is considered the most important extrinsic factor that affects solubility. Autoreactivity. Generally, it is thought that autoreactive clones should be eliminated during ontogeny by negative selection, however it has become clear that many human naturally occurring antibodies with autoreactive properties persist in adult mature repertoires, and the autoreactivity may enhance the antiviral function of many antibodies to pathogens. It has been noted that HCDR3 loops in antibodies during early B cell development are often rich in positive charge and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). One can test a given antibody for autoreactivity by assessing the level of binding to human origin cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometric cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity also can be surveyed using assessment of binding to tissues in tissue arrays. Preferred residues (“Human Likeness”). B cell repertoire deep sequencing of human B cells from blood donors is being performed on a wide scale in many recent studies. Sequence information about a significant portion of the human antibody repertoire facilitates statistical assessment of antibody sequence features common in healthy humans. With knowledge about the antibody sequence features in a human recombined antibody variable gene reference database, the position specific degree of “Human Likeness” (HL) of an antibody sequence can be estimated. HL has been shown to be useful for the development of antibodies in clinical use, like therapeutic antibodies or antibodies as vaccines. The goal is to increase the human likeness of antibodies to reduce potential adverse effects and anti-antibody immune responses that will lead to significantly decreased efficacy of the antibody drug or can induce serious health implications. One can assess antibody characteristics of the combined antibody repertoire of three healthy human blood donors of about 400 million sequences in total and created a novel “relative Human Likeness” (rHL) score that focuses on the hypervariable region of the antibody. The rHL score allows one to easily distinguish between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in human repertoires. D. Single Chain Antibodies A single chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification

[0039] 38 4890-9150-3059, v.1 usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using Protein L since Protein L interacts with the variable region of kappa light chains. Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single- chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 106different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VHC terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin / avidin, which permit the combination of two antibodies. In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge). Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different

[0040] 39 4890-9150-3059, v.1 analogs can be created. To link two different compounds in a step-wise manner, hetero- bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation. An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent). It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic / preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents. Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site. The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue. In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0041] 40 4890-9150-3059, v.1 U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and / or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug. U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. E. Multispecific Antibodies In certain embodiments, antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti-pathogen arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and Fc gamma RIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess a pathogen-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab′)2bispecific antibodies). WO 96 / 16673 describes a bispecific anti-ErbB2 / anti-Fc gamma RIII antibody and U.S. Patent 5,837,234 discloses a bispecific anti-ErbB2 / anti-Fc gamma RI antibody. A bispecific anti-ErbB2 / Fc alpha antibody is shown in WO98 / 02463. U.S. Patent 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy

[0042] 41 4890-9150-3059, v.1 chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93 / 08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991). According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination. In a particular embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94 / 04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). According to another approach described in U.S. Patent 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains

[0043] 42 4890-9150-3059, v.1 (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent 4,676,980), and for treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent 4,676,980, along with a number of cross-linking techniques. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Techniques exist that facilitate the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol.16, 677–681 (1998). doi:10.1038 / nbt0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol.,

[0044] 43 4890-9150-3059, v.1 148(5):1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994). In a particular embodiment, a bispecific or multispecific antibody may be formed as a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Patents 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264; Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences. Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60, 1991; Xu et al., Science, 358(6359):85-90, 2017). A multivalent antibody may be internalized (and / or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen

[0045] 44 4890-9150-3059, v.1 binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH- CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides contemplated here comprise a light chain variable region and, optionally, further comprise a CL domain. Charge modifications are particularly useful in the context of a multispecific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi- / multispecific antigen binding molecules with a VH / VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015 / 150447, particularly the examples therein, incorporated herein by reference in its entirety). Accordingly, in particular embodiments, an antibody comprised in the therapeutic agent comprises (a) a first Fab molecule which specifically binds to a first antigen (b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, wherein the first antigen is an activating T cell antigen and the second antigen is a target cell antigen, or the first antigen is a target cell antigen and the second antigen is an activating T cell antigen; and wherein i) in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the

[0046] 45 4890-9150-3059, v.1 amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index); or ii) in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index). The antibody may not comprise both modifications mentioned under i) and ii). The constant domains CL and CH1 of the second Fab molecule are not replaced by each other (i.e., remain unexchanged). In another embodiment of the antibody, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). In a further embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). In a particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). In a more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to

[0047] 46 4890-9150-3059, v.1 Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). In an even more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). F. Chimeric Antigen Receptors Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell, with transfer of their coding sequence facilitated by retroviral vectors. In this way, a large number of target-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach show efficacy. The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g., neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19. The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows to the scFv to orient in different directions to enable antigen binding.

[0048] 47 4890-9150-3059, v.1 The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal. Type I proteins are in fact two protein domains linked by a transmembrane alpha helix in between. The cell membrane lipid bilayer, through which the transmembrane domain passes, acts to isolate the inside portion (endodomain) from the external portion (ectodomain). It is not so surprising that attaching an ectodomain from one protein to an endodomain of another protein results in a molecule that combines the recognition of the former to the signal of the latter. Ectodomain. A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence usually works fine. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in a scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used The antigen recognition domain is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g., CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact, almost anything that binds a given target with high affinity can be used as an antigen recognition region. A spacer region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgG1. Alternatives include the CH2CH3region of immunoglobulin and portions of CD3. For most scFv based constructs, the IgG1 hinge suffices. However, the best spacer often has to be determined empirically. Transmembrane domain. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Generally, the transmembrane domain from the most membrane proximal component of the endodomain is used. Interestingly, using the CD3-zeta transmembrane domain may result in incorporation of the artificial TCR into the native TCR a factor that is dependent on the presence of the native CD3-zeta transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly expressed, stable receptor.

[0049] 48 4890-9150-3059, v.1 Endodomain. This is the "business-end" of the receptor. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. "First-generation" CARs typically had the intracellular domain from the CD3 ξ- chain, which is the primary transmitter of signals from endogenous TCRs. "Second- CARs add intracellular signaling domains from various costimulatory protein receptors CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, "third-generation" CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further augment potency. G. ADCs Antibody Drug Conjugates or ADCs are a new class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with infectious disease. ADCs are complex molecules composed of an antibody (a whole mAb or an antibody fragment such as a single-chain variable fragment, or scFv) linked, via a stable chemical linker with labile bonds, to a biological active cytotoxic / anti-viral payload or drug. Antibody Drug Conjugates are examples of bioconjugates and immunoconjugates. By combining the unique targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates allow sensitive discrimination between healthy and diseased tissue. This means that, in contrast to traditional systemic approaches, antibody-drug conjugates target and attack the infected cell so that healthy cells are less severely affected. In the development ADC-based anti-tumor therapies, an anticancer drug (e.g., a cell toxin or cytotoxin) is coupled to an antibody that specifically targets a certain cell marker (e.g., a protein that, ideally, is only to be found in or on infected cells). Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cell or impairs viral replication. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other agents.

[0050] 49 4890-9150-3059, v.1 A stable link between the antibody and cytotoxic / anti-viral agent is a crucial aspect of an ADC. Linkers are based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (noncleavable) and control the distribution and delivery of the cytotoxic agent to the target cell. Cleavable and noncleavable types of linkers have been proven to be safe in preclinical and clinical trials. Brentuximab vedotin includes an enzyme-sensitive cleavable linker that delivers the potent and highly toxic antimicrotubule agent Monomethyl auristatin E or MMAE, a synthetic antineoplastic agent, to human specific CD30-positive malignant cells. Because of its high toxicity MMAE, which inhibits cell division by blocking the polymerization of tubulin, cannot be used as a single-agent chemotherapeutic drug. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor or TNF receptor) proved to be stable in extracellular fluid, cleavable by cathepsin and safe for therapy. Trastuzumab emtansine, the other approved ADC, is a combination of the microtubule-formation inhibitor mertansine (DM- 1), a derivative of the Maytansine, and antibody trastuzumab (Herceptin® / Genentech / Roche) attached by a stable, non-cleavable linker. The availability of better and more stable linkers has changed the function of the chemical bond. The type of linker, cleavable or noncleavable, lends specific properties to the cytotoxic (anti-cancer) drug. For example, a non-cleavable linker keeps the drug within the cell. As a result, the entire antibody, linker and cytotoxic agent enter the targeted cancer cell where the antibody is degraded to the level of an amino acid. The resulting complex – amino acid, linker and cytotoxic agent – now becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in the host cell where it releases the cytotoxic agent. Another type of cleavable linker, currently in development, adds an extra molecule between the cytotoxic / anti-viral drug and the cleavage site. This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics. Researchers are also developing a new method of peptide cleavage based on Edman degradation, a method of sequencing amino acids in a peptide. Future direction in the development of ADCs also include the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and α emitting immunoconjugates and antibody-conjugated nanoparticles. H. BiTES Bi-specific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies that are investigated for the use as anti-cancer drugs. They direct a host's immune

[0051] 50 4890-9150-3059, v.1 system, more specifically the T cells' cytotoxic activity, against infected cells. BiTE is a registered trademark of Micromet AG. BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other to an infected cell via a specific molecule. Like other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTEs form a link between T cells and target cells. This causes T cells to exert cytotoxic / anti-viral activity on infected cells by producing proteins like perforin and granzymes, independently of the presence of MHC I or co-stimulatory molecules. These proteins enter infected cells and initiate the cell's apoptosis. This action mimics physiological processes observed during T cell attacks against infected cells. I. Intrabodies In a particular embodiment, the antibody is a recombinant antibody that is suitable for action inside of a cell – such antibodies are known as “intrabodies.” These antibodies may interfere with target function by a variety of mechanism, such as by altering intracellular protein trafficking, interfering with enzymatic function, and blocking protein-protein or protein-DNA interactions. In many ways, their structures mimic or parallel those of single chain and single domain antibodies, discussed above. Indeed, single-transcript / single-chain is an important feature that permits intracellular expression in a target cell, and also makes protein transit across cell membranes more feasible. However, additional features are required. The two major issues impacting the implementation of intrabody therapeutic are delivery, including cell / tissue targeting, and stability. With respect to delivery, a variety of approaches have been employed, such as tissue-directed delivery, use of cell-type specific promoters, viral-based delivery and use of cell-permeability / membrane translocating peptides. With respect to the stability, the approach is generally to either screen by brute force, including methods that involve phage display and may include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement / modification. An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to subcellular regions such as the

[0052] 51 4890-9150-3059, v.1 cytoplasm, nucleus, mitochondria and ER have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997). By virtue of their ability to enter cells, intrabodies have additional uses that other types of antibodies may not achieve. In the case of the present antibodies, the ability to interact with the MUC1 cytoplasmic domain in a living cell may interfere with functions associated with the MUC1 CD, such as signaling functions (binding to other molecules) or oligomer formation. In particular, it is contemplated that such antibodies can be used to inhibit MUC1 dimer formation. J. Purification In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques. In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be

[0053] 52 4890-9150-3059, v.1 changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide. Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies are bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.). Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity. It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS / PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. III. Active / Passive Immunization and Treatment / Prevention of [INSERT] virus Infection A. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising anti-influenz B virus antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal,

[0054] 53 4890-9150-3059, v.1 vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation. Active vaccines are also envisioned where antibodies like those disclosed are produced in vivo in a subject at risk of influenza B virus infection. Such vaccines can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by intradermal and intramuscular routes are contemplated. The vaccine could alternatively be administered by a topical route directly to the mucosa, for example, by nasal drops, inhalation, by nebulizer, or via intrarectal or vaginal delivery. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for

[0055] 54 4890-9150-3059, v.1 intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable. Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. 2. ADCC Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or fragments thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. By “antibody having increased / reduced antibody dependent cell-mediated cytotoxicity (ADCC)” is meant an antibody having increased / reduced ADCC as determined by any suitable method known to those of ordinary skill in the art. As used herein, the term “increased / reduced ADCC” is defined as either an increase / reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or a reduction / increase in the concentration of antibody, in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The increase / reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using

[0056] 55 4890-9150-3059, v.1 the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the increase in ADCC mediated by an antibody produced by host cells engineered to have an altered pattern of glycosylation (e.g., to express the glycosyltransferase, GnTIII, or other glycosyltransferases) by the methods described herein, is relative to the ADCC mediated by the same antibody produced by the same type of non-engineered host cells. 3. CDC Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is the processes in the immune system that kill pathogens by damaging their membranes without the involvement of antibodies or cells of the immune system. There are three main processes. All three insert one or more membrane attack complexes (MAC) into the pathogen which cause lethal colloid-osmotic swelling, i.e., CDC. It is one of the mechanisms by which antibodies or antibody fragments have an anti-viral effect. IV. Antibody Conjugates Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non- limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin. Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948,

[0057] 56 4890-9150-3059, v.1 and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents. In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III). In the case of radioactive isotopes for therapeutic and / or diagnostic application, one might mention astatine211,14carbon,51chromium,36chlorine,57cobalt,58cobalt, copper67,152Eu, gallium67,3hydrogen, iodine123, iodine125, iodine131, indium111,59iron,32phosphorus, rhenium186, is often being preferred are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA). Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red. Additional types of antibodies contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a

[0058] 57 4890-9150-3059, v.1 chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten- based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate. Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents. Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate. In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector

[0059] 58 4890-9150-3059, v.1 or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O’Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation. V. Immunodetection Methods In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting influenza B virus and its associated antigens. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate / desired reactivity profiles. Other immunodetection methods include specific assays for determining the presence of influenza B virus in a subject. A wide variety of assay formats are contemplated, but specifically those that would be used to detect influenza B virus in a fluid obtained from a subject, such as saliva, blood, plasma, sputum, semen or urine. In particular, semen has been demonstrated as a viable sample for detecting influenza B virus (Purpura et al., 2016; Mansuy et al., 2016; Barzon et al., 2016; Gornet et al., 2016; Duffy et al., 2009; CDC, 2016; Halfon et al., 2010; Elder et al.2005). The assays may be advantageously formatted for non-healthcare (home) use, including lateral flow assays (see below) analogous to home pregnancy tests. These assays may be packaged in the form of a kit with appropriate reagents and instructions to permit use by the subject of a family member. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of influenza B virus antibodies directed to specific parasite epitopes in samples also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing influenza B virus and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0060] 59 4890-9150-3059, v.1 These methods include methods for purifying influenza B virus or related antigens from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the influenza B virus or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the influenza B virus antigen immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column. The immunobinding methods also include methods for detecting and quantifying the amount of influenza B virus or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing influenza B virus or its antigens and contact the sample with an antibody that binds influenza B virus or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing influenza B virus or influenza B virus antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine. Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to influenza B virus or antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art. The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary

[0061] 60 4890-9150-3059, v.1 immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected. Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired. One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible. Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method

[0062] 61 4890-9150-3059, v.1 up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. A. ELISAs Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used. In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the influenza B virus or influenza B virus antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-influenza B virus antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-influenza B virus antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. In another exemplary ELISA, the samples suspected of containing the influenza B virus or influenza B virus antigen are immobilized onto the well surface and then contacted with the anti- influenza B virus antibodies of the disclosure. After binding and washing to remove non- specifically bound immune complexes, the bound anti-influenza B virus antibodies are detected. Where the initial anti-influenza B virus antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-influenza B virus antibody, with the second antibody being linked to a detectable label.

[0063] 62 4890-9150-3059, v.1 Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand. “Under conditions effective to allow immune complex (antigen / antibody) formation” means that the conditions preferably include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in the reduction of nonspecific background. The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C or may be overnight at about 4°C or so. Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined. To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development

[0064] 63 4890-9150-3059, v.1 upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween). After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of influenza B virus antibodies in sample. In competition-based assays, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample. Here, the inventor proposes the use of labeled influenza B virus monoclonal antibodies to determine the amount of influenza B virus antibodies in a sample. The basic format would include contacting a known amount of influenza B virus monoclonal antibody (linked to a detectable label) with influenza B virus antigen or particle. The influenza B virus antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample. B. Western Blot The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / non-denaturing

[0065] 64 4890-9150-3059, v.1 conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing. The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension. In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non- specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using

[0066] 65 4890-9150-3059, v.1 labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies. C. Lateral Flow Assays Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized and costly equipment, though many laboratory-based applications exist that are supported by reading equipment. Typically, these tests are used as low resources medical diagnostics, either for home testing, point of care testing, or laboratory use. A widely spread and well-known application is the home pregnancy test. The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt- sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the final porous material – the wick – that simply acts as a waste container. Lateral Flow Tests can operate as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Patent 6,485,982.

[0067] 66 4890-9150-3059, v.1 D. Immunohistochemistry The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990). Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings. Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted. E. Immunodetection Kits In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect influenza B virus or influenza B virus antigens, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to influenza B virus or influenza B virus antigen, and optionally an immunodetection reagent. In certain embodiments, the influenza B virus antibody may be pre-bound to a solid support, such as a column matrix and / or well of a microtiter plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0068] 67 4890-9150-3059, v.1 Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure. The kits may further comprise a suitably aliquoted composition of the influenza B virus or influenza B virus antigens, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. F. Vaccine and Antigen Quality Control Assays The present disclosure also contemplates the use of antibodies and antibody fragments as described herein for use in assessing the antigenic integrity of a viral antigen in a sample. Biological medicinal products like vaccines differ from chemical drugs in that they cannot normally be characterized molecularly; antibodies are large molecules of significant complexity and have the capacity to vary widely from preparation to preparation. They are also administered to healthy individuals, including children at the start of their lives, and thus a strong emphasis must be placed on their quality to ensure, to the greatest extent possible, that they are efficacious in preventing or treating life-threatening disease, without themselves causing harm. The increasing globalization in the production and distribution of vaccines has opened new possibilities to better manage public health concerns but has also raised questions about the equivalence and interchangeability of vaccines procured across a variety of sources. International standardization of starting materials, of production and quality control testing, and the setting of high expectations for regulatory oversight on the way these products are manufactured and used, have thus been the cornerstone for continued success. But it remains a

[0069] 68 4890-9150-3059, v.1 field in constant change, and continuous technical advances in the field offer a promise of developing potent new weapons against the oldest public health threats, as well as new ones - malaria, pandemic influenza, and HIV, to name a few - but also put a great pressure on manufacturers, regulatory authorities, and the wider medical community to ensure that products continue to meet the highest standards of quality attainable. Thus, one may obtain an antigen or vaccine from any source or at any point during a manufacturing process. The quality control processes may therefore begin with preparing a sample for an immunoassay that identifies binding of an antibody or fragment disclosed herein to a viral antigen. Such immunoassays are disclosed elsewhere in this document, and any of these may be used to assess the structural / antigenic integrity of the antigen. Standards for finding the sample to contain acceptable amounts of antigenically correct and intact antigen may be established by regulatory agencies. Another important embodiment where antigen integrity is assessed is in determining shelf-life and storage stability. Most medicines, including vaccines, can deteriorate over time. Therefore, it is critical to determine whether, over time, the degree to which an antigen, such as in a vaccine, degrades or destabilizes such that is it no longer antigenic and / or capable of generating an immune response when administered to a subject. Again, standards for finding the sample to contain acceptable amounts of antigenically intact antigen may be established by regulatory agencies. In certain embodiments, viral antigens may contain more than one protective epitope. In these cases, it may prove useful to employ assays that look at the binding of more than one antibody, such as 2, 3, 4, 5 or even more antibodies. These antibodies bind to closely related epitopes, such that they are adjacent or even overlap each other. On the other hand, they may represent distinct epitopes from disparate parts of the antigen. By examining the integrity of multiple epitopes, a more complete picture of the antigen’s overall integrity, and hence ability to generate a protective immune response, may be determined. Antibodies and fragments thereof as described in the present disclosure may also be used in a kit for monitoring the efficacy of vaccination procedures by detecting the presence of protective influenza B virus antibodies. Antibodies, antibody fragment, or variants and derivatives thereof, as described in the present disclosure may also be used in a kit for monitoring vaccine manufacture with the desired immunogenicity.

[0070] 69 4890-9150-3059, v.1 VI. Examples The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – Materials & Methods Cell lines. The variant Madin-Darby canine kidney (MDCK) cell line MDCK-SIAT1 1 that was derived by stable transfection of MDCK cells with the cDNA of human 2,6- sialtransferase (SIAT1) was obtained from Sigma-Aldrich (catalog # 05071502-1VL). MDCK- SIAT1 cells were cultured in Dulbecco Minimal Essential Medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% penicillin- streptomycin and incubated at 37°C in 5% CO2. ExpiCHO (hamster, female origin) and FreeStyle 293F cell lines were purchased (Thermo Fisher Scientific) and cultured according to the manufacturer’s protocol. Drosophila S2 cells (Thermo Fisher Scientific catalog # 72851- 4003) were cultured in suspension at 37°C shaking at 125 RPM in Schneider’s Drosophila Medium (Thermo Fisher Scientific, catalog # 21720001) supplemented with 10% fetal bovine serum. The primary tracheal cells were purchased from Marsico Lung Institute Tissue Procurement and Cell Culture Core Facility, North Carolina School of Medicine, Chapel Hill, NC. All cell lines were tested for mycoplasma monthly, and all samples were negative. Human subject. The participating individual was a 48-year-old woman, vaccinated intramuscularly with 2018-2019 Influenza Vaccine (Flucelvax Quadrivalent; Seqirus, Inc.). Peripheral blood and bone marrow aspirate specimens were obtained from this individual after written informed consent. The study was approved by the Vanderbilt University Medical Center Institutional Review Board. Animal model. BALB / c mice were purchased from Jackson Laboratories (Bar Harbor). Breeding, maintenance, and experimentation complied with Vanderbilt Institutional Animal Care and Use Committee regulations. Details of mouse husbandry can be found in the “In vivo protection study” subsection. Viruses. B / Hawaii / 01 / 2018 (NA D197N) (Victoria), B / Florida / 78 / 2015 (Victoria), B / Maryland / 15 / 2016 (Victoria), B / Missouri / 12 / 2018 (NA D197E) (Victoria),

[0071] 70 4890-9150-3059, v.1 B / Jiangsu / 10 / 2003 (Yamagata), B / Indiana / 17 / 2017 (NA I221T) (Yamagata), B / Oklahoma / 10 / 2018 (NA D197N) (Yamagata), B / Wisconsin / 01 / 2010 (Yamagata), B / Wisconsin / 10 / 2016 (NA I221V) (Yamagata), (International Reagent Resource [IRR]) were propagated and titered in monolayer cultures of MDCK-SIAT1 cells. Cells were maintained in Dulbecco’s Modified Eagle Medium (GIBCO DMEM, Invitrogen) supplemented with 10% FBS, 1% penicillin, and streptomycin incubated at 37°C in 5% CO2. B / New York / PV01181 / 2018 (Victoria lineage) and B / New York / PV00094 / 2017 (Yamagata lineage) were kindly provided by Dr. Ted Ross (University of Georgia) and passaged in embryonated chicken eggs as previously described (Brauer & Chen, 2015). All viruses were manipulated under BSL-2 conditions. Isolation of peripheral blood mononuclear cells and B cell sorting. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using SepMate PBMC isolation tubes (StemCELL, catalog # 85450). PBMCs were then enriched for plasmablasts using negative selection with a paramagnetic bead kit that depleted irrelevant cell populations according to manufacturer protocol (StemCELL, catalog # 19409-A05U). Enriched cell populations were stained with anti-CD19-FITC antibody 1:20 dilution (eBioscience, catalog # 11-0199-42), anti-CD27-APC antibody 1:20 dilution (BD, catalog # 558664), and anti-CD38- PE 1:25 dilution (BD, catalog # 555460) antibody in RoboSep buffer (StemCELL, catalog # 20104) on ice for 1 hr. After staining, cells were centrifuged and resuspended in RoboSep containing DAPI as a viability dye. Viable CD19LowCD27highCD38highcells were sorted into sequencing buffer and plasmablasts were carried through single-cell RNA sequencing. Single- cell RNA capture was performed using the 10X Genomics Chromium platform with enrichment using the 5′ VDJ amplification kit (10X Genomics) according to the manufacturer’s instructions. Amplicons were sequenced on an Illumina NovaSeq 6000, and data were processed using the CellRanger software v3.1.0 (10X Genomics). Freshly collected bone marrow aspirate was washed with RoboSep buffer and filtered through a 70-micron strainer to remove unwanted bone shards and stromal clots that would interfere with downstream processing steps. Bone marrow plasma cells then were enriched using a paramagnetic bead kit and negative-selection cocktail that depleted irrelevant cell populations (StemCELL, catalog # 19409-A05U). Cells then were resuspended in a staining cocktail containing anti-CD19-FITC antibody 1:20 dilution (eBioscience, catalog # 11-0199- 42), anti-CD138-APC antibody 1:20 dilution (BioLegend, catalog # 52307), and anti-CD38- PE antibody 1:25 dilution (BD, catalog # 555460) in RoboSep buffer (StemCELL, catalog #

[0072] 71 4890-9150-3059, v.1 20104) on ice for 1 hr. After staining, cells were centrifuged and resuspended in RoboSep buffer containing DAPI as a viability dye. Viable CD19+CD138+CD38+cells and CD19- CD138+CD38+cells were sorted into sequencing buffer in a two-way sort. Antibody gene sequence analysis. B cells of interest were processed through the 10X Genomics workflow, and sequences were processed through Cell Ranger. All sequences then were analyzed using PyIR software (Soto et al., 2020) to determine the V gene, J gene, and the third complementarity determining region (CDR3) of each antibody variable region, as previously described (Soto et al., 2019). Specific combinations of V and J genes and CDR3s were designated V3J clonotypes, and for each of these clonotypes the inventor searched to determine if clonotypes occurred in multiple tissue sites by searching for exact sequence matches of CDR3 amino acid sequence in the bone marrow and plasmablast populations. MAb generation. Sequences of mAbs that had been synthesized as cDNAs (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (designated as pTwist- mCis_G1) were used for mAb secretion in mammalian cell culture. This plasmid IgG expression vector contains an enhanced 2A sequence and GSG amino acid linker that allows the simultaneous expression of mAb heavy and light chain genes from a single construct upon transfection (Gilchuk et al., 2020; Zost et al., 2020). The inventor previously described the microscale expression of mAbs 1 mL ExpiCHO cultures in 96-well plates. For larger-scale mAb expression, the inventor performed transfection (1 to 300 mL per antibody) of Chinese hamster ovary cell (Wagner et al., 2002) cultures using the Gibco ExpiCHO Expression System and protocol for 50 mL mini bioreactor tubes (Corning), as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe resin (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein BioSolutions). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-450-kDa centrifugal filter units (Millipore Sigma) and stored at 4°C until use. Purified mAbs were tested routinely for endotoxin levels (found to be less than 30 EU per mg IgG for mouse studies). Endotoxin testing was performed using the PTS201F cartridge (Charles River), with a sensitivity range from 10 to 0.1 EU per mL, and an Endosafe Nexgen-MCS instrument (Charles River). Expression and purification of NA antigens. Soluble, recombinant NA antigens were expressed and purified as previously described (Serris et al., 2020). Genes encoding the influenza B / Iowa / 06 / 2017 rNA (GenBank: CY221704.1) and influenza B / Singapore / INFTT- 16-0610 / 2016 (GSAID: EPI_ISL_226481) were codon-optimized for Drosophila cell expression and cloned into a plasmid (pT350) containing an MT promoter, BiP signal sequence,

[0073] 72 4890-9150-3059, v.1 and C-terminal double strep tag. Drosophila S2 cells were transfected with the NA and pCoBlast (Thermo Fisher Scientific) plasmids at a 19:1 ratio, respectively, and stably transfected cells were selected using 25 μg / mL of blasticidin. Stable cell lines were maintained in Schneider’s Drosophila Medium supplemented with 25 μg / mL blasticidin, grown in shaker flasks to a density of 1 x 107cells / mL, and induced with 4 μM 760 CdCl2. S2 cell supernatant was collected after five days, supplemented with 10 μg / mL of avidin, and purified through a StrepTrap HP column (Cytiva). The sample was purified further by size-exclusion chromatography on a HiLoad 16 / 600 Superdex column (Cytiva). Enzyme-linked immunosorbent assay (ELISA).384-well plates were coated with 1 μg / mL of purified rNA protein overnight at 4°C in 1× phosphate buffered saline (PBS). Plates were incubated with blocking buffer (2% non-fat dry milk, 2% goat serum, and 0.1% Tween- 20 in PBS) for 1 hr at room temperature. Diluted primary antibodies were added to appropriate wells and incubated for 1 hr at room temperature. The bound antibodies were detected using goat anti-human IgG conjugated with horseradish peroxidase (HRP) (Southern Biotech, catalog # 2040-05, lot B3919-XD29, 1:5,000 dilution) and a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, catalog # T0440). Color development was monitored, 1M hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose–response assays, serial dilutions of purified mAbs were applied to the wells in triplicate, and antibody binding was detected, as detailed above. EC50values for binding were determined using Prism software v.8.0 (GraphPad) after log transformation of the mAb concentration using sigmoidal dose–response nonlinear regression analysis. ELISAs were performed in technical triplicate and biological duplicate. Enzyme-linked lectin assay (ELLA).384-well flat-bottom microtiter plates (Thermo Fisher Scientific) were coated with 25 µL / well fetuin (Sigma) at a concentration of 25 µg / mL in 1× PBS without CaCl2or MgCl2at 4°C for at least 18 hr. Plates were washed three times with PBS-T. Three-fold serial mAb dilution was performed in a 96-well plate using sample diluent (Dulbecco's PBS [DPBS] with CaCl2and MgCl2, 1% BSA, 0.5% Tween-20) starting at 20 µg / mL. 25 µL of mAb from the dilution plate was transferred to a fetuin-coated plate in triplicate wells. Next, 25 µL of the diluted virus was added to all wells except the virus-free column.25 µL of sample diluent was added to the virus-free control column. The virus dilution was determined by selecting the dilution factor of the virus that yields the max optical density. Plates were placed in a humidified incubator at 33°C for 18 hr. The following day, the plates were washed six times, and 100 µL / well of peanut agglutinin (PNA)-HRP (Sigma) was added

[0074] 73 4890-9150-3059, v.1 at 1 mg / mL in PBS with 1% BSA. After a 2-hr-incubation at room temperature, the plates were developed with 25 µL TMB substrate (Thermo Fisher Scientific). Color development was monitored, 1M hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose–response assays, serial dilutions of purified mAbs were applied to the wells in triplicate, and inhibition was detected, as detailed above. Half maximal inhibitory concentrations (IC50) values for neutralization were determined using Prism v.8.0 software (GraphPad) after log transformation of the mAb concentration using sigmoidal dose–response nonlinear regression analysis. The data were analyzed using Excel (Microsoft) and Prism (GraphPad) software. The IC50 value was defined as the concentration of mAb at which 50% of the NA activity was inhibited compared to the negative control (virus with no mAb). Assays were performed in technical triplicate and biological duplicate. Neuraminidase inhibition measured by NA-Fluor assay. The NA-Fluor Influenza Neuraminidase Inhibitor Kit (Thermo Fisher, catalog # 4457091) was used to quantify the inhibition of NA activity (cleavage of a small chemiluminescent substrate MUNANA) in the presence of NA-mAbs. Before performing assays, each virus was tittered to determine the optimum dilution. The experiments were performed according to the manufacturer’s protocol. mAbs were diluted to a concentration of 20 µg / mL, and a serial three-fold dilution was performed. 6.25 µL from each dilution were transferred to a black, flat-bottom 384-well cell culture plate and mixed with 6.25 µL / well of the virus at a predetermined concentration for 30 min at 33°C. NA-Fluor Substrate (12.5 µL / well) was added after the incubation, and the plates were incubated at 33°C for 1 hr. NA-Fluor Stop solution (25 µL / well) was added to the plates. The chemiluminescent signal was detected by a microtiter plate reader (Bio-Tek) by exciting at 360 nm and detecting emission at 450 nm. Data were analyzed using Excel and Prism. The IC50value was defined as the concentration of mAb at which 50% of the NA activity was inhibited compared to the negative control (virus with no mAb). Assays were performed in technical triplicate and biological duplicate. Competition-binding ELISA. Wells of 384-well microtiter plates were coated with purified recombinant influenza B / Iowa / 06 / 2017 rNA1protein at 4°C overnight. Plates were blocked with 2% non-fat dry milk and 2% goat serum in DPBS containing 0.05% Tween-20 for 1 hr and washed three times. Each antibody was diluted to a concentration of 10 μg / mL, and 20 µL was added to each well. Next, biotinylated antibodies were diluted to 10 μg / mL and 5 μL added to the primary antibody solution without washing. Biotinylated antibody binding was detected with horseradish peroxidase-conjugated avidin (Sigma) and developed with TMB

[0075] 74 4890-9150-3059, v.1 substrate. The reaction was quenched with 1N hydrochloric acid once color was developed. Absorbance was measured at 450 nm using a spectrophotometer. Antibodies were determined to be competing if the % blocking was below 70%, and not competing >70%. ELISAs were performed in technical triplicate and biological duplicate. Egress inhibition assay. MDCK cells were seeded in plain Dulbecco Modified Eagle Medium (GIBCO DMEM, Thermo Fisher Scientific) containing 10% FBS in 96-well plates overnight. The cells were washed three times with virus growth medium (VGM); DMEM with 2% BSA and 2 mg / mL TPCK-treated trypsin (Sigma-Aldrich), and 100 µL of one multiplicity of infection (MOI) of virus in VGM added to the cells and incubated for 3 hr at 33°C in 5% CO2. The cells then were washed with VGM again and replenished with VGM containing three-fold serial dilutions of mAbs starting at the highest concentration of mAbs tested (10 µg / mL) or equimolar. The plates were incubated for 21 hr at 33°C in 5% CO2, and the supernatants were collected for performing the HA assay. For HA assay, the inventor used turkey red blood cells (LAMPIRE Biological Products, catalog # 7209403) that were washed and diluted to 0.5% vol / vol in PBS. A volume of 50 µL of the supernatants was incubated with 50 µL of the 0.5% turkey red blood cells in v-bottom plates for 1 hr at 4°C. The IC100 values were defined as the lowest antibody concentration added to virus-inoculated MDCK cells corresponding to the absence of virus in supernatant, measure by hemadsorption of red blood cells. Assays were performed in technical duplicate and biological duplicate. Neutralization assay based on a real time cell analysis (RTCA) impedance assay. A high-throughput RTCA assay that quantifies virus-induced cytopathic effect (CPE) was used to test antibody-mediated virus neutralization under BSL-2 conditions, using a general approach previously described for other viruses (Suryadevara et al., 2022). Viruses were titrated and tested by RTCA on MDCK-SIAT1 cell culture monolayers to determine the concentration that elicited complete CPE at 72 hrs post-inoculation, and an MOI of 0.1 was chosen to standardize across strains.50 μL of Opti-MEMTMI medium supplemented with 1% penicillin and streptomycin was added to each well of a 96-well E-plate (Agilent) to establish the background reading, and then a 50 μL volume of MDCK-SIAT1 cells in suspension (30,000 cells / well) was seeded into each well to allow the cells to adhere. Plates were incubated at room temperature for 30 min and then placed on the xCELLigence RTCA analyzer (formerly ACEA Biosciences, now Agilent). Cellular impedance was measured every 15 min. A suspension containing an MOI of 0.1 of virus was mixed 1:1 with mAb in duplicate in a total volume of 100 μL using Opti-MEM I medium supplemented with 1% penicillin and streptomycin and incubated for 1 hr at 33°C in 5% CO2. At 16 to 18 hrs after seeding the cells, the virus-mAb

[0076] 75 4890-9150-3059, v.1 mixtures were added to the cells. Wells containing virus only (no mAb added) or cells only (no virus or mAb added) were included as controls. Plates were measured every 15 min for 72 hrs post-inoculation to assess for inhibition of CPE as a marker of virus neutralization. Cellular index1values at the endpoint (~72 hrs after virus inoculation) were determined using the RTCA software version 2.1.0 (Agilent). Percent neutralization was calculated as the CI in the presence of mAb divided by the values from the cells-only (no-CPE control) wells. The background value was subtracted using the virus-only (maximum CPE) control wells. Half maximal inhibitory concentration (IC50) values were calculated by nonlinear regression analysis using Prism software version 9 (GraphPad). Assays were performed in technical duplicate and biological duplicate. Isolation and culture of primary respiratory epithelial cells. The primary tracheal cells were purchased from the MLI Tissue Procurement and Cell Culture Core Facility at the UNC School of Medicine, Chapel Hill, NC. Cells were seeded into T75 flasks and cultured in PneumaCult-EX Plus Basal Medium supported with 50× medium supplement and incubated at 37°C in 5% CO2 until the cell culture reached 80% confluency. Cells were trypsinized and seeded into Transwells with 0.4 µm diameter pores (Corning) at a density of 1.2 × 105cells per well, and both apical and basal sides were supplemented with PneumaCult-EX Plus Basal Medium. The medium was changed every other day until cells reached 100% confluency (3 to 4 days). Apical medium was lifted after the cells reached 100% confluency. Cells were fed from the basal side with PneumaCult-ALI medium supplemented with 10× PneumaCult ALI maintenance supplement, heparin, and hydrocortisone stock solution. The medium was changed from the basal side every two days, and the mucus washed off the apical surface every five days. After 3 to 4 weeks of differentiation, the cells were prepared for use in assays. Early attachment events inhibition assay. Differentiated primary respiratory cells were washed with PBS and inoculated with an MOI of 0.1 of influenza B / New York / 2017 (Yamagata lineage) that had been incubated for 1 hr with either PBS alone, an IgG isotype control to an heterologous antigen (mAb DENV r2D22 to dengue virus envelope protein) (10 μg / mL), zanamivir (1 µg / mL), mAb FluB-393 (10 μg / mL), mAb FluB-400 (10 μg / mL), or the positive control mAb r1G05 (10 μg / mL). The inoculum was washed away after one hr, and eight hours later, the cells were fixed by adding 3.7% paraformaldehyde to the basal and apical surface. Cells were permeabilized by adding 0.1% TritonX for 20 minutes, washed with PBS, and blocked with a 2% BSA solution in PBS. Staining was performed by adding the primary antibodies, anti-NP (IRR, catalog # FR-1218), and anti-E-cadherin (Cell Signaling Technology,

[0077] 76 4890-9150-3059, v.1 catalog # 3195S), both diluted 1:100 in 1% BSA PBS Tween. Cells were incubated on a shaker for 30 minutes at room temperature and washed with PBS. Secondary antibodies were added, Secondary NP: Alexa Fluor 568 (red) (Thermo Scientific, catalog # A-11004), secondary E- cadherin: Alexa Fluor Plus 488 (green) (Thermo Scientific, catalog # A32731TR), both diluted 1:1,000 in 1% BSA PBS Tween. Cells were incubated for 30 minutes at room temperature on a shaker. Cells were washed with PBS, and the membrane was removed and mounted on a glass coverslip with Prolong™ Diamond Antifade Mountant (Thermo Scientific, catalog # P36961). Images were captured on Zeiss 710, and 9 representative fields were captured. The experiment was performed in duplicate. Testing of protective efficacy against B / New York / 2018 in a BALB / c mouse model. Six- to eight-week-old female BALB / c mice were purchased from The Jackson Laboratory (strain 000651). Mice were housed in groups of up to 5 mice per cage at 18 to 24°C ambient temperatures and 40 to 60% humidity. Mice were fed a 20% protein diet (PicoLab 5053, Purina) and maintained on a 12 hr light–dark cycle (06:00 to 18:00). Food and water were available ad libitum. Mice were housed in individually ventilated cage racks with negative pressure ventilation and air filtering. To assess the protective efficacy of mAbs, mice were inoculated by the intraperitoneal route with individual mAbs 24 hr before (prophylaxis) or 12 hr after (therapy) virus administration. Human anti-dengue virus mAb DENV r2D22 served as an antibody control treatment. In ABSL-2 facilities, sevoflurane anesthetized mice were inoculated i.n. with 105FFU of the respective virus strain in 50 µL of sterile PBS. Mice were weighed and monitored daily for morbidity, and those losing over 30% of initial body weight were humanely euthanized as per IACUC requirements. Lung tissues were collected at 3 d and 6 d post-infection (n=3). Measuring lung viral burden. The left lung lobe was collected and homogenized with high-impact zirconia beads (Benchmark, catalog # D1032-10) in a refrigerated Bead Blaster for 3 min (Benchmark, catalog # D2400-R) in 1 mL of DMEM medium. Tissue homogenates were clarified by centrifugation at 10,000 rpm for 5 min and stored at −80°C. For the plaque assay, homogenates were diluted serially tenfold and applied to MDCK-SIAT1 cell culture monolayers in 12-well plates with DMEM supplemented with 1% penicillin, streptomycin, and 1 µg / mL of TPCK-treated trypsin. Plates were incubated at 33°C for 1 hr. Cells were then overlaid with 1.2% methylcellulose in DMEM supplemented with 1% penicillin, streptomycin, and 1 µg / mL of TPCK-treated trypsin. Plates were collected 72 hr later by removing overlays and fixed with 4% PFA in PBS for 20 min at ambient temperature. After removing the 4% PFA, plaques were visualized by adding 0.5 mL per well 0.05% crystal violet in 20% methanol for

[0078] 77 4890-9150-3059, v.1 20 min at ambient temperature. Excess crystal violet was washed away with PBS, and plaques were counted. Histopathology. Mice were euthanized, and tissues were collected before lung inflation and fixation. The left lung lobe was tied off at the left main bronchus and collected for viral quantification. The right lung lobe was insufflated with 1 mL of 10% neutral buffered formalin using a 3-mL syringe and needle inserted into the trachea. For fixation after infection, insufflated lungs were kept in a 10% neutral buffered formalin for 48 hrs before processing. Tissues were embedded in paraffin and sectioned at 5 µm followed by staining with hematoxylin and eosin. Tissue sections were visualized using an Olympus BX53 microscope equipped with an Olympus UC90 camera. A board-certified veterinary pathologist scored the sections. Epitope mapping using saturation alanine scanning mutagenesis. Epitope mapping was performed essentially as described previously (Davidson & Doranz, 2014) using an influenza B (strain B / Colorado / 06 / 2017) NA head region shotgun mutagenesis mutation library, made using a full-length expression construct for NA. Each of 368 residues of the head region (between residues 76 and 466) were mutated individually to alanine, and alanine residues to serine. The mutant library was arrayed in 384-well microplates, transiently transfected into HEK293T cells, and allowed to express for 22 hrs. Cells were then incubated with antibodies at concentrations pre-determined using an independent binding titration curve on cells expressing wild type NA. Cells were incubated with MAbs diluted in PBS plus calcium and magnesium (PBS++), 10% normal goat serum (Sigma). Antibodies were detected using 3.75 μg / mL of Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) in 10% normal goat serum. Cells were washed three times with PBS, and mean cellular fluorescence was detected using a high-throughput Intellicyte iQue flow cytometer (Sartorius). Antibody reactivity against each mutant NA clone was calculated relative to wild- type NA reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type NA-transfected controls. Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test MAb but supported reactivity of other anti-NA antibodies. This counter-screen strategy facilitates the exclusion of NA protein mutants that are locally misfolded or have an expression defect. Negative stain electron microscopy. Immune complexes were prepared by incubating recombinant NA protein based on the sequence of B / Iowa / 9 / 2017 with either mAb FluB-393, FluB-400, or 2D10 at a 1:4 molar ratio overnight at 4°C. The resulting samples were deposited at a concentration of 20 μg / mL on a carbon-coated 400 mesh copper grids (Electron

[0079] 78 4890-9150-3059, v.1 Microscopy Sciences) that had been glow discharged for 25 s. After 10 s, the sample was blotted off and subsequently stained twice with a 2% w / v uranyl formate solution for 45 s each. The samples were then imaged at 52,000× magnification (2.06 Å per pixel) at 120 kV, using a Tecnai Spirit T12 microscope equipped with an Eagle CCD 4k camera (FEI). The defocus was set to -1.5 μm. Micrographs were collected using Leginon (Potter et al., 1999). Single particles were picked using DoG Picker (Voss et al., 2009) and processed with Appion (Lander et al., 2009) with 2D processing completed in Relion 3.0. Cryo-EM sample preparation. Immune complexes were prepared by incubating recombinant NA protein based on the sequence of B / Iowa / 9 / 2017 with either FluB-393, FluB- 400, or 2D10 at 1:4 molar ratio overnight at 4°C before cryo-grid preparation.0.1% w / v octyl- beta-glucoside detergent was added to the complex to aid in particle tumbling. The final concentration of sample was 0.4 mg / mL on the grid. A Vitrobot Mark IV system was used for the preparation of cryoEM grids. The settings were as follows: temperature inside the chamber was 25°C, humidity was 100%, blotting force was 1, wait time was 5 s, blotting time was varied within a 3.5 to 5.5 s range. 3 μL of sample was added to plasma cleaned 1.2 / 1.3 copper Quantifoil 300 mesh grid. The plasma cleaning step was performed in the Solarus 950 plasma system (Gatan) with Ar / O2gas mix for 25 s. The sample was blotted off for 4 s and the grids were plunge-frozen into liquid-nitrogen-cooled liquid ethane. Cryo-EM data collection, processing, and model building. Cryo grids of NA / FluB- 393 and NA / FluB-400 complexes were imaged at 190,000× nominal magnification using a Falcon 4i camera on a Glacios microscope at 200 kV. Automated image collection was performed using EPU from Thermo Fisher. For both NA / FluB-393 and NA / FluB-400, a subset of images was collected at a 30° tilt along the z-axis to obtain a higher distribution of particle views. Images were aligned, dose-weighted, and Contrast Transfer Function (CTF)-corrected in the CryoSPARC Live™ software platform, with automated image collection also performed using Smart EPU software (Thermo Fisher). Cryo grids of 2D10 complexes were imaged at a nominal magnification of 36,000× using a Talos Arctica electron microscope (FEI) with a CETA 4k CMOS camera. Micrographs were collected, aligned, and CTF-corrected using Leginon, MotionCor2 in Appion, and Patch-CTF in CryoSPARC, respectively. Data processing for all three datasets was carried out in CryoSPARC v4.1.1. Briefly, particles were picked using templates generated from their respective negative stain datasets. Clean particle stacks were selected through reference-free 2D classification which were then used to generate a 3D reference model from ab initio refinement, which was then used for 3D refinement.

[0080] 79 4890-9150-3059, v.1 NA / FluB-393 underwent 2D class rebalancing, using a rebalance factor of 0.7 and 10 superclasses, before 3D refinement. Final maps were generated with C4 symmetry, per-particle CTF refinement using default parameters, followed by CryoSPARC local refinement. Final maps had a global resolution between 2.7Å and 3.36Å. Antibody Fv models were generated using Abody builder, and the NAB model was built using Rosetta. The models were built into cryoEM maps using Rosetta Relax (DiMaio et al., 2015; Afonine et al., 2018) with manual adjustment with Coot (Emsley et al., 2010). Example 2 - Results Isolation and characterization of NA-reactive mAbs that recognize two binding sites on the IBV NA protein. A 47-year-old female was vaccinated in March of 2019 with the Flucelvax™ quadrivalent influenza vaccine, a surface antigen inactivated vaccine prepared in cell cultures. The vaccine administered contained surface antigens from the following viruses: A / Singapore / GP1908 / 2015 IVR-180 (H1N1) (an A / Michigan / 45 / 2015-like virus); A / North Carolina / 04 / 2016 (H3N2) (an A / Singapore / INFIMH-16-0019 / 2016-like virus); B / Iowa / 06 / 2017 (a B / Colorado / 06 / 2017-like virus); and B / Singapore / INFTT-16-0610 / 2016 (a B / Phuket / 3073 / 2013-like virus). Seven days following vaccination, peripheral blood was drawn, and peripheral blood mononuclear cells (PBMCs) were isolated for the enrichment of plasmablasts via flow cytometry (data not shown). Fifty-seven days after vaccination, a bone marrow aspirate was taken, and long-lived plasma cells (LLPCs) were sorted from the suspension of bone marrow cells using flow cytometric sorting (FIGS. 1A-B). Donor serum was screened for antigen reactivity by ELISA against B / Iowa / 06 / 2017 using sera collected before or after vaccination (data not shown). The antibody variable genes in individual PBMCs or LLPCs were sequenced using a Chromium single-cell encapsulated system (10X Genomics), which yielded 1,262 or 7,445 paired antibody heavy- and light-chain sequences for the PBMC or LLPC populations, respectively. These two populations were evaluated for shared use of immunoglobulin heavy chain V, D, and J genes and identical (100%) immunoglobulin heavy- chain complementarity-determining region 3 (HCDR3) amino acid sequences. Sixty-four antibody clones were identified in both compartments and were chosen for microscale expression of recombinant IgG1 format antibodies (data not shown). Of the 64 clones tested, 17 mAbs bound to antigens in the 2019-2020 Fluzone vaccine (which matched those of the Flucelvax vaccine she received) and bound to recombinantly expressed NA protein based on the sequence of influenza B / Singapore / INFTT-16-0610 / 2016 (B Yamagata lineage) virus. These 17 mAbs belonged to two clonally expanded families. One lineage was encoded by

[0081] 80 4890-9150-3059, v.1 antibody genes IGHV4-39, IGKV1-5, and has an HCDR3 amino acid sequence of ARRPTHFDFWKTFDY (with 12 antibodies designated FluB-387 through FluB-398). The second lineage was encoded by antibody genes IGHV5-51, IGKV3-20, and has an HCDR3 amino acid sequence of (with 5 antibodies designated FluB-399 to FluB-403) (FIG.1B). Upon further screening, this panel of mAbs demonstrated cross-reactive binding to recombinantly expressed influenza B / Iowa / 06 / 2017 (Victoria [V]) and B / Singapore / 2016 (Yamagata [Y]) NA protein when tested by ELISA. Analysis of the number of major antigenic sites recognized by these mAbs was performed using competition-binding in ELISA with recombinant NA protein antigen based on the NA sequence of influenza B / Iowa / 06 / 2017. These studies revealed the presence of two competition-binding groups, which was consistent with the fact that the panel of mAbs contained clones representing two clonal families. One clonal family, comprising mAbs FluB-399 through FluB-403, competed unidirectionally with the antibody 1G05, a previously reported mAb that recognizes the IBV NA active site. This finding suggests that this family of mAbs also may bind in or near the IBV NA active site (FIGS.2A-B). To test if this pattern of binding to NA was germline-encoded, the inventor generated germline revertant (GR) clones and tested for binding of those GR clones to NA in the ELISA. The studies revealed loss of detectable binding for both FluB-393-GR and FluB-400-GR, suggesting that the detectable binding affinity of these clones was achieved through somatic hypermutation (data not shown). Epitope mapping with alanine-scanning mutagenesis. The inventor determined the NA residues critical for binding by mAbs from the different lineages. MAbs were screened for binding to an alanine-scan mutant library of the NA head region. For the IGHV4-39-encoded lineage that includes the mAb FluB-393, residues E338 and K272 were critical for binding. E338 was a dominant residue for IGHV4-39-encoded mAbs, as it proved critical for binding of all 6 MAbs from this lineage that the inventor tested (data not shown). K272 also was critical for 5 of the 6 mAbs. The exception was FluB-398, which is a clone whose sequence is most closely related to the inferred germline sequence; this mAb was isolated from the peripheral blood memory B cell compartment. An ELISA was performed with two recombinant NA antigens, and the binding curves demonstrate that this mAb specifically recognizes B / Iowa / 06 / 2017 (V) and not B / Singapore / INFTT-16-0610 / 2016 (Y). This observation suggests that this clonal family underwent somatic hypermutation to acquire mutations that achieved breadth of binding across both Victoria and Yamagata lineages. These additional residues identified were Y296 (critical for FluB-389) and G335 (critical for FluB-398 and

[0082] 81 4890-9150-3059, v.1 FluB-389) (data not shown). These residues were consistent with the essential residues of contact defined subsequently by cryo-electron microscopy, as described below. For the IGHV5-51-encoded lineage mAbs FluB-400 and FluB-401, alanine scanning library loss-of-binding screening identified active site residues R272 and R374 as critical for binding (data not shown). R272 and R374 also were identified as contact residues by cryo-EM analysis of FluB-400 and previously for other influenza B NA active site mAbs (Madsen et al., 2020). NA-reactive mAbs inhibit IBV NA enzymatic activity. To down-select from the panel of 17 mAbs so that the inventor could identify one lead mAb from each clonal family, an enzyme-linked lectin assay (ELLA)- neuraminidase inhibition (NI) (ELLA-NI) was performed as a surrogate assay for NA sialidase activity. The ELLA-NI tet uses lectins that bind to sialic acid carbohydrates, which are exposed on several glycosylated proteins (i.e., NA) of the influenza virus surface. Lectin binds to the glycans and forms a complex (i.e., lectin- NA), which is detected as a readout of a colorimetric signal generated by the enzymatic breakdown of a suitable substrate. The intensity of the signal helps quantify the percent inhibition of NA enzymatic activity by mAbs. The panel of 17 mAbs was screened for inhibition of NA enzymatic activity against nine viruses spanning the Victoria and Yamagata lineages. FluB-393 and FluB-400 demonstrated the highest potency in their respective clonal families (data not shown). For further characterization, four functional assays (ELLA-NI, NA-Fluor assay, egress inhibition assay, and a real-time cell analysis [RTCA] neutralization assay) were performed with FluB-393 and FluB-400. Two previously described active-site-specific IBV mAbs (r1G05, r2E0124) and an isotype-matched control IgG mAb to a heterologous target (rDENV-2D22 recognizing dengue virus envelope protein) (Alwis et al., 2012) also were included (FIG.3A). Since the ELLA uses a large-molecule substrate, the observed inhibition of NA enzymatic activity may or may not indicate direct recognition of the active site by the antibody. It is plausible that some antibodies block NA activity in the ELLA-NI test through a mechanism of steric hindrance, without directly binding to the active site. Thus, the inventor next performed an NA-Fluor™ influenza neuraminidase assay, which is a direct, functional enzyme assay that uses the small substrate 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA). Antibodies active in the NA-Fluor assay likely bind the NA active site directly. The inventor tested representative mAbs in the NA-Fluor assay against 14 viruses representing both IBV lineages, which allowed functional discrimination of antibodies that bind at the NA active site. It is desirable that new antibody therapeutics for NA should be active against IBV strains that

[0083] 82 4890-9150-3059, v.1 are resistant to currently approved small molecule inhibitors. It should be noted that four of the viruses the inventor used in this assay contain mutations in the active site that confer oseltamivir resistance (B / Indiana / 17 / 2017 (I221T), B / Oklahoma / 10 / 2018 (NA D197N), B / Wisconsin / 10 / 2016 (NA I221V), and B / Hawaii / 01 / 2018 (NA D197N)). FluB-400 and the positive control mAbs inhibited NA enzymatic activity against all IBV strains tested, whereas FluB-393 did not. This finding indicates that FluB-393 inhibits NA activity by binding to the NA protein at an epitope outside of the active site, perhaps by steric interference for access to the active site, as observed in the ELLA-NI test. The sialidase activity of the IBV NA protein contributes to the efficiency of viral egress from infected cells. NA cleaves sialic acids from cellular receptors and newly synthesized HA and NA proteins on budding virions, which were sialylated as part of the glycosylation events in the host cell (McAuley et al., 2019). To address whether FluB-393 or FluB-400 inhibit viral egress, the inventor performed virus egress inhibition assays, as previously described (Bangaru et al., 2018). MDCK-SIAT1 cells were plated, grown to near confluence, and inoculated with IBV at an MOI of 1. Three hours following inoculation, the unattached virus was removed by extensive washing, and medium containing mAbs was added to each well. MAbs r1G05 and r2E01 were included as positive controls for NA inhibition, although the egress inhibition activity for these mAbs was not reported. IBV-inoculated cells with the mAb-containing medium were incubated at 33°C for 18 hours. The supernatant was then collected and tested for the presence of virus particles by HAI assay. The inventor used three representative IBVs to assess the breadth of egress inhibition activity in this assay: two Victoria lineage viruses (B / Hawaii / 01 / 2018 (NA D197N) and B / New York / PV00081 / 18, and the Yamagata lineage virus B / New York / PV00094 / 2017. FluB-393, FluB-400, and r2E01 inhibited viral egress of all the IBV strains tested, whereas r1G05 only inhibited B / New York / PV00094 / 2017 (Y). Next, the inventor tested for inhibition of viral replication in a cell-based bioassay using Real-Time Cell Analysis (RTCA), which uses label-free cellular impedance to continuously monitor cytopathic effects of virus. Representative viruses from Yamagata or Victoria lineages were tested, and B / Hawaii / 01 / 2018 (NA D197N) (Wagner et al., 2002) was used to assess inhibition against a virus with a NA active site mutation related to oseltamivir resistance (Oakley et al., 2010). Based on these results, FluB-400 neutralized viruses from both IBV lineages, whereas FluB-393 and r1G05 only neutralized B / New York / PV00094 / 2017 (Y). r2E01 did not neutralize any of the IBV strains tested. Altogether, these results demonstrate that FluB-393 inhibits NA enzymatic activity, although it does not bind directly to the NA active site. This finding suggests that FluB-393

[0084] 83 4890-9150-3059, v.1 engages a nearby epitope that allows for steric inhibition of NA enzymatic activity and viral egress. FluB-393 exhibits Yamagata-lineage-specific neutralization. In contrast, FluB-400 inhibits NA sialidase activity and blocks viral egress by exerting its action through direct binding to the NA active site. FluB-400 neutralized all the IBV strains tested. The NA-reactive mAbs used for comparative purposes, r1G05, and r2E01, inhibited NA enzymatic activity in the ELLA and NA-Fluor assays, consistent with previously described findings (Madsen et al., 2018). FluB-400 restricts early attachment events on primary human respiratory epithelial cells. NA not only contributes to release of progeny virions from host cells to infect new cells, but also some studies suggest that NA may play a role in viral attachment and entry. Therefore, the inventor next tested whether human anti-NA antibodies can inhibit early stages the virus life cycle. The inventor used an in vitro air-liquid interface (ALI) culture system of differentiated primary human tracheal epithelial cells to serve as a surrogate for the human respiratory epithelium (FIG. 3C). In this system, cells are exposed to air on one side of the membrane, while on the other, the cells are maintained in contact with the growth medium. To assess NA inhibition, a suspension of B / New York / PV00094 / 2017 (Y) virus prepared at a concentration to deliver a MOI of 0.1 was incubated with mAbs at an antibody concentration of 10 µg / mL. The mixtures of virus + mAb then were added to cells for 1 hr. IBV particles that did not attach to the cells were removed by extensive washing. Cells then were incubated for 8 hrs, a period long enough to allow for viral attachment and entry but not for a complete viral replication cycle (Smet et al., 2022). Cells were fixed and stained for E-cadherin (to demarcate the tight junctions of the cells) and IBV nucleoprotein (NP) (to identify sites of IBV replication). Images were captured by confocal microscopy (FIG.3D). NP was detected in the images captured for the FluB-393 and negative control (rDENV-2D22) treatment groups but not for the FluB-400- treated cultures. Thus, the imaging data show that only FluB-400 prevents early attachment events. Cryo-EM analysis of FluB-393 or FluB-400 Fab molecules in complex with recombinant IBV NA protein. To determine the antigenic sites recognized by the two lineages of NA mAbs, the inventor first performed negative stain electron microscopy with B / Iowa / 6 / 2017 complexed with either FluB-393 or FluB-400. FluB-400 bound in a manner consistent with other active-site-specific NA mAbs, as expected from the mutagenesis studies. In contrast, FluB-393 bound laterally at the "corner" of the NA tetramer.

[0085] 84 4890-9150-3059, v.1 To map the epitopes targeted by these clonal families at high-resolution, the inventor solved cryo-EM structures of FluB-393 and FluB-400 with B / Iowa / 6 / 2017. FluB-393 makes multiple contacts with the corner of the NA using the antibody loops LCDR1, LCDR3, HCDR2 and HCDR3. FIG. 6 highlights the interactions formed with NA residues K272 and D338, which were identified as critical for binding in the alanine scanning mutagenesis studies. The backbone amide of K272 forms an H-bond to the side carboxylate of D56 in HCDR2, which also hydrogen bonds to the side-chain hydroxyl of T54 in HCDR2, which itself H-bonds to the side chain amine of K272. The side chain carboxylate of NA residue D338 forms multiple hydrogen bonds with LCDR3 – the backbone carbonyls of Y91 and Y94, and the indole amide of W96. FluB-400 binds in a manner common to other NA active-site-specific mAbs, with HCDR3 extending into the NA active site and HCDR1, LCDR1 and LCDR2 interacting with residues at the periphery of entrance to the active site. FIGS. 7A-G shows how D100B in the HCDR3 forms an H-bond to NA residues R272 and R374, which were shown important for binding by mutagenesis studies and contacts another active site residue Y409. R374 also forms a stacked cation-pi interaction with Y100. The HCDR3 also disrupts other active site residues, such as the displacement of R224 by R100D, the side chain of which also binds to the carboxylate of E276. Identification of IBV NA antibody lineage with shared gene usage and binding site. FluB-393 and its clonally related mAbs are encoded by IGHV4-39 and IGKV1-05. Previously, Madsen et al. described mAb 2D10, an IBV-specific NA antibody that exhibits NAI activity in the ELLA and NA-Fluor assay and is also encoded by IGHV4-39 and IGKV1-5. Negative stain EM analysis indicated that 2D10 binds at the corner of NA at the 330-loop. To investigate if any elements of this interaction were similar in the FluB-393 molecular mode or recognition of NA, the inventor solved a cryo-EM structure. The structure confirmed that 2D10 also forms contacts with the 330-loop. Interestingly, however, none of the contact residues are conserved between FluB-393 and 2D10, despite the fact that the heavy or light chain variable fragments (Fv) have 76% or 91% identity, respectively. FluB-393 and FluB-400 broadly protect against IBV in vivo. The inventor next evaluated the in vivo efficacy of FluB-393 and FluB-400 in prophylactic and treatment models against two IBV viruses, one Victoria lineage IBV (B / New York / PV00081 / 181) and one Yamagata lineage IBV (B / New York / PV00094 / 2017). Six to eight-week-old female BALB / c mice were inoculated intranasally (IN) with 105PFU / mouse. Body weight and survival were measured 14 days post-inoculation, with a humane endpoint set at 70% of the initial body weight.

[0086] 85 4890-9150-3059, v.1 In the B / New York / PV00081 / 18 prophylaxis model, FluB-393 and FluB-400 were administered a single 10 mg / kg dose of mAb by the IP route 12 hours before IN viral inoculation. Both mAbs completely protected against mortality (100%) and reduced weight loss compared to the isotype-matched negative control treatment group 86dministered rDENV- 2D22 (0% survival) (FIGS.4A-F). Lungs were collected 3 or 6 days post-inoculation (dpi) to quantify infectious virus particles in a plaque assay. The mean lung viral titer at 3 dpi for the FluB-393 or FluB-400 treatment groups was 105log10 PFU / mL and decreased to below the detection limit by 6 dpi (FIG. 4C and FIG. 4F). In addition, lungs collected 6 dpi were processed, embedded, and analyzed for histopathology by hematoxylin and eosin and RNAScopeTMviral NP RNA staining (FIG.4H). Based on the findings from those assessments, the lungs of FluB-400-treated mice were the least affected by IBV inoculation, with a minimal % affected tissue area, low level of bronchiolar epithelial erosion, and low extent of peribronchiolar or interstitial inflammation (data not shown). FluB400 treatment markedly reduced the visually detectable viral RNA signal in the lung tissue. In the B / New York / PV00094 / 2017 (Y) prophylaxis model, FluB-393, FluB-400, and r1G05 protected 80% (n = 4 / 5) of the mice compared to the isotype-matched negative control treatment group, (0% survival with rDENV-2D22 treatment) (FIG. 4D and FIG. 4G). These findings show that FluB-393 and FluB-400 protect against IBV infection and disease in vivo and suggest that these mAbs may more potently act against Victoria lineage strain viruses. Intranasally administered FluB-400 treatment protects against IBV in a lethal model. Intranasal delivery of neuraminidase antibodies could allow for multiple mechanisms of action at the respiratory interface, both through absorption into the bloodstream to prevent viral egress and blocking NA-mediated enzymatic cleavage of the virus from mucins in the airway. To assess the efficacy of topically applied (IN-administered) anti-NA mAbs, the inventor established a treatment model for lethal B / New York / PV00081 / 18 infection. The virus was administered by the IN route 24 hrs before mAb administration at a single dose of 5 mg / kg by IP injection or by large volume IN administration in anesthetized mice. In the IP treatment groups, FluB-393 and FluB-400 each protected 60% (n = 3 / 5) of the mice. The positive control mAb treatment group administered mAb r1G05, protected 40% (n = 2 / 5) of the mice. In contrast, none (n = 0 / 5) of the isotype control rDENV-2D22-treated mice survived beyond 8 dpi. In the IN route treatment groups, FluB-400 mediated survival for 60% of the mice treated, the same % protection as IP. In contrast, FluB-393 and the isotype-matched negative control mAb rDENV-2D22 did not protect (0% survival) against IBV lethal challenge (FIG. 5B). Further histopathologic investigation was performed for mice treated with FluB-400 by the IN

[0087] 86 4890-9150-3059, v.1 route, revealing less detectable viral RNA signal in the lung tissue than for the isotype-mtached negative control treatment group (FIG. 5C). These findings show that NA active site mAbs (i.e., FluB-400) can be delivered by the IN route to treat IBV infection. NA mAb Fc-mediated effector functions partially contribute to protection against IBV infection and disease. Fc effector functions of influenza-specific mAbs have been described in the literature, highlighting their ability to engage Fc receptors and trigger antibody-dependent cellular cytotoxicity (ADCC) or other immune effector mechanisms (Vanderven & Kent, 2020). The inventor sought to determine if Fc-mediated effector functions contribute to the protection against IBV infection in vivo mediated by FluB-393 and FluB-400. To assess these effects, the inventor generated IgG Fc variant molecules with LALA-PG mutations (L234A / L235A - P329G) for FluB-393 and FluB-400. LALA-PG mutations abrogate binding to Fcγ receptors and complement protein 1q (C1q) (Schlothauer et al., 2016), but wh showed that the variant IgGs for FluB-393 and FluB-400 maintain IBV NA reactivity (data not shown). To test the contribution of Fc-mediated functions in vivo, FluB-393 or FluB- 400 were administered by IP injection at a 5 mg / kg dose 24 hrs before IN inoculation of B / New York / PV00081 / 18. In both treatment groups, reduced efficacy was observed for the LALA-PG variant. For the FluB-393 LALA-PG treatment group, 20% of the mice survived compared to 60% of the mice administered FluB-393 wild-type Fc IgG1. For the FluB-400 LALA-PG treatment group, none of the mice survived compared to 60% of the mice administered FluB- 400 wild-type Fc IgG1. The body weights of the mice corresponded with survival. The observed reduction in vivo efficacy for the LALA-PG Fc mutant antibodies directed to NA suggests that Fc-mediated effector functions contribute to in vivo protection against IBV (data not shown). Example 3 - Discussion Here, the inventor probed the human antibody response to IBV by isolating NA-specific human mAbs from a healthy individual who received the Flucelvax™ quadrivalent influenza vaccine. He identified two clonally expanded families from this panel of mAbs and selected a representative mAb from each family to further delve into the structure, function, and in vivo protection of IBV NA mAbs. These studies are necessary to help guide universal influenza vaccination efforts and propose alternative mAb delivery methods for treating influenza infections.

[0088] 87 4890-9150-3059, v.1 Previous literature describes IBV NA active site mAbs (Madsen et al., 2020); however, little is known about human NA corner-binding mAbs. Furthermore, the inventors’ current understanding is based on reported protective murine mAbs (Wohlbold et al., 2017). Due to this limitation, the inventor used what is known for IAV NA mAbs, which supports the mAb- mediated mechanisms of ALI neutralization and IN delivery (Smet et al., 2022; Vigil et al., 2020; Limberis et al., 2013; Vonarburg et al., 2019). With this knowledge, the inventor applied these applications to evaluate the mAb-mediated mechanisms of IBV NA mAbs. Other groups found that influenza vaccination induces a plasma cell response specific to the IBV NA (Piepenbrink et al., 2019). In the case of the blood sample used here, the inventor observed IBV NA protein reactivity based on detecting a serum antibody response via ELISA before and after vaccination. This finding suggests that the individual was previously exposed to an unknown source of IBV NA. These results indicate that the quadrivalent influenza vaccine can elicit a robust IBV NA B cell memory response, with immunodominant responses focused on the NA active site or corner. The clonally expanded NA active site family of mAbs (i.e., FluB-400) demonstrated higher potency against the recombinantly expressed IBV Victoria lineage (B / Iowa / 06 / 2017) NA protein by ELISA compared to the IBV Yamagata strain (B / Singapore / 2016) tested. FluB- 400 maintained functionality by inhibiting NA enzymatic activity, viral egress, and virus- induced cytopathic effect against the Victoria and Yamagata viruses tested. Furthermore, FluB- 400 blocked IBV early attachment events in the surrogate air-liquid-interface culture system of the human respiratory epithelium. When FluB-400 was administered IN or IP, in vivo efficacy (60 to 100%) was observed against both IBV Victoria and Yamagata lineages. In comparison, the NA corner binding family of mAbs (i.e., FluB-393) potently bound recombinantly expressed IBV Victoria (B / Iowa / 06 / 2017) or Yamagata (B / Singapore / 2016) strains tested by ELISA. FluB-393 inhibited NA enzymatic activity, and viral egress was also observed for both IBV lineages. However, FluB-393 mediated neutralization of virus-induced cytopathic effect was only observed for the IBV Yamagata (B / New York / PV0094 / 2017) strain tested. In addition, FluB-393 did not prevent early IBV attachment events in the surrogate air- liquid-interface culture system of the human respiratory epithelium. When FluB-393 was administered IP, but not IN, in vivo efficacy (60 to 100%) was observed against viruses of both IBV Victoria and Yamagata lineages. The FluB-393 clonal family of mAbs is encoded by IGHV4-39 and IGKV1-05. These genes also encode a previously described IBV NA-specific mAb, 2D10 (Madsen et al., 2020).

[0089] 88 4890-9150-3059, v.1 Based on ELLA and NA-Fluor assays, 2D10 inhibits NA enzymatic activity, which is distinct (?) from the FluB-393 data observed here. Historically, mAbs are administered via intravenous infusion or intramuscular injection. However, delivery of mAbs to the respiratory mucosa presents two possible benefits: first, it delivers the mAb to the site of viral replication, and second, that nebulization rather than injection is a less invasive procedure (Liang et al., 2020). Since pediatric patients are often infected with IBV, less invasive delivery options, such as nebulization, are preferred (Zhao et al., 2021). ALI cultures are often used to study the behavior and function of cells in a more realistic respiratory environment than traditional cell cultures, typically grown in a completely submerged environment (Si et al., 2021; Coles et al., 2020; Chen & Schoen, 2019; Riet et al., 2020; Huh et al., 2010; Wu et al., 2016). Upon inhalation, IBV must migrate through a thick mucus layer to reach the respiratory epithelium. However, IBV is restricted in its migration due to the recognition of sialylated mucins by HA, which traps IBV in the mucosa. IBV can escape this through cleavage of sialylated mucins by NA, which releases IBV and enables migration through the mucus layer (Yang et al., 2014; Vries et al., 2020). Thus, the prevention of NA enzymatic activity to help trap IBV in the respiratory mucosa is one of the reasons NA inhibitors are an attractive class of drugs to deliver to the mucosal surface.

[0090] 89 4890-9150-3059, v.1 TABLE 1: NUCLEOTIDE SEQUENCES FOR ANTIBODY VARIABLE REGION Clone Seq ID Chain Variable Sequence Region CAGATGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTG G T A C C A G G T C C C A T C G C C C C T G C C G G T A C C C C G C G G G G T A C G C C T G C C T G A C 4890-9150-3059, v. 1 TCAAGAGATGATTCAAAAACCACGCTGTATCTGCAAATGAACAGCCTGAAAACC GAGGACACAGCCGTGTATTACTGTACCACGCCCCGTCGTTTAGGCTACACCGCA GGGTTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCA C G C G G A G G T A G C C G C A C G G G T A C C A G A C C C G G T A C A C C G C G G A G G T A C C G C T A T C G C T 4890-9150-3059, v. 1 TACAGTGGGAGCATCGACTACAACCCGTCCCTCAAGAGTCGAGTTTCCATGTCA GTAGACACGTCCAAGAATCAGTTCTCCCTGAAGCTGAGCTCTATGACTGCCGCA GACACGGCCGTGTATTACTGTGCCAGGGGGTATTACGATTTTTGGAGCGACGTT GGGCACTACTACTACTACATGGACTTCTGGGGCAAAGGGACCACGGTCACCGTC C G G C A C G G C G C C C G C G G C G G T C A G C G G C A C G G C G C C C C T G C C G G T A A G C G C G G A

[0091] 924890-9150-3059, v. 1 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTG AGACTCTCCTGTGAAGCCTCTGGATTCACCTTCAGCAGCTATGGCGTACACTGG GTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGATGGCTTTTATATCATATGAT h AAATAAAATATAT AAT T A ATTATATT AA C C G G T G C G G G T C A C A C G C G G C G G A C G C C C A C G G T C G T A A C G T A A C G T C G G A C C A C C 934890-9150-3059, v. 1 CAGGTCCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGCCTATGTCATCCACTGG GTCCGCCAGGCTCCAGGCGAGGGGCTGGAGTGGGTGGCAACTATATCATATGAT h AATAAAATATAT AAT TA ATTA TT AA C C G G T G T G C G A C C A C G C G G A G G T A C C G G T G A C G G T A G T C A T A T G C G T C G C C A C G G

[0092] 944890-9150-3059, v. 1 GAGACGTGGGACAGCAGCACTGGCGTCTTCGGAACTGGGACCATGGTCACCGTC CTA CAGGTCACCTTGAAGGAGTCTGGGCCTGCGCTGGTGGAACCCACCCAGACCCTC G T C T T G G T G A C C G C C C C G G T G A C G G T A C C A C G T G A G G T A C A C C G C G G A G G C C A G A C G T 4890-9150-3059, v. 1 CTCACCATCAGTAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCGC CGTAGCAACTGGCCTCCGTGGACGTTCGGCCCAGGGACCAAGGTGGAAATCAAA CAACTGCAGCTGCAGGAGTGGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG G C C A G A C G T G A G G T C A C C G C G G G G C G C C C C T G C C G G T G C C C C C T G C C C G T C G A G G T G 4890-9150-3059, v. 1 TCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCA GTTTATTACTGTCAGCAATATTATGATACTCCTCCCACTTTCGGCGGAGGGACC AAGGTGGAGATCAAA G G T G C C C G C G G A T G A C C A C G C G G C G G T A C T T A T G T G G G T A C C G C C T T G G G T A C T A T G 4890-9150-3059, v. 1 CAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGTTCTGGGACAGAATTCACT CTCACAATCAGCAACCTGCAGCCTGAAGATTTTGCGACTTATTACTGTCAACAA CTTGATACTTACCCTCCCATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA G G C G C C C G C G G A G G C G C G C C C G G G C G T C G C C G C G G C G T C G T C C T G C C C G T C G C C C C T G 4890-9150-3059, v. 1 GCCTCCCTGACCATCTCTGGGCTCCAGGCTGACGACGAGGCTGATTATTACTGC AGTTCATATACAACGACCCACACCTGGGTTTTCGGCGGAGGGACCAGGCTGACC GTCCTT C G T C G C C C C T G C C G G T A C C C G C G G G G T A C T A C G T G G G T A C T C C G C G G A G G C A C C G G T 4890-9150-3059, v. 1 TACTGGGCATCTTCCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGG TCTGGGACAGACTTCACTCTCACCATCAACAGCCTGCAGGCTGAGGATGTGGCA GTTTATTACTGTCAGCAATATTATAGTCCGCCGTACACTTTTGGCCAGGGGACC AAGTTGGAGATCAAA G G T A C G C G C G G A G G A C C T G C A C G G G G T A C A C A T G T G G G T C A T C G C G G G G T A A G C G C 4890-9150-3059, v. 1 TCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACTCTG ACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGG GATAGTGATAGTGTTTCTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA G C G A C G C C G G C A C G G T C A T G G T G A G G T T C C T A C C C G G G T C G T G C G C G G G G A G C G

[0093] 1014890-9150-3059, v. 1 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAGAGA GCCACCCTCTCCTGCAGGGCCAGTCAGAGTCTTAGCAACTACTACTTAGCCTGG TACCAACACAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTCCAAC 118 light A AT AT A A TTAT AT TT AAATTC G A G G T G A T C C G C G G T G A C C C C A C G G T G G C C A C G C G G A G G T A C A C G G C A C G G T A C T 1024890-9150-3059, v.1 GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGA GTCACCATCACTTGCCGGGCCAGTGAGAGTATTAGTAGTTGGTTGGCCTGGTAT CAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTAGTTTA 128 light AAAT T ATAA TTA AT ATT AAAATTAT G A G G T C A A A T A T A G G T C A A A T A T A G G T C A A A T G T A G G T C A A A T G T A G G T C A A A T 4890-9150-3059, v.1 CAGCAGAAGCCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTAGTTTA GAGAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACTGAATTCACT CTCACCATCAGCAGTCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAA TATAATGTTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA G G T C A A A T G T A G G T C A A A T G T A G G T C A A A T G T A G G T C A A A T A T A G G T C A A A T G T 1044890-9150-3059, v. 1 CTCACCATCAACAGTCTGCAGCCTGATGATTTTGCAACTTATTACTGCCTCCAA TATAATATTTATTCGTGGACGTTCGGCCCAGGGACCAAGGTGGAAATCAAA CAGGTGCAGCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG G T C A A A T A T A G G T C A A A T A T A G G T C C A C A G C C G A G G T C C A C A G C C G A G G T C C A C A G C C 4890-9150-3059, v. 1 ACTCTCACCATCAGCAGGCTGGAGCCTGAAGACTTTGCAGTGTATTTTTGTCAG CACTATGGTACCTCACCCGGGACTTTCGGCGGAGGGACCAAGGTAGAGATCAAA GGTGTGCAGCTGGTGCAGTCTGGAGCAGAAGTGAAAAAGCCCGGGGAGTCTCTG G T C C A C A G C C G A G G T C C A C A G C C G A

[0094] 1064890-9150-3059, v. 1 TABLE 2: PROTEIN SEQUENCES FOR ANTIBODY VARIABLE REGION Clone Seq ID Chain Variable Sequence Region QMQLVESGGGVVQPGRSLRLSCVASGFSFSSYGMYWVRQAPGKGLEWVAVTSFD G Q T D Y S T D Y P D V N T K A P L Y H N K D Y Q T D S P L D C Y T

[0095] 1074890-9150-3059, v. 1 QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGDYYCSWIRQPPGKGLEWIGNFY 181 heavy YSGSIDYNPSLKSRVSMSVDTSKNQFSLKLSSMTAADTAVYYCARGYYDFWSDV GHYYYYMDFWGKGTTVTVSS FlB4 K T D F P V Y Q Q T N Y S T Y W P L D C I G Y V P K W T N T R R Y R N T

[0096] 1084890-9150-3059, v. 1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSAYVIHWVRQAPGEGLEWVATISYD 201 heavy GSKTYYADSVRGRFTLSRDNSKNTLYLQMNSLRPEDAAVYYCAKDDSAYCDGDC YFDYWGQGTLVTVSS FlB 4 I G K A P L D C L T S D L Y R P V D A I T D A I T G A R K D L P L H W R K H W 4890-9150-3059, v. 1 222 lightDIVLTQSPATLSLSPGERATLSCRASQSISSYLGWYQQKPGQAPRLIVHDASKRATGVPARFSASGSGTDFTLTISSLEPEDFAVYYCQRRSNWPPWTFGPGTKVEIKQLQLQESGPGLVKPSDTLSLTCTVSGGSVTNRTYYWTWIRQPPGKGLEWIASLY D P N Y S T S D S T D R I T S D P L K A P T G G L D C Y K T F L K N I P L

[0097] 1104890-9150-3059, v. 1 QVQLVQSGAEMKKPGSSVKVSCKASGDSFSSIPLTWVRQTPGHGFEWLGRIVPV 243 heavy YNTINHAQRFHGRITISADESTNTAYLELSSLRADDTAVYYCARDLVHRRFDPW FluB-199 GQGTLVTVSS P L Y R P D G T T D Y S T D A S T N Y P S I R S N P L D C I T H W P L K D

[0098] 1114890-9150-3059, v. 1 264 lightSYDLTQPSSVSVSPGQTASITCSGDQLGDKYACWYQQKPGQSPVVVIFLDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSNIVVVGGGTKLTVLEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVSGISWN K L Y V P Y W P L Q W K T Y F I G H S S S G P I W N K Y S P K Y P K A 4890-9150-3059, v. 1 286 lightSYVLTQPPSVSVAPGQTAGITCGGDNIGSKSLHWYQQKPGQAPVLVVYEDSDRPSGIPERFSGSNSGNTATLTISGVEAGDEADYYCQVWDSSSAHVVFGGGTKLTVLQMQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALMSYD A R T D P L K S K L S K L S K L S K L S K L S K L S K L S K L S K

[0099] 1134890-9150-3059, v. 1 308 lightDIQMTQSPSTLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKVLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNVYSWTFGQGTKVEIKQVQLVESGPGLVKPSETLSLTCTVSGDSVSDTSYFWGWIRQPPGKGLEWIGSVS K L S K L S K L G R S K G R S K G R S K V R S K G R S K

[0100] 1144890-9150-3059, v. 1 TABLE 3: HEAVY CHAIN CDR SEQUENCES Clone CDRH1 CDRH2 CDRH3 F F Y T FD KK AKE EAH D T DF

[0101] 1154890-9150-3059, v.1 GYTFSAYY INPNNGGT ARDMRPYSKYNYYGMDV FluB-50

[0102] 1164890-9150-3059, v. 1 GFSVKTFGYH VDWDDDQ ARSHIYPYSDAFDI FluB-117

[0103] 1174890-9150-3059, v. 1 GFRFSRYS ISETGRTI ARDEDTPLVRAFDL FluB-168

[0104] 1184890-9150-3059, v. 1 GFTFDDYA ISWNSGSI AKDVAGFGYAAKFTSGPDNDFVMDV FluB-240

[0105] 1194890-9150-3059, v. 1 GGSVSDTTYF VSYTGDN ARRPTHFDFWKTFDY FluB-387

[0106] 1204890-9150-3059, v. 1 GYTFPIYW IYPGDSDT ARHSEPWYPDHRFWPPYPFDI FluB-400

[0107] 1214890-9150-3059, v. 1 TABLE 4: LIGHT CHAIN CDR SEQUENCES Clone CDRL1 CDRL2 CDRL3 IANY EDN YDNNNVV

[0108] 1224890-9150-3059, v. 1 SSDVGGYNY DVS SSYTSNSTWV FluB-50

[0109] 1234890-9150-3059, v. 1 QPVLYESNNKHY WAS QQYRSPSWS FluB-117

[0110] 1244890-9150-3059, v. 1 QAISSY ATS QQLDTYPPIT FluB-168

[0111] 1254890-9150-3059, v. 1 QSVGPY SAS QQSYSILT FluB-240

[0112] 1264890-9150-3059, v. 1 QSINSW KAS QQYNVYSWT FluB-387

[0113] 1274890-9150-3059, v. 1 QTVSSGY GAS QHHGTSPGT FluB-400 * * * * * * * * * * * * * * * * * All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0114] 1284890-9150-3059, v. 1 VII. REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. U.S. Patent 3,817,837 U.S. Patent 3,850,752 U.S. Patent 3,939,350 U.S. Patent 3,996,345 U.S. Patent 4,196,265 U.S. Patent 4,275,149 U.S. Patent 4,277,437 U.S. Patent 4,366,241 U.S. Patent 4,472,509 U.S. Patent 4,554,101 U.S. Patent 4,680,338 U.S. Patent 4,816,567 U.S. Patent 4,867,973 U.S. Patent 4,938,948 U.S. Patent 5,021,236 U.S. Patent 5,141,648 U.S. Patent 5,196,066 U.S. Patent 5,563,250 U.S. Patent 5,565,332 U.S. Patent 5,856,456 U.S. Patent 5,880,270 U.S. Patent 6,485,982 “Antibodies: A Laboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988. Abbondanzo et al., Am. J. Pediatr. Hematol. Oncol.12(4): 480-489, 1990. Allred et al., Arch. Surg.125(1), 107-113: 1990. Atherton et al., Biol. of Reproduction 32: 155-171, 1985. Barzon et al., Euro Surveill.11: 21(32), 2016.

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[0118] 1324890-9150-3059, v. 1

Claims

WHAT IS CLAIMED IS:

1. A method of detecting an influenza B virus infection in a subject comprising: (a) contacting a sample from said subject with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) detecting influenza B virus in said sample by binding of said antibody or antibody fragment to an influenza B virus antigen in said sample.

2. The method of claim 1, wherein said sample is a body fluid.

3. The method of claims 1-2, wherein said sample is blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces.

4. The method of claims 1-3, wherein detection comprises ELISA, RIA, lateral flow assay or Western blot.

5. The method of claims 1-4, further comprising performing steps (a) and (b) a second time and determining a change in influenza B virus antigen levels as compared to the first assay.

6. The method of claims 1-5, wherein the antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

7. The method of claims 1-5, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone- paired variable sequences as set forth in Table 1.

8. The method of claims 1-5, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1.1334890-9150-3059, v.

19. The method of claims 1-5, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

10. The method of claims 1-5, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

11. The method of claims 1-5, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

12. The method of claims 1-11, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

13. A method of treating a subject infected with influenza B virus, or reducing the likelihood of infection of a subject at risk of contracting influenza B virus, comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

14. The method of claim 13, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.

15. The method of claim 13-14, the antibody or antibody fragment is encoded by clone- paired light and heavy chain variable sequences having 95% identity to as set forth in Table 1.

16. The method of claim 13-14, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone- paired sequences from Table 1.

17. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.1344890-9150-3059, v.

118. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

19. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

20. The method of claims 13-19, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

21. The method of claims 13-20, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

22. The method of claims 13-19, wherein said antibody is a chimeric antibody or a bispecific antibody.

23. The method of claim 13-22, wherein said antibody or antibody fragment is administered prior to infection or after infection.

24. The method of claim 13-23, wherein said subject is a pregnant female, a sexually active female, or a female undergoing fertility treatments.

25. The method of claim 13-24, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.1354890-9150-3059, v.

126. A monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

27. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.

28. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.

29. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1.

30. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

31. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

32. The monoclonal antibody of claims 26-31, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

33. The monoclonal antibody of claims 26-31, wherein said antibody is a chimeric antibody, or is bispecific antibody.

34. The monoclonal antibody of claim 26-33, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase1364890-9150-3059, v. 1therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

35. The monoclonal antibody of claim 26-34, wherein said antibody or antibody fragment further comprises a cell penetrating peptide and / or is an intrabody.

36. A hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

37. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone- paired sequences from Table 1.

38. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 1.

39. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone-paired variable sequences from Table 1.

40. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

41. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 2.1374890-9150-3059, v.

142. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

43. The hybridoma or engineered cell of claims 36-42, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment.

44. The hybridoma or engineered cell of claim 36-43, wherein said antibody is a chimeric antibody or a bispecific antibody.

45. The hybridoma or engineered cell of claim 36-43, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

46. The hybridoma or engineered cell of claim 36-45, wherein said antibody or antibody fragment further comprises a cell penetrating peptide and / or is an intrabody.

47. A vaccine formulation comprising one or more antibodies or antibody fragments characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

48. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences according to clone- paired sequences from Table 1.

49. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.1384890-9150-3059, v.

150. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1.

51. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences according to clone- paired sequences from Table 2.

52. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

53. The vaccine formulation of claims 47-52, wherein at least one of said antibody fragments is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment.

54. The vaccine formulation of claims 47-52, wherein at least one of said antibodies is a chimeric antibody or is bispecific antibody.

55. The vaccine formulation of claims 47-54, wherein at least one of said antibodies is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

56. The vaccine formulation of claims 47-55, wherein at least one of said antibodies or antibody fragments further comprises a cell penetrating peptide and / or is an intrabody.

57. A vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment according to claims 26-34.1394890-9150-3059, v.

158. The vaccine formulation of claim 57, wherein said expression vector(s) is / are Sindbis virus or VEE vector(s).

59. The vaccine formulation of claims 57-58, formulated for delivery by needle injection, jet injection, or electroporation.

60. The vaccine formulation of claim 57, further comprising one or more expression vectors encoding for a second antibody or antibody fragment, such as a distinct antibody or antibody fragment of claims 26-34.

61. A method of reducing the risk of serious illness, hospitalization or death in a “high risk” subject infected with or at risk of infection with influenza B virus comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

62. The method of claim 61, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.

63. The method of claim 61-62, the antibody or antibody fragment is encoded by clone- paired light and heavy chain variable sequences having 95% identity to as set forth in Table 1.

64. The method of claim 61-62, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone- paired sequences from Table 1.

65. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

66. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.1404890-9150-3059, v.

167. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

68. The method of claims 61-67, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment.

69. The method of claims 61-68, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and / or increase therapeutic efficacy, such as a LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

70. The method of claims 61-67 wherein said antibody is a chimeric antibody or a bispecific antibody.

71. The method of claim 61-70, wherein said antibody or antibody fragment is administered prior to infection or after infection.

72. The method of claim 61-71, wherein said subject is a under the age of 12, over the age of 60, is immunocompromised, or suffers from an underlying medical condition increasing risk of serious illness, hospitalization or death resulting from viral infection.

73. The method of claim 61-72, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

74. The method of claim 61, wherein the antibody or antibody reduces severity of disease and / or hospitalization as compared to an untreated control.1414890-9150-3059, v.

175. The method of claim 61, wherein the antibody or antibody fragment reduces risk of death as compared to an untreated control.

76. A method of determining the antigenic integrity, correct conformation and / or correct sequence of an influenza B virus antigen comprising: (a) contacting a sample comprising said antigen with a first antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) determining antigenic integrity, correct conformation and / or correct sequence of said antigen by detectable binding of said first antibody or antibody fragment to said antigen.

77. The method of claim 76, wherein said sample comprises recombinantly produced antigen.

78. The method of claim 76, wherein said sample comprises a vaccine formulation or vaccine production batch.

79. The method of claims 76-78, wherein detection comprises ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining.

80. The method of claims 76-79, wherein the first antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

81. The method of claims 76-79, wherein said first antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1.

82. The method of claims 76-79 wherein said first antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone- paired sequences as set forth in Table 1.1424890-9150-3059, v.

183. The method of claims 76-79, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

84. The method of claims 76-79, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

85. The method of claims 76-79, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

86. The method of claims 76-85, wherein the first antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

87. The method of claims 76-86, further comprising performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time.

88. The method of claims 76-87, further comprising: (c) contacting a sample comprising said antigen with a second antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (d) determining antigenic integrity of said antigen by detectable binding of said second antibody or antibody fragment to said antigen.

89. The method of claim 88, wherein the second antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

90. The method of claim 89, wherein said second antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1.1434890-9150-3059, v.

191. The method of claim 89, wherein said second antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1.

92. The method of claims 89, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

93. The method of claim 89, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone- paired sequences from Table 2.

94. The method of claim 89, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

95. The method of claim 89, wherein the second antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

96. The method of claim 89, further comprising performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.1444890-9150-3059, v. 1

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