Broadly reactive antibodies against influenza b virus

Abstract

The present disclosure relates to antibodies that bind to and neutralize influenza B virus and methods of use thereof.

Description

[0001] Priority Declaration

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 515,279, filed July 24, 2023, the entire contents of which are incorporated herein by reference.

[0003] References to sequence lists

[0004] This application contains a sequence list XML, which has been submitted electronically and is incorporated herein by reference in its entirety. The XML sequence list was created on July 23, 2024, named VBLTP0321WO.xml, and has a size of 718,732 bytes. background Technical Field

[0005] This disclosure generally pertains to the fields of medicine, infectious diseases, and immunology. More specifically, this disclosure relates to human antibodies that bind to influenza B virus. Background Technology

[0006] Influenza B virus (IBV) is a member of the order Orthomyxoviridae. These viruses contain a negative-sense, single-stranded RNA genome with eight gene segments encoding ten viral proteins. Among these viral proteins, the surface contains neuraminidase (NA), hemagglutinin (HA), NB, and BM2 proteins. HA is the most abundant and immunodominant surface glycoprotein on influenza viruses, enabling the virus to bind to sialic acid on host cells (Wagner et al., 2002; Gaymard et al., 2016). The second most abundant protein, NA, is an enzymatically cleaved tetrameric protein of sialic acid. Several potential mechanisms for inhibiting NA function to protect the host have been proposed, and antibodies have been used to investigate these mechanisms (Schneider & Coughlan, 2023). First, some anti-NA antibodies inhibit NA-mediated enzymatic cleavage of the virus from airway mucin (Matrosovich et al., 2004; Cohen et al., 2013). This activity prevents the virus from effectively penetrating the airway mucus layer to infect the underlying airway epithelial cells and also inhibits viral transmission by preventing virion particles from detaching from the mucin covering infected cells. Second, anti-NA antibodies binding to NA on virion particles inhibit HA binding to sialic acid, hindering viral attachment and entry into host cells. Anti-NA antibodies can also mediate Fc-dependent protective mechanisms (DiLillo et al., 2016) that eliminate virus-infected cells, including by synergistically promoting this activity with HA stem antibodies (Momont et al., 2023). Finally, by blocking NA enzyme activity during viral egress, NA antibodies prevent the cleavage of viral aggregates and inhibit viral release and subsequent dissemination within the host (McAuley et al., 2019; Chen et al., 2018). Historically, the focus on vaccine-induced immune responses against hemagglutination inhibition (HAI) has contributed to our understanding of current humoral responses to influenza viruses and provided protective relevance for regulatory purposes in hemagglutination inhibition (HAI) assays. However, recent literature suggests that natural influenza virus infection induces distinct immune responses in which antibodies recognize viral proteins distributed across several antigenic targets, including the NA. However, the NA content is uncontrolled in most influenza subunit protein vaccines, and therefore many existing vaccines may contain suboptimal concentrations of key protective antigens against influenza immune responses, particularly NA (Chen et al., 2018).

[0007] Of circulating influenza viruses, IBV causes a median case burden rate of 23% (Zaraket et al., 2021). IBV was first described in 1940 and subsequent sequence analyses have shown that it circulates in two distinct genetic lineages named Victoria and Yamagata (Palese & Young, 1982). The respective dominance of these two lineages varies between influenza seasons; however, the lack of detection of Yamagata lineage strains in clinical samples in recent years suggests that this lineage may have been eradicated following 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 annually and disproportionately affect pediatric and older populations (Sharma et al., 2019), including immunocompromised individuals. Preventive or therapeutic options available for IBV are limited.

[0008] Clinically, it is difficult to distinguish patients with IBV infection from those with influenza A virus (IAV) infection (Liu et al., 2019). The presented syndrome typically includes cough, fever, congestion, vomiting, and body aches. In severe IBV cases, the clinical syndrome can progress to bronchopneumonia with relatively frequent secondary bacterial lung infections (Taubenberger & Morens, 2008). During the COVID-19 pandemic, co-infection with IBV and SARS-CoV-2 has led to severe illness due to limited therapeutic options. Therefore, there is an unmet medical need for better prevention and treatment of IBV infection.

[0009] Most currently approved influenza vaccines contain HA proteins from both IBV lineages, but they may not fully protect vaccinated individuals from the disease. Currently, FDA-approved treatments for influenza exist, including M2 inhibitors that work only against IAV and a class of small-molecule NA inhibitors that work against both IAV and IBV (including zanamivir, oseltamivir, laninamivir, and peramivir) (Heneghan et al., 2016). While these treatments are helpful for early-stage administration and mild infections, they offer limited benefit in later stages of infection. Furthermore, single amino acid mutations in NA can reduce efficacy or lead to resistance to these drugs. Finally, small-molecule NA inhibitors are generally less effective at treating IBV infections compared to IAV infections. In summary, this situation creates a need to develop 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). Therefore, improved approaches to treating influenza B remain needed, particularly in these high-risk categories. Summary of the Invention

[0010] Therefore, according to this disclosure, a method for detecting influenza B virus infection in a subject is provided, comprising (a) contacting a sample from the subject with an antibody or antibody fragment having clone-paired heavy chain CDR sequences and light chain CDR sequences from Tables 3 and 4, respectively; and (b) detecting influenza B virus in the sample by the binding of the antibody or antibody fragment to influenza B virus antigen in the sample. The sample may be bodily fluids, including blood, sputum, tears, saliva, mucus or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, transudate, tissue scraping, or feces. Detection may include ELISA, RIA, lateral flow assay, or Western blot. The method may also include repeating steps (a) and (b) and determining the change in influenza B virus antigen levels compared to the first assay. Antibodies or antibody fragments may be encoded by the clone-paired variable sequences shown in Table 1, or by light and heavy chain variable sequences having 70%, 80%, or 90% identity with the clone-paired variable sequences shown in Table 1, or by light and heavy chain variable sequences having 95% identity with the clone-paired sequences shown in Table 1. Antibodies or antibody fragments may contain light and heavy chain variable sequences based on the clone-paired sequences from Table 2, or may contain light and heavy chain variable sequences having 70%, 80%, or 90% identity with the clone-paired sequences from Table 2, or may contain light and heavy chain variable sequences having 95% identity with the clone-paired sequences from Table 2. Antibody fragments may be recombinant scFv (single-chain variable fragment) antibodies, Fab fragments, F(ab')2 fragments, or Fv fragments.

[0011] In another embodiment, a method is provided for treating a subject infected with influenza B virus or reducing the likelihood of infection in a subject at risk of influenza B virus infection, comprising delivering to the subject an antibody or antibody fragment having clonal-paired heavy chain CDR sequences and light chain CDR sequences respectively from Tables 3 and 4. The antibody or antibody fragment may be encoded by clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences shown in Table 1. The antibody or antibody fragment may contain light chain and heavy chain variable sequences according to clonal-paired sequences from Table 2, or may contain light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired sequences from Table 2, or may contain light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG or recombinant IgG antibody or antibody fragment containing an Fc moiety mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or improve therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or modified with glycans to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or expression in cell lines modified with a defined glycosylation pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may be administered before or after infection. The recipient may be a pregnant female, a sexually active female, or a female undergoing fertility treatment. Delivery may include administration of the antibody or antibody fragment, or genetic delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

[0012] In another embodiment, a monoclonal antibody is provided, wherein the antibody or antibody fragment is characterized by clonal-paired heavy chain CDR sequences and light chain CDR sequences respectively from Tables 3 and 4. The antibody or antibody fragment may be encoded by clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences shown in Table 1. The antibody or antibody fragment may comprise light chain and heavy chain variable sequences according to clonal-paired sequences from Table 2, or may comprise light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired sequences from Table 2, or may comprise light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG or recombinant IgG antibody or antibody fragment containing an Fc moiety mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or improve therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or modified with glycans to alter (eliminate or enhance) FcR interactions, such as through enzymatic or chemical addition or removal of glycans, or expression in cell lines modified with a defined glycosylation pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may also contain a cell-penetrating peptide and / or be an intracellular antibody.

[0013] In another embodiment, a hybridoma or engineered cell encoding an antibody or antibody fragment is provided, wherein the antibody or antibody fragment is characterized by clonal pairing of heavy chain CDR sequences and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clonal pairing variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal pairing variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal pairing sequences shown in Table 1. The antibody or antibody fragment may comprise light chain and heavy chain variable sequences according to clonal pairing sequences from Table 2, or may comprise light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal pairing sequences from Table 2, or may comprise light chain and heavy chain variable sequences having 95% identity with the clonal pairing sequences from Table 2. The antibody fragment may be a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG or recombinant IgG antibody or antibody fragment containing an Fc moiety mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or improve therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or modified with glycans to alter (eliminate or enhance) FcR interactions, such as through enzymatic or chemical addition or removal of glycans, or expression in cell lines modified with a defined glycosylation pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may also contain a cell-penetrating peptide and / or be an intracellular antibody.

[0014] In another embodiment, a vaccine formulation comprising one or more antibodies or antibody fragments is provided, said antibodies or antibody fragments being characterized by clonal pairings of heavy chain CDR sequences and light chain CDR sequences respectively from Tables 3 and 4. One or more antibodies or antibody fragments may be encoded by clonal pairings of variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal pairings of variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal pairings of variable sequences shown in Table 1. One or more antibodies or antibody fragments may comprise light chain and heavy chain variable sequences according to clonal pairing sequences from Table 2, or may comprise light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal pairing sequences from Table 2, or may comprise light chain and heavy chain variable sequences having 95% identity with the clonal pairing sequences from Table 2. One or more antibody fragments may be recombinant scFv (single-chain variable fragment) antibodies, Fab fragments, F(ab')2 fragments, or Fv fragments. One or more antibodies may be IgG or recombinant IgG antibodies or antibody fragments comprising an Fc moiety mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or improve therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or modified with glycans to alter (eliminate or enhance) FcR interactions, such as by enzymatic or chemical addition or removal of glycans, or by expression in cell lines modified with a defined glycosylation pattern. The antibody may be a chimeric antibody or a bispecific antibody. At least one of the antibodies or antibody fragments may also comprise a cell-penetrating peptide and / or be an intracellular antibody.

[0015] In another embodiment, a vaccine formulation is provided comprising one or more expression vectors encoding a first antibody or antibody fragment, which is an antibody or antibody fragment defined herein. The expression vector may be a Sindbis virus or VEE vector. The vaccine formulation of the claims may be formulated for delivery by needle injection, jet injection, or electroporation. The vaccine formulation may also comprise one or more expression vectors encoding a second, different antibody or antibody fragment.

[0016] In another embodiment, a method is provided for reducing the risk of severe illness, hospitalization, or death in individuals infected with influenza B virus or at "high-risk" individuals at risk of influenza B virus infection, comprising delivering to the individuals an antibody or antibody fragment having clonal-paired heavy chain CDR sequences and light chain CDR sequences respectively from Tables 3 and 4. The antibody or antibody fragment may be encoded by clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences shown in Table 1. The antibody or antibody fragment may contain light chain and heavy chain variable sequences according to clonal-paired sequences from Table 2, or may contain light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired sequences from Table 2, or may contain light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences from Table 2. The antibody fragment may be a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG or recombinant IgG antibody or antibody fragment containing an Fc moiety mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or enhance therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or modified with glycans to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or expressed in a cell line modified with a defined glycosylation pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may be administered before or after infection. Subjects may be under 12 years of age or over 60 years of age, may be immunocompromised, or may have underlying conditions that increase the risk of severe illness, hospitalization, or death from viral infection. Delivery may include administration of the antibody or antibody fragment, or genetic delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. Compared with untreated controls, antibodies or antibodies may reduce the severity of disease and / or hospitalization, and / or reduce the risk of death compared with untreated controls.

[0017] In another embodiment, a method is provided for determining the antigenic integrity, correct conformation, and / or correct sequence of an influenza B virus antigen, comprising (a) contacting a sample containing the antigen with a first antibody or antibody fragment having clonal pairings of heavy chain CDR sequences and light chain CDR sequences from Tables 3 and 4, respectively; and (b) determining the antigenic integrity, correct conformation, and / or correct sequence of the antigen by the detectable binding of the first antibody or antibody fragment to the antigen. The sample may contain recombinant antigen or a vaccine formulation or vaccine production batch. Detection may include ELISA, RIA, Western blot, biosensors using surface plasmon resonance or biolayer interferometry, or flow cytometry staining. The first antibody or antibody fragment may be encoded by clonal pairings of variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal pairings of variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal pairings of variable sequences shown in Table 1. The first antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone-paired sequences from Table 2, and may comprise light and heavy chain variable sequences having 70%, 80%, or 90% identity with the clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity with the clone-paired sequences from Table 2. The first antibody fragment may be a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The method may also include repeating steps (a) and (b) to determine the antigenic stability of the antigen over time.

[0018] The method may further include (c) contacting a sample containing the antigen with a second antibody or antibody fragment having clonal-paired heavy chain CDR sequences and light chain CDR sequences from Tables 3 and 4, respectively; and (d) determining the antigenic integrity of the antigen by detectable binding of the second antibody or antibody fragment to the antigen. The second antibody or antibody fragment may be encoded by clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired variable sequences shown in Table 1, or by light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences shown in Table 1. The second antibody or antibody fragment may contain light chain and heavy chain variable sequences according to clonal-paired sequences from Table 2, or may contain light chain and heavy chain variable sequences having 70%, 80%, or 90% identity with the clonal-paired sequences from Table 2, or may contain light chain and heavy chain variable sequences having 95% identity with the clonal-paired sequences from Table 2. The second antibody fragment may be a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The method may also include repeating steps (c) and (d) to determine the antigenic stability of the antigen over time.

[0019] When used in conjunction with the term "comprising / including" in the claims and / or description, the use of a noun without a quantifier may mean "one / type," but it also includes the meanings of "one / type or more / types," "at least one / type," and "one / type or more than one / type." The word "about" means 5% plus or minus the stated number.

[0020] It is anticipated that any method or composition described herein can be practiced in relation to any other method or composition described herein. Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples point to some specific embodiments of this disclosure, they are given by way of illustration only, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art based on such detailed description. Attached Figure Description

[0021] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments shown herein.

[0022] Figure 1A To B. The donor of the vaccine produced a norepinephrine (NA) response to influenza B. Figure 1A Study design. Intramuscular administration of the quadrivalent 2018-19 seasonal influenza vaccine Flucelvax.TM Subsequently, studies were conducted on healthy adult individuals. Blood and bone marrow aspirates were collected after vaccination for cells secreting antibodies against IBV, and the resulting mAbs from single cells were expressed in micro-levels. A pie chart illustrates that 17 out of a total of 64 clones secreted antibodies that reacted with the IBV NA protein. Figure 1B A phylogenetic tree of antibody variable genes encoding mAbs, and a table of variable gene usage. (Using V) H Germ genes were used as outgroups to construct phylogenetic tree clonal families on the heavy chain VDJ-REGION using neighbor-joining. Alignment was performed in MUSCLE. The tree was inferred using PHYLIP and visualized using FigTree. Circles correspond to tissue samples from which sequences were isolated: plasmablasts (PB) or long-lived plasma cells (LLPC) in bone marrow, or both.

[0023] Figures 2A to B. mAbs from two clonal families bind to NAs from two IBV lineages and identify different binding sites. (Figure 2A) Binding of 16 NA-reactive mAbs, positive control mAb (+)r1G05, or isotype-matched negative control (-)r2D22 to recombinant NAs from specified IBV strains in ELISA. mAb (+)r1G05 was used as a reference mAb to identify the NA active site for competitive binding. In competitive ELISA, the binding of the reference mAb to B / Iowa / 06 / 2017 (V) was measured in the presence of saturated concentrations of competitor mAbs and normalized relative to the binding observed in the presence of dengue virus mAb (-)r2D22. Black indicates complete competition (binding of reference mAb <70%), and white indicates no competition (binding of reference mAb >71%). Orange boxes indicate clonal amplification families of mAbs that bind to the active site as indicated by competition with (+)r1G05. The light pink boxes indicate mAb families that recognize different antigenic sites through clonal amplification. Data represent two independent experiments. (Figure 2B) Binding to influenza B virus NA protein (half-maximum effective concentration [EC50]). 50 [Value; ng / mL]

[0024] Figures 3A to C. Anti-NA human mAbs broadly inhibit viral activity. (Figure 3A) Heatmap indicating mAb efficacy: enzyme-linked lectin assay (ELLA; IC50) 50 Value); NA-Fluor determination (IC50) 50 Value); Excretion assay (IC) 100Value); real-time cell analysis (RTCA) neutralization (IC) 50 Values). Data represent two experiments. (Fig. 3B). Primary human tracheal respiratory epithelial cells were seeded at 0.1 MOI and cultured at the air-liquid interface with 10 µg / mL anti-NA mAb, isotype-matched negative control mAb, or virus-only conditions. Incubation time was limited to 8 hpi to allow attachment but not expulsion. Cells were fixed and stained with anti-NP mAb (red) and anti-e-cadherin antibody (green). (Fig. 3C). Images obtained as shown in Fig. 3B were captured on a Zeiss 710 confocal microscope. Data represent 9 fields of view and two experiments were performed.

[0025] Figures 4A to D. Anti-NA mAb mediates protection against lethal IBV challenge in mice. (Figure 4A) Study design. BALB / c mice were intraperitoneally (IP) inoculated 12 hours prior to viral challenge with either: 10 mg / kg of anti-NA mAb, a (-)r2D22 control mAb responsive to an irrelevant antigen (dengue virus), or a control recombinant mAb (+)r1G05 recognizing IBV NA. Mice were intranasally (IN) challenged with a lethal dose of the designated IBV virus, and protection was monitored daily. Weights are expressed as group mean ± SEM (Figures 4B to C). The dashed lines below indicate the endpoint of no recovery threshold (weight loss >30%) and euthanasia. Survival curves were estimated using the Kaplan-Meier method (Figures 4B to C). Survival in each group treated with anti-NA or (+)r1G05 mAb was compared to the dengue virus mAb control group using the log-rank (Mantel-Cox) test. n = 5 mice / group. (Figure 4D) Histopathology and RNAScope demonstrate the efficacy of FluB antibody-mediated targeting of viral replication in disease and tissues.

[0026] Figures 5A to D. Inhalational delivery of anti-influenza active site mAbs is effective. (Figure 5A) Study design. BALB / c mice were inoculated with a lethal dose of influenza B / New York / PV01181 / 2018 virus via intranasal (IN) route, and one day later (d1) were inoculated with a designated anti-NA mAb, (-)r2D22 control mAb, or (+)r1G05 positive control mAb via IN or intraperitoneal (IP) route, and protection was monitored (Figures 5C and 5E). Weights (Figures 5B and 5D) represent group mean ± SEM. The dashed lines below indicate the endpoint of no recovery threshold (weight loss >30%) and euthanasia. Survival curves were estimated using the Kaplan-Meier method and compared using the Mantel-Cox test as shown (Figures 5B and 5C). Data represent one experiment, n = 5 mice / group. (Figure 5D) Histopathology of the lungs showed a reduction in virus mediated by FluB-400.

[0027] Figure 6 Cryo-EM reconstruction of NA-FluB-400. Cryo-EM reconstruction of Fab FluB-400 (top left) complexed with NA. One NA tetramer (grey) bound to four Fabs. Overall structure of FluB-400 bound to NA (top right). Specific interaction between FluB-400 and NA (bottom).

[0028] Figures 7A to G. Cryo-EM reconstruction of NA-FluB-393 and NA-2D10. (Figure 7A) IGHV and IGLV gene usage and HCDR3 amino acid sequences of mAbs FluB-393 (SEQ ID NO: 811) and 2D10 (SEQ ID NO: 812). Cryo-EM reconstruction of Fab FluB-393 and 2D10 complexed with NA (Figures 7B to C). One NA tetramer (grey) binds to four Fabs. (Figure 7D) The common loop is targeted by three mAbs: orange FluB-393, blue 2D10, and red NA-63. (Figures 7E to F) Specific interaction of FluB-393 with NA. (Figure 7G) Specific interaction of 2D10 with NA. Detailed Implementation

[0029] As discussed above, influenza B virus is less dangerous than its closely related counterpart, influenza A virus, but still poses a significant health risk to some individuals. Here, the inventors describe the isolation of human mAbs recognizing IBV NA from circulating blood plasmablasts or long-lived plasma cells in bone marrow of individuals following vaccination with a 2018-2019 quadrivalent influenza vaccine. Antibody variable gene sequences were obtained from isolated B cell clones, and a group of 17 antibodies reacting with IBV NA proteins was expressed using selected mAbs. The sequences of these mAbs fall into two clonal amplification families, with representative clones named FluB-393 or FluB-400. FluB-400 mAb recognizes contact residues within the NA active site, exhibiting potent inhibition of NA enzyme activity and preventing IBV attachment to primary human respiratory epithelial cells in cultures. When administered via systemic (intraperitoneal) or topical (intranasal) routes, FluB-400 protects mice from airway attack by real IBV virus. These and other aspects of this disclosure are described in detail below.

[0030] I. Influenza B virus

[0031] Influenza B virus is the only species in the genus *Betainfluenzavirus* within the family Orthomyxoviridae. It is known to infect only humans and seals. This limited host range, in contrast to the pandemics caused by morphologically similar influenza A viruses, is clearly the reason for the lack of related pandemics, as both viruses mutate through antigenic drift and reassortment. Based on the antigenic properties of the surface glycoprotein hemagglutinin, two co-circulating lineages of influenza B virus are known. These two lineages are referred to as B / Yamagata / 16 / 88-like virus and B / Victoria / 2 / 87-like virus. The quadrivalent influenza vaccine, licensed by the CDC, is currently designed to provide protection against both co-circulating lineages and has shown to be more effective than the previous trivalent vaccine in preventing influenza caused by influenza B virus.

[0032] To further mitigate the virus's impact, in humans, influenza B virus evolves more slowly than influenza A and more quickly than influenza C. The mutation rate of influenza B virus is 2 to 3 times slower than that of influenza A. However, influenza B virus is recognized as causing significant morbidity and mortality worldwide, and significantly impacting adolescents and school-aged children. Due to COVID-19 pandemic measures, the B / Yamagata lineage may become extinct in 2020 / 2021.

[0033] The influenza B virus has an enveloped capsid, and its virion consists of an envelope, matrix proteins, a nucleoprotein complex, a nucleocapsid, and a polymerase complex. Influenza B virus virions can have a spherical, irregular, or filamentous appearance and are surrounded by a membrane. The membrane contains viral HA, NA, NB, and BM2, which are membrane-integrated. Influenza A and B viruses cannot be distinguished based on their morphology or size, as determined by electron microscopy. The average size of spherical influenza B virions of the prototype B / Lee / 40 strain grown in embryonic chicken eggs is 137 ± 27 nm. Eight viral negative-strand RNA gene segments with different conformations, along with viral nucleoproteins and polymerase, exist within the influenza B virus virion as viral RNPs. Purified virions show that each spherical virion contains 400 to 500 spike-like projections corresponding to viral glycoproteins. M1 is the most abundant virion component, followed by NP, HA, and NA, with the lowest levels of NB and P proteins.

[0034] It is estimated that subtypes of influenza A virus diverged 2,000 years ago. Influenza A and B viruses are estimated to have diverged from a single ancestor approximately 4,000 years ago, while the ancestors of influenza A and B viruses, as well as the ancestor of influenza C virus, are estimated to have diverged from a common ancestor approximately 8,000 years ago. Metatranscriptomics studies have also identified closely related influenza B-like viruses, such as Wuhan spiny eel influenza virus, and influenza B-like viruses in various vertebrate species, such as salamanders and fish.

[0035] II. Monoclonal Antibodies and Their Production

[0036] "Isolated antibody" is an antibody that has been isolated and / or recovered from components of its native environment. Contaminant components of its native environment are substances that can interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein- or non-protein-based solutes. In some specific embodiments, the antibody is purified to: (1) greater than 95% by weight of the antibody as determined by the Lowry method, and most particularly greater than 99% by weight; (2) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) homogenized by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Since at least one component of the antibody's native environment will be absent, isolated antibodies include in situ antibodies from recombinant cells. However, generally, isolated antibodies are prepared by at least one purification step.

[0037] A basic tetrameric antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 basic tetrameric units and the J chain. In the case of IgG, the tetrameric unit is typically about 150,000 Daltons. Each L chain is linked to the H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable region (V) at its N-terminus. H ), followed by three constant structural domains (C) for each α and γ chain. H ), and for μ and the same type are four Cs H Structural domain. Each L-chain has a variable region (V) at its N-end. L ), followed by a constant structural domain (C) at its other end. L V L With V H Alignment, and C L With the first constant structural domain of the heavy chain (C H1 Alignment. It is assumed that specific amino acid residues form interfaces between the variable regions of the light and heavy chains. V H and V L The antibodies pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6.

[0038] L-chains from any vertebrate species can be based on their constant structural domains (C L The amino acid sequence of is assigned to one of two distinct types (called κ and λ). This is based on the constant structural domain (C) of its heavy chain. H Immunoglobulins can be classified into different classes or isotypes based on their amino acid sequences. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with heavy chains designated α, δ, ε, γ, and μ, respectively. Based on C... H With relatively minor differences in sequence and function, its γ and α classes are further subdivided into subclasses, with humans expressing the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0039] The term "variability" refers to the fact that certain segments of the V domain vary significantly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. Instead, the V region consists of relatively invariant stretches of 15 to 30 amino acids called framework regions (FRs), which are separated by extremely variable shorter regions, each 9 to 12 amino acids long, called "hypervariant regions." The variable regions of both the natural heavy and light chains each contain four FRs linked by three hypervariant regions connected by forming loops. These four FRs primarily adopt a β-sheet configuration and, in some cases, form part of a β-sheet structure. The hypervariable regions in each chain are tightly held together by FRs and, together with hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as enabling antibodies to participate in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0040] When used in this document, the term "hypervariate region" refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariate regions typically contain amino acid residues from the "complementarity-determining region" or "CDR" (e.g., V...). L Near residues approximately 24 to 34 (L1), 50 to 56 (L2), and 89 to 97 (L3) in the middle, and V HNear residues approximately 31 to 35 (H1), 50 to 65 (H2), and 95 to 102 (H3) in 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 the “hypervariant ring” (e.g., V). L Residues at positions 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H Residues at positions 26–32 (H1), 52–56 (H2), and 95–101 (H3) in the cyclotomic ring, when numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901–917 (1987)); and / or those residues from the “hypervariant ring” / CDR (e.g., V L Residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in the middle, and V H Residues 27-38 (H1), 56-65 (H2), and 105-120 (H3) in the antibody, when numbered according to the IMGT numbering system; Lefranc, MP 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: V L 28, 36 (L1), 63, 74 to 75 (L2) and 123 (L3) in the middle, and V sub H 28, 36 (H1), 63, 74 to 75 (H2) and 123 (H3), when according to AHo number; Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001)).

[0041] "German nucleic acid residues" refer to nucleic acid residues naturally present in germline genes that encode constant or variable regions. "German genes" are the DNA found in germ cells (i.e., cells destined to become eggs or sperm). "German mutation" refers to a heritable change in specific DNA that occurs in a germ cell or fertilized egg at the single-cell stage, and when passed on to offspring, such a mutation is incorporated into every cell of the body. German mutations contrast with somatic mutations acquired in a single somatic cell. In some cases, nucleotides in the germline DNA sequence encoding variable regions are mutated (i.e., somatic mutations) and replaced with different nucleotides.

[0042] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that the antibodies constituting the population are identical except for a possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody preparations which contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies available in this disclosure can be prepared by the hybridoma method originally described by Kohler et al., Nature, 256:495 (1975), or by single-cell sorting of antigen-specific B cells, antigen-specific plasmablasts in response to infection or immunization, or by recombinant DNA methods in bacterial, eukaryotic, or plant cells after capturing linked heavy and light chains from a large collection of sorted antigen-specific cells (see, for example, U.S. Patent 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries, for example, using techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0043] A. General Method

[0044] It should be understood that monoclonal antibodies binding to the influenza B virus will have a variety of applications. These include the production of diagnostic kits for the detection and diagnosis of influenza B virus infection and for its treatment. In these cases, such antibodies can be linked to diagnostic or therapeutic agents, used as capture or competing agents in competitive assays, or used alone without any additional reagent linked to them. As discussed further below, antibodies can be mutated or modified. Methods for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; U.S. Patent 4,196,265).

[0045] Methods for producing monoclonal antibodies (MAb) generally begin along the same route as methods for preparing polyclonal antibodies. The first step in both methods is to immunize a suitable host or identify an individual immunized due to prior natural infection or vaccination with a licensed or experimental vaccine. As is known in the art, the immunogenicity of a given composition used for immunization can vary. Therefore, it is often necessary to enhance the host's immune system, such as by conjugating a peptide or polypeptide immunogen to a carrier. Some 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. Methods for conjugating peptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Also as well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by using a nonspecific stimulant of the immune response (referred to as an adjuvant). Exemplary and preferred adjuvants in animals include complete Freund's adjuvant (containing a nonspecific stimulant of the immune response to kill Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant, and in humans, combinations of alum, CpG, MFP59, and immunostimulatory molecules (“adjuvant systems”, such as AS01 or AS03). It is possible to induce influenza B virus-specific B cells through other experimental inoculation methods, including nanoparticle vaccines, or delivery of DNA or RNA genes in physical delivery systems (such as lipid nanoparticles or on gold bio-ballistic beads), and delivery of gene-encoded antigens using needles, gene guns, or percutaneous electroporation devices. The antigen gene may also be carried by replicative or replication-defective viral vectors such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alpha virus replicons, or alternatively, virus-like particles.

[0046] In the case of human antibodies against naturally occurring pathogens, a suitable approach is to identify individuals who have been exposed to the pathogen, such as those diagnosed with the disease or vaccinated to develop protective immunity against it, or to test the safety or efficacy of experimental vaccines. Circulating antipathogen antibodies can be detected, and antibodies encoding or generating B cells can subsequently be obtained from antibody-positive individuals.

[0047] The amount of immunogen composition used to generate polyclonal antibodies varies depending on the nature of the immunogen and the animals used for immunization. The immunogen can be administered via various routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored by collecting blood from immunized animals at different time points after immunization. Second and booster injections can also be given. The booster immunization and titration process is repeated until the appropriate titer is obtained. When the desired immunogenicity level is obtained, blood can be collected from the immunized animals and the serum can be separated and stored, and / or the animals can be used to generate MAb.

[0048] Following immunization, somatic cells with antibody-producing potential, particularly B lymphocytes (B cells), are selected for the MAb production protocol. These cells can be obtained from biopsied spleen, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs such as the lungs or GI tracts, or from circulating blood. Antibody-producing B lymphocytes from the immunized animal or immunized human are then fused with immortalized myeloma cells, typically immortalized myeloma cells of the same species as the immunized animal or human or human / mouse chimeric cells. Myeloma cell lines suitable for hybridoma-producing fusion procedures are preferably antibody-inhibiting, have high fusion efficiency, and are enzyme-deficient, which then prevents them from growing in certain selective media that only support the growth of the desired fused cells (hybridomas). As known to those skilled in the art, any of a variety of myeloma cells can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells.

[0049] Methods for generating antibody-producing spleen cells or antibody-producing lymph node cells to hybridize myeloma cells typically involve mixing somatic cells and myeloma cells at a 2:1 ratio in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion, although this ratio can vary from about 20:1 to about 1:1. In some cases, transformation of human B cells with Epstein-Barr virus (EBV) as an initial step increases B cell size, thereby enhancing fusion with relatively large myeloma cells. EBV transformation efficiency has been enhanced by using CpG and Chk2 inhibitor drugs in the transformation medium. Alternatively, human B cells can be activated by co-culturing transfected cell lines expressing CD40 ligand (CD154) in a medium containing additional soluble factors such as IL-21 and human B cell activating factor (BAFF) (a type II member of the TNF superfamily). Kohler and Milstein (1975; 1976) described fusion methods using Sendai virus, and Gefter et al. (1977) described fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG. Electro-induced fusion methods are also suitable (Goding, pp. 71–74, 1986), and some methods are more efficient (Yu et al., 2008). Fusion procedures are typically performed at a low frequency (approximately 1 × 10⁻⁶). -6 Up to 1×10 -8 Viable hybrids are produced, but with optimized procedures, fusion efficiencies approaching 1 / 200 can be achieved (Yu et al., 2008). However, the relatively low fusion efficiency is not problematic because, by culturing in a selective medium, the live fusion hybrids are distinguished from the parental unfused cells (especially unfused myeloma cells that would normally divide indefinitely). Selective media are typically tissue media containing reagents that block de novo nucleotide synthesis. Exemplary and preferred reagents are aminopterin, methotrexate, and diazoserine. Aminopterin and methotrexate block the de novo synthesis of both purines and pyrimidines, while diazoserine blocks only purine synthesis. When using aminopterin or methotrexate, hypoxanthine and thymidine are added to the medium as a source of nucleotides (HAT medium). When using diazoserine, hypoxanthine is added to the medium. If the B cell source is an EBV-transformed human B cell line, ouabain is added to eliminate EBV-transformed lines that have not fused with myeloma.

[0050] The preferred selection medium is HAT or HAT containing ouabain. Only cells capable of the nucleotide rescue pathway can survive in HAT medium. Myeloma cells are deficient in key enzymes of the rescue pathway (e.g., hypoxanthine phosphoribosyl transferase, HPRT) and therefore cannot survive. B cells can perform this pathway, but they have a limited lifespan in culture and typically die within about two weeks. Therefore, only cells that can survive in the selection medium are those heterozygotes formed from myeloma and B cells. When the source of B cells used for fusion is an EBV-transformed B cell line, ouabain can also be used for drug selection of heterozygotes because EBV-transformed B cells are susceptible to drug killing, while the myeloma mates used for selection are resistant to ouabain.

[0051] Culture provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed as follows: cells are cultured by monoclonal dilution in a microtiter plate, followed by testing the desired reactivity of individual clonal supernatants (after approximately two to three weeks). Assays should be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, etc. The selected hybridomas are then serially diluted or sorted into single cells by flow cytometry and cloned into a separate antibody-producing cell line, which can then be proliferated indefinitely to provide mAbs. Cell lines can be used for mAb production in two basic ways. Hybridoma samples can be injected (usually into the peritoneal cavity) into animals (e.g., mice). Optionally, the animal is primed with hydrocarbons, particularly oils (e.g., pterostilbene (tetramethylpentadecane)) prior to injection. When using human hybridomas in this manner, injection into immune-compromising mice (e.g., SCID mice) is ideal to prevent tumor rejection. The animal is injected with a tumor that secretes specific monoclonal antibodies produced by the fusion cell hybrid. Body fluids from the animal (e.g., serum or ascites) can then be extracted to provide high concentrations of MAb. Alternatively, a single cell line can be cultured in vitro, from which high concentrations of MAb can be readily obtained by naturally secreting the MAb into the culture medium. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. The cell lines can be adapted for growth in serum-free media to optimize the ability to recover high-purity human monoclonal immunoglobulins.

[0052] If desired, the mAbs produced by any of these methods can be further purified using filtration, centrifugation, and various chromatographic methods (e.g., FPLC or affinity chromatography). Fragments of the monoclonal antibodies of this disclosure can be obtained from purified monoclonal antibodies by methods including digestion with enzymes (e.g., pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Alternatively, the monoclonal antibody fragments covered by this disclosure can be synthesized using an automated peptide synthesizer.

[0053] The use of molecular cloning methods to generate monoclonal antibodies was also considered. Single B cells labeled with the target antigen can be physically sorted using paramagnetic bead selection or flow cytometry, and RNA can then be isolated from the single cells, with antibody genes amplified by RT-PCR. Alternatively, antigen-specific, large-scale sorted cell populations can be isolated in microvesicles, and matched heavy and light chain variable genes can be recovered from single cells using physical ligation of heavy and light chain amplicones or co-barcoding of heavy and light chain genes from the vesicles. Matched heavy and light chain genes from single cells can also be obtained from antigen-specific B cell populations by treating cells with cell-penetrating nanoparticles carrying RT-PCR primers and barcodes for labeling transcripts with one barcode per cell. Antibody variable genes can also be isolated from RNA extraction from hybridoma lines, and antibody genes can be obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, an assembled immunoglobulin phage plasmid library can be prepared from RNA isolated from cell lines, and phage plasmids expressing suitable antibodies can be selected by panning using viral antigens. The advantage of this method over conventional hybridoma technology is that it can generate and screen approximately 10 hybridomas in a single round. 4 The increased number of antibodies, and the generation of new specificities through the combination of H and L chains, further enhances the chances of discovering suitable antibodies.

[0054] Other U.S. patents (each incorporated herein by reference) that teach the generation of antibodies usable in this disclosure include U.S. Patent 5,565,332, which describes the generation of chimeric antibodies using a combination method; U.S. Patent 4,816,567, which describes the preparation of recombinant immunoglobulins; and U.S. Patent 4,867,973, which describes antibody-therapeutic agent conjugates.

[0055] B. Antibodies disclosed in this publication

[0056] Antibodies according to this disclosure can be defined by their binding specificity in a first instance. By assessing the binding specificity / affinity of a given antibody using techniques known to those skilled in the art, they can determine whether such an antibody falls within the scope of the claims of this invention. For example, the epitope to which a given antibody binds may consist of a single, continuous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) located within the antigen molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of multiple discontinuous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope).

[0057] A variety of techniques known to those skilled in the art can be used to determine whether an antibody interacts with “one or more amino acids” within a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays, such as those described in Antibodies, Harlow, and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Cross-blocking can be measured in a variety of binding assays, such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, high-resolution electron microscopy using single-particle reconstruction, cryoEM, or tomography, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer, 2000, Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids within a peptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium-labeling the target protein, followed by binding an antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo deuterium-to-hydrogen reverse exchange at a slower rate than those within amino acids not part of the interface. As a result, the amino acids forming part of the protein / antibody interface retain deuterium and thus exhibit relatively higher quality compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring, 1999, Analytical Biochemistry 267: 252-259; Engen and Smith, 2001, Anal. Chem. 73: 256A-265A. When antibodies neutralize influenza B virus, antibody-escape mutant organisms can be isolated by propagating the influenza B virus in vitro or in animal models in the presence of high antibody concentrations. Sequence analysis of the influenza B virus gene encoding the antigen targeted by the antibody revealed mutations conferring antibody escape, indicating residues in the epitope or residues that allosterically affect the epitope structure.

[0058] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from either consecutive amino acids or non-consecutive amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three amino acids with a unique spatial conformation, and more commonly at least five or eight to ten amino acids.

[0059] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen based on the similarity of their binding properties to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, which is incorporated herein by reference in its entirety). Each category reflects a unique epitope that is distinctly different from or partially overlaps with the epitopes represented by another category. This technique enables rapid filtering of genetically identical antibodies, allowing characterization to focus on genetically unique antibodies. When applied to hybridoma screening, MAP can help identify rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to classify the antibodies of this disclosure into groups of antibodies that bind to different epitopes.

[0060] This disclosure includes antibodies that can bind to the same epitope or a portion of an epitope. Similarly, this disclosure includes antibodies that compete with any specific exemplary antibody described herein for binding to a target or fragment thereof. Whether an antibody binds to the same epitope as a reference antibody or competes for binding with it can be readily determined using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to the target under saturation conditions. Next, the ability of the test antibody to bind to the target molecule is evaluated. If the test antibody is able to bind to the target molecule after 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 cannot bind to the target molecule after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0061] To determine whether an antibody competes with a reference antibody against influenza B virus for binding, the binding method described above is performed in two directions: In the first direction, the reference antibody is bound to the influenza B virus antigen under saturation conditions, and then the binding of the test antibody to the influenza B virus molecule is evaluated. In the second direction, the test antibody is bound to the influenza B virus antigen molecule under saturation conditions, and then the binding of the reference antibody to the influenza B virus molecule is evaluated. If only the first (saturated) antibody can bind to the influenza B virus in both directions, it can be concluded that the test antibody and the reference antibody compete for binding to the influenza B virus. As will be understood by those skilled in the art, the antibody competing for binding with the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0062] If one of two antibodies competitively inhibits (blocks) the binding of the other to an antigen, then both antibodies bind to the same or overlapping epitopes. That is, an excess of 1, 5, 10, 20, or 100 times that of one antibody will inhibit the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have overlapping epitopes.

[0063] Further routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding to the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric hindrance (or other phenomena) is the cause of the lack of observed binding. These experiments 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 using EM or crystallography can also demonstrate whether two competing antibodies recognize the same epitope.

[0064] In another aspect, monoclonal antibodies are provided having clonal pairs of CDRs derived from the heavy and light chains, as shown in Tables 3 and 4, respectively. Such antibodies can be produced using the methods described herein through clones discussed below in the Examples section.

[0065] In another respect, antibodies may be defined by variable sequences that contain additional "framework" regions. These are provided in Tables 1 and 2, encoding or representing complete variable regions. Furthermore, antibody sequences may differ from these sequences, optionally using methods discussed in more detail below. For example, a nucleic acid sequence may differ from those listed above in the following ways: (a) the variable region may be separable from the constant structural domains of the light and heavy chains; (b) the nucleic acid may differ from those listed above without affecting the residues it encodes; (c) the nucleic acid may differ from those listed above in terms of a given percentage, such as 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; (d) the nucleic acid may differ from those listed above due to its ability to hybridize under high-strength conditions, as exemplified by low-salt and / or high-temperature conditions, such as those 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 acid may differ from those listed above in terms of a given percentage, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; or (f) the amino acid may differ from those listed above by allowing conserved substitutions (discussed below). The above applies to the nucleic acid sequences shown in Table 1 and the amino acid sequences shown in Table 2.

[0066] When comparing polynucleotide and polypeptide sequences, two sequences are considered "identical" if the sequences of nucleotides or amino acids are identical when the maximum correspondence is found during alignment, as described below. Comparisons between two sequences are typically performed by comparing sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions (typically 30 to about 75, or 40 to about 50), where, after optimal alignment of the two sequences, the sequence can be compared to a reference sequence with the same number of consecutive positions.

[0067] The optimal alignment of sequences for comparison can be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) in the Lasergene suite of bioinformatics software with default parameters. This procedure embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345–358; Hein J. (1990) Unified Approach to Alignment and Phylogeny, pp. 626–645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, DG and Sharp, PM (1989) CABIOS 5:151–153; Myers, EW and Muller W. (1988) CABIOS 4:11–17; Robinson, ED (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0068] Alternatively, the best alignment of sequences for comparison can be performed using: the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482; the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; the similarity retrieval method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; or by computer implementation or inspection of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.).

[0069] A specific example of an algorithm suitable for determining the percentage of sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, 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 the percentage of sequence identity of polynucleotides and peptides of this disclosure. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. The rearrangement nature of antibody sequences and the variable length of each gene require multiple rounds of BLAST searching to find a single antibody sequence. Moreover, the artificial assembly of different genes is difficult and error-prone. The sequence analysis tool IgBLAST (accessible via ncbi.nlm.nih.gov / igblast / ) identifies matches with germline V, D, and J genes, provides details of rearranged junctions, and describes the IgV domain framework and complementarity-determining regions. IgBLAST can analyze nucleotide or protein sequences, process sequences in batches, and allows simultaneous searches of germline gene databases and other sequence databases to minimize the chance of missing potentially best-matched germline V genes.

[0070] In an illustrative example, for a nucleotide sequence, the cumulative score can be calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). Word hits will stop expanding in each direction if: the cumulative alignment score decreases by an amount X from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored 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) defaults to using a word length (W) of 11, an expectation (E) of 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) for alignment, with (B) of 50, an expectation (E) of 10, M = 5, N = -4, and comparisons on both strands.

[0071] For amino acid sequences, a scoring matrix can be used to calculate cumulative scores. Word hits will stop expanding in each direction if: the cumulative alignment score decreases by an amount X from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the alignment sensitivity and speed.

[0072] In one approach, a “sequence identity percentage” is determined by comparing two best-aligned sequences across a comparison window of at least 20 positions. This comparison window contains a portion of a polynucleotide or polypeptide sequence that may contain 20% or less, typically 5% to 15%, or 10% to 12% of additions or deletions (i.e., gaps) compared to a reference sequence (which contains no additions or deletions) for best-alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to generate a number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage.

[0073] Another way to define antibodies is as “derivatives” of any of the antibodies and their antigen-binding fragments described below. The term “derivative” refers to an antibody or its antigen-binding fragment that binds specifically to an antigen but contains 1, 2, 3, 4, 5, or more amino acid substitutions, additions, deletions, or modifications relative to the “parent” (or wild-type) molecule. Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, variants having altered CH1, hinge, CH2, CH3, or CH4 regions to form antibodies, for example, variant Fc regions exhibiting enhanced or impaired effector or binding characteristics. The term "derivative" also encompasses non-amino acid modifications, such as amino acids that can be glycosylated (e.g., having altered contents of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-hydroxyacetic acid neuraminic acid, etc.), acetylated, PEGylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. In some embodiments, the altered carbohydrate modification modifies one or more of the following: antibody solubilization, promotion of subcellular transport and secretion of antibodies, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In one specific embodiment, the altered carbohydrate modification enhances antibody-mediated effector function relative to antibodies lacking carbohydrate modification. Carbohydrate modifications that lead to antibody-mediated alterations in effector function are well known in the art (see, for example, Shields, RL et al. (2002), J. Biol. Chem. 277(30): 26733-26740; Davies J. et al. (2001), Biotechnology & Bioengineering 74(4): 288-294). Methods for altering carbohydrate content are known to those skilled in the art, see, for example, Wallick, SC et al. (1988), J. Exp. Med. 168(3): 1099-1109; Tao, MH et al. (1989), J. Immunol. 143(8): 2595-2601; Routledge, EG et al. (1995), Transplantation 60(8):847-53; Elliott, S. et al. (2003), Nature Biotechnol. 21:414-21; Shields, RL et al. (2002), J. Biol. Chem. 277(30):26733-26740).

[0074] It can produce derived antibodies or antibody fragments with modified sequences or glycosylation states to confer preferred levels of activity in the following: antibody-dependent cytotoxicity (ADCC), antibody-dependent 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.

[0075] Derived antibodies or antibody fragments can be chemically modified using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and tunicamycin metabolic synthesis. In one embodiment, the antibody derivative will have a function similar to or the same as the parent antibody. In another embodiment, the antibody derivative will exhibit altered activity relative to the parent antibody. For example, the derived antibody (or a fragment thereof) may bind more tightly to its epitope or be more resistant to proteolysis compared to the parent antibody.

[0076] C. Modification of antibody sequences

[0077] In various embodiments, the sequence of the identified antibody may be modified for a variety of reasons, such as improving expression, improving cross-reactivity, or reducing off-target binding. Modified antibodies can be prepared using any technique known to those skilled in the art, including expression via standard molecular biotechnology or chemical synthesis of peptides. Methods for recombinant expression are described elsewhere in this document. The following is a general discussion of relevant target techniques for antibody modification.

[0078] Hybridomas can be cultured, cells lysed, and total RNA extracted. Random hexamers and RT can be used together to generate cDNA copies of the RNA, followed by PCR using a multiplex mixture of PCR primers intended to amplify all variable gene sequences. PCR products can be cloned into the pGEM-T Easy vector and then sequenced using standard vector primers via automated DNA sequencing. Binding and neutralization assays can be performed using antibodies collected from hybridoma supernatant and purified via FPLC using a G protein column.

[0079] Recombinant full-length IgG antibodies can be generated by subcloning the heavy and light chain Fv DNA from the cloning vector into an IgG plasmid vector, transfecting it into 293 (e.g., Freestyle) cells or CHO cells, and collecting and purifying the antibody from the 293 or CHO cell supernatant. Other suitable host cell systems include bacteria (e.g., E. coli), insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco with or without human-like glycans), algae, or various non-human transgenic environments such as mice, rats, goats, or cattle.

[0080] The expression of antibody-encoding nucleic acids was also considered for both subsequent antibody purification and host immunization. The antibody-encoding sequence can be RNA, such as native RNA or modified RNA. Modified RNA considered certain chemical modifications that confer increased stability and low immunogenicity to mRNA, thereby promoting the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) is superior to several other nucleoside modifications and combinations thereof in terms of translational capability. In addition to shutting down immuno / eIF2α phosphorylation-dependent translational repression, the incorporated N1mΨ nucleotide significantly alters the dynamics of translation by increasing ribosomal pauses and density on mRNA. Increased ribosomal loading on modified mRNA makes them more readily initiated by promoting ribosomal recycling or de novo ribosomal recruitment on the same mRNA. Such modifications can be used to enhance antibody expression in vivo after RNA inoculation. RNA, whether native or modified, can be delivered as naked RNA or in delivery carriers such as lipid nanoparticles.

[0081] Alternatively, DNA encoding antibodies can be used for the same purpose. The DNA is contained in an expression cassette that includes a promoter active in the host cell to which it is designed. The expression cassette is advantageously contained in a reproducible vector, such as a conventional plasmid or microvector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses. Replicons encoding antibody genes, such as alphavirus replicons based on VEE virus or Sindbis virus, are also considered. Delivery of such vectors, when desired for in vivo expression, can be performed via needle-through intramuscular, subcutaneous, or intradermal routes, or via percutaneous electroporation.

[0082] The rapid availability of antibodies produced using the same host cells and cell culture processes as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a general method for rapidly producing small amounts (up to 50 g) of antibodies in CHO cells using co-transfectants cultured in CDACF medium. While slightly slower than a true transient system, its advantages include higher product concentrations and the use of the same host and process as the production cell lines. An example of the growth and productivity of a GS-CHO library expressing model antibodies in a single-use bioreactor is as follows: a harvest antibody concentration of 2 g / L was achieved within 9 weeks post-transfection in a single-use bag bioreactor culture (5 L working volume) in fed-batch mode.

[0083] The antibody molecule will contain fragments, for example, generated by proteolytic cleavage of mAb (e.g., F(ab'), F(ab')2) or, for example, single-chain immunoglobulins that can be generated recombinantly. The F(ab') antibody derivative is monovalent, while the F(ab')2 antibody derivative is divalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands, to form a "chimeric" binding molecule. Clearly, such a chimeric molecule may contain substituents capable of binding to different epitopes of the same molecule.

[0084] In some relevant embodiments, the antibody is a derivative of the disclosed antibody, for example, an antibody containing a CDR sequence identical to the CDR sequence in the disclosed antibody (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, modifications may be desired, such as introducing conserved changes into the antibody molecule. When making such changes, the hydropathic index of the amino acids may be considered. The importance of the amino acid hydropathic index in conferring interacting biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0085] It should also be understood in the art that similar amino acid substitutions can be made efficiently based on hydrophilicity. U.S. Patent 4,554,101 (incorporated herein by reference) claims that the maximum local average hydrophilicity of a protein (e.g., governed by the hydrophilicity of its neighboring amino acids) is related to the biological properties 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: aspartic acid (+3.0 ± 1), glutamic acid (+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), and proline (-0.5 ± 1). 1) Alanine (-0.5) and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5) and tyrosine (-2.3).

[0086] It should be understood that amino acids can be replaced with another amino acid having similar hydrophilicity, resulting in a biologically or immunologically modified protein. In such modifications, the substitution of amino acids with hydrophilicity values ​​within ±2 is preferred, particularly those within ±1, and even more particularly preferred those within ±0.5.

[0087] As summarized above, amino acid substitutions are typically based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions for several of the aforementioned features are expected to be well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0088] This disclosure also considers isotype modification. By modifying the Fc region to have different isotypes, different functions can be achieved. For example, changing to IgG1 can improve antibody-dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve titer.

[0089] As an alternative or supplement, it may be useful to combine amino acid modifications with one or more other amino acid modifications that alter the C1q binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of the IL-23p19 binding molecule. Particularly interesting binding peptides may be those that bind to C1q and exhibit complement-dependent cytotoxicity. Peptides with pre-existing C1q binding activity may be modified, optionally also having CDC-mediating capabilities, to enhance one or both of these activities. Amino acid modifications that alter C1q and / or modify their complement-dependent cytotoxic function are described, for example, in WO / 0042072, which is incorporated herein by reference.

[0090] The Fc region of an antibody with altered effector functions can be designed, for example, by modifying C1q binding and / or FcγR binding, thereby altering CDC and / or ADCC activities. An "effector function" is responsible for activating or deactivating biological activity (e.g., in a subject). Some 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; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require an Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays (e.g., Fc binding assay, ADCC assay, CDC assay, etc.).

[0091] For example, variant Fc regions that can produce antibodies with improved C1q binding and improved FcγRIII binding (e.g., with both improved ADCC activity and improved CDC activity). Alternatively, if it is desired to reduce or eliminate effector function, variant Fc regions can be modified to have reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities can be enhanced, and optionally, the other activity is also reduced (e.g., generating Fc region variants with improved ADCC activity but reduced CDC activity, or vice versa).

[0092] FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the nascent Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to FcRn has 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). Numerous methods are known to lead to increased half-life (Kuo and Aveson, (2011)), including techniques involving amino acid modifications such as alanine scanning mutagenesis, random mutagenesis, and screening to assess binding to the nascent Fc receptor (FcRn) and / or in vivo behavior. Post-mutation computational strategies can also be used to select one of the amino acid mutations for further mutation.

[0093] Therefore, this disclosure provides variants of antigen-binding proteins having optimal binding with FcRn. In one specific embodiment, said variant of the antigen-binding protein includes at least one amino acid modification in the Fc region of said antigen-binding protein, wherein said modification is selected from positions 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, and 276 of the Fc region, compared to said parent polypeptide. 284th, 285th, 288th, 289th, 290th, 291st, 292nd, 294th, 297th, 298th, 299th, 301st, 302nd, 303rd, 305th, 307th, 308th, 309th, 311th, 315th, 317th, 320th, 322nd, 325th, 327th, 330th, 332nd, 334th, 335th, 338th, 340th, 342nd, 3rd 43rd, 345th, 347th, 350th, 352nd, 354th, 355th, 356th, 359th, 360th, 361st, 362nd, 369th, 370th, 371st, 375th, 378th, 380th, 382nd, 384th, 385th, 386th, 387th, 389th, 390th, 392nd, 393rd, 394th, 395th, 396th, 397th, 398th, 399th Positions 400, 401, 403, 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, wherein the amino acid number in the Fc region is the EU index number in Kabat. In another aspect of this disclosure, the modification is M252Y / S254T / T256E.

[0094] In addition, several publications describe methods for obtaining physiologically active molecules with modified half-lives, see, for example, Kontermann (2009), by introducing FcRn-binding peptides into the molecule, or by fusing the molecule with an antibody that retains FcRn binding affinity but has greatly reduced affinity for other Fc receptors, or by fusing the molecule with the FcRn-binding domain of an antibody.

[0095] Derived antibodies can be used to alter the half-life (e.g., serum half-life) of parental antibodies in mammals, particularly humans. Such alterations can result in a half-life greater than 15 days, preferably greater than 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 2 months, 3 months, 4 months, or 5 months. The increased half-life of the antibodies or fragments of this disclosure in mammals, preferably humans, results in higher serum titers of the antibodies or antibody fragments in mammals, and thus reduces the frequency of administration of the antibodies or antibody fragments and / or the concentration of the antibodies or antibody fragments to be administered. Antibodies or fragments of the antibody or antibody fragment with an increased in vivo half-life can be produced using techniques known to those skilled in the art. For example, antibodies or fragments of the antibody or antibody fragment with an increased in vivo half-life can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor.

[0096] Beltramello et al. (2010) previously reported a modification that neutralizes mAbs (due to their tendency to enhance dengue virus infection) by replacing leucine residues at positions 1,3 and 1,2 of the CH2 domain (based on the IMGT unique numbering of the C domain) with alanine residues. This modification (also known as the “LALA” mutation) eliminates antibodies binding to FcγRI, FcγRII, and FcγRIIIa, as described by Hescell et al. (2007). The neutralizing and enhancing abilities of the variant and the unmodified recombinant mAb against the four dengue virus serotypes were compared. The LALA variant retained the same neutralizing activity as the unmodified mAb, but showed no enhancing activity whatsoever. Therefore, the LALA mutation, with this property, is considered in the context of currently published antibodies.

[0097] Glycosylation alteration. One specific embodiment of this disclosure is an isolated monoclonal antibody or antigen-binding fragment thereof comprising a substantially homogeneous glycan free of sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, which may be linked to the heavy chain or light chain constant region, respectively. The aforementioned substantially homogeneous glycan may be covalently linked to the heavy chain constant region.

[0098] Another embodiment of this disclosure comprises a mAb having a novel Fc glycosylation pattern. The isolated monoclonal antibody or its antigen-binding fragment is present in a substantially homogeneous composition represented by GNGN or G1 / G2 glycoforms. Fc glycosylation plays a crucial role in the antiviral and anticancer properties of therapeutic mAbs. This disclosure is consistent with recent studies showing enhanced cell-mediated viral suppression against lentiviral viruses in vitro with fucose-free anti-HIV mAbs. This embodiment of the disclosure, with a homogeneous glycan lacking a core fucose, shows more than a two-fold increase in protection against specific viruses. Elimination of the core fucose significantly improves ADCC activity of mAbs mediated by natural killer (NK) cells, but shows the opposite effect on ADCC activity in polymorphonuclear cells (PMNs).

[0099] Compared to antibodies that do not have a substantially homogeneous GNGN glycoform and those that have glycoforms containing G0, G1F, G2F, GNF, GNGNF, or GNGNFX, isolated monoclonal antibodies or their antigen-binding fragments comprising a substantially homogeneous composition represented by GNGN or G1 / G2 glycoforms exhibit increased binding affinity for FcγRI and FcγRIII. In one embodiment of this disclosure, the antibody is in a concentration of 1 × 10⁻⁶. -8 M or smaller Kd dissociates from FcγRI and at 1×10 -7 M or smaller Kd dissociates from FcγRIII.

[0100] Glycosylation in the Fc region is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation involves the attachment of one of the sugars—N-acetylgalactosamine, galactose, or xylose—to a hydroxy amino acid (most commonly serine or threonine), although 5-hydroxyproline or 5-hydroxylysine can also be used. The recognition sequences used for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequence are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. Therefore, the presence of either of these peptide sequences in the polypeptide creates a potential glycosylation site.

[0101] Glycosylation patterns can be altered, for example, by deleting one or more glycosylation sites present in the polypeptide and / or adding one or more glycosylation sites not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody (for N-linked glycosylation sites) is conveniently achieved by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences. An exemplary glycosylation variant has an amino acid substitution of the heavy chain residue Asn 297. This alteration can also be made by adding one or more serine or threonine residues to the sequence of the original polypeptide or by replacing the sequence of the original polypeptide with one or more serine or threonine residues (for O-linked glycosylation sites). Alternatively, changing Asn 297 to Ala can remove one of the glycosylation sites.

[0102] In some embodiments, the antibody is expressed in cells expressing β(1,4)-N-acetylglucosamine transferase III (GnT III), such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for generating antibodies in this manner 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 using genome editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to enhance, reduce, or eliminate certain post-translational modifications, such as glycosylation. For example, CRISPR technology can be used to eliminate genes encoding glycosylation enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies.

[0103] Elimination of monoclonal antibody protein sequence liability. Modifiable antibody gene sequences derived from human B cells can be used to enhance their manufacturability and safety. Potential protein sequence liability can be identified by searching for sequence motifs associated with sites containing:

[0104] 1) Unpaired Cys residues,

[0105] 2) N-linked glycosylation,

[0106] 3) Deamidation of Asn,

[0107] 4) Asp heterogeneity,

[0108] 5) SYE is truncated.

[0109] 6) Met oxidation,

[0110] 7) Trp oxidation,

[0111] 8) N-terminal glutamic acid,

[0112] 9) Integral binding,

[0113] 10) CD11c / CD18 combination, or

[0114] 11) Fragmentation.

[0115] Such motifs can be eliminated by altering the gene encoding the cDNA of recombinant antibodies.

[0116] Protein modification work in the development of therapeutic antibodies has clearly revealed that certain sequences or residues are associated with differences in solubility (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 suggests 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.

[0117] Stability. Antibodies can be modified to enhance their biophysical properties. The average apparent melting temperature can be used; increasing the temperature causes the antibody to unfold, thus determining relative stability. Differential scanning calorimetry (DSC) measures the heat capacity (C) of a molecule as a function of temperature. p (The heat required to raise the temperature of this molecule by one degree). The thermal stability of antibodies can be studied using DSC. DSC data for mAbs are particularly interesting because they sometimes resolve the unfolding of individual domains within the mAb structure, resulting in up to three peaks in the thermogram (from Fab, C...). H 2 and C H3. Unfolding of the Fab domain. Generally, the unfolding of the Fab domain produces the strongest peak. DSC spectra and relative stability of the Fc region reveal characteristic differences among human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). Circular dichroism (CD) (performed using a CD spectrometer) can also be used to determine the average apparent unfolding temperature. Far-UV CD spectra of the antibody are measured in increments of 0.5 nm in the range of 200 to 260 nm. The final spectrum is determined as the average of 20 cumulative measurements. Residue ellipticity values ​​can be calculated after background subtraction. Thermal unfolding of the antibody (0.1 mg / mL) can be monitored at 235 nm at temperatures ranging from 25°C to 95°C with a heating rate of 1°C / min. Dynamic light scattering (DLS) can be used to assess the tendency to aggregate. DLS is used to characterize the size of various particles, including proteins. If the system is not dimensionally dispersed, the average effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of the surface structure, and the particle concentration. Since DLS essentially measures the fluctuations in the intensity of scattered light caused by the particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays particle groups of different diameters. Stability studies can be conveniently performed using DLS. DLS measurements of a sample can indicate whether particles aggregate over time or with temperature by determining whether the hydrodynamic radius of the particles increases. If particles aggregate, larger groups of particles with larger radii can be seen. Temperature-dependent stability can be analyzed by controlling the in-situ temperature. Capillary electrophoresis (CE) includes validated methods for determining antibody stability characteristics. The iCE method can be used to resolve antibody protein charge variants caused by deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other protein changes that can lead to changes in the protein pI. The Protein Simple Maurice instrument can be used to evaluate each expressed antibody protein in a capillary column (cIEF) via high-throughput, free-solution isoelectric focusing (IEF). Full-column UV absorbance detection can be performed every 30 seconds to monitor molecules focused at their isoelectric point (pI) in real time. This method combines the high resolution of traditional gel IEF with the quantification and automation advantages of column-based separation, while eliminating the need for a transfer step. This technique provides reproducible, quantitative analysis of the identity, purity, and heterogeneity profiles of expressed antibodies.The results determined the charge heterogeneity and molecular size of the antibody, with detection sensitivity as low as 0.7 µg / mL in both absorbance and native fluorescence detection modes.

[0118] Solubility. An intrinsic solubility score can be determined for antibody sequences. The intrinsic solubility score can be calculated using CamSolIntrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The solubility score can be calculated by evaluating the amino acid sequence of residues 95 to 102 (Kabat number) in HCDR3 of each antibody fragment, such as scFv, using an online program. Solubility can also be determined using laboratory techniques. Several techniques exist, including adding lyophilized protein to a solution until the solution becomes saturated and reaches its solubility limit, or concentrating it by ultrafiltration in a microconcentrator with a suitable molecular weight cutoff. The most straightforward method is to induce amorphous precipitation, which uses methods involving protein precipitation with ammonium sulfate to measure protein solubility (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information about relative solubility values. Ammonium sulfate precipitation produces a solution containing a precipitate with clearly defined aqueous and solid phases, and requires a relatively small amount of protein. Solubility measurements using ammonium sulfate-induced amorphous precipitation can also be readily performed at various pH values. Protein solubility is highly pH-dependent, and pH is considered the most important external factor affecting solubility.

[0119] Autoreactivity. It is generally believed that autoreactive clones should be eliminated through negative selection during ontogeny; however, it is clear that many naturally occurring antibodies with autoreactive properties persist in the adult mature library, and that autoreactivity can enhance the antiviral function of many antibodies against pathogens. It has been noted that the HCDR3 loop in antibodies is often positively charged and exhibits an autoreactive pattern during early B cell development (Wardemann et al., Science 301, 1374-1377, 2003). The autoreactivity of a given antibody can be tested by assessing the level of binding to human-derived cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometry cell surface staining (using suspended Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity can also be examined by assessing binding to tissues in tissue arrays.

[0120] Preferred Residues (“Human Likeness”). In many recent studies, large-scale deep sequencing of human B cell libraries derived from blood donors is underway. Sequence information about key portions of human antibody libraries facilitates statistical evaluation of antibody sequence characteristics common in healthy individuals. Using knowledge of antibody sequence characteristics from a human recombinant antibody variable gene reference database, the positional specificity of “Human Likeness” (HL) of antibody sequences can be estimated. HL has been shown to be useful for developing antibodies for clinical use, such as therapeutic antibodies or antibodies as vaccines. The aim is to improve antibody human likeness to reduce potential adverse effects and anti-antibody immune responses that could significantly reduce the efficacy of antibody drugs or induce serious health effects. Antibody characterization of combined antibody libraries from three healthy human blood donors, totaling approximately 400 million sequences, has been evaluated, and a new “Relative Human Likeness” (rHL) score focusing on hypervariable regions of antibodies has been created. The rHL score makes it easy to distinguish human sequences (positive score) from non-human sequences (negative score). Antibodies can be engineered to eliminate residues uncommon in human libraries.

[0121] D. Single-chain antibody

[0122] Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains linked together by short (typically serine or glycine) linkers. While these chimeric molecules remove the constant region and introduce linker peptides, they retain the specificity of the original immunoglobulin. This modification generally does not alter specificity. Historically, these molecules were developed to facilitate phage display, where it is convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be generated directly from subclonal heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region present in complete antibody molecules and therefore lack common binding sites (e.g., protein A / G) used for antibody purification. These fragments are often purified / fixed using protein L because protein L interacts with the variable region of the κ light chain.

[0123] Flexible linkers are typically composed of amino acid residues that promote helical and turn-like structures (e.g., alanine, serine, and glycine). However, other residues can also function well. Tang et al. (1996) used phage display as a method for rapidly selecting specific linkers for single-chain antibodies (scFv) from a protein linker library. Random linker libraries were constructed in which genes for variable domains of the heavy and light chains were linked by segments encoding 18-amino acid polypeptides with variable compositions. The scFv library (approximately 5 × 10⁻⁶) was displayed on filamentous phages. 6(1054 different members) and affinity selection was performed using haptens. The selected variants showed significantly enhanced binding activity while retaining considerable sequence diversity. Subsequent screening of 1054 individual variants yielded catalytically active scFvs produced efficiently in soluble form. Sequence analysis revealed that the only common feature of the selected tethers was: V H The two residues following the C-terminus are conserved proline in the linker, and there are abundant arginine and proline at other positions.

[0124] The recombinant antibodies disclosed herein may also involve sequences or portions that allow for receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow for multimerization with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chain may be modified with an agent that allows for the combination of two antibodies (e.g., biotin / avidin).

[0125] In one independent implementation, a single-chain antibody can be generated by linking the receptor light chain and heavy chain using a non-peptide linker or chemical unit. Typically, the light and heavy chains are generated in different cells, purified, and then linked together in a suitable manner (i.e., the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a suitable chemical bridge).

[0126] Crosslinking agents are used to form molecular bridges that bind the functional groups of two different molecules together; for example, stabilizers and coagulants. However, dimers or polymers that can produce the same analogues or heteropolymer complexes containing different analogues are also considered. To link two different compounds in a stepwise manner, hetero-bifunctional crosslinking agents can be used, which eliminate the unwanted formation of homopolymers.

[0127] An exemplary hetero-bifunctional crosslinking agent comprises two reactive groups: one reacting with a primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimide, halogen, etc.). Through the primary amine reactive group, the crosslinking agent can react with a lysine residue of a protein (e.g., a selected antibody or fragment), and through the thiol reactive group, the crosslinking agent already attached to the first protein reacts with a cysteine ​​residue (free thiol group) of another protein (e.g., a selector).

[0128] Preferably, crosslinking agents with reasonable stability in the blood are used. Various types of disulfide-containing linkers are known to be successfully used for conjugating targeting agents and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds have been shown to provide greater stability in vivo, thereby preventing the release of the targeting peptide before reaching its site of action. Therefore, these linkers constitute a group of conjugating agents.

[0129] Another cross-linking agent is SMPT, a bifunctional cross-linking agent containing a disulfide bond that is "sterically hindered" by the adjacent benzene ring and methyl group. The steric hindrance of the disulfide bond is thought to protect it from attack by thiolate anions (such as glutathione) that can be present in tissues and blood, and thus helps prevent the conjugate from uncoupling before the linked drug is delivered to the target site.

[0130] Like many other known crosslinking agents, SMPT crosslinking agents are capable of crosslinking functional groups such as the SH group of cysteine ​​or primary amines (e.g., the ε-amino group of lysine). Another possible type of crosslinking agent includes iso-bifunctional photoreactive azidobenzenes containing disulfide bonds that can be cleaved, such as sulfosuccinimidyl-2-(p-azidosalicylic acid)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with the primary amine group, and the azidobenzene (after photodecomposition) reacts nonselectively with any amino acid residue.

[0131] In addition to hindered crosslinking agents, non-hindered crosslinking agents can also be used. Other available crosslinking agents, without considering the inclusion or formation of protected disulfides, include SATA, SPDP, and 2-iminothionane (Wawrzynczak & Thorpe, 1987). The use of such crosslinking agents is well known in the art. Another embodiment involves the use of flexible joints.

[0132] U.S. Patent 4,680,338 describes bifunctional linkers that can be used to generate conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing unstable bonds that can be cleaved under a variety of mild conditions. Such linkers are particularly useful because the target drug can bind directly to the linker, and its cleavage leads to the release of the active agent. Particular uses include adding free amino or free thiol groups to proteins such as antibodies or drugs.

[0133] U.S. Patent 5,856,456 provides peptide linkers for linking polypeptide components to prepare fusion proteins (e.g., single-chain antibodies). The linker is up to about 50 amino acids in length; it comprises a charged amino acid (preferably arginine or lysine) followed by a proline, appearing at least once; and is characterized by higher stability and reduced aggregation. U.S. Patent 5,880,270 discloses amino-oxygenated linkers that can be used in a variety of immunodiagnostic and separation techniques.

[0134] E. Multispecific antibodies

[0135] In some embodiments, the antibodies of this disclosure are bispecific or multispecific. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. An exemplary bispecific antibody may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a second antigen binding site. Alternatively, an antipathogen arm may be combined with an arm that binds to triggering molecules on leukocytes, such as T-cell receptor molecules (e.g., CD3) or Fc receptors (FcγRs) of IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to concentrate and localize cellular defense mechanisms to infected cells. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies have a pathogen-binding arm and an arm that binds to cytotoxic agents, such as saporin, interferon-α, vinca alkaloids, ricin A chain, methotrexate, or a radioactive isotope hapten. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). WO 96 / 16673 describes a bispecific anti-ErbB2 / anti-FcγRIII antibody, and U.S. Patent 5,837,234 discloses a bispecific anti-ErbB2 / anti-FcγRI antibody. WO 98 / 02463 shows a bispecific anti-ErbB2 / Fcα antibody. U.S. Patent 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody.

[0136] Methods for preparing bispecific antibodies are known in the art. The conventional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random sorting of the immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually accomplished by affinity chromatography, is quite cumbersome and yields low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0137] Depending on the method, an antibody variable region with desired binding specificity (antibody-antigen combination site) is fused to an immunoglobulin constant domain sequence. Preferably, the fusion is with the Ig heavy chain constant domain (containing the hinge region, C... H2 District and C H3 The process is performed on at least a portion of the region. Preferably, the first heavy chain constant region (C) containing the sites necessary for light chain bonding is included.H1 The DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into suitable host cells. In some embodiments where unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody, this provides greater flexibility in adjusting the relative proportions of the three polypeptide fragments. However, when expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratios do not significantly affect the yield of the desired chain combination, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector.

[0138] In one specific embodiment of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain in one arm having a first binding specificity and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (providing a second binding specificity). This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from undesirable combinations of immunoglobulin chains, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy separation method. This method is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0139] According to another method described in U.S. Patent 5,731,168, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers recovered from recombinant cell cultures. Preferred interfaces include C... H3 At least a portion of the 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 (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensating "cavity" of the same or similar size as the large side chains is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers rather than other undesirable end products (e.g., homodimers).

[0140] Bispecific antibodies include cross-linked antibodies or "heteroconjugated" antibodies. For example, one antibody in a heteroconjugation may be conjugated to antibiotin, and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (US Patent 4,676,980) and for the treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US Patent 4,676,980, as well as numerous cross-linking techniques.

[0141] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, chemical bonding can be used to prepare bispecific antibodies. Brennan et al., Science, 229: 81 (1985) described such an operation in which intact antibodies are cleaved by proteolytic hydrolysis to generate F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize the ortho-dithiol and prevent the formation of intermolecular disulfides. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One Fab'-TNB derivative is then converted back to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a reagent for the selective immobilization of enzymes.

[0142] Techniques exist to facilitate the direct recovery of Fab'-SH fragments from *E. coli*, which can be chemically conjugated to form bispecific antibodies. Shalaby et al., *J. Exp. Med.*, 175: 217-225 (1992) described the production of the humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was individually secreted from *E. coli* and directionally chemically conjugated in vitro to form bispecific antibodies. The bispecific antibodies thus formed can bind to cells overexpressing the ErbB2 receptor and normal human T cells, and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0143] Several techniques for the direct preparation and isolation of bispecific antibody fragments from recombinant cell cultures 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 generated using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' moieties of two different antibodies via gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, which is then oxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The “dual antibody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. The fragments contain antibodies that bind to V via a linker. L Connected V H The joint is too short to allow pairing between two structural domains on the same chain. Therefore, a segment of V... H and V L The structural domain is forced to interact with the complementary V of another segment. L and V H Domain pairing forms two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0144] In one specific implementation, bispecific or multispecific antibodies can be formed as a DOCK-AND-LOCK. TM (DNL) TMThe complex (see, for example, U.S. Patents 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the respective embodiments of which are incorporated herein by reference). Generally, this technique utilizes a specific and high-affinity binding interaction between the dimerization and docking domain (DDD) sequence of the regulatory (R) subunit 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 can be linked to any protein, peptide, or other molecule. Because the DDD sequence spontaneously dimers and binds to the AD sequence, this technique allows for the formation of complexes between any chosen molecules that can be linked to either the DDD or AD sequence.

[0145] Antibodies with more than two valent antigens are considered. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60, 1991; Xu et al., Science, 358(6359):85-90, 2017). Multivalent antibodies can be internalized (and / or catabolized) more rapidly by the expression of the antigen to which the antibody binds than bivalent antibodies. The antibodies of this disclosure can be multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen-binding sites, which can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may contain a dimerizing domain and three or more antigen-binding sites. Preferred dimerizing domains contain an Fc region or a hinge region (or consist of an Fc region or a hinge region). In this case, the antibody will contain an Fc region and three or more antigen-binding sites at the N-terminus of the Fc region. Preferred multivalent antibodies herein contain three to about eight, but preferably four, antigen-binding sites (or consist of them). Multivalent antibodies comprise at least one polypeptide chain (and preferably two polypeptide chains), wherein the one or more polypeptide chains contain two or more variable regions. For example, one or more polypeptide chains may contain VD1-(X1). n -VD2-(X2) n-Fc, where VD1 is the first variable region, VD2 is the second variable region, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, one or more polypeptide chains may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibodies described herein preferably also comprise at least two (and preferably four) light chain variable region polypeptides. The multivalent antibodies described herein may, for example, comprise about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides considered herein comprise light chain variable regions and optionally also comprise C L Structural domain.

[0146] Charge modification is particularly useful in the case of multispecific antibodies, where amino acid substitutions in the Fab molecule lead to a reduction in the mismatch between the light chain and the mismatched heavy chain (Bence-Jones-type byproducts). This can occur in the production of Fab-based bispecific / multispecific antigen-binding molecules, one or more of the binding arms of the molecule having VH / VL exchange (see also PCT Publication No. WO 2015 / 150447, in particular the examples therein, which are incorporated herein by reference in their entirety).

[0147] Therefore, in some specific implementations, the antibodies contained in the therapeutic agent include:

[0148] (a) The first Fab molecule that specifically binds to the first antigen.

[0149] (b) A second Fab molecule that specifically binds to the second antigen, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged.

[0150] Wherein the first antigen is an activated 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 activated T cell antigen; and

[0151] in:

[0152] i) In the constant domain CL of the first Fab molecule in a), the 124th amino acid is replaced by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the first Fab molecule in a), the 147th or 213th amino acid is replaced by a negatively charged amino acid (according to Kabat EU index number); or

[0153] ii) In the constant structural domain CL of the second Fab molecule in b), the 124th amino acid is replaced by a positively charged amino acid (according to the Kabat number), and in the constant structural domain CH1 of the second Fab molecule in b), the 147th or 213th amino acid is replaced by a negatively charged amino acid (according to the Kabat EU index number).

[0154] The antibody may not contain the two modifications mentioned in i) and ii). The constant domains CL and CH1 of the second Fab molecule do not substitute for each other (i.e., remain unexchanged).

[0155] In another embodiment of the antibody, in the constant domain CL of the first Fab molecule in a), the 124th amino acid is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat number) (in a preferred embodiment, it is independently replaced by lysine (K) or arginine (R)), and in the constant domain CH1 of the first Fab molecule in a), the 147th or 213th amino acid is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

[0156] In another embodiment, in the constant domain CL of the first Fab molecule in a), the 124th amino acid is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first Fab molecule in a), the 147th amino acid is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering).

[0157] In one specific embodiment, in the constant structural domain CL of the first Fab molecule in a), the 124th amino acid is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in a preferred embodiment, independently replaced by lysine (K) or arginine (R)) and the 123rd amino acid is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in a preferred embodiment, independently replaced by lysine (K) or arginine (R)), and in the constant structural domain CH1 of the first Fab molecule in a), the 147th amino acid is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the 213th amino acid is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0158] In a more specific embodiment, in the constant domain CL of the first Fab molecule in a), the 124th amino acid is replaced by lysine (K) (according to Kabat number) and the 123rd amino acid is replaced by either lysine (K) or arginine (R) (according to Kabat number), and in the constant domain CH1 of the first Fab molecule in a), the 147th amino acid is replaced by glutamic acid (E) (according to Kabat EU index number) and the 213th amino acid is replaced by glutamic acid (E) (according to Kabat EU index number).

[0159] In one or more specific embodiments, in the constant domain CL of the first Fab molecule in a), the 124th amino acid is replaced by lysine (K) (according to Kabat number) and the 123rd amino acid is replaced by arginine (R) (according to Kabat number), and in the constant domain CH1 of the first Fab molecule in a), the 147th amino acid is replaced by glutamic acid (E) (according to Kabat EU index number) and the 213th amino acid is replaced by glutamic acid (E) (according to Kabat EU index number).

[0160] F. Chimeric antigen receptor

[0161] Artificial T-cell receptors (also known as chimeric T-cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs)) are modified receptors that can be specifically transferred to immune effector cells. Typically, these receptors are used to specifically transfer monoclonal antibodies to T cells, facilitating the transfer of their coding sequences via retroviral vectors. In this way, a large number of target-specific T cells can be generated for adoptive cell transfer. Phase I clinical trials of this method have demonstrated efficacy.

[0162] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which is fused to both the transmembrane and intracellular domains of CD3-ζ. Such molecules result in the transmission of a ζ signal in response to the scFv's recognition of its target. An example of such a construct is 14g2a-ζ, a fusion of a hybridoma 14g2a (which recognizes disialotetrahexosylganglioside GD2) scFv. When T cells express this molecule (typically via transduction through an oncoreactive vector), they recognize and kill GD2-expressing target cells (e.g., neuroblastoma cells). To target malignant B cells, researchers have redirected T cell specificity using a chimeric immune receptor specific to the B-lineage molecule CD19.

[0163] The variable portions of the immunoglobulin heavy and light chains fuse via flexible linkers to form a scFv. This scFv is preceded by a signal peptide to direct nascent proteins to the endoplasmic reticulum for subsequent surface expression (which is cleaved). Flexible spacers allow the scFv to orient itself in different directions, enabling antigen binding. The transmembrane domain is a typical hydrophobic α-helix of the primitive molecule, usually derived from the intracellular signal transduction domain, which protrudes into the cell and delivers the desired signal.

[0164] Type I proteins are actually two protein domains connected by a transmembrane α-helix. The transmembrane domain passes through the cell membrane lipid bilayer, which serves to separate the inner portion (intracellular domain) from the outer portion (extracellular domain). Unsurprisingly, linking the extracellular domain of one protein with the intracellular domain of another creates a molecule that combines the recognition of the former with the signal of the latter.

[0165] Extracellular domain. The signal peptide directs nascent proteins 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 generally functions well. Typically, the signal peptide natively linked to the most N-terminal component is used (e.g., in scFv with a light chain-linker-heavy chain orientation, the native signal peptide of the light chain is used).

[0166] Antigen recognition domains are typically scFvs. However, many alternatives exist. Antigen recognition domains derived from the α and β single chains of the natural T-cell receptor (TCR) have been described, such as those with simple extracellular domains (e.g., the CD4 extracellular domain that recognizes HIV-infected cells) and more distinctive recognition components such as linked cytokines (which lead to recognition of cells carrying cytokine receptors). In fact, almost anything that binds to a given target with high affinity can be used as an antigen recognition region.

[0167] The spacer connects the antigen-binding domain to the transmembrane domain. It should be flexible enough to allow the antigen-binding domain to orient itself in different directions to facilitate antigen recognition. The simplest form is the hinge region derived from IgG1. Alternatives include the CH2CH3 region of immunoglobulins and portions of CD3. For most scFv-based constructs, the IgG1 hinge is sufficient. However, the best spacer often must be determined empirically.

[0168] Transmembrane domains. Transmembrane domains are hydrophobic α-helices that span the membrane. Typically, transmembrane domains derived from the intracellular domain closest to the membrane are used. Interestingly, the use of the CD3-ζ transmembrane domain can lead to the incorporation of artificial TCRs into native TCRs, a factor dependent on the presence of charged aspartic acid residues in the native CD3-ζ transmembrane domain. Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain produces a well-expressed, stable receptor.

[0169] Intracellular domain. This is the "business-end" of the receptor. After antigen recognition, the receptor aggregates and signals are transmitted to the cell. The most commonly used intracellular domain component is CD3ζ, which contains 3 ITAMs. This transmits activation signals to T cells after antigen binding. CD3ζ may not provide a fully effective activation signal and requires additional co-stimulatory signaling.

[0170] First-generation CARs typically possess an intracellular domain from the CD3 ξ-chain, which is the primary signaling transducer from the endogenous TCR. Second-generation CARs add intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the CAR's cytoplasmic tail to provide additional signaling to T cells. Preclinical studies have shown that second-generation CAR designs enhance the antitumor activity of T cells. Recently, third-generation CARs have combined multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance potency.

[0171] G. ADC

[0172] Antibody-drug conjugates, or ADCs, are a new class of highly potent biological drugs designed to treat people with infectious diseases through targeted therapies. An ADC is a complex molecule consisting of an antibody (whole mAb or antibody fragment, such as a single-chain variable fragment or scFv) linked to a bioactive cytotoxic / antiviral payload or drug via a stable chemical linker with unstable bonds. Antibody-drug conjugates are examples of both biological and immunoconjugates.

[0173] By combining the unique targeting capabilities of monoclonal antibodies with the cancer-killing abilities of cytotoxic drugs, antibody-drug conjugates allow for the sensitive differentiation between healthy and diseased tissues. This means that, unlike traditional systemic approaches, antibody-drug conjugates target and attack infected cells, thus minimizing the impact on healthy cells.

[0174] In the development of antibody-drug conjugates (ADCs) for anticancer therapy, anticancer drugs (such as cell toxins) are conjugated to antibodies that specifically target certain cellular markers (e.g., proteins ideally found only in or on infected cells). The antibodies track these proteins in vivo and attach themselves to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers signaling in tumor cells, which then absorb or internalize the antibody along with the cytotoxic agent. After ADC internalization, the cytotoxic drug is released and kills cells or impairs viral replication. Due to this targeting, the drug ideally has fewer side effects and provides a wider therapeutic window compared to other agents.

[0175] Stable linkers between antibodies and cytotoxic / antiviral agents are a key aspect of ADCs. Linkers are based on chemical motifs including disulfides, hydrazones, or peptides (cleavable) or thioethers (uncleavable) and control the distribution and delivery of the cytotoxic agent to target cells. Both cleavable and uncleavable linkers have been demonstrated to be safe in preclinical and clinical trials. Brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers the potent and highly toxic anti-microtubule agent monomethyl auristatin E (MMAE), a synthetic antitumor agent, to human-specific CD30-positive malignant cells. MMAEs cannot be used as single-agent chemotherapeutic agents due to their high toxicity (they inhibit cell division by blocking microtubule polymerization). However, the combination of an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor or TNF receptor) and an MMAE has been shown to be stable in extracellular fluid, cleavable by cathepsins, and safe for therapeutic use. Trastuzumab emtansine, another approved ADC, is a combination of the microtubule formation inhibitor maytansine (DM-1) (a derivative of maytansine) and the antibody trastuzumab (Herceptin® / Genentech / Roche) linked by a stable, non-cleavable linker.

[0176] The availability of better and more stable linkers has altered the function of chemical bonds. The type of linker (cleavable or uncleavable) provides specific properties for cytotoxic (anticancer) drugs. For example, an uncleavable linker retains the drug inside the cell. As a result, the entire antibody, linker, and cytotoxic agent enter the target cancer cell, where the antibody is degraded to the amino acid level. The resulting complex (amino acid, linker, and cytotoxic agent) now becomes the active drug. Conversely, a cleavable linker is catalyzed by enzymes in the host cell, where the cytotoxic agent is released.

[0177] Another type of cleavable linker currently under development adds an extra molecule between the cytotoxic / antiviral drug and the cleavage site. This linker technology allows researchers to create ADCs with greater flexibility without worrying about altering the cleavage kinetics. Researchers are also developing novel methods for peptide cleavage based on Edman degradation, which involves sequencing the amino acids in the peptide. Future directions for ADC development also include the development of site-specific conjugations (TDCs) to further improve stability and therapeutic index, as well as alpha-emitting immunoconjugates and antibody-conjugated nanoparticles.

[0178] H. BiTE

[0179] Bi-specific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies that have been investigated for use as anticancer drugs. They guide the host's immune system to more specifically target the cytotoxic activity of T cells against infected cells. BiTE is a registered trademark of Micromet AG.

[0180] BiTE is a fusion protein composed of two single-chain variable fragments (scFv) from different antibodies, or amino acid sequences from four different genes on a single polypeptide chain of approximately 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, while the other binds to infected cells via a specific molecule.

[0181] Like other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTE forms a junction between T cells and target cells. This allows T cells to exert cytotoxic / antiviral activity against infected cells independently of the presence of MHC I or co-stimulatory molecules by producing proteins such as perforin and granzymes. These proteins enter the infected cells and trigger apoptosis. This action mimics the physiological processes observed during T cell attack on infected cells.

[0182] I. Intracellular antibodies

[0183] In one specific implementation, the antibody is a recombinant antibody adapted to function within cells—such antibodies are called “intracellular antibodies.” These antibodies can interfere with targeting function through a variety of mechanisms, such as altering intracellular protein transport, interfering with enzyme function, and blocking protein-protein or protein-DNA interactions. In many respects, their structure mimics or resembles those of the single-chain and single-domain antibodies discussed above. Indeed, single transcript / single strand is an important feature, allowing for intracellular expression in the target cell and also making protein transmembrane transport more feasible. However, additional features are required.

[0184] Two main issues affecting the implementation of intracellular antibody therapy are delivery (including cell / tissue targeting) and stability. Regarding delivery, various approaches have been employed, such as tissue-directed delivery, the use of cell type-specific promoters, virus-based delivery, and the use of cell permeability / membrane translocation peptides. Regarding stability, methods typically involve brute-force screening, including approaches involving phage display and potentially including the development of sequence maturation or shared sequences, or more targeted modifications such as the insertion of stable sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide substitutions / modifications.

[0185] Another feature that may be required for intracellular antibodies is an intracellular targeting signal. Vectors that can target intracellular antibodies (or other proteins) to subcellular regions (such as the cytoplasm, nucleus, mitochondria, and ER) have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997).

[0186] By virtue of their ability to enter cells, intracellular antibodies possess additional uses that other types of antibodies may not be able to achieve. In the case of the antibodies of the present invention, the ability to interact with the cytoplasmic domains of MUC1 in living cells can interfere with functions associated with MUC1CD, such as signal transduction (binding to other molecules) or oligomer formation. In particular, such antibodies are contemplated for use in inhibiting the formation of MUC1 dimers.

[0187] J. Purification

[0188] In some embodiments, the antibodies of this disclosure may be purified. As used herein, the term "purified" is intended to refer to a composition separable from other components, wherein the protein is purified to any degree relative to its naturally available state. Thus, purified protein also refers to a protein removed from its naturally occurring environment. When the term "substantially purified" is used, this designation refers to a composition in which a protein or peptide forms the major component of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0189] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, the coarse separation of the cellular environment into peptide and non-peptide fractions. After separating the peptide from other proteins, chromatographic and electrophoretic techniques can be used to further purify the target peptide to achieve partial or complete purification (or purification to homogenization). Analytical methods particularly suitable for preparing pure peptides include ion exchange chromatography, size exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric aggregation. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, or by thermal denaturation followed by centrifugation; gel filtration, reversed-phase chromatography, hydroxyapatite chromatography, and affinity chromatography; and combinations of such techniques with other techniques.

[0190] In purifying the antibodies of this disclosure, it is desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide can be purified from other cellular components using an affinity column that binds to a labeled portion of the polypeptide. As is generally known in the art, it is considered that the order of the purification steps can be altered, or certain steps can be omitted, while still obtaining a method suitable for preparing substantially purified proteins or peptides.

[0191] Typically, complete antibodies are fractionated using reagents that bind to the Fc portion of the antibody (i.e., protein A). Alternatively, an antigen can be used to simultaneously purify and select suitable antibodies. Such methods typically use a selector bound to a support (e.g., column, filter, or bead). The antibody is then bound to the support, contaminants are removed (e.g., washed away), and the antibody is released by applying conditions (salt, heat, etc.).

[0192] Based on this disclosure, those skilled in the art will know of various methods for quantifying the degree of purification of proteins or peptides. These include, for example, determining the specific activity of an active fraction, or assessing the amount of peptide within a fraction by SDS / PAGE analysis. Another method for assessing fraction purity is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and thus calculate the purity. Of course, the actual unit used to express the amount of activity will depend on the specific assay technique chosen after purification and whether the expressed protein or peptide exhibits detectable activity.

[0193] It is known that the migration of peptides can be altered, sometimes significantly, by different SDS / PAGE conditions (Capaldi et al., 1977). Therefore, it should be understood that the apparent molecular weight of purified or partially purified expression products can change under different electrophoretic conditions.

[0194] III. Active / passive immunization and treatment / prevention of [INSERT] viral infection

[0195] A. Formulation and application

[0196] This disclosure provides pharmaceutical compositions comprising an antibody against influenza B virus and an antigen for generating the antibody against influenza B virus. Such compositions comprise a preventative or therapeutically effective amount of the antibody or a fragment or peptide immunogen thereof, and a pharmaceutically acceptable carrier. In one specific embodiment, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals and more particularly for use in humans. The term "carrier" refers to a diluent, excipient, or transporter administered with the therapeutic agent. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a specific carrier when the pharmaceutical composition is administered intravenously. Saline solutions, dextran solutions, and glycerol solutions are also used 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc.

[0197] If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Some examples of suitable pharmaceutical agents are described in Remington's Pharmaceutical Sciences. Such compositions will contain a prophylactic or therapeutically effective amount of an antibody or its fragment (preferably in purified form) and a suitable amount of carrier to provide the patient with a form suitable for appropriate administration. The formulation should be suitable for the route of administration, which may be oral, intravenous, intra-arterial, buccal, intranasal, nebulized, bronchial inhalation, rectal, vaginal, surface, or delivered via mechanical ventilation.

[0198] Active vaccines are also envisioned, in which antibodies, like those disclosed, are generated in the body of individuals at risk of influenza B virus infection. Such vaccines can be formulated for parenteral administration, for example, for injection via intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Intradermal and intramuscular administration are considered. Alternatively, the vaccine can be applied directly to mucous membranes via surface routes, such as via nasal drops, inhalation, nebulizer, or rectal or vaginal delivery. Pharmaceutically acceptable salts include acid salts and salts formed with: inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0199] Passive transfer of antibodies, known as artificially acquired passive immunization, typically involves intravenous or intramuscular injection. Antibodies can be in the form of human or animal blood plasma or serum, as intravenously or intramuscularly combined human immunoglobulin (IVIG), as high-titer human IVIG or IG derived from immunized donors or those recovered from disease, and as monoclonal antibodies (MAbs). Such immunization usually lasts only a short period and carries the potential risk of hypersensitivity reactions and serum sickness, particularly from gamma globulins of non-human origin. However, passive immunization provides immediate protection. Antibodies are formulated in a carrier suitable for injection (i.e., sterile and injectable).

[0200] Generally, the components of the compositions disclosed herein are provided separately or mixed together in unit dosage forms, for example, as dried lyophilized powders or anhydrous concentrates in sealed containers (e.g., ampoules or sachets) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, a single ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0201] The compositions disclosed herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those that form with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and those that form with cations, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0202] 2. ADCC

[0203] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells to which antibodies or fragments of them containing an Fc region typically bind specifically via a protein portion at the N-terminus of that Fc region. "Antibody with enhanced / reduced antibody-dependent cell-mediated cytotoxicity (ADCC)" means an antibody that exhibits enhanced / reduced ADCC as determined by any suitable method known to those skilled in the art.

[0204] As used herein, the term “enhanced / decreased ADCC” is defined as: an increase / decrease in the number of target cells lysed within a given time period by means of ADCC as defined above, at a given concentration of antibody in the medium surrounding the target cells; and / or a decrease / increase in the antibody concentration required to achieve the lysis of a given number of target cells within a given time period by means of ADCC in the medium surrounding the target cells. An enhancement / decrease in ADCC is relative to ADCC mediated by the same antibody produced from the same type of host cells (but unmodified) using the same standard methods of production, purification, formulation, and storage (which are known to those skilled in the art). For example, an enhancement of ADCC mediated by an antibody produced from a host cell modified by the methods described herein to have a modified glycosylation pattern (e.g., expressing glycosyltransferase GnTIII or other glycosyltransferases) is relative to ADCC mediated by the same antibody produced from the same type of unmodified host cells.

[0205] 3. CDC

[0206] Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is a process in the immune system that kills pathogens by disrupting their membranes without the involvement of antibodies or cells from the immune system. There are three main processes. All three processes involve the insertion of one or more membrane attack complexes (MACs) into the pathogen, causing lethal colloidal-osmotic swelling, i.e., CDC. It is one of the mechanisms by which antibodies or antibody fragments exert antiviral activity.

[0207] IV. Antibody conjugates

[0208] The antibodies disclosed herein can be conjugated to at least one reagent to form antibody conjugates. To enhance the potency of antibody molecules as diagnostic or therapeutic agents, they are routinely conjugated, covalently bound, or complexed with at least one desired molecule or moiety. Such a molecule or moiety can be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Some non-limiting examples of effector molecules already conjugated to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any part that can be detected using an assay. Some non-limiting examples of reporter molecules already conjugated to antibodies include enzymes, radiolabeled molecules, haptens, fluorescently labeled molecules, phosphorescent molecules, chemiluminescent molecules, chromophores, photosynthetic molecules, colored particles, or ligands, such as biotin.

[0209] Antibody conjugates are generally preferred as diagnostic agents. Antibody diagnostic agents typically fall into two categories: those used for in vitro diagnostics, such as for various immunoassays, and those used for in vivo diagnostic protocols commonly referred to as "antibody-guided imaging." Many suitable imaging agents are known in the art, as are methods of conjugating them to antibodies (see, for example, U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging component used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

[0210] In the case of paramagnetic ions, examples of ions include chromium (III), manganese (II), iron (III), 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 available in other cases (e.g., X-ray imaging) include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0211] In the case of radioisotopes used for therapeutic and / or diagnostic applications, astatine may be mentioned. 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 Eu, Gallium 67 , 3 hydrogen, iodine 123 ,iodine 125 ,iodine 131 ,indium111 , 59 iron, 32 Phosphorus, rhenium 186 ,rhenium 188 , 75 selenium, 35 sulfur, technetium 99m and / or yttrium 90 In some implementations, it is generally preferred to use 125 I, and technetium is also generally preferred. 99m and / or indium 111 Because of its low energy and suitability for long-distance detection. The radiolabeled monoclonal antibodies of this disclosure can be produced according to methods known in the art. For example, the monoclonal antibody can be iodinated by contacting sodium iodide and / or potassium iodide with a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). The monoclonal antibodies according to this disclosure can be produced using technetium via a ligand exchange process. 99m Labeling can be performed, for example, by reducing pertechnetium with a stannous solution, chelating the reduced technetium onto a Sephadex column, and then applying an antibody to the column. Alternatively, direct labeling techniques can be used, such as by incubating pertechnetium, a reducing agent (e.g., SNCl2), a buffer solution (e.g., sodium potassium phthalate solution), and an antibody. The intermediate functional group typically used to bind the radioactive isotope, present as a metal ion, to the antibody is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0212] Fluorescent labels considered 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.

[0213] Other types of antibodies considered in this disclosure are those primarily intended for in vitro use, wherein the antibody is linked to a second binding ligand and / or an enzyme (enzyme tag) that produces a colored product upon contact with a chromogenic substrate. Some examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase, or glucose oxidase. Preferred second binding ligands are biotin, as well as avidin and streptavidin compounds. The use of such tagging is well known to those skilled in the art and is 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.

[0214] Another known method for site-specific binding of molecules to antibodies involves reacting the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with an amino acid at the antigen-binding site, thereby disrupting that site and blocking the specific antigen reaction. However, this can be disadvantageous because it leads to the loss of antigen binding via the antibody conjugate.

[0215] Molecules containing azido groups can also be used to form covalent bonds with proteins via reactive azine intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2-azido and 8-azido analogs 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). 2-azidonucleotides and 8-azidonucleotides have also been used to map the nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binders.

[0216] Several methods for linking or conjugating antibodies to their conjugated moieties are known in the art. Some linking methods involve the use of metal chelate complexes, which employ, for example, organic chelating agents linked to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (US Patents 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Conjugates with fluorescein-labeled antibodies are prepared in the presence of these conjugating agents or by reaction with isothiocyanates. In US Patent 4,938,948, imaging of breast tumors was achieved using monoclonal antibodies and a linker such as methyl-p-hydroxybenzoimino ester or N-succinimide-3-(4-hydroxyphenyl)propionate was used to bind the detectable imaging moieties to the antibody.

[0217] In other embodiments, immunoglobulin derivatization is considered by selectively introducing thiol groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method have been disclosed to exhibit improved lifetime, specificity, and sensitivity (US Patent 5,196,066, which is incorporated herein by reference). Site-specific conjugations of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to sugar residues in the Fc region, have also been disclosed in the literature (O'Shannessy et al., 1987). This method has been reported to produce promising antibodies currently under clinical evaluation for diagnostic and therapeutic purposes.

[0218] V. Immunological detection methods

[0219] In other embodiments, this disclosure relates to immunoassay 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 conventional sense, another use would be for quality control and monitoring of vaccines and other viral reservoirs, where antibodies according to this disclosure can be used to assess the amount or integrity (i.e., long-term stability) of antigens in the virus. Alternatively, the method can be used to screen for appropriate / desired reactivity profiles of multiple antibodies.

[0220] Other immunoassay methods include specific assays for determining the presence of influenza B virus in subjects. A wide variety of assay formats have been considered, but specifically those for detecting influenza B virus in fluids obtained from subjects, such as saliva, blood, plasma, sputum, semen, or urine. In particular, semen has been shown to be 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). Assays can be advantageously formatted for non-healthcare (home) use, including lateral flow assays similar to home pregnancy tests (see below). These assays can be packaged together as kits with appropriate reagents and instructions to allow use by family members.

[0221] Some immunoassay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioassay, fluorescence immunoassay, chemiluminescence assay, bioluminescence assay, and Western blotting. In particular, competitive assays for detecting and quantifying influenza B virus antibodies against specific parasitic epitopes in samples have been provided. The procedures for various available immunoassay methods have been described in scientific literature such as Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, immunobinding methods involve obtaining a sample suspected of containing influenza B virus and contacting the sample with a primary antibody according to this disclosure, as appropriate, under conditions that effectively allow for immune complex formation.

[0222] These methods include those for purifying influenza B virus or related antigens from samples. Antibodies are preferably attached to a solid support, such as a column matrix, and samples suspected of containing influenza B virus or antigenic components are applied to immobilized antibodies. Unwanted components are washed off the column, allowing influenza B virus antigens to immunorecombine with the immobilized antibodies, which are then collected by removing the organism or antigen from the column.

[0223] Immunobinding methods also include methods for detecting and quantifying the amount of influenza B virus or related components in a sample, and for detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing influenza B virus or its antigens is obtained, and the sample is contacted with an antibody bound to influenza B virus or its components. The amount of immune complexes formed under specific conditions is then detected and quantified. For antigen detection, the biological sample analyzed can be any sample suspected of containing influenza B virus or influenza B virus antigens, such as tissue sections or specimens, homogenized tissue extracts, biological fluids including blood and serum, or secretions such as feces or urine.

[0224] The process of contacting the selected biological sample with the antibody under effective conditions for a sufficient period 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 long enough for the antibody to form an immune complex (i.e., bind to) the present influenza B virus or antigen. After this time, the sample-antibody composition, such as tissue sections, ELISA plates, dot blots, or Western blots, is typically washed to remove any non-specifically bound antibody species, thus allowing only those antibodies specifically bound to the primary immune complexes to be detected.

[0225] Generally, the detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are typically based on the detection of labels or markers, such as those that are radiolabeled, fluorescent, biolabeled, or enzyme-labeled. Patents relating to 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, as is known in the art, additional advantages can be found by using a second binding ligand, such as a second antibody, and / or a biotin / antibiotin ligand binding arrangement.

[0226] The antibody used for detection can be conjugated to a detectable label, which is then simply detected, allowing determination of the amount of primary immune complex in the composition. Alternatively, the antibody bound to the primary immune complex can be detected by a second binding ligand having binding affinity for the first antibody. In these cases, the second binding ligand can be conjugated to the detectable label. The second binding ligand itself is typically an antibody, which may therefore be referred to as the "second" antibody. The primary immune complex is contacted with the labeled second binding ligand or antibody under effective conditions for a time sufficient to allow for the formation of a secondary immune complex. The secondary immune complex is then typically washed to remove any non-specifically bound labeled second antibody or ligand, and any remaining label in the secondary immune complex is subsequently detected.

[0227] Other methods include detecting primary immune complexes via a two-step approach. As described above, a second binding ligand (e.g., an antibody) with binding affinity to the antibody is used to form secondary immune complexes. After washing, the secondary immune complexes are again contacted under effective conditions with a third binding ligand or antibody with binding affinity to the second antibody for a time sufficient to allow for the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is then linked to a detectable label, thereby allowing the detection of the resulting tertiary immune complexes. The system can provide signal amplification if desired.

[0228] An immunoassay method uses two different antibodies. A first biotinylated antibody is used to detect a target antigen, and subsequently a second antibody is used to detect biotin linked to a biotin complex. In this 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 a sequential solution of streptoavidin (or anti-Biotin), biotinylated DNA, and / or complementary biotinylated DNA, wherein each step adds an additional biotin site to the antibody / antigen complex. The amplification steps are repeated until a suitable amplification level is reached, at which point the sample is incubated in a solution containing a second-step antibody targeting biotin. This second-step antibody is labeled, for example, enzyme-labeled, and the enzyme can be used to detect the presence of the antibody / antigen complex via tissue enzymology using a chromogenic substrate. After appropriate amplification, a macroscopically visible conjugate is produced.

[0229] Another known immunoassay method utilizes immunoPCR (polymerase chain reaction). The PCR method is similar to the Cantor method before incubation with biotinylated DNA; however, as an alternative to incubation with multiple rounds of streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed away with a low-pH or high-salt buffer, releasing the antibody. The resulting wash solution is then used for PCR with appropriate primers and suitable controls. At least in theory, the enormous amplification capacity and specificity of PCR can be used to detect single antigen molecules.

[0230] A. ELISA

[0231] Immunoassays, in their simplest and most direct sense, are binding assays. Some preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily understood that detection is not limited to such techniques, and Western blots, dot blots, FACS analysis, etc., can also be used.

[0232] In one exemplary ELISA, the antibody of this disclosure is immobilized on a selected surface exhibiting protein affinity, such as wells in a polystyrene microtiter plate. A test composition suspected of containing influenza B virus or influenza B virus antigen is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding a separate anti-influenza B virus antibody linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection can also be achieved by adding a second anti-influenza B virus antibody, followed by the addition of a third antibody with binding affinity to the second antibody, wherein the third antibody is linked to a detectable label.

[0233] In another exemplary ELISA, a sample suspected of containing influenza B virus or influenza B virus antigen is immobilized on the surface of a well and subsequently contacted with an anti-influenza B virus antibody of this disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-influenza B virus antibody is detected. When the initial anti-influenza B virus antibody is conjugated to a detectable label, the immune complex can be detected directly. Similarly, a second antibody having binding affinity to the first anti-influenza B virus antibody can be used to detect the immune complex, wherein the second antibody is conjugated to a detectable label.

[0234] Regardless of the form used, ELISA shares certain common characteristics, such as coating, incubation and binding, washing to remove non-specifically bound substances, and detection of bound immune complexes. These are described below.

[0235] In plates coated with antigens or antibodies, the wells are typically incubated overnight or for a specified period with a solution of the antigen or antibody. The wells are then washed to remove any incompletely adsorbed material. Any remaining usable surface in the wells is then “coated” with a nonspecific protein that is antigenically neutral relative to the test antiserum. These include bovine serum albumin (BSA), casein, or milk powder solutions. Coating allows for the closure of nonspecific adsorption sites on the immobilized surface and thus reduces background caused by nonspecific binding of the antiserum to the surface.

[0236] In ELISA, it may be more common to use secondary or tertiary detection methods rather than direct manipulation. Therefore, after the protein or antibody binds to the well, the well is coated with a non-reactive substance to reduce background, and washed to remove unbound material, the immobilized surface is brought into contact with the biological sample to be tested under conditions that effectively allow for the formation of immune complexes (antigen / antibody). The detection of these immune complexes then requires a labeled second binding ligand or antibody, and a second binding ligand or antibody conjugated with a labeled third antibody or third binding ligand.

[0237] "Under conditions that effectively allow for the formation of immune complexes (antigen / antibody)" means that these conditions preferably include diluting the antigen and / or antibody with a solution (e.g., BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween). These added reagents also tend to help reduce nonspecific background.

[0238] "Suitable conditions" also means that incubation is carried out at a temperature or time sufficient to allow for effective binding. The incubation step is usually carried out at a temperature preferably between about 25°C and 27°C for about 1 to 2 to 4 hours, or overnight at about 4°C.

[0239] In ELISA, after all incubation steps, the contacted surfaces are washed to remove uncomplexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween or borate buffer. Even trace amounts of immune complexes can be identified after the formation of specific immune complexes between the test sample and the initially bound material, followed by washing.

[0240] To provide a detection method, the second or third antibody has an associated label to allow detection. Preferably, this is an enzyme that produces color after incubation with a suitable chromogenic substrate. Thus, for example, it is desirable to contact or incubate the primary and secondary immune complexes with antibodies conjugated to urease, glucose oxidase, alkaline phosphatase, or catalase at a time and under conditions that favor the occurrence of further immune complex formation (e.g., incubation for 2 hours at room temperature in a solution containing PBS, such as PBS-Tween).

[0241] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of labeling is quantified, for example, by incubation with a chromogenic substrate (e.g., urea or bromocresol purple or 2,2'-azido-di-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) or H2O2 (in the case of peroxidase as the enzyme label)). Quantification is then achieved by measuring the intensity of the resulting color, for example, using a visible spectrophotometer.

[0242] In another embodiment, this disclosure contemplates the use of a competitive approach. This is particularly useful for detecting influenza B virus antibodies in samples. In a competition-based assay, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, a quantifiable loss of signal indicates the amount of unknown antibody or analyte in the sample.

[0243] Here, the inventors propose using labeled influenza B virus monoclonal antibodies to determine the amount of influenza B virus antibodies in a sample. The basic approach involves contacting a known amount of influenza B virus monoclonal antibody (linked to a detectable label) with influenza B virus antigen or particles. The influenza B virus antigen or organism is preferably attached to a support. After the labeled monoclonal antibody binds to the support, the sample is added and incubated under conditions that allow any unlabeled antibody in the sample to compete with and thus replace the labeled monoclonal antibody. By measuring the lost or remaining label (and subtracting it from the initial amount of bound label), the amount of unlabeled antibody bound to the support can be determined, and thus the amount of antibody present in the sample.

[0244] B. Western Imprint

[0245] Western blotting (or protein immunoblotting) is an analytical technique used to detect specific proteins in a given tissue homogenate or extract sample. It uses gel electrophoresis to separate native or denatured proteins by peptide length (denaturing conditions) or by the protein's 3-D structure (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), on which antibodies specific to the target protein are used for detection.

[0246] Samples can be taken from whole tissues or from cell cultures. In most cases, solid tissues are first mechanically disrupted using a stirrer (for larger sample volumes), a homogenizer (for smaller volumes), or by acoustic treatment. Cells can also be ruptured using one of the aforementioned mechanical methods. However, it should be noted that bacterial, viral, or environmental samples can be sources of proteins, and therefore Western blotting is not limited to cell studies. A variety of detergents, salts, and buffers can be used to promote cell lysis and protein dissolution. Protease and phosphatase inhibitors are typically added to prevent the sample from being digested by its own enzymes. Tissue preparation is usually performed at low temperatures to avoid protein denaturation.

[0247] Proteins in a sample are separated using gel electrophoresis. Protein separation can be achieved by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample preparation and the properties of the gel. This is a very useful method for identifying proteins. Two-dimensional (2-D) gels can also be used, which disperse proteins from a single sample in two dimensions. Proteins are separated in the first dimension according to their isoelectric point (the pH at which a protein has a neutral net charge), and in the second dimension according to their molecular weight.

[0248] To facilitate antibody detection of proteins, they are transferred from the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is placed in a buffer solution, which moves to the paper via capillary action, thus transferring the proteins. Another method for transferring proteins is called electroblotting, which uses an electric current to pull the proteins from the gel to the PVDF or nitrocellulose membrane. The proteins move from the gel to the membrane while maintaining their organization as they did in the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). These two membranes were chosen because of their non-specific protein-binding properties (i.e., equally good binding to all proteins). Protein binding is based on hydrophobic interactions as well as charged interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF but are more brittle and do not withstand repeated probes well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once the transfer is complete, the protein is detected indirectly using either a labeled primary antibody or an unlabeled primary antibody, followed by indirect detection using labeled protein A or a second labeled antibody that binds to the Fc region of the primary antibody.

[0249] C. Sideflow measurement

[0250] Lateral flow assays, also known as lateral flow immunochromatography, are simple devices designed to detect the presence (or absence) of a target analyte in a sample (matrix) without requiring specialized and expensive equipment, although many lab-based applications supported by reading devices exist. These tests are typically used for low-resource medical diagnostics, for home testing, point-of-care testing, or for laboratory use. A widely used and well-known application is home pregnancy testing.

[0251] This technology is based on a series of capillary beds, such as porous paper sheets or sintered polymers. Each of these elements has the ability to spontaneously transport fluids (e.g., urine). The first element (sample pad) acts as a sponge and holds excess sample fluid. Once soaked, the fluid migrates to a second element (conjugation pad) in which the manufacturer has stored so-called conjugates, which are bioactive particles in a dried form in a salt-sugar matrix (see below). This matrix contains all the substances that ensure an optimized chemical reaction between the target molecule (e.g., antigen) and its chemical conjugate (e.g., antibody) immobilized on the particle surface. As the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles, and the sample and conjugate mix as they flow through the porous structure in a combined transport action. In this way, the analyte binds to the particles as it further migrates through a third capillary bed. This material has one or more regions (often called strips) on which the manufacturer has immobilized a third molecule. When the sample-conjugate mixture reaches these strips, the analyte is already bound to the particle, and the third “capture” molecule binds to the complex. After a period of time, as more and more fluid has passed through the strips, particles accumulate and the strip regions change color. Typically, there are at least two strips: one (control) captures any particles, thus indicating that the reaction conditions and technique are working well; the second contains specific capturing molecules and captures only those particles that have already immobilized analyte molecules. After passing through these reaction zones, the fluid enters the final porous material, the wick, which only acts as a waste container. Sideflow testing can be performed as a competitive assay or a sandwich assay. Sideflow testing is disclosed in U.S. Patent 6,485,982.

[0252] D. Immunohistochemistry

[0253] The antibodies disclosed herein can also be used in combination with fresh frozen and / or formalin-fixed paraffin-embedded tissue blocks prepared for immunohistochemistry (IHC) studies. Methods for preparing tissue blocks from these particulate samples have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0254] In short, frozen sections can be prepared by: rehydrating 50 ng of frozen “pulverized” tissue in phosphate-buffered saline (PBS) in a small plastic capsule at room temperature; precipitating the particles by centrifugation; resuspending it in viscous embedding medium (OCT); inverting the capsule and / or precipitating again by centrifugation; flash freezing in isopentane at -70°C; cutting the plastic capsule and / or removing the frozen tissue cylinder; fixing the tissue cylinder onto a cryostat chuck; and / or cutting 25 to 50 consecutive sections from the capsule. Alternatively, the entire frozen tissue sample can be used for serial section cutting.

[0255] Permanent sections can be prepared using a similar method, which involves rehydrating 50 mg of sample in a plastic microcentrifuge tube; precipitation; resuspending in 10% formalin for 4 hours; washing / precipitation; resuspending in warm 2.5% agar; precipitation; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; immersing and / or embedding the block in paraffin; and / or cutting up to 50 consecutive permanent sections. Similarly, the entire tissue sample can be replaced.

[0256] E. Immunoassay kit

[0257] In other embodiments, this disclosure relates to immunoassay kits for use with the immunoassay methods described above. Since antibodies can be used to detect influenza B virus or influenza B virus antigens, the kit may contain antibodies. The immunoassay kit therefore contains, in a suitable container, a first antibody that binds to influenza B virus or influenza B virus antigen and optionally, an immunoassay reagent.

[0258] In some implementations, influenza B virus antibodies may be pre-bound to a solid support, such as a column matrix and / or the wells of a microtiter plate. The immunoassay reagents in the kit may take any of a variety of forms, including those detectable markers associated with or linked to a given antibody. Detectable markers associated with or linked to a second binding ligand are also considered. Some exemplary second ligands are those second antibodies that have binding affinity for the first antibody.

[0259] Other suitable immunoassay reagents for use in the kits of the present invention include two-component reagents comprising a second antibody having binding affinity to a first antibody and a third antibody having binding affinity to the second antibody, said third antibody being linked to a detectable marker. As described above, many exemplary markers are known in the art, and all such markers may be used in conjunction with this disclosure.

[0260] The kit may also contain appropriate aliquots of a composition of influenza B virus or influenza B virus antigen, whether labeled or unlabeled, as used to prepare a standard curve for detection assays. The kit may contain antibody-labeled conjugates in fully conjugated form, as intermediates, or as separate components to be conjugated by the kit user. Kit components may be packaged in aqueous media or in lyophilized form.

[0261] The container device of the kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container device in which antibodies can be placed, or preferably appropriately aliquoted. The kits of this disclosure typically also include means for sealing and restricting the containment of antibodies, antigens, and any other reagents for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0262] F. Vaccine and Antigen Quality Control Assay

[0263] This disclosure also considers the use of antibodies and antibody fragments, as described herein, for assessing the antigenic integrity of viral antigens in samples. Biological products such as vaccines differ from chemical drugs in that they are generally not characterizable at the molecular level; antibodies are large molecules of significant complexity and have the capacity to vary widely due to their preparation. They are also administered to healthy individuals, including newborns, and therefore their quality must be highly valued to ensure, to the greatest extent possible, their effectiveness in preventing or treating life-threatening diseases without causing harm themselves.

[0264] The increasing globalization of vaccine production and distribution opens new possibilities for better managing public health issues, but also raises questions about the equivalence and interchangeability of vaccines procured from multiple sources. Therefore, international standardization of starting materials, production, and quality control testing, along with high expectations for the management and oversight of how these products are prepared and used, have been cornerstones of continued success. However, this remains an evolving field, and ongoing technological advancements in this area offer the prospect of developing potent new weapons against both old and emerging public health threats (malaria, pandemic influenza, and HIV, to name a few), but also place immense pressure on manufacturers, regulatory bodies, and the broader medical community to ensure that products continue to meet the highest achievable quality standards.

[0265] Therefore, antigens or vaccines can be obtained from any source or at any point during the preparation process. Thus, the quality control process can begin with the preparation of samples for an immunoassay to identify the binding of the antibodies or fragments disclosed herein to the viral antigen. Such immunoassays are disclosed elsewhere in this document, and any of these can be used to assess the structural / antigenic integrity of the antigen. The criteria used to determine if a sample contains an acceptable amount of the correct and intact antigen can be established by the regulatory agency.

[0266] Another important implementation method for assessing antigen integrity is determining shelf life and storage stability. Most drugs, including vaccines, deteriorate over time. Therefore, it is crucial to determine the extent to which antigens (such as those in vaccines) degrade or become unstable over time, to the point that they no longer possess antigenicity and / or the ability to elicit an immune response when administered to a subject. Again, the criteria used to detect intact antigens in samples containing acceptable amounts of antigenicity can be established by regulatory agencies.

[0267] In some implementations, the viral antigen may contain more than one protective epitope. In these cases, assays observing the binding of more than one antibody (e.g., antibodies 2, 3, 4, 5, or even more) can prove useful. These antibodies bind to closely related epitopes, such that they are adjacent to or even overlap each other. Alternatively, they may represent different epitopes from different parts of the antigen. By examining the integrity of multiple epitopes, a more complete picture of the overall integrity of the antigen can be determined, and thus the ability to generate a protective immune response can be determined.

[0268] The antibodies and fragments thereof described in this disclosure may also be used in kits for monitoring the efficacy of vaccination programs by detecting the presence of protective influenza B virus antibodies. The antibodies, antibody fragments, or variants and derivatives thereof described in this disclosure may also be used in kits for monitoring the preparation of vaccines with desired immunogenicity.

[0269] Example

[0270] The following embodiments are included to illustrate some preferred embodiments. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in the implementation of the embodiments, and therefore can be considered to constitute preferred modes for their practice. However, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed in this disclosure and still obtain the same or similar results without departing from the spirit and scope of this disclosure.

[0271] Example 1 - Materials & Methods

[0272] Cell line. The Madin-Darby canine kidney (MDCK) cell line MDCK-SIAT1, a variant derived from MDCK cells stably transfected with human 2,6-sialyltransferase (SIAT1) cDNA, was obtained from Sigma-Aldrich (catalog number 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) and FreeStyle 293F cell lines (Thermo Fisher Scientific) were purchased and cultured according to the manufacturer's protocol. Drosophila S2 cells (Thermo Fisher Scientific catalog number 72851-4003) were cultured in suspension at 37°C with shaking at 125 RPM in Schneider Drosophila medium (Thermo Fisher Scientific catalog number 21720001) supplemented with 10% fetal bovine serum. Primary tracheal cells were purchased from the Marsico Lung Institute Tissue Procurement and Cell Culture Core Facility, North Carolina School of Medicine, Chapel Hill, NC. All cell lines were tested monthly for mycoplasma, and all samples were negative.

[0273] Subjects. The participants were 48-year-old women who had received the 2018-2019 influenza vaccine (Flucelvax quadrivalent; Seqirus, Inc.) intramuscularly. Peripheral blood and bone marrow aspirate samples were obtained from the individuals after obtaining written informed consent. This study was approved by the Vanderbilt University Medical Center Institutional Review Board.

[0274] Animal model. BALB / c mice were purchased from Jackson Laboratories (Bar Harbor). Breeding, maintenance, and experimentation were conducted in accordance with the guidelines of the Vanderbilt Institution's Animal Care and Use Committee. Detailed information on mouse husbandry can be found in the "In vivo protection studies" section.

[0275] 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), 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 multiplied and titrated in monolayer cultures of MDCK-SIAT1 cells. Cells were maintained in Dulbecco modified Eagle medium (GIBCO DMEM, Invitrogen) supplemented with 10% FBS, 1% penicillin, and streptomycin, and incubated at 37°C in 5% CO2. Lines 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 embryo-bearing chicken eggs as previously described (Brauer & Chen, 2015). All viruses were handled under BSL-2 conditions.

[0276] Isolation and B-cell sorting of peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using SepMate PBMC isolation tubes (StemCELL, catalog 85450). Plasma blasts from PBMCs were then enriched using a negative selection kit with paramagnetic beads, according to the manufacturer's protocol (StemCELL, catalog 19409-A05U), which depletes unrelated cell populations. The enriched cell populations were stained on ice for 1 hour 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 antibody 1:25 dilution (BD, catalog 555460) in RoboSep buffer (StemCELL, catalog 20104). After staining, the cells were centrifuged and resuspended in RoboSep containing DAPI as the active dye. Live CD19 cells were then... 低 CD27 高 CD38 高 Cells were sorted into sequencing buffer, and single-cell RNA sequencing was performed on plasmablasts. Single-cell RNA capture was performed using the 10X Genomics Chromium platform, and enrichment was performed 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 CellRanger software v3.1.0 (10X Genomics).

[0277] Freshly collected bone marrow aspirate was washed with RoboSep buffer and filtered through a 70-micron filter to remove unwanted bone fragments and matrix clots that could interfere with downstream processing steps. Bone marrow plasma cells were then enriched using a paramagnetic bead kit for depleting unrelated cell populations and a negative selection mixture (StemCELL, catalog 19409-A05U). Cells were then resuspended on ice in a staining mixture containing 1:20 dilutions of anti-CD19-FITC antibody (eBioscience, catalog 11-0199-42), anti-CD138-APC antibody (BioLegend, catalog 52307), and anti-CD38-PE antibody (BD, catalog 555460) in RoboSep buffer (StemCELL, catalog 20104). After staining, cells were centrifuged and resuspended in RoboSep containing DAPI as the active dye. Viable CD19 cells were then...+ CD138 + CD38 + Cells and CD19 - CD138 + CD38 + Cells were sorted into sequencing buffer using bidirectional sorting.

[0278] Antibody gene sequence analysis. Target B cells were processed using the 10X Genomics workflow, and sequences were processed using CellRanger. All sequences were then analyzed using PyIR software (Soto et al., 2020) to identify the V gene, J gene, and third complementarity determining region (CDR3) for each antibody variable region, as previously described (Soto et al., 2019). Specific combinations of the V and J genes and CDR3 were designated as V3J clonal types, and for each of these clonal types, the inventors determined whether the clonal type was present in multiple tissue sites by searching for precise sequence matches of the CDR3 amino acid sequence in bone marrow and plasmablast populations.

[0279] MAb generation. mAb sequences, synthesized as cDNA (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (named pTwist-mCis_G1), were used for mAb secretion in mammalian cell cultures. This plasmid IgG expression vector contains an enhanced 2A sequence and a GSG amino acid linker, which allows simultaneous expression of mAb heavy and light chain genes by a single construct at transfection (Gilchuk et al., 2020; Zost et al., 2020). The inventors previously described small-scale expression of mAbs in 1 mL of ExpiCHO culture in 96-well plates. For larger-scale mAb expression, the inventors used the GibcoExpiCHO expression system and a protocol for 50 mL microbioreactor tubes (Corning) as described by the supplier, to transfect Chinese hamster ovary cell (Wagner et al., 2002) cultures (1 to 300 mL of each antibody). 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). The purified mAb was buffer-exchanged in PBS, concentrated using an Amicon Ultra-4 50-kDa centrifugal filter unit (Millipore Sigma), and stored at 4°C until use. Endotoxin levels of the purified mAb were routinely tested (found to be below 30 EU / mg IgG in mouse studies). Endotoxin detection was performed using a PTS201F card (Charles River) (sensitivity range 10 to 0.1 EU / mL) and an Endosafe Nexgen-MCS instrument (Charles River).

[0280] Expression and purification of NA antigens. Soluble recombinant NA antigens were expressed and purified as previously described (Serris et al., 2020). Genes encoding 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 expression in Drosophila cells and cloned into a plasmid (pT350) containing the MT promoter, BiP signal sequence, and C-terminal double-stranded mycotoxin tag. Drosophila S2 cells were transfected with NA and PCoBlast (Thermo Fisher Scientific) plasmids at a ratio of 19:1, and stably transfected cells were selected using 25 μg / mL blast fungicide. Stable cell lines were maintained in Schneider Drosophila medium supplemented with 25 μg / mL blast fungicide and grown in shake flasks to a height of 1 × 10⁶ cells / year. 7 S2 cells were induced at a density of 10 cells / mL using 4 μM 760 CdCl2. After five days, S2 cell supernatant was collected, supplemented with 10 μg / mL avidin, and purified using a StrepTrap HP column (Cytiva). The sample was further purified by size exclusion chromatography on a HiLoad 16 / 600 Superdex column (Cytiva).

[0281] Enzyme-linked immunosorbent assay (ELISA). A 384-well plate was coated overnight at 4°C with 1 μg / mL purified rNA protein in 1× phosphate-buffered saline (PBS). The plate was incubated with blocking buffer (2% skim milk powder, 2% goat serum, and 0.1% Tween-20 in PBS) at room temperature for 1 hour. Diluted primary antibody was added to appropriate wells and incubated at room temperature for 1 hour. The bound antibody was detected using horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Southern Biotech, catalog 2040-05, batch B3919-XD29, 1:5,000 dilution) and 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 absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose-response assays, serial dilutions of the purified mAb were applied to three wells, and antibody binding was detected, as detailed above. After logarithmically transforming the mAb concentration using sigmoidal dose-response nonlinear regression analysis, the binding EC50 was determined using Prism software v.8.0 (GraphPad). 50 Value. ELISA is performed in triplicate technically and in duplicate biologically.

[0282] Enzyme-linked lectin assay (ELLA). A 384-well flat-bottom microtiter plate (Thermo Fisher Scientific) was coated at 4°C with 25 μl / well of fetoglobulin (Sigma) at a concentration of 25 μg / mL in 1× PBS without CaCl2 or MgCl2 for at least 18 hours. The plate was washed three times with PBS-T. Triple-fold serial mAb dilutions were performed in 96-well plates starting at 20 μg / mL using sample dilution buffer (Dulbecco's PBS [DPBS] containing CaCl2 and MgCl2, 1% BSA, 0.5% Tween-20). 25 μL of mAb from the dilution plate was transferred in triplicate to the fetoglobulin-coated plate. Next, 25 μL of diluted virus was added to all wells except the virus-free column. 25 μL of sample dilution buffer was added to the virus-free control column. The viral dilution was determined by selecting the viral dilution factor that produced the maximum optical density. The plates were placed in a humidified incubator at 33°C for 18 hours. The next day, the plates were washed six times and 100 μL / well of peanut agglutinin (PNA)-HRP (Sigma) at a concentration of 1 mg / mL in PBS containing 1% BSA was added. After incubation at room temperature for 2 hours, the plates were developed with 25 μL of TMB substrate (Thermo Fisher Scientific). Color development was monitored, and 1M hydrochloric acid was added to stop the reaction. The absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose-response assays, serial dilutions of the purified mAb were applied to the wells in triplicate, and inhibition was assessed as detailed above. After logarithmic transformation of the mAb concentrations using sigmoidal dose-response nonlinear regression analysis, the half-maximal inhibitory concentration (IC50) was determined using Prism v.8.0 software (GraphPad). 50 The values ​​were analyzed using Excel (Microsoft) and Prism (GraphPad) software. 50 The value was defined as the mAb concentration at which 50% of NA activity was inhibited compared to the negative control (virus without mAb). Assays were performed in technical triplicate and biological duplicate.

[0283] Neuraminidase inhibition was measured using the NA-Fluor assay. The NA-Fluor Influenza Neuraminidase Inhibitor Kit (Thermo Fisher, catalog number 4457091) is used to quantify the inhibition of NA activity (cleavage of the small chemiluminescent substrate MUNANA) in the presence of NA-mAb. Before the assay, each virus was titrated to determine the optimal dilution. The experiment was performed according to the manufacturer's protocol. The mAb was diluted to a concentration of 20 μg / mL and a series of ternary dilutions were performed. 6.25 μL from each dilution was transferred to a black flat-bottomed 384-well cell culture plate and mixed with 6.25 μL / well of the pre-determined concentration of virus at 33°C for 30 min. After incubation, NA-Fluor substrate (12.5 μL / well) was added, and the plate was incubated at 33°C for 1 h. NA-Fluor stop solution (25 μL / well) was added to the plate. The chemiluminescence signal was detected by a microtiter plate reader (Bio-Tek) by excitation at 360 nm and emission detection at 450 nm. Data were analyzed using Excel and Prism. IC 50 The value was defined as the mAb concentration at which 50% of NA activity was inhibited compared to the negative control (virus without mAb). Assays were performed in technical triplicate and biological duplicate.

[0284] Competitive combination ELISA. The wells of a 384-well microtiter plate were filled with purified recombinant influenza B virus. / Iowa / 06 / 2017rNA 1 The protein was coated overnight at 4°C. The plate was blocked for 1 hour with 2% skim milk powder and 2% goat serum in DPBS containing 0.05% Tween-20, and washed three times. Each antibody was diluted to a concentration of 10 μg / mL and 20 μL was added to each well. Next, the biotinylated antibody was diluted to 10 μg / mL, and 5 μL was added to the primary antibody solution without washing. The binding of the biotinylated antibody was detected with horseradish peroxidase-conjugated avidin (Sigma) and developed with TMB substrate. Once developed, the reaction was quenched with 1N hydrochloric acid. The absorbance was measured at 450 nm using a spectrophotometer. If the blocking percentage was less than 70%, the antibody was considered competitive; if it was non-competitive, it was >70%. ELISA was performed in triplicate technically and in duplicate biologically.

[0285] Egress inhibition assay. MDCK cells were seeded overnight in 96-well plates in standard Dulbecco modified Eagle medium (GIBCO DMEM, Thermo Fisher Scientific) containing 10% FBS. Cells were washed three times with virus growth medium (VGM) (DMEM containing 2% BSA and 2 mg / mL TPCK-treated trypsin (Sigma-Aldrich), and 100 μL of virus at one multiple of infection (MOI) in VGM was added to the cells. The plates were incubated at 33°C in 5% CO2 for 3 hours. Cells were then washed again with VGM and replenished with VGM containing serially diluted mAbs (10 μg / mL) or equimolar concentrations, starting from the highest concentration of the test mAb (10 μg / mL). The plates were incubated at 33°C in 5% CO2 for 21 hours, and the supernatant was collected for HA assay. For the HA assay, the inventors used turkey red blood cells (LAMPIRE Biological Products, catalog number 7209403), washed them, and diluted them to 0.5% vol / vol in PBS. 50 μL of the supernatant was incubated with 50 μL of 0.5% turkey red blood cells in a V-shaped plate at 4°C for 1 hour. IC50 100 The value is defined as the lowest antibody concentration added to virus-inoculated MDCK cells corresponding to the absence of virus in the supernatant, and is measured by hemocytosis on red blood cells. The determination is performed in technical duplicate and biological duplicate.

[0286] Neutralization assays based on real-time cell analysis (RTCA) impedance measurement. High-throughput RTCA analysis of quantitative virus-induced cytopathic effect (CPE) was used to test antibody-mediated virus neutralization under BSL-2 conditions, employing general methods 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 concentrations inducing complete CPE at 72 hours post-inoculation, and an MOI of 0.1 was selected to normalize all strains. 50 μL of Opti-MEM supplemented with 1% penicillin and streptomycin was used. TMMedium I was added to each well of a 96-well E plate (Agilent) to establish background readings, followed by seeding each well with 50 μL of MDCK-SIAT1 cell suspension (30,000 cells / well) to induce cell adhesion. The plate was incubated at room temperature for 30 minutes and then placed on an xCELLigence RTCA analyzer (formerly ACEABI Biosciences, now Agilent). Cell impedance was measured every 15 minutes. Using Opti-MEM I medium supplemented with 1% penicillin and streptomycin, a 1:1 mixture of virus suspension and mAb (0.1 MOI) was prepared in duplicate, with a total volume of 100 μL, and incubated at 33°C in 5% CO2 for 1 hour. The virus-mAb mixture was added to the cells 16–18 hours after seeding. Wells containing only virus (without mAb) or only cells (without virus or mAb) served as controls. The plate was measured every 15 minutes for 72 hours post-inoculation to assess inhibition of CPE, a marker of virus neutralization. Cell indexes at the endpoint (approximately 72 hours post-inoculation) were determined using RTCA software version 2.1.0 (Agilent). 1 Values. The neutralization percentage was calculated as the CI in the presence of mAb divided by the value from cell-only (no CPE control) wells. Background values ​​were subtracted from virus-only (maximum CPE) control wells. The half-maximum inhibitory concentration (IC50) was calculated using Prism software version 9 (GraphPad) via nonlinear regression analysis. 50 Value. Measurements are performed in technical duplicate and biological duplicate.

[0287] Isolation and culture of primary respiratory epithelial cells. 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 supplemented with 50× medium supplement, and incubated at 37°C in 5% CO2 until the cell culture reached 80% confluence. Cells were trypsinized and cultured at 1.2 × 10⁻⁶ ppm. 5Cells were seeded at a density of 100 cells / well in Transwell (Corning) wells with 0.4 μm diameter wells, with both the apical and basal sides supplemented with PneumaCult-EX Plus basal medium. The medium was changed every other day until 100% confluence was achieved (3 to 4 days). After 100% confluence, the apical medium was removed. 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 was washed off the apical surface every five days. Cells were prepared for analysis after 3 to 4 weeks of differentiation.

[0288] Early attachment event inhibition assay. Differentiated primary respiratory cells were washed with PBS and inoculated with 0.1 MOI influenza B / New York / 2017 (Yamagata lineage) virus, which had been incubated for 1 h with PBS alone, an IgG isotype control against a heterologous antigen (mAb DENV r2D22 against dengue virus envelope protein) (10 μg / mL), zanamivir (1 μg / mL), mAb FluB-393 (10 μg / mL), mAb FluB-400 (10 μg / mL), or a positive control mAb r1G05 (10 μg / mL). After one hour, the inoculum was washed off, and after 8 hours, the cells were fixed by adding 3.7% paraformaldehyde to the substrate and apical surfaces. Cells were permeabilized by adding 0.1% Triton X for 20 min, washed with PBS, and blocked with 2% BSA solution in PBS. Staining was performed by adding primary antibodies, anti-NP (IRR, catalog number FR-1218), and anti-E-cadherin (CellSignaling Technology, catalog number 3195S), both diluted 1:100 in 1% BSA PBS Tween. Cells were incubated on a shaker at room temperature for 30 minutes and washed with PBS. Secondary antibodies were added: secondary NP: Alexa Fluor 568 (red) (Thermo Scientific, catalog number A-11004), and secondary E-cadherin: Alexa FluorPlus 488 (green) (Thermo Scientific, catalog number A32731TR), both diluted 1:1,000 in 1% BSA PBS Tween. Cells were incubated on a shaker at room temperature for 30 minutes. Cells were washed with PBS, and membranes were removed and cells were treated with Prolong. TMThe Diamond Antifade Mountant (Thermo Scientific, catalog number P36961) was used to fix the device onto a glass coverslip. Images were captured on a Zeiss 710, capturing nine representative fields of view. The experiment was performed in duplicate.

[0289] Testing of protective efficacy against dengue virus B / New York / 2018 in a BALB / c mouse model. Female BALB / c mice aged six to eight weeks were purchased from Jackson Laboratories (strain 000651). Mice were housed in groups of up to five mice per cage at ambient temperatures of 18°C ​​to 24°C and humidity of 40% to 60%. Mice were fed a 20% protein diet (PicoLab5053, Purina) and maintained on a 12-hour light-dark cycle (06:00 to 18:00). Food and water were readily available. Mice were housed in individually ventilated cages with negative pressure ventilation and air filtration. To assess the protective efficacy of the mAb, mice were inoculated with a single mAb via intraperitoneal route 24 hours before (prophylaxis) or 12 hours after (treatment) administration of the virus. Human anti-dengue virus mAb DENV r2D22 was used as an antibody control. At the ABSL-2 facility, 10 μL of sterile PBS was used to prepare the mAb. 5 The corresponding viral strain of FFU was intranasally inoculated into mice anesthetized with sevoflurane. Mice were weighed daily and morbidity was monitored. Mice that lost more than 30% of their initial body weight were humanely euthanized according to IACUC guidelines. Lung tissue was collected at 3 and 6 days post-infection (n=3).

[0290] Pulmonary viral load was measured. Left lung lobes were collected and homogenized for 3 minutes in 1 mL DMEM medium with high-impact zirconia beads (Benchmark, catalog number D1032-10) using a cryo-bead mill (Benchmark, catalog number D2400-R). The tissue homogenate was clarified by centrifugation at 10,000 rpm for 5 minutes and stored at -80°C. For plaque assays, the homogenate was serially diluted tenfold and applied to MDCK-SIAT1 cell culture monolayers in 12-well plates supplemented with 1% penicillin, streptomycin, and 1 μg / mL TPCK-treated trypsin. The plates were incubated at 33°C for 1 hour. Cells were then covered with 1.2% methylcellulose in DMEM supplemented with 1% penicillin, streptomycin, and 1 μg / mL TPCK-treated trypsin. After 72 hours, the plates were collected by removing the covering and fixed with 4% PFA in PBS at ambient temperature for 20 minutes. After removing 4% PFA, plaques were visualized by adding 0.5 mL / well of 0.05% crystal violet in 20% methanol solution for 20 minutes at ambient temperature. Excess crystal violet was washed away with PBS, and plaques were counted.

[0291] Histopathology. Mice were euthanized, and tissues were collected before lung inflatation and fixation. The left lung lobe was ligated at the left main bronchus and collected for viral quantification. 1 mL of 10% neutral buffered formalin was blown into the right lung lobe using a 3-mL syringe and a needle inserted into the trachea. For fixation after infection, the inflated lung was kept in 10% neutral buffered formalin for 48 hours before processing. Tissues were embedded in paraffin and sectioned at 5 μm, then stained with hematoxylin and eosin. Tissue sections were visualized using an Olympus BX53 microscope equipped with an Olympus UC90 camera. Sections were graded by a board-certified veterinary pathologist.

[0292] Epitope mapping was performed using saturated alanine scanning mutagenesis. Epitope mapping was performed essentially as previously described (Davidson & Doranz, 2014) using a shotgun mutagenesis mutant library of the NA head region of influenza B (strain B / Colorado / 06 / 2017), prepared using a full-length expression construct of NA. Each of the 368 residues (residues 76 to 466) in the head region was mutated to alanine, and the alanine residues were mutated to serine. The mutant library was arranged in 384-well microplates, transiently transfected into HEK293T cells, and expressed for 22 hours. The cells were then incubated with a predetermined concentration of antibody, the concentration of which was predetermined using independent binding titration curves on cells expressing wild-type NA. The cells were incubated with MAb diluted in PBS with calcium and magnesium (PBS++) and 10% normal goat serum (Sigma). Antibody detection was performed using a secondary antibody conjugated with Alexa-Fluor-488 at 3.75 μg / mL in 10% normal goat serum (Jackson Immuno Research Laboratories). Cells were washed three times with PBS, and mean cell fluorescence was detected using a high-throughput Intellicyte iQue flow cytometer (Sartorius). Antibody reactivity against wild-type NA was calculated by subtracting the signal from the simulated transfection control and normalizing the signal from the wild-type NA transfection control. If a mutation within a clone did not support reactivity to the tested MAb but supported reactivity to other anti-NA antibodies, it was identified as essential to the mAb epitope. This reverse screening strategy facilitated the exclusion of NA protein mutants with local misfolding or defective expression.

[0293] Negative staining electron microscopy. Immune complexes were prepared by incubating recombinant NA protein based on the B / Iowa / 9 / 2017 sequence with mAb FluB-393, FluB-400, or 2D10 at a 1:4 molar ratio overnight at 4°C. The resulting sample was deposited at a concentration of 20 μg / mL onto a carbon-coated 400-mesh copper grid (Electron Microscopy Sciences) that had undergone glow discharge for 25 seconds. After 10 seconds, the sample was aspirated and subsequently stained twice with 2% w / v uranyl formate solution for 45 seconds each time. The staining was then performed using a Tecnai Spirit T12 microscope equipped with an Eagle CCD 4k camera (FEI) at 120 kV and a magnification of 52,000× (2.06). The sample was imaged using pixels. The defocus was set to -1.5 μm. Micrographs were collected using Leginon (Potter et al., 1999). Individual particles were picked up using a DoG Picker (Voss et al., 2009) and processed using Appion (Lander et al., 2009), with 2D processing performed in Relion 3.0.

[0294] Cryo-EM Sample Preparation. Prior to cryo-EM grid preparation, an immune complex was prepared by incubating a recombinant NA protein based on the B / Iowa / 9 / 2017 sequence with FluB-393, FluB-400, or 2D10 at a 1:4 molar ratio overnight at 4°C. 0.1% w / v octyl-β-glucan detergent was added to the complex to aid particle tumbling. The final sample concentration on the grid was 0.4 mg / mL. A Vitrobot Mark IV system was used to prepare the cryo-EM grid. The settings were as follows: zone chamber temperature 25°C, humidity 100%, blotting force 1, waiting time 5 seconds, and blotting time varying from 3.5 to 5.5 seconds. 3 μL of sample was added to a plasma-cleaned 1.2 / 1.3 copper Quantifoil 300-mesh grid. The plasma cleaning step was performed for 25 seconds in a Solarus 950 plasma system (Gatan) with an Ar / O2 gas mixture. The sample was aspirated for 4 seconds, and the grid was then immersed in liquid nitrogen-cooled liquid ethane for freezing.

[0295] Cryo-EM data collection, processing, and modeling. Low-temperature grids of the 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 acquisition was performed using an EPU from Thermo Fisher. For both NA / FluB-393 and NA / FluB-400, subsets of images were acquired at a 30° tilt along the z-axis to obtain a more comprehensive view of the particle distribution. (Cryo-EM data was collected, processed, and modeled using CryoSPARC Live.) TMImage alignment, dose weighting, and contrast transfer function (CTF) correction were performed on the software platform, and automated image acquisition was also performed using Smart EPU software (Thermo Fisher). The cryogenic mesh of the 2D10 complex was imaged using a Talos Arctica electron microscope (FEI) with a CETA 4k CMOS camera at a nominal magnification of 36,000×. Micrographs were acquired, aligned, and CTF corrected using Leginon, MotionCor2 in Appion, and Patch-CTF in CryoSPARC, respectively. Data processing for all three datasets was performed in CryoSPARC v4.1.1. In short, particles were selected using templates generated from their respective negative staining datasets. Pure particle stacks were selected through reference-free 2D classification, and then these pure particle stacks were used to refine a 3D reference model from scratch, which was then used for 3D refinement. NA / FluB-393 underwent 2D classification rebalancing with a rebalancing factor of 0.7 and 10 superclasses before 3D refinement. The final image was generated using C4 symmetry, with per-particle CTF refinement using default parameters, followed by CryoSPARC local refinement. The final image has a global resolution of 2.7. Up to 3.36 Antibody Fv models were generated using the Abody builder, and NAB models were built using Rosetta. The models were then plotted on cryoEM plots using Rosetta Relax (DiMaio et al., 2015; Afonine et al., 2018) and manually tuned using Coot (Emsley et al., 2010).

[0296] Example 2 - Results

[0297] Isolation and characterization of NA-reactive mAbs identifying two binding sites on the IBV NA protein. A 47-year-old woman was vaccinated with Flucelvax in March 2019. TMThe quadrivalent influenza vaccine is an inactivated surface antigen vaccine prepared in cell culture. The administered vaccine contains surface antigens from the following viruses: A / Singapore / GP1908 / 2015 IVR-180 (H1N1) (A / Michigan / 45 / 2015-like virus), A / North Carolina / 04 / 2016 (H3N2) (A / Singapore / INFIMH-16-0019 / 2016-like virus), B / Iowa / 06 / 2017 (B / Colorado / 06 / 2017-like virus), and B / Singapore / INFTT-16-0610 / 2016 (B / Phuket / 3073 / 2013-like virus). On day seven post-vaccination, peripheral blood was drawn, and peripheral blood mononuclear cells (PBMCs) were isolated to enrich plasmablasts via flow cytometry (data not shown). On day 57 post-vaccination, bone marrow aspirate was collected, and long-lived plasma cells (LLPCs) were sorted from the bone marrow cell suspension using flow cytometry. Figure 1A To B). Donor sera were screened for antigen reactivity to B / Iowa / 06 / 2017 by ELISA using sera collected before or after vaccination (data not shown). Antibody variable genes in individual PBMCs or LLPCs were sequenced using a chromium single-cell encapsulation system (10X Genomics), yielding 1,262 or 7,445 antibody heavy and light chain sequences for the PBMC or LLPC population, respectively. The shared use of immunoglobulin heavy chain V, D, and J genes in both populations, as well as the same (100%) immunoglobulin heavy chain complementarity-determining region 3 (HCDR3) amino acid sequence, was evaluated. Sixty-four antibody clones were identified in both compartments and selected for small-scale expression of recombinant IgG1 antibody (data not shown). Of the 64 tested clones, 17 mAbs bound to antigens from the 2019–2020 Fluzone vaccine (which matched those antigens in the Flucelvax vaccine she received) and to recombinantly expressed NA proteins based on the influenza B / Singapore / INFTT-16-0610 / 2016 (B Yamagata lineage) viral sequence. These 17 mAbs belonged to two clone-amplified families. One lineage was encoded by antibody genes IGHV4-39 and IGKV1-5 and possessed… The HCDR3 amino acid sequence (named as FluB-387 to FluB-398, 12 antibodies). The second lineage is encoded by antibody genes IGHV5-51 and IGKV3-20, and possesses the HCDR3 amino acid sequence (named as FluB-399 to FluB-403, 5 antibodies). Figure 1B Following further screening, this group of mAbs exhibited cross-reactive binding to recombinantly expressed influenza B / Iowa / 06 / 2017 (Victoria [V]) and B / Singapore / 2016 (Yamagata [Y]) NA proteins when tested by ELISA. The number of major antigenic sites recognized by these mAbs was analyzed using competitive binding with recombinant NA protein antigens based on the NA sequence of influenza B / Iowa / 06 / 2017 in ELISA. These studies revealed the presence of two competing binding groups, consistent with the fact that the mAb group contains clones representing two clonal families. One clonal family, comprising mAbs FluB-399 to FluB-403, unidirectionally competes with antibody 1G05 (a previously reported mAb recognizing the IBVNA active site). This finding suggests that this mAb family may also bind to or near the IBVNA active site (Figures 2A-B). To test whether this NA binding pattern is germline-encoded, the inventors generated germline revertant (GR) clones and tested the binding of these GR clones to NA in ELISA. These studies revealed a loss of detectable binding in FluB-393-GR and FluB-400-GR, indicating that the detectable binding affinity of these clones was achieved through somatic hypermutation (data not shown).

[0298] Epitope mapping was performed using alanine scanning mutagenesis. The inventors identified NA residues crucial for binding to mAbs from different lineages. mAbs binding to a library of alanine scanning mutants in the NA head region were screened. For the IGHV4-39-encoded lineage containing mAb FluB-393, residues E338 and K272 were crucial for binding. For IGHV4-39-encoded mAbs, E338 was the dominant residue because it proved crucial for binding to all six mAbs from this lineage tested by the inventors (data not shown). K272 was also crucial for five of these six mAbs. An exception was FluB-398, the clone whose sequence was most closely related to the inferred germline sequence; this mAb was isolated from a peripheral blood memory B cell compartment. ELISA was performed using two recombinant NA antigens, and the binding curves showed that the mAb specifically recognized B / Iowa / 06 / 2017(V) but not B / Singapore / INFTT-16-0610 / 2016(Y). This observation indicates that this clonal family has undergone somatic high mutation rates to acquire mutations that enable binding breadth across both the Victoria and Yamagata lineages. These additional residues identified were Y296 (essential for FluB-389) and G335 (essential for both FluB-398 and FluB-389) (data not shown). These residues are consistent with contact-essential residues subsequently defined by cryo-electron microscopy, as described below.

[0299] For the lineage mAbs FluB-400 and FluB-401 encoded by IGHV5-51, alanine scanning library binding deletion screening identified active site residues R272 and R374 as crucial for binding (data not shown). R272 and R374 were also identified as contact residues by cryo-EM analysis of FluB-400 and previous analyses of other influenza B NA active site mAbs (Madsen et al., 2020).

[0300] NA-reactive mAbs inhibit IBV NA enzyme activity. To select downwards from a group of 17 mAbs, allowing the inventors to identify a lead mAb from each clonal family, an enzyme-linked lectin assay (ELLA)-neuraminidase inhibition (NI) (ELLA-NI) was performed as an alternative assay for NA sialylase activity. The ELLA-NI assay uses a lectin that binds to sialic acid carbohydrates exposed on several glycosylated proteins (i.e., NA) on the surface of the influenza virus. The lectin binds to the glycan and forms a complex (i.e., lectin-NA), which is detected as a colorimetric signal generated by the enzymatic degradation of a suitable substrate. The intensity of the signal helps quantify the percentage inhibition of NA enzyme activity by the mAb. A group of 17 mAbs was screened for inhibition of NA enzyme activity in nine viruses across the Victoria and Yamagata lineages. FluB-393 and FluB-400 have shown the highest potency in their respective clone families (data not shown).

[0301] For further characterization, four functional assays (ELLA-NI, NA-Fluor assay, excretion inhibition assay, and real-time cell analysis [RTCA] neutralization assay) were performed using FluB-393 and FluB-400. Two previously described active site-specific IBV mAbs (r1G05, r2E01) were also included. 24 (and an isotype-matched control IgG mAb targeting a heterologous target (rDENV-2D22, which recognizes the dengue virus envelope protein) (Alwis et al., 2012) (Figure 3A). Because ELLA uses a large molecular substrate, the observed inhibition of NA enzyme activity may or may not indicate direct antibody recognition of the active site. Logically, in the ELLA-NI assay, some antibodies block NA activity through steric hindrance mechanisms rather than directly binding to the active site. Therefore, the inventors subsequently performed NA-Fluor... TMThe influenza neuraminidase assay is a direct functional enzyme assay using the small substrate 2'-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (MUNANA). Antibodies active in the NA-Fluor assay may directly bind to the NA active site. In the NA-Fluor assay, the inventors tested representative mAbs against 14 viruses representing two IBV lineages, allowing for functional differentiation of antibodies binding at the NA active site. It is desired that novel antibody therapeutics against NA be active against IBV strains resistant to currently approved small molecule inhibitors. It should be noted that the four viruses used by the inventors in this assay contain mutations at the active site that confer resistance to oseltamivir (B / Indiana / 17 / 2017 (I221T), B / Oklahoma / 10 / 2018 (NAD197N), B / Wisconsin / 10 / 2016 (NA I221V), and B / Hawaii / 01 / 2018 (NA D197N)). FluB-400 and the positive control mAb inhibited NA enzyme activity against all tested IBV strains, while FluB-393 did not. This finding suggests that FluB-393 inhibits NA activity by binding to NA proteins at epitopes outside the active site, possibly through spatial interference with proximity to the active site, as observed in the ELLA-NI assay.

[0302] The sialidase activity of the IBV NA protein promotes the efficiency of viral shedding from infected cells. NA cleaves sialic acid from cell receptors and newly synthesized HA and NA proteins on budding virions, which are sialylated as part of a glycosylation event in the host cell (McAuley et al., 2019). To determine whether FluB-393 or FluB-400 inhibits viral shedding, the inventors performed a viral shedding inhibition assay as previously described (Bangaru et al., 2018). MDCK-SIAT1 cells were plate-seeded and allowed to grow to near confluence, then inoculated with IBV at an MOI of 1. Three hours after inoculation, unattached virus was removed by thorough washing, and medium containing mAb was added to each well. mAbr1G05 and r2E01 were used as positive controls for NA inhibition, although the shedding inhibition activity of these mAbs has not been reported. IBV-inoculated cells were incubated with medium containing mAb at 33°C for 18 hours. The supernatant was then collected, and the presence of viral particles was tested using a HAI assay. The inventors used three representative IBVs to evaluate the breadth of expulsion inhibition activity in this assay: two Victoria lineage viruses (B / Hawaii / 01 / 2018 (NA D197N) and B / New York / PV00081 / 18) and one Yamagata lineage virus (B / New York / PV00094 / 2017). FluB-393, FluB-400, and r2E01 inhibited viral expulsion from all tested IBV strains, while r1G05 inhibited only B / New York / PV00094 / 2017 (Y).

[0303] Next, the inventors used real-time cell analysis (RTCA) to test the inhibition of viral replication in a cell-based bioassay, which used label-free cellular impedance to continuously monitor the cytopathic effects of the virus. Representative viruses from the Yamagata or Victoria lineages were tested, and inhibition against viruses with NA active site mutations associated with oseltamivir resistance was evaluated using B / Hawaii / 01 / 2018 (NA D197N) (Wagner et al., 2002) (Oakley et al., 2010). Based on these results, FluB-400 neutralized viruses from both IBV lineages, while FluB-393 and r1G05 neutralized only B / New York / PV00094 / 2017 (Y). r2E01 did not neutralize any of the tested IBV strains.

[0304] In summary, these results indicate that FluB-393 inhibits NA enzyme activity, although it does not bind directly to the NA active site. This finding suggests that FluB-393 binding allows for spatial inhibition of NA enzyme activity and viral shedding from nearby epitopes. FluB-393 exhibits Yamagata lineage-specific neutralization. In contrast, FluB-400 exerts its action by directly binding to the NA active site to inhibit NA sialidase activity and block viral shedding. FluB-400 neutralizes all tested IBV strains. NA-reactive mAbs (r1G05 and r2E01) used for comparative purposes inhibited NA enzyme activity in ELLA and NA-Fluor assays, consistent with previously described findings (Madsen et al., 2018).

[0305] FluB-400 restricts early attachment events on primary human respiratory epithelial cells. Not only does NA (virion nucleoside) facilitate the release of progeny virions from host cells to infect new cells, but some studies have also shown that NA plays a role in viral attachment and entry. Therefore, the inventors next tested whether a human anti-NA antibody could inhibit the early stages of the viral life cycle.

[0306] The inventors used an in vitro air-liquid interface (ALI) culture system of differentiated primary human tracheal epithelial cells as a substitute for human respiratory epithelium (Fig. 3C). In this system, one side of the cell membrane is exposed to air, while the other side remains in contact with the growth medium. To assess NA inhibition, a suspension of B / New York / PV00094 / 2017(Y) virus prepared at a delivery MOI of 0.1 was incubated with mAb at a concentration of 10 µg / mL. The virus + mAb mixture was then added to the cells and incubated for 1 hour. Unattached IBV particles were removed by thorough washing. The cells were then incubated for 8 hours, a period long enough to allow viral attachment and entry, but not the complete viral replication cycle (Smet et al., 2022). The cells were fixed and stained for E-cadherin (to distinguish tight junctions of cells) and IBV nucleoprotein (NP) (to identify sites of IBV replication). Images were captured by confocal microscopy (Fig. 3D). NPs were detected in images captured from both the FluB-393 and negative control (rDENV-2D22) treatment groups, but not in images captured from cultures treated with FluB-400. Therefore, the imaging data suggest that only FluB-400 prevented early attachment events.

[0307] Frozen-EM analysis of complexes of FluB-393 or FluB-400 Fab molecules with recombinant IBV NA protein. To determine the antigenic sites recognized by these two NA mAb lineages, the inventors first performed negative staining electron microscopy on B / Iowa / 6 / 2017 complexed with FluB-393 or FluB-400. FluB-400 bound in a manner consistent with other active site-specific NA mAbs, as expected from mutagenesis studies. In contrast, FluB-393 bound laterally at the "corner" of the NA tetramer.

[0308] To map the epitopes targeted by these clonal families at high resolution, the inventors resolved the frozen EM structures of FluB-393 and FluB-400 from B / Iowa / 6 / 2017. FluB-393 was obtained by multiple contacts of the antibody loops LCDR1, LCDR3, HCDR2, and HCDR3 with the NA at their angles. Figure 6 The interactions with NA residues K272 and D338 were highlighted, as these residues were identified as crucial for binding in the alanine scanning mutagenesis study. The main-chain amide of K272 forms an H bond with the side carboxylate group of D56 in HCDR2, which in turn hydrogen-bonds with the side-chain hydroxyl group of T54 in HCDR2, which itself is hydrogen-bonded with the side-chain amine of K272. The side-chain carboxylate group of NA residue D338 forms multiple hydrogen bonds with the main-chain carbonyl groups of LCDR3—Y91 and Y94—and the indoleamide of W96. FluB-400 binds in a manner common to other NA active site-specific mAbs, where HCDR3 extends into the NA active site, and HCDR1, LCDR1, and LCDR2 interact with residues around the active site inlet. Figures 7A to G illustrate how D100B in HCDR3 forms H bonds with NA residues R272 and R374, and contacts another active site residue Y409, whose binding has been shown to be important through mutagenesis studies. R374 also forms a stacked cation-π interaction with Y100. HCDR3 also perturbs other active site residues, such as the substitution of R224 due to R100D, whose side chain also binds to the carboxylate group of E276.

[0309] Identification of IBV NA antibody lineages with shared gene usage and binding sites. FluB-393 and its clone-associated mAbs are encoded by IGHV4-39 and IGKV1-05. Previously, Madsen et al. described mAb 2D10 (an IBV-specific NA antibody) which exhibited NAI activity in both ELLA and NA-Fluor assays and was also encoded by IGHV4-39 and IGKV1-5. Negative staining EM analysis showed that 2D10 binds at the 330-loop at the corner of the NA. To investigate whether any elements of this interaction are similar in the FluB-393 molecular pattern or in NA recognition, the inventors resolved the frozen EM structure. The structure confirmed that 2D10 also forms a contact with the 330-loop. However, interestingly, no contact residue is conserved between FluB-393 and 2D10, despite the fact that the heavy chain or light chain variable fragment (Fv) has 76% and 91% identity, respectively.

[0310] FluB-393 and FluB-400 exhibit broad in vivo protection against IBV. The inventors then evaluated the in vivo efficacy of FluB-393 and FluB-400 against two IBV prophylactic and therapeutic models: one Victoria lineage IBV (B / New York / PV00081 / 18). 1 ) and a Yamagata lineage IBV (B / New York / PV00094 / 2017). Six- to eight-week-old female BALB / c mice were fed 10 5 PFU was administered intranasally (IN) to mice. Body weight and survival were measured 14 days post-inoculation, with the humane endpoint set at 70% of initial body weight.

[0311] In the B / New York / PV00081 / 18 prophylaxis model, FluB-393 and FluB-400 were administered via IP at a single 10 mg / kg dose of mAbs 12 hours prior to IN virus inoculation. Both mAbs completely averted mortality (100%) and reduced weight loss compared to the isotype-matched negative control group treated with rDENV-2D22 (0% survival) (Figures 4A to F). Lungs were collected at 3 or 6 days post-inoculation (dpi) for quantification of infectious viral particles in plaque assays. The mean lung viral titer at 3 dpi was 10 in the FluB-393 or FluB-400 treatment groups. 5 log 10 PFU / mL, and decreased below the detection limit by 6 dpi (Figures 4C and 4F). Additionally, lung samples collected at 6 dpi were processed, embedded, and analyzed using hematoxylin and eosin, as well as RNAScope. TMHistopathological analysis was performed using viral NP RNA staining (Fig. 4H). Based on findings from those assessments, mice treated with FluB-400 showed minimal impact from IBV inoculation, with the smallest affected tissue area, low levels of bronchiolar epithelial erosion, and low degrees of peribronchiolar or interstitial inflammation (data not shown). FluB400 treatment significantly reduced visually detectable viral RNA signaling in lung tissue. In the B / New York / PV00094 / 2017(Y) prophylaxis model, FluB-393, FluB-400, and r1G05 protected 80% (n = 4 / 5) of mice compared to the isotype-matched negative control group (0% survival with rDENV-2D22 treatment) (Figs. 4D and 4G). These findings demonstrate that FluB-393 and FluB-400 provide in vivo protection against IBV infection and disease and suggest that these mAbs are more effective against Victoria lineage strains of the virus.

[0312] Intranasal administration of FluB-400 provided protection against IBV in a lethal model. Intranasal delivery of neuraminidase antibodies allows for multiple mechanisms of action at the respiratory interface: by absorption into the bloodstream to prevent viral shedding, and by blocking NA-mediated enzymatic cleavage of the virus from airway mucins. To evaluate the efficacy of surface-applied (IN) anti-NA mAb, the inventors established a treatment model for lethal IBV infection (B / New York / PV00081 / 18). In anesthetized mice, the virus was administered via the IN route 24 hours prior to either intraperitoneal injection (IP) or a single large-volume IN administration at a dose of 5 mg / kg. In the IP-treated group, FluB-393 and FluB-400 each protected 60% (n = 3 / 5) of the mice. The positive control mAb treatment group, administered with mAb r1G05, protected 40% (n = 2 / 5) of the mice. In contrast, none of the isotype-matched mice treated with rDENV-2D22 (n = 0 / 5) survived for more than 8 dpi. In the IN pathway treatment group, FluB-400 mediated 60% survival in treated mice, the same percentage of protection as IP. In contrast, FluB-393 and the isotype-matched negative control mAb rDENV-2D22 did not provide protection against (0% survival) lethal IBV challenge (Figure 5B). Further histopathological studies of FluB-400-treated mice via the IN pathway revealed lower detectable viral RNA signaling in lung tissue compared to the isotype-matched negative control group (Figure 5C). These findings suggest that the NA active site mAb (i.e., FluB-400) can treat IBV infection via IN pathway delivery.

[0313] The Fc-mediated effector function of NA mAbs contributes to the defense against IBV infection and disease. The Fc effector function of influenza-specific mAbs has been described in the literature, highlighting their ability to bind to Fc receptors and induce antibody-dependent cytotoxicity (ADCC) or other immune effector mechanisms (Vanderven & Kent, 2020). The inventors sought to determine whether the Fc-mediated effector function contributes to the defense against IBV infection mediated by FluB-393 and FluB-400 in vivo. To evaluate these effects, the inventors generated IgG Fc variant molecules targeting FluB-393 and FluB-400 with the LALA-PG mutation (L234A / L235A -P329G). The LALA-PG mutation eliminates binding to the Fcγ receptor and complement protein 1q (C1q) (Schlothauer et al., 2016), but wh indicated that variant IgGs targeting FluB-393 and FluB-400 maintained IBV NA reactivity (data not shown). To test the contribution of Fc-mediated function in vivo, FluB-393 or FluB-400 was administered via intraperitoneal injection at a dose of 5 mg / kg 24 hours prior to INB / New York / PV00081 / 18. Reduced potency of the LALA-PG variant was observed in both treatment groups. For the FluB-393 LALA-PG treatment group, 20% of mice survived compared to 60% of mice treated with FluB-393 wild-type FcIgG1. In the FluB-400 LALA-PG treatment group, no mice survived compared to the 60% survival rate of mice treated with FluB-400 wild-type Fc IgG1. Mouse weight was consistent with survival. The observed reduced in vivo potency of the LALA-PG Fc mutant antibody against NA suggests that Fc-mediated effector function contributes to in vivo resistance against IBV (data not shown).

[0314] Example 3 - Discussion

[0315] Here, the inventors transfer NA-specific human mAbs from individuals who have received Flucelvax. TM Antibody responses to IBV were investigated by isolating and phasing from healthy individuals vaccinated with the quadrivalent influenza vaccine. The inventors identified two clonal amplification families from this mAb group and selected representative mAbs from each family to further investigate the structure, function, and in vivo protection of IBV NA mAbs. These studies are essential for guiding universal influenza vaccination efforts and for developing alternative mAb delivery methods for treating influenza infections.

[0316] Previous literature has described IBV NA active site mAbs (Madsen et al., 2020); however, little is known about human NA stalk-binding mAbs. Additionally, the current understanding of the present inventors is based on the reported protective murine mAbs (Wohlbold et al., 2017). Due to this limitation, the present inventors used known IAV NA mAbs, which support the mechanisms of mAb-mediated ALI neutralization and IN delivery (Smet et al., 2022; Vigil et al., 2020; Limberis et al., 2013; Vonarburg et al., 2019). Based on this understanding, the present inventors applied these applications to evaluate the mechanisms of mAb-mediated IBV NA mAbs.

[0317] Other groups have found that influenza vaccination induces a plasma cell response specific for IBV NA (Piepenbrink et al., 2019). In the case of the blood samples used herein, the present inventors observed the IBV NA protein reactivity based on serum antibody responses detected by ELISA before and after vaccination. This finding indicates that the individual was previously exposed to IBV NA of unknown origin. These results suggest that the quadrivalent influenza vaccine can elicit a robust IBV NA B cell memory response, with the immunodominant response concentrated at the NA active site or stalk.

[0318] By ELISA, the clonally expanded NA active site family of mAbs (i.e., FluB-400) showed higher potency against recombinantly expressed IBV Victoria lineage (B / Iowa / 06 / 2017) NA protein compared to the tested IBV Yamagata strain (B / Singapore / 2016). Against the tested Victoria and Yamagata viruses, FluB-400 maintained functionality by inhibiting NA enzymatic activity, virus egress, and virus-induced cytopathic effects. Additionally, in an alternative air-liquid interface culture system of human respiratory epithelium, FluB-400 blocked early attachment events of IBV. In vivo efficacy (60% to 100%) against both IBV Victoria and Yamagata lineages was observed when FluB-400 was administered IN or IP.

[0319] In contrast, the mAb's NA horn-binding family (i.e., FluB-393) potently bound recombinantly expressed test IBV Victoria (B / Iowa / 06 / 2017) or Yamagata (B / Singapore / 2016) strains, as detected by ELISA. FluB-393 inhibited NA enzyme activity, and viral shedding was also observed for both IBV lineages. However, neutralization of the FluB-393-mediated virus-induced cytopathic effect was only observed in the test IBV Yamagata (B / New York / PV0094 / 2017) strain. Furthermore, FluB-393 did not prevent early IBV attachment events in alternative air-liquid interface culture systems for human respiratory epithelial cells. When FluB-393 was administered via IP instead of IN, in vivo potency (60% to 100%) against both IBV Victoria and Yamagata lineages was observed.

[0320] The FluB-393 clonal family of mAbs is encoded by IGHV4-39 and IGKV1-05. These genes also encode the previously described IBV NA-specific mAb, 2D10 (Madsen et al., 2020). Based on ELLA and NA-Fluor assays, 2D10 inhibits NA enzyme activity, which differs (?) from the FluB-393 data observed here.

[0321] Historically, mAbs have been administered via intravenous infusion or intramuscular injection. However, delivering mAbs directly to the respiratory mucosa offers two potential benefits: first, it delivers the mAb to the site of viral replication; and second, nebulization, rather than injection, is a less invasive procedure (Liang et al., 2020). Given the frequent presence of IBV infection in pediatric patients, less invasive delivery options such as nebulization are preferred (Zhao et al., 2021).

[0322] Compared to conventional cell cultures that are typically grown in a fully submerged environment, ALI cultures are often used to study cell behavior and function in a more realistic respiratory system setting (Si et al., 2021; Coles et al., 2020; Chen & Schoen, 2019; Riet et al., 2020; Huh et al., 2010; Wu et al., 2016). Once inhaled, IBV is bound to migrate across the thick mucus layer to reach the respiratory epithelium. However, IBV's migration is restricted due to the recognition of sialylated mucin by HA, which traps IBV in the mucosa. IBV can escape this restriction by cleaving sialylated mucin with NA, thereby releasing IBV and allowing it to migrate across the mucus layer (Yang et al., 2014; Vries et al., 2020). Therefore, inhibiting NA enzyme activity to help trap IBV in the respiratory mucosa is one reason why NA inhibitors have become an attractive class of drugs for delivery to the mucosal surface.

[0323] Table 1: Nucleotide sequence of antibody variable region

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341] Table 2: Protein sequences of antibody variable regions

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350] Table 3: Heavy Chain CDR Sequences

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] Table 4: Light Chain CDR Sequences

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] According to this disclosure, all compositions and methods disclosed and claimed herein can be performed and implemented without excessive experimentation. Although the compositions and methods of this disclosure have been described according to some preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the compositions and methods described herein, as well as the steps or order of steps, without departing from the concept, spirit, and scope of this disclosure. More specifically, it will be apparent that certain reagents, which are relevant in both chemical and physiological respects, can be substituted for the reagents described herein, while achieving the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0368] VII. References

[0369] The following references are incorporated herein by reference in particular for providing exemplary operations or other details that supplement those operations or details set forth herein.

[0370]

[0371]

[0372]

[0373]

Claims

1. Methods for detecting influenza B virus infection in subjects, including: (a) Contact a sample from the object with an antibody or antibody fragment having clone-paired heavy chain CDR sequences and light chain CDR sequences from Tables 3 and 4, respectively; as well as (b) Detecting influenza B virus in the sample by binding the antibody or antibody fragment to the influenza B virus antigen in the sample.

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

3. The method according to claims 1 to 2, wherein the sample is blood, sputum, tears, saliva, mucus or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, transudate, tissue scrapings or feces.

4. The method of claims 1 to 3, wherein the detection includes ELISA, RIA, lateral flow assay, or Western blot.

5. The method of claims 1 to 4, further comprising repeating steps (a) and (b) and determining the change in influenza B virus antigen levels compared to the first determination.

6. The method of claims 1 to 5, wherein the antibody or antibody fragment is encoded by the clonal pairing variable sequence shown in Table 1.

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

8. The method of claims 1 to 5, wherein the antibody or antibody fragment is encoded by a light chain and heavy chain variable sequence having 95% identity with the clone-paired sequences shown in Table 1.

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

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

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

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

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

14. The method of claim 13, wherein the antibody or antibody fragment is encoded by a clonal pair of light and heavy chain variable sequences shown in Table 1.

15. The method of claims 13 to 14, wherein the antibody or antibody fragment is encoded by a light chain and heavy chain variable sequence paired with a clone having 95% identity as shown in Table 1.

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

17. The method of claim 13, wherein the antibody or antibody fragment comprises a light chain and heavy chain variable sequence according to the clone-paired sequences from Table 2.

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

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

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

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

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

23. The method of claims 13 to 22, wherein the antibody or antibody fragment is administered before or after infection.

24. The method of claims 13 to 23, wherein the subject is a pregnant female, a sexually active female, or a female undergoing fertility treatment.

25. The method of claims 13 to 24, wherein delivery comprises administration of an antibody or antibody fragment, or genetic delivery using an RNA or DNA sequence or vector encoding said antibody or antibody fragment.

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

27. The monoclonal antibody of claim 26, wherein the antibody or antibody fragment is encoded by a variable sequence of light and heavy chains according to the clonal pairing sequences from Table 1.

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

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

30. The monoclonal antibody of claim 26, wherein the antibody or antibody fragment comprises a light chain and heavy chain variable sequence according to the cloning pairing sequences from Table 2.

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

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

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

34. The monoclonal antibody of claims 26 to 33, wherein the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc moiety, the Fc moiety being mutated to alter (eliminate or enhance) FcR interaction, increase half-life and / or increase therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE or LS mutations; or being modified with glycans to alter (eliminate or enhance) FcR interaction, such as enzymatic or chemical addition or removal of glycans or expression in a cell line modified with a defined glycosylation pattern.

35. The monoclonal antibody of claims 26 to 34, wherein the antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intracellular antibody.

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

37. The hybridoma or modified cell of claim 36, wherein the antibody or antibody fragment is encoded by a variable sequence of light and heavy chains according to the cloning pairing sequences from Table 1.

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

39. The hybridoma or modified cell of claim 36, wherein the antibody or antibody fragment is encoded by a light chain and a heavy chain variable sequence having 95% identity with the clone-paired variable sequence from Table 1.

40. The hybridoma or modified cell of claim 36, wherein the antibody or antibody fragment comprises a light chain and heavy chain variable sequence according to the cloning pairing sequence from Table 2.

41. The hybridoma or modified cell of claim 36, wherein the antibody or antibody fragment is encoded by a light chain and a heavy chain variable sequence having at least 70%, 80%, or 90% identity with the clone-paired variable sequences from Table 2.

42. The hybridoma or modified cell of claim 36, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity with the clone-paired sequences from Table 2.

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

44. The hybridoma or modified cell of claims 36 to 43, wherein the antibody is a chimeric antibody or a bispecific antibody.

45. The hybridoma or modified cell of claims 36 to 43, wherein the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc moiety, the Fc moiety being mutated to alter (eliminate or enhance) FcR interaction, increase half-life, and / or enhance therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or being modified with glycans to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition or removal of glycans or expression in a cell line modified with a defined glycosylation pattern.

46. ​​The hybridoma or modified cell of claims 36 to 45, wherein the antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intracellular antibody.

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

48. The vaccine formulation of claim 47, wherein at least one of the antibodies or antibody fragments is encoded by a light chain and heavy chain variable sequence according to the cloning pairing sequences from Table 1.

49. The vaccine formulation of claim 47, wherein at least one of the antibodies or antibody fragments is encoded by a light chain and a heavy chain variable sequence having at least 70%, 80%, or 90% identity with the clone-paired sequences from Table 1.

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

51. The vaccine formulation of claim 47, wherein at least one of the antibodies or antibody fragments comprises a light chain and a heavy chain variable sequence according to the cloning pairing sequences from Table 2.

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

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

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

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

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

57. A vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment, wherein the first antibody or antibody fragment is as described in claims 26 to 34.

58. The vaccine formulation of claim 57, wherein the expression vector is a Sindbis virus or a VEE vector.

59. The vaccine formulation of claims 57 to 58, wherein it is 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 a second antibody or antibody fragment, the second antibody or antibody fragment being, for example, the antibody or antibody fragments of different claims 26 to 34.

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

62. The method of claim 61, wherein the antibody or antibody fragment is encoded by a clonal pair of light and heavy chain variable sequences shown in Table 1.

63. The method of claims 61 to 62, wherein the antibody or antibody fragment is encoded by a light chain and heavy chain variable sequence paired with a clone having 95% identity as shown in Table 1.

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

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

66. The method of claim 61, wherein the antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80%, or 90% identity with the clone-paired sequences from Table 2.

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

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

69. The method of claims 61 to 68, wherein the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc moiety, the Fc moiety being mutated to alter (eliminate or enhance) FcR interaction, increase half-life, and / or enhance therapeutic efficacy, such as LALA, LALA-PG, N297, GASD / ALIE, DHS, YTE, or LS mutations; or modified with glycans to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition or removal of glycans or expression in a cell line modified with a defined glycosylation pattern.

70. The method of claims 61 to 67, wherein the antibody is a chimeric antibody or a bispecific antibody.

71. The method of claims 61 to 70, wherein the antibody or antibody fragment is administered before or after infection.

72. The method of claims 61 to 71, wherein the subject is less than 12 years of age or older than 60 years of age, is immunocompromised, or has an underlying condition that increases the risk of serious illness, hospitalization, or death due to viral infection.

73. The method of claims 61 to 72, wherein delivery comprises administration of an antibody or antibody fragment, or genetic delivery using an RNA or DNA sequence or vector encoding said antibody or antibody fragment.

74. The method of claim 61, wherein the antibody or antibody reduces the severity of the disease and / or hospitalization compared to an untreated control.

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

76. Methods for determining the antigenic integrity, correct conformation, and / or correct sequence of influenza B virus antigens, including: (a) Contacting a sample containing the antigen with a first antibody or antibody fragment having clone-paired heavy chain CDR sequences and light chain CDR sequences from Tables 3 and 4, respectively; and (b) The antigenic integrity, correct conformation and / or correct sequence of the antigen are determined by the detectable binding of the first antibody or antibody fragment to the antigen.

77. The method of claim 76, wherein the sample comprises a recombinant antigen.

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

79. The method of claims 76 to 78, wherein the detection comprises ELISA, RIA, Western blot, biosensors using surface plasmon resonance or biolayer interferometry, or flow cytometry staining.

80. The method of claims 76 to 79, wherein the first antibody or antibody fragment is encoded by the clonal pairing variable sequence shown in Table 1.

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

82. The method of claims 76 to 79, wherein the first antibody or antibody fragment is encoded by a light chain and heavy chain variable sequence having 95% identity with the clone-paired sequences shown in Table 1.

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

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

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

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

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

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

89. The method of claim 88, wherein the second antibody or antibody fragment is encoded by the clonal pairing variable sequence shown in Table 1.

90. The method of claim 89, wherein the second antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity with the clone-paired variable sequences shown in Table 1.

91. The method of claim 89, wherein the second antibody or antibody fragment is encoded by a light chain and heavy chain variable sequence having 95% identity with the clone-paired sequences shown in Table 1.

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

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

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

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

96. The method of claim 89, further comprising repeating steps (c) and (d) to determine the antigenic stability over time.

Images