Human monoclonal antibodies against enterovirus D68.

Abstract

The present disclosure relates to antibodies that bind to enterovirus D68 (EV-D68) and methods of use thereof.

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of priority to U.S. Provisional Application Nos. 62 / 878,955, 62 / 899,503, and 63 / 047,330, filed July 26, 2019, September 12, 2019, and July 2, 2020, respectively, the entire contents of each of which are incorporated herein by reference.

[0002] 1. Field of the Disclosure The present disclosure relates generally to the fields of medicine, infectious diseases, and immunology. More specifically, the present disclosure relates to human antibodies that bind to enterovirus D68, and methods of using such antibodies to diagnose and treat enterovirus D68 infection. [Background technology]

[0003] 2. Background Enterovirus-D68 (EV-D68) is a positive-sense, single-stranded RNA virus in the genus Enterovirus, family Picornaviridae. Other common human pathogens within this genus include poliovirus, echovirus, coxsackievirus, rhinovirus, and other numbered enteroviruses, such as enterovirus-A71 (Zell et al., 2017). These viruses are structurally and genetically similar but cause a wide variety of pediatric illnesses, including neonatal sepsis, myocarditis, poliomyelitis, meningitis, respiratory tract infections, and hand, foot, and mouth disease (HMD). Like rhinoviruses, EV-D68 is primarily an acid-labile respiratory pathogen that replicates best at 33°C (Oberste et al., 2004). These similarities are such that a strain of EV-D68 was even first reported as rhinovirus 87 (Blomqvist et al., 2002).

[0004] From its first identification in a child with pneumonia in California in 1962 (Schieble et al., 1967) through the turn of the century, EV-D68 was detected only rarely (Khetsuriani et al., 2006). Since then, EV-D68 has been recognized as an increasingly important pathogen due to its global detection in outbreaks of respiratory illness, primarily in children (Xiang and Wang, 2016). In the United States, the largest known outbreak to date occurred in 2014, with 1,152 confirmed cases across all states except Alaska (Oermann et al., 2015). This number likely significantly underestimates the actual number of EV-D68 cases in 2014, as mild upper respiratory tract infections are unlikely to result in the specialized testing required to detect this virus. Children with asthma experienced particularly severe infections (Biggs et al., 2017), although one center study found that EV-D68 was less severe than non-EV-D68 enterovirus / rhinovirus infections in asthmatic children (Overdahl et al., 2016). Over half of hospitalized patients were admitted to the intensive care unit, 80% received supplemental oxygen, 23% required noninvasive ventilation, 8% required intubation and mechanical ventilation, and 1% died (Midgley et al., 2015).

[0005] Concurrent with the 2014 outbreak, a small cluster of pediatric patients with acute-onset flaccid paralysis and cranial nerve dysfunction was identified in Colorado (Messacar et al., 2015). This syndrome, termed acute flaccid myelitis (AFM), is typically defined as a poliomyelitis-like illness with asymmetric flaccid limb weakness and primarily gray matter myelitis seen on spinal cord imaging (Division of Viral Diseases, CDC, 2018). Since the 2014 outbreak, many more cases and clusters of AFM have been linked to EV-D68. To date, 74 AFM cases from any patient source that tested positive for EV-D68 have been identified across six continents (Messacar et al., 2018). Although the US Centers for Disease Control and Prevention (CDC) has not officially recognized EV-D68 as a proven cause of AFM (Nat'l Center for Immun.Resp.Disease), many experts find the preponderance of evidence compelling enough to consider the relationship between the two to be causal (Messacar et al. 2018). As the global outbreak continues, the World Health Organization's Research and Development Blueprint now designates EV-D68 as a major public health risk (Ann.Rev.of Diseases, 2018).

[0006] Because EV-D68 has only recently emerged as a pathogen of major importance, most early studies focused on defining the biology of the virus rather than characterizing immune responses. Therefore, humoral immunity studies against EV-D68 are only just beginning. The role of serum antibodies in protection against other Enteroviruses varies. For example, three doses of inactivated poliovirus vaccine reach 100% induction of serum neutralizing antibodies and are 80–96% effective in preventing paralytic poliomyelitis, but vaccination fails to induce nasal or duodenal IgA (Sutter et al., 2018) and does not completely prevent intestinal or nasopharyngeal poliovirus shedding (Vidor et al., 2018). Studies of rhinovirus infection have shown that humoral immunity to certain serotypes of the virus cannot reliably protect against homotypic virus reinfection within several months (Howard et al., 2016). These differences in the degree of protection associated with antibody responses are likely due to the different sites of pathogenesis for these viruses, i.e., secondary neuronal spread after initial enteral infection with poliovirus, compared with localized respiratory tract infection with rhinovirus. Because EV-D68 infection can cause disease within the respiratory tract and is associated with disease in the central nervous system, the role of antibodies in protection and pathogenesis is likely complex. A better understanding of this role will aid in the development of vaccines and treatments for EV-D68 infection. Summary of the Invention

[0007] overview Thus, the present disclosure provides a method for detecting enterovirus D68 (EV-D68) infection in a subject, comprising: (a) contacting a sample obtained from the subject with an antibody or antibody fragment having a heavy chain CDR sequence and a light chain CDR sequence clone-paired with a clone in Table 3 and Table 4, respectively; and (b) detecting EV-D68 in the sample by binding the antibody or antibody fragment to an EV-D68 antigen in the sample. The sample can be a bodily fluid, such as blood, sputum, tears, saliva, mucous membranes or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissue, urine, exudates, filtrates, tissue scrapings, respiratory droplets or aerosols, or feces. Detection can include, for example, ELISA, RIA, lateral flow assay, Western blot, or the like. The method may further comprise performing steps (a) and (b) again and determining a change in EV-D68 antigen levels compared to the first assay.

[0008] The antibody or antibody fragment may be encoded by the variable sequences paired with the clones in Table 1, by light and heavy chain variable sequences that are 70%, 80%, or 90% identical to the variable sequences paired with the clones in Table 1, or by light and heavy chain variable sequences that are 95% identical to the sequences paired with the clones in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences as per the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences that are 70%, 80%, or 90% identical to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences that are 95% identical to the sequences paired with the clones in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment.

[0009] In another aspect, the disclosure provides a method of treating a subject infected with enterovirus D68 (EV-D68) or reducing the likelihood of infection in a subject at risk for EV-D68, comprising delivering to the subject an antibody or antibody fragment having heavy and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. The antibody or antibody fragment can be encoded by variable sequences paired with a clone in Table 1, by light and heavy chain variable sequences with 70%, 80%, or 90% identity to the variable sequences paired with a clone in Table 1, or by light and heavy chain variable sequences with 95% identity to the sequences paired with a clone in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences with 70%, 80% or 90% identity to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences with 95% identity to the sequences paired with the clones in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment or Fv fragment. The antibody includes, but is not limited to, IgG, or an Fc portion mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or to increase therapeutic effect, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, or Glycan Enzymatic or chemical addition or removal or expression in engineered cell lines with defined glycosylation patterns The antibody may be of any isotype, including recombinant IgG antibodies or antibody fragments that contain glycans modified to alter (eliminate or enhance) FcR interactions, such as: The antibody may be a chimeric antibody or a bispecific antibody.

[0010] The antibody or antibody fragment can be administered before or after infection, for example, less than about 7 days, less than about 5 days, less than about 3 days, less than about 2 days, or less than about 1 day after infection. The treatment can treat pulmonary and / or neurological infection, or the treatment reduces pulmonary and / or neurological infection. Delivery can include systemic administration of the antibody or antibody fragment, such as by aerosol delivery, or genetic delivery by an RNA sequence or DNA sequence or vector encoding the antibody or antibody fragment. The method can further include treating the subject with a second therapy, for example, an antiviral therapy or an anti-inflammatory therapy.

[0011] In yet another aspect, a monoclonal antibody is provided, wherein the antibody or antibody fragment is characterized by heavy chain and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. The antibody or antibody fragment can be encoded by a variable sequence paired with a clone in Table 1, by a light chain and a heavy chain variable sequence that are 70%, 80%, or 90% identical to the variable sequence paired with a clone in Table 1, or by a light chain and a heavy chain variable sequence that are 95% identical to the sequence paired with a clone in Table 1. The antibody or antibody fragment can comprise a light chain and a heavy chain variable sequence according to the sequence paired with a clone in Table 2, or can comprise a light chain and a heavy chain variable sequence that are 70%, 80%, or 90% identical to the sequence paired with a clone in Table 2, or can comprise a light chain and a heavy chain variable sequence that are 95% identical to the sequence paired with a clone in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. Antibodies include, but are not limited to, IgG, or an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or to increase therapeutic effect, such as LALA, N297, GASD / ALIE, YTE, DHS, or LS mutations, or Glycan Enzymatic or chemical addition or removal or expression in engineered cell lines with defined glycosylation patternsThe antibody or antibody fragment may be of any isotype, including recombinant IgG antibodies or antibody fragments containing modified glycans to alter (eliminate or enhance) FcR interaction, such as IgG antibodies or antibody fragments. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may further comprise a cell-penetrating peptide and / or be an intrabody.

[0012] A hybridoma or engineered cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by heavy and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. The hybridoma or engineered cell may encode an antibody or antibody fragment encoded by the variable sequences paired with a clone in Table 1, by light and heavy chain variable sequences that have 70%, 80%, or 90% identity to the variable sequences paired with a clone in Table 1, or by light and heavy chain variable sequences that have 95% identity to the sequences paired with a clone in Table 1. The hybridoma or engineered cell may comprise an antibody or antibody fragment comprising light and heavy chain variable sequences according to the sequences paired with the clones in Table 2, or comprising light and heavy chain variable sequences with 70%, 80% or 90% identity to the sequences paired with the clones in Table 2, or comprising light and heavy chain variable sequences with 95% identity to the sequences paired with the clones in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment or Fv fragment. The antibody may be an IgG, or may comprise an Fc portion mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or to increase therapeutic effect, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, or Glycan Enzymatic or chemical addition or removal or expression in engineered cell lines with defined glycosylation patternsThe antibody or antibody fragment may be a recombinant IgG antibody or antibody fragment that contains modified glycans to alter (eliminate or enhance) FcR interaction, such as: (I) a recombinant IgG antibody or antibody fragment that contains modified glycans to alter (eliminate or enhance) FcR interaction, such as (II ...

[0013] Also provided are vaccine formulations comprising one or more antibodies or antibody fragments characterized by heavy and light chain CDR sequences paired with the clones of Tables 3 and 4, respectively. The antibodies or antibody fragments can be encoded by the variable sequences paired with the clones listed in Table 1, by light and heavy chain variable sequences that have 70%, 80%, or 90% identity to the variable sequences paired with the clones listed in Table 1, or by light and heavy chain variable sequences that have 95% identity to the sequences paired with the clones listed in Table 1. The antibodies or antibody fragments can comprise light and heavy chain variable sequences as per the sequences paired with the clones of Table 2, or can comprise light and heavy chain variable sequences that have 70%, 80%, or 90% identity to the sequences paired with the clones of Table 2, or can comprise light and heavy chain variable sequences that have 95% identity to the sequences paired with the clones of Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment or an Fv fragment. The antibody may be an IgG, or may contain an Fc portion mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or to increase therapeutic effect, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, or Glycan Enzymatic or chemical addition or removal or expression in engineered cell lines with defined glycosylation patterns The antibody or antibody fragment may be a recombinant IgG antibody or antibody fragment that contains modified glycans to alter (eliminate or enhance) FcR interaction, such as: (I) a recombinant IgG antibody or antibody fragment that contains modified glycans to alter (eliminate or enhance) FcR interaction, such as (II ...

[0014] Yet another embodiment includes a vaccine formulation comprising one or more expression vectors encoding an antibody or antibody fragment characterized by heavy and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. The antibody or antibody fragment can be encoded by the variable sequences paired with the clone in Table 1, by light and heavy chain variable sequences having 70%, 80%, or 90% identity to the variable sequences paired with the clone in Table 1, or by light and heavy chain variable sequences having 95% identity to the sequences paired with the clone in Table 1. The antibody or antibody fragment can comprise light and heavy chain variable sequences as the sequences paired with the clone in Table 2, or can comprise light and heavy chain variable sequences having 70%, 80%, or 90% identity to the sequences paired with the clone in Table 2, or can comprise light and heavy chain variable sequences having 95% identity to the sequences paired with the clone in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment or an Fv fragment. The antibody may be an IgG, or may contain an Fc portion mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or to increase therapeutic effect, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, or Glycan Enzymatic or chemical addition or removal or expression in engineered cell lines with defined glycosylation patternsThe vaccine preparation may be a recombinant IgG antibody or antibody fragment containing modified glycans to alter (eliminate or enhance) FcR interaction, such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG15, IgG16, IgG17, IgG18, IgG19, IgG19, IgG19, IgG19, IgG18, IgG19, IgG19, IgG19, IgG20, IgG21, IgG21, IgG22, IgG23, IgG24, IgG25, IgG26, IgG27, IgG28, IgG29, IgG20, IgG21, IgG20, IgG21, IgG21, IgG22, IgG23, IgG24, IgG25, IgG26, IgG27, IgG28, IgG29, IgG30, IgG31, IgG32, IgG33, IgG34, IgG35, IgG45, IgG46, IgG47, IgG48, IgG49, IgG48, IgG49, IgG49, IgG41, IgG41, IgG42, IgG43, IgG44, IgG45, IgG45, IgG46, IgG47, IgG48, IgG49, IgG41, IgG41, IgG42, IgG43, IgG44, IgG45, IgG46, IgG47, IgG48, IgG49, IgG41, IgG The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences that have 70%, 80% or 90% identity to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences that have 95% identity to the sequences paired with the clones in Table 2.

[0015] Still further embodiments include methods of protecting the health of the placenta and / or fetus of a pregnant subject infected with or at risk of infection with enterovirus D68, comprising delivering to the subject an antibody or antibody fragment having heavy and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. The antibody or antibody fragment can be encoded by variable sequences paired with a clone in Table 1, by light and heavy chain variable sequences with 70%, 80%, or 90% identity to the variable sequences paired with a clone in Table 1, or by light and heavy chain variable sequences with 95% identity to the sequences paired with a clone in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences with 70%, 80% or 90% identity to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences with 95% identity to the sequences paired with the clones in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment or Fv fragment. The antibody may be an IgG, or may comprise an Fc portion mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or to increase therapeutic effect, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, or Glycan Enzymatic or chemical addition or removal or expression in engineered cell lines with defined glycosylation patterns The antibody or antibody fragment may be a recombinant IgG antibody or antibody fragment that contains modified glycans to alter (eliminate or enhance) FcR interaction, such as: (I) a recombinant IgG antibody or antibody fragment that contains modified glycans to alter (eliminate or enhance) FcR interaction, such as (II ...

[0016] The antibody or antibody fragment can be administered before or after infection, for example, less than about 7 days, less than about 5 days, less than about 3 days, less than about 2 days, or less than about 1 day after infection. The subject can be a pregnant woman, a sexually active woman, or a woman undergoing infertility treatment. Delivery can include administration of the antibody or antibody fragment, or genetic delivery by an RNA sequence, DNA sequence, or vector encoding the antibody or antibody fragment. The antibody or antibody fragment can increase placental size compared to an untreated control. The antibody or antibody fragment can reduce fetal viral load and / or pathology compared to an untreated control.

[0017] In yet another embodiment, a method for determining the antigenic integrity, precise conformation, and / or precise sequence of an enterovirus D68 antigen is provided, comprising: (a) contacting a sample containing the antigen with a first antibody or antibody fragment having a heavy chain CDR sequence and a light chain CDR sequence paired with a clone in Table 3 and Table 4, respectively; and (b) determining the antigenic integrity, precise conformation, and / or precise sequence of the antigen by detectable binding of the first antibody or antibody fragment to the antigen. The sample may contain a recombinantly produced antigen or may contain a vaccine formulation or vaccine production batch. Detection may include ELISA, RIA, Western blot, biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometry staining.

[0018] The first antibody or antibody fragment is encoded by the variable sequences paired with the clones listed in Table 1, by light chain and heavy chain variable sequences that have 70%, 80%, or 90% identity to the variable sequences paired with the clones listed in Table 1, or by light chain and heavy chain variable sequences that have 95% identity to the sequences paired with the clones listed in Table 1. The first antibody or antibody fragment may comprise light chain and heavy chain variable sequences as the sequences paired with the clones in Table 2, or may comprise light chain and heavy chain variable sequences that have 70%, 80%, or 90% identity to the sequences paired with the clones in Table 2, or may comprise light chain and heavy chain variable sequences that have 95% identity to the sequences paired with the clones in Table 2. The first antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The method may further comprise performing steps (a) and (b) again to determine the antigenic stability of the antigen over time.

[0019] The method may further include (c) contacting a sample containing the antigen with a second antibody or antibody fragment having heavy and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively, and (d) determining the antigen integrity of the antigen by detectable binding of the second antibody or antibody fragment to the antigen. The second antibody or antibody fragment is encoded by the variable sequences paired with the clone in Table 1, by light and heavy chain variable sequences having 70%, 80%, or 90% identity to the variable sequences paired with the clone in Table 1, or by light and heavy chain variable sequences having 95% identity to the sequences paired with the clone in Table 1. The second antibody or antibody fragment may comprise light and heavy chain variable sequences according to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences with 70%, 80%, or 90% identity to the sequences paired with the clones in Table 2, or may comprise light and heavy chain variable sequences with 95% identity to the sequences paired with the clones in Table 2. The second antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. The method may include performing steps (c) and (d) again to determine the antigenic stability of the antigen over time.

[0020] Also provided is a human monoclonal antibody or antibody fragment, or a hybridoma or engineered cell producing same, which binds to EV-D68 across at least two viral clades.

[0021] Additional embodiments include human monoclonal antibodies or antibody fragments, or hybridomas or engineered cells producing same, which bind to EV-D68 VP1, VP2, and VP3. The antibody may bind to the EV-D68 VP1 DE loop, and / or the EV-D68 VP2 EE loop, and / or the EV-D68 VP3 N-terminal loop.

[0022] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The word "about" means ±5% of the stated number.

[0023] [The present invention 1001] A method of treating a subject infected with enterovirus D68 (EV-D68) or reducing the likelihood of infection in a subject at risk of contracting EV-D68, comprising delivering to the subject an antibody or antibody fragment having heavy chain and light chain CDR sequences clone-paired with a clone in Table 3 and Table 4, respectively. [The present invention 1002] 1001. The method of claim 1001, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence paired with a clone as set forth in Table 1. [The present invention 1003] The method of any of claims 1001 to 1002, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence paired with a clone having 95% identity to those listed in Table 1. [The present invention 1004] The method of any of claims 1001 to 1002, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80%, or 90% identity to the sequences paired with the clones of Table 1. [The present invention 1005] 1001. The method of claim 1001, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the sequences paired with the clones of Table 2. [The present invention 1006] 1001. The method of claim 1001, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to the sequences paired with the clones of Table 2. [The present invention 1007] 1001. The method of claim 1001, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [The present invention 1008] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The method of any of claims 1001 to 1007, wherein the antibody is a chimeric antibody or a bispecific antibody. [The present invention 1009] The antibody IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, to increase half-life and / or to increase therapeutic effect, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as by enzymatic or chemical addition or removal of glycans, or expression in an engineered cell line with a defined glycosylation pattern; Any of the methods 1001 to 1008 of the present invention. [The present invention 1010] The method of any of claims 1001 to 1009, further comprising the step of treating said subject with a second therapy, eg, an antiviral therapy or an anti-inflammatory therapy. [The present invention 1011] The method of any of claims 1001 to 1010, wherein said antibody or antibody fragment is administered before or after infection. [The present invention 1012] The method of any of claims 1001 to 1011, wherein said treating treats a pulmonary infection and / or a neurological infection, or wherein said treating reduces a pulmonary infection and / or a neurological infection. [The present invention 1013] The method of any of claims 1001 to 1012, wherein the delivering step comprises administration of an antibody or antibody fragment, or genetic delivery by an RNA sequence or DNA sequence or vector encoding the antibody or antibody fragment. [The present invention 1014] 1. A method for detecting enterovirus D68 (EV-D68) infection in a subject, the method comprising the steps of: (a) contacting a sample obtained from the subject with an antibody or antibody fragment having heavy chain and light chain CDR sequences paired with a clone of Table 3 and Table 4, respectively; and (b) detecting EV-D68 in the sample by binding the antibody or antibody fragment to an EV-D68 antigen in the sample. [The present invention 1015] 1015. The method of claim 1014, wherein the sample is a body fluid. [The present invention 1016] 1014. The method of claim 1014, wherein said sample is blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, filtrate, respiratory droplets or aerosol, tissue scraping, or feces. [The present invention 1017] 17. The method of any of claims 1014 to 1016, wherein detection comprises ELISA, RIA, lateral flow assay, or Western blot. [The present invention 1018] 18. The method of any of claims 1014 to 1017, further comprising the step of performing steps (a) and (b) again and determining a change in EV-D68 antigen levels compared to the first assay. [The present invention 1019] 19. The method of any of claims 1014 to 1018, wherein the antibody or antibody fragment is encoded by a variable sequence paired with a clone described in Table 1. [The present invention 1020] Any of the methods of claims 1014 to 1018, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that have 70%, 80%, or 90% identity to the variable sequences paired with the clones listed in Table 1. [The present invention 1021] 19. The method of any of claims 1014 to 1018, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones listed in Table 1. [The present invention 1022] 19. The method of any of claims 1014 to 1018, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the sequences paired with the clones in Table 2. [The present invention 1023] 19. The method of any of claims 1014 to 1018, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to the sequences paired with the clones of Table 2. [The present invention 1024] 19. The method of any of claims 1014 to 1018, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [The present invention 1025] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The method of any one of claims 1014 to 1024, wherein the antibody is a fragment or an Fv fragment. [The present invention 1026] A monoclonal antibody, wherein the antibody or antibody fragment is characterized by heavy chain CDR sequences and light chain CDR sequences paired with the clones of Table 3 and Table 4, respectively. [The present invention 1027] The monoclonal antibody of the present invention 1026, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence according to the clone-paired sequences of Table 1. [The present invention 1028] 1026. The monoclonal antibody of the present invention, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 70%, 80% or 90% identity to the sequences paired with the clones of Table 1. [The present invention 1029] 1026. The monoclonal antibody of the invention, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 95% identity to the sequences paired with the clones of Table 1. [The present invention 1030] The monoclonal antibody of the present invention 1026, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone-paired sequences of Table 2. [The present invention 1031] 1026. The monoclonal antibody of the present invention, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [The present invention 1032] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The monoclonal antibody of any one of the monoclonal antibodies 1026 to 1031 of the present invention, which is a fragment or Fv fragment. [The present invention 1033] The monoclonal antibody of any one of claims 1026 to 1031, wherein the antibody is a chimeric antibody or a bispecific antibody. [The present invention 1034] The antibody IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, to increase half-life and / or to increase therapeutic effect, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as by enzymatic or chemical addition or removal of glycans, or expression in an engineered cell line with a defined glycosylation pattern; The monoclonal antibody of any one of 1026 to 1033 of the present invention. [This invention 1035] The monoclonal antibody of any one of 1026 to 1034, wherein the antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intrabody. [The present invention 1036] A hybridoma or engineered cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by heavy chain and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. [This invention 1037] 1036. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence according to the clone-paired sequences of Table 1. [The present invention 1038] 1036. The hybridoma or engineered cell of the present invention, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 70%, 80%, or 90% identity to the variable sequences paired with the clones of Table 1. [This invention 1039] 1036. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to the variable sequences paired with the clones of Table 1. [The present invention 1040] 1036. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the clone-paired sequences of Table 2. [This invention 1041] 1036. The hybridoma or engineered cell of the present invention, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 70%, 80%, or 90% identity to the variable sequences paired with the clones of Table 2. [The present invention 1042] 1036. The hybridoma or engineered cell of the present invention, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [This invention 1043] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The hybridoma or engineered cell of any one of claims 1036 to 1042, which is a fragment or Fv fragment. [This invention 1044] The hybridoma or engineered cell of any of claims 1036 to 1043, wherein the antibody is a chimeric antibody or a bispecific antibody. [This invention 1045] The antibody IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, to increase half-life and / or to increase therapeutic effect, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or expression in an engineered cell line with a defined glycosylation pattern; The hybridoma or engineered cell of any one of 1036 to 1043 of the present invention. [The present invention 1046] 1046. The hybridoma or engineered cell of any one of claims 1036 to 1045, wherein said antibody or antibody fragment further comprises a cell-penetrating peptide and / or is an intrabody. [This invention 1047] A vaccine formulation comprising one or more antibodies or antibody fragments characterized by heavy chain and light chain CDR sequences paired with the clones of Tables 3 and 4, respectively. [This invention 1048] 1047. The vaccine formulation of the invention, wherein at least one of said antibodies or antibody fragments is encoded by a light chain variable sequence and a heavy chain variable sequence according to the clone-paired sequences of Table 1. [This invention 1049] 1047. The vaccine formulation of the present invention, wherein at least one of said antibodies or antibody fragments is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 70%, 80% or 90% identity to the sequences paired with the clones of Table 1. [The present invention 1050] 1047. The vaccine formulation of the present invention, wherein at least one of said antibodies or antibody fragments is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 95% identity to the sequences paired with the clones of Table 1. [This invention 1051] 1047. The vaccine formulation of the invention, wherein at least one of said antibodies or antibody fragments comprises a light chain variable sequence and a heavy chain variable sequence according to the clone-paired sequences of Table 2. [This invention 1052] 1047. The vaccine formulation of the invention, wherein at least one of said antibodies or antibody fragments comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [This invention 1053] At least one of the antibody fragments is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The vaccine preparation of any one of claims 1047 to 1052, which is a fragment or Fv fragment. [This invention 1054] The vaccine preparation of any of claims 1047 to 1052, wherein at least one of the antibodies is a chimeric antibody or a bispecific antibody. [This invention 1055] The antibody IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, to increase half-life and / or to increase therapeutic effect, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or expression in an engineered cell line with a defined glycosylation pattern; A vaccine preparation according to any one of claims 1047 to 1054 of the present invention. [This invention 1056] The vaccine formulation of any of claims 1047 to 1055, wherein at least one of said antibodies or antibody fragments further comprises a cell-penetrating peptide and / or is an intrabody. [This invention 1057] A vaccine formulation comprising one or more expression vectors encoding the first antibody or antibody fragment of any of 1026 to 1034 of the present invention. [This invention 1058] 1057. The vaccine formulation of the present invention, wherein said expression vector is a Sindbis virus vector or a VEE vector. [This invention 1059] 9. The vaccine formulation of any of claims 1057 to 1058, formulated for delivery by needle injection, jet injection, or electroporation. [The present invention 1060] The vaccine formulation of claim 1057, further comprising one or more expression vectors encoding a second antibody or antibody fragment, eg, a distinct antibody or antibody fragment of any of claims 1026 to 1034. [This invention 1061] A method for protecting the health of the placenta and / or fetus of a pregnant subject infected with or at risk of being infected with enterovirus D68, comprising delivering to the subject an antibody or antibody fragment having heavy chain and light chain CDR sequences paired with a clone in Table 3 and Table 4, respectively. [This invention 1062] 1062. The method of claim 1061, wherein said antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence paired with a clone as set forth in Table 1. [This invention 1063] 1063. The method of any of claims 1061 to 1062, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence paired with a clone having 95% identity to those listed in Table 1. [This invention 1064] Any of the methods of claims 1061 to 1062, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to the sequences paired with the clones in Table 1. [This invention 1065] 1062. The method of claim 1061, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the sequences paired with the clones of Table 2. [The present invention 1066] 1061. The method of claim 1061, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 70%, 80% or 90% identity to the sequences paired with the clones of Table 2. [This invention 1067] 1061. The method of claim 1061, wherein said antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [The present invention 1068] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The method of any one of claims 1061 to 1067, wherein the antibody is a fragment or an Fv fragment. [The present invention 1069] The antibody IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations, to increase half-life and / or to increase therapeutic effect, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or expression in an engineered cell line with a defined glycosylation pattern; Any of the methods of the present invention 1061 to 1068. [The present invention 1070] The method of any of claims 1061 to 1067, wherein the antibody is a chimeric antibody or a bispecific antibody. [This invention 1071] The method of any of claims 1061 to 1070, wherein said antibody or antibody fragment is administered before or after infection. [This invention 1072] 1072. The method of any of claims 1061 to 1071, wherein said subject is a pregnant woman, a sexually active woman, or a woman undergoing infertility treatment. [This invention 1073] 1073. The method of any of claims 1061 to 1072, wherein the delivery comprises administration of an antibody or antibody fragment, or genetic delivery by an RNA sequence or DNA sequence or vector encoding the antibody or antibody fragment. [This invention 1074] The method of claim 1061, wherein the antibody or antibody fragment increases placental size relative to an untreated control. [This invention 1075] The method of claim 1061, wherein the antibody or antibody fragment reduces viral load and / or pathology in the fetus compared to an untreated control. [This invention 1076] 1. A method for determining the antigenic integrity, the correct conformation and / or the correct sequence of an enterovirus D68 antigen, comprising the steps of: (a) contacting a sample containing the antigen with a first antibody or antibody fragment having heavy chain and light chain CDR sequences paired with clones in Tables 3 and 4, respectively; and (b) determining the antigenic integrity, correct conformation and / or correct sequence of said antigen by detectable binding of said first antibody or antibody fragment to said antigen. [This invention 1077] 1076. The method of claim 1076, wherein said sample comprises a recombinantly produced antigen. [This invention 1078] 1076. The method of claim 1076, wherein said sample comprises a vaccine formulation or a vaccine production batch. [This invention 1079] 1079. The method of any of claims 1076 to 1078, wherein detection comprises ELISA, RIA, Western blot, biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometry staining. [The present invention 1080] 1079. The method of any of claims 1076 to 1079, wherein said first antibody or antibody fragment is encoded by a variable sequence paired with a clone listed in Table 1. [This invention 1081] 1076. The method of any of claims 1076 to 1079, wherein said first antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80%, or 90% identity to the variable sequences paired with the clones listed in Table 1. [This invention 1082] 1076. The method of any of claims 1076 to 1079, wherein said first antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones listed in Table 1. [This invention 1083] 1079. The method of any of claims 1076 to 1079, wherein said first antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the sequences paired with the clones in Table 2. [This invention 1084] 1076. The method of any of claims 1076 to 1079, wherein said first antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 70%, 80%, or 90% identity to the sequences paired with the clones in Table 2. [This invention 1085] 1079. The method of any of claims 1076 to 1079, wherein said first antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [The present invention 1086] The first antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The method of any one of claims 1076 to 1085, wherein the antibody is a fragment or an Fv fragment. [This invention 1087] The method of any of claims 1076 to 1086, further comprising the step of performing steps (a) and (b) again to determine the antigen stability of said antigen over time. [This invention 1088] Any of the methods of claims 1076 to 1087, further comprising the steps of: (c) contacting the sample containing the antigen with a second antibody or antibody fragment having heavy chain and light chain CDR sequences paired with the clones of Tables 3 and 4, respectively; and (d) determining the antigenic integrity of said antigen by detectable binding of said second antibody or antibody fragment to said antigen. [This invention 1089] The method of claim 1088, wherein said second antibody or antibody fragment is encoded by a variable sequence paired with a clone listed in Table 1. [The present invention 1090] 1089. The method of claim 1089, wherein said second antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 70%, 80%, or 90% identity to the variable sequences paired with the clones listed in Table 1. [This invention 1091] 1089. The method of claim 1089, wherein said second antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones listed in Table 1. [This invention 1092] 1089. The method of claim 1089, wherein said second antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence according to the sequences paired with the clones of Table 2. [This invention 1093] 1089. The method of claim 1089, wherein said second antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 70%, 80%, or 90% identity to the sequences paired with the clones of Table 2. [This invention 1094] 1089. The method of claim 1089, wherein said second antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to the sequences paired with the clones of Table 2. [This invention 1095] The second antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 1089. The method of claim 1089, wherein the antibody is a fragment or an Fv fragment. [This invention 1096] 1089. The method of claim 1089, further comprising the step of performing steps (c) and (d) again to determine the antigenic stability of said antigen over time. [This invention 1097] A human monoclonal antibody or antibody fragment, or a hybridoma or engineered cell producing the same, wherein the antibody binds to EV-D68 across at least two viral clades. [This invention 1098] A human monoclonal antibody or antibody fragment, or a hybridoma or engineered cell that produces the same, wherein the antibody binds to EV-D68 VP1, VP2 and VP3. [This invention 1099] 1098. A human monoclonal antibody or antibody fragment of the present invention, or a hybridoma or engineered cell producing the same, wherein the antibody binds to the EV-D68 VP1 DE loop. [The present invention 1100] 1098. A human monoclonal antibody or antibody fragment of the present invention, or a hybridoma or engineered cell producing the same, wherein the antibody binds to the EV-D68 VP2 EE loop. [The present invention 1101] 1098. A human monoclonal antibody or antibody fragment of the present invention, or a hybridoma or engineered cell producing the same, wherein the antibody binds to the EV-D68 VP3 N-terminal loop. [The present invention 1102] A human monoclonal antibody or antibody fragment of the present invention, or a hybridoma or engineered cell producing the same, wherein the antibody binds to (a) the EV-D68 VP3 N-terminal loop and the EV-D68 VP2 EE loop, or (b) the EV-D68 VP2 EE loop and the EV-D68 VP1 DE loop, or (c) the EV-D68 VP1 DE loop and the EV-D68 VP3 N-terminal loop, or (d) the EV-D68 VP3 N-terminal loop, the EV-D68 VP1 DE loop, and the EV-D68 VP2 EE loop. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] [Figure 1] Figure 1: Antibody isolation. Sixty-four human monoclonal antibodies were isolated and screened using an indirect ELISA in which live virus isolates from the 2014 EV-D68 outbreak were directly coated onto ELISA plates. Any hits were then fused with myeloma cells to create hybridomas that would theoretically continue to divide indefinitely. After the antibodies were purified, several in vitro characterization steps were performed to define their phenotype. [Figure 2] Figure 2: In vitro antibody neutralization. In vitro neutralization is performed using the cell culture 50% infectious dose, or CCID50, assay. Some antibodies are very potent, some are still very potent, and others are weak, with approximately 1 / 3 of the mAbs showing some detectable neutralizing activity. [Figure 3-1]Figure 3: Cross-reactive binding between clades. To compare across all mAbs, a heatmap was generated using the 50% effective concentrations, or EC50s, from the ELISA. Some bind very well to all clades, while some have significant dropouts, typically in the distant A2 clade. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4-1] Figure 4: Competition reveals at least four major surface epitopes. We used Pearson correlation to generate a relatedness value as a colored readout to the clusters. This relatedness is useful for sorting antibodies, but it is not an actual interference percentage. We have now described three to four phenotypes that may be related to each other. This allows for the identification of candidates for clinical development. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 5] Figure 5: Clinical Candidates. In addition to neutralization, additional characterization revealed additional clinical candidates. mAb 159 is highly potent and does not bind to mock preparations of virus, yet does not cross-react by ELISA or CCID50. mAb 105 is still highly potent, but cross-reacts very well. [Figure 6]Figure 6A-C: Experiment NIA-1849. Lung virus titers in EV-D68-infected AG129 mice treated with EV-D68-228. Treatment with EV-D68-228 significantly reduced lung virus titers on days 1 (Figure 6A), 3 (Figure 6B), and 5 (Figure 6C) postinfection. In mice treated with 10, 3, or 1 mg / kg of EV-D68-228, lung virus titers were not detected on days 1, 3, or 5 postinfection. Treatment with IVIg was only able to reduce lung virus titers on day 1 postinfection. Guanidine significantly reduced lung virus titers on days 1 and 3 postinfection, but not on day 5. **P<0.01, ****P<0.0001 compared to placebo-treated mice. [Figure 7] Figures 7A-C: Experiment NIA-1849. Blood viral titers in EV-D68-infected AG129 mice treated with EV-D68-228. Treatment with EV-D68-228 mAb reduced blood viral titers on days 1 (Figure 7A), 3 (Figure 7B), and 5 (Figure 7C) post-infection. Treatment with IVIg significantly reduced blood viral titers on days 1, 3, and 5 post-infection. Guanidine reduced blood viral titers on days 1 and 5 post-infection, but not on day 3. **P<0.01, ***P<0.001, ****P<0.0001 compared with placebo-treated mice. [Figure 8] Figure 8: Experiment NIA-1849. Lung concentrations of IL-1α and IL-1β from EV-D68-infected AG129 mice treated with EV-D68-228. Treatment with EV-D68-228 significantly reduced IL-1α and IL-1β concentrations on day 3 post-infection compared to placebo-treated mice. Treatment with IVIg or guanidine did not significantly reduce lung concentrations of IL-1α or IL-1β. ****P<0.0001 compared to placebo-treated mice. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 9]Figure 9: Experiment NIA-1849. Lung concentrations of IL-6 and MCP-1 from EV-D68-infected AG129 mice treated with EV-D68-228. Treatment with EV-D68-228 significantly reduced IL-6 and MCP-1 concentrations on day 3 post-infection compared to placebo-treated mice. Treatment with IVIg or guanidine significantly reduced lung concentrations of MCP-1 on day 3 post-infection compared to placebo-treated mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 10] Figure 10: Experiment NIA-1849. Lung concentrations of RANTES from EV-D68-infected AG129 mice treated with EV-D68-228. Treatment with EV-D68-228 significantly reduced RANTES concentrations on days 3 and 5 post-infection compared to placebo-treated mice. Treatment with IVIg or guanidine did not significantly reduce lung concentrations of RANTES on any day post-infection. **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 11] Figure 11: Experiment NIA-1849. Lung concentrations of IL-2, IL-3, and IL-4 from EV-D68-infected AG129 mice treated with EV-D68-228. No significant changes in IL-2, IL-3, or IL-4 concentrations were observed after infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 12] Figure 12: Experiment NIA-1849. Lung concentrations of IL-5, IL-10, and IL-12p70 from EV-D68-infected AG129 mice treated with EV-D68-228. No significant changes in IL-5, IL-10, or IL-12p70 concentrations were observed after infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 13]Figure 13: Experiment NIA-1849. Lung concentrations of IL-17, IFNγ, and TNFα from EV-D68-infected AG129 mice treated with EV-D68-228. No significant changes in IL-17, IFNγ, or TNFα concentrations were observed after infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 14] Figure 14: Experiment NIA-1849. Lung concentrations of MIP-1α and GM-CSF from EV-D68-infected AG129 mice treated with EV-D68-228. No significant changes in MIP-1α or GM-CSF concentrations were observed after infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 15] Figure 15: Experiment NIA-1850. Survival rate of 10-day-old AG129 mice infected with EV-D68 and treated with EV-D68-228. (n=6 mice / group). Treatment with EV-D68-228 at doses of 10, 3, or 1 mg / kg completely protected the mice from death. IVIg at a dose of 10 mg / kg protected 4 out of 6 mice from death. Guanidine at a dose of 100 mg / kg / day completely protected the mice from death. All 6 placebo-treated mice died from infection. *P<0.05, **P<0.01 compared with placebo-treated mice. [Figure 16] Figure 16: Experiment NIA-1850. Mean body weight of EV-D68-infected 10-day-old AG129 mice treated with EV-D68-228. Treatment with doses of 10, 3, or 1 mg / kg protected mice from infection-associated weight loss. Treatment with IVIg at a dose of 10 mg / kg also protected mice from weight loss. Mice treated with guanidine at a dose of 100 mg / kg / day were also protected from weight loss. *P<0.05, ***P<0.001, ****P<0.0001 compared with placebo-treated mice. [Figure 17]Figures 17A-C: Experiment NIA-1850. Blood viral titers in 10-day-old AG129 mice infected with EV-D68 and treated with EV-D68-228. Mice treated with EV-D68-228 at doses of 10, 3, or 1 mg / kg had no detectable blood viral titers on days 1 (Figure 17A), 3 (Figure 17B), or 5 (Figure 17C) post-infection. Treatment with IVIg at a dose of 10 mg / kg significantly reduced blood viral titers on days 1, 3, and 5 post-infection. Treatment with guanidine at a dose of 100 mg / kg / day also significantly reduced blood viral titers on days 1, 3, and 5 post-infection. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with placebo-treated mice. [Figure 18] Figure 18: Experiment NIA-1850. Neurological scores of 10-day-old AG129 mice infected with EV-D68 and treated with EV-D68-228. Treatment with EV-D68-228 at doses of 10, 3, or 1 mg / kg prevented clinical signs of paralysis, as measured by neurological score. Treatment with IVIg at a dose of 10 mg / kg reduced neurological scores on days 3, 4, and 5 post-infection. No neurological scores were observed in mice treated with 100 mg / kg / day of guanidine. **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 19]Figures 19A-B: Experiment NIA-1869. Lung viral titers in EV-D68-infected AG129 mice treated post-infection with EV-D68-228. Treatment with EV-D68-228 significantly reduced lung viral titers at days 3 (Figure 19A) and 5 (Figure 19B) post-infection. In mice treated with 10 mg / kg EV-D68-228, lung viral titers were not detected within 24 hours post-infection. Treatment with EV-D68-228 at doses of 10 and 1 mg / kg at 48 hours post-infection significantly reduced lung viral titers at days 3 and 5 post-infection. Treatment with IVIg did not reduce lung viral titers when treated 24 hours post-infection. (****P<0.0001 compared with placebo-treated mice). [Figure 20] Figures 20A-B: Experiment NIA-1869. Blood viral titers in EV-D68-infected AG129 mice treated post-infection with EV-D68-228. Treatment with EV-D68-228 mAb reduced blood viral titers at days 3 (Figure 20A) and 5 (Figure 20B) post-infection. Treatment with EV-D68-228 up to 48 hours post-infection reduced blood viral titers to below the limit of detection at days 3 and 5 post-infection. Treatment with IVIg significantly reduced blood viral titers at days 3 and 5 post-infection. (*P<0.05, ****P<0.0001 compared with placebo-treated mice). [Figure 21] Figure 21: Experiment NIA-1869. Lung concentrations of IL-1α and IL-1β from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. Treatment with EV-D68-228 significantly reduced IL-1α and IL-1β concentrations on days 3 and 5 post-infection compared to placebo-treated mice. Treatment with IVIg significantly reduced lung concentrations of IL-1α or IL-1β only on days 3 and 5 post-infection. (*P<0.05, **P<0.01, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 22]Figure 22: Experiment NIA-1869. Lung concentrations of IL-5 and IL-6 from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. Treatment with EV-D68-228 up to 48 hours post-infection significantly reduced IL-5 and IL-6 concentrations at day 3 post-infection compared to placebo-treated mice. Treatment with IVIg 24 hours post-infection significantly reduced lung concentrations of IL-5 and IL-6 at day 3 post-infection. (**P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 23] Figure 23: Experiment NIA-1849. Lung concentrations of MCP-1 and RANTES from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. Treatment with EV-D68-228 within 48 hours of infection significantly reduced MCP-1 and RANTES concentrations on days 3 and 5 post-infection compared to placebo-treated mice. Treatment with IVIg 24 hours post-infection significantly reduced lung concentrations of MCP-1 on day 3 and RANTES on days 3 and 5 post-infection. (**P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 24] Figure 24: Experiment NIA-1869. Lung concentrations of IL-2, IL-3, and IL-4 from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. No significant changes in IL-2, IL-3, or IL-4 concentrations were observed following infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 25]Figure 25: Experiment NIA-1869. Lung concentrations of IL-10, IL-12p70, and IL-17 from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. No significant changes in IL-5, IL-10, or IL-12p70 concentrations were observed following infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 26] Figure 26: Experiment NIA-1869. Lung concentrations of IFNγ and TNFα from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. No significant changes in IFNγ or TNFα concentrations were observed following infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 27] Figure 27: Experiment NIA-1869. Lung concentrations of MIP-1α and GM-CSF from EV-D68-infected AG129 mice treated post-infection with EV-D68-228. No significant changes in MIP-1α or GM-CSF concentrations were observed after infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 28]Figure 28: Experiment NIA-1870. Survival rate of 10-day-old AG129 mice infected with EV-D68 and treated post-infection with EV-D68-228. (n = 6 mice / group, n = 7 mice / group treated 120 hours post-infection). Treatment with EV-D68-228 at a dose of 10 mg / kg completely protected mice from death at 24 hours post-infection. Treatment with EV-D68-228 at 48 hours post-infection protected 4 of 6 mice from death. Only 1 of 6 mice treated with EV-D68-228 72 hours post-infection survived the infection. Although none of the 7 mice in the group treated 120 hours post-infection with EV-D68-228 survived, the survival curve differed from placebo-treated mice due to a delayed day of death. IVIg at a dose of 10 mg / kg protected all six mice from death when treated 24 hours after infection. All six placebo-treated mice died from infection (*P<0.05, **P<0.01, ***P<0.001 compared with placebo-treated mice). [Figure 29] Figure 29: Experiment NIA-1870. Mean body weight of EV-D68-infected 10-day-old AG129 mice treated post-infection with EV-D68-228. Treatment with EV-D68-228 at a dose of 10 mg / kg 24 or 48 hours post-infection protected mice from infection-associated weight loss. Treatment with IVIg at a dose of 10 mg / kg 24 hours post-infection did not protect mice from weight loss. (*P<0.05, **P<0.01 compared with placebo-treated mice). [Figure 30]Figures 30A-C: Experiment NIA-1870. Blood virus titers of 10-day-old AG129 mice infected with EV-D68 and treated post-infection with EV-D68-228. Blood virus titers are shown on days 1 (Figure 30A), 3 (Figure 30B), or 5 (Figure 30C) post-infection. Treatment with EV-D68-228 at a dose of 10 mg / kg significantly reduced blood virus titers on days 3 and 5 post-infection when administered 24, 48, or 72 hours post-infection. IVIg at a dose of 10 mg / kg administered 24 hours post-infection also reduced blood virus titers on days 3 and 5 post-infection. (*P<0.05, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). [Figure 31] Figures 31A-B: Experiment NIA-1870. Neurological scores of 10-day-old AG129 mice infected with EV-D68 and treated post-infection with EV-D68-228. Neurological scores are shown on days 2 and 3 post-infection (Figure 31A) and days 4 and 5 post-infection (Figure 31B). Treatment with EV-D68-228 at a dose of 10 mg / kg 24 hours post-infection prevented clinical signs of paralysis on days 3, 4, and 5 post-infection, as measured by neurological score. Treatment with IVIg at a dose of 10 mg / kg 24 hours post-infection reduced neurological scores on days 3, 4, and 5 post-infection (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). Sample names in the graphs correspond from left to right with a top-to-bottom vertical description. [Figure 32-1] Figure 32: Competitive binding groups of mAbs from EV-D68-immune human subjects. Relatedness scores were generated from competitive binding ELISAs with B1 clade EV-D68 isolates and used to cluster the mAbs into four competitive binding groups, designated 1 to 4. Clone numbers in red or blue are potently neutralizing mAbs, and blue clone names indicate two mAbs tested in detail in subsequent figures. [Figure 32-2] See description of Figure 32-1. [Figure 32-3] See description of Figure 32-1. [Figure 32-4] See description of Figure 32-1. [Figure 33-1] Figures 33A-C: Neutralization potency and binding capacity of human mAbs. (Figure 33A) MAbs were ranked within competitive binding groups (Comp. group, where the fifth group represents a singleton residual collection) by IC50 values ​​in a CCID50 neutralization assay using B1 clade isolates. We also tested the 21 most potently neutralizing mAbs for neutralization of D clade and Fermon (Fer.) isolates. ">" indicates no neutralization detected when tested at concentrations up to 50 μg / mL. Blank cells indicate not tested. Clone numbers in red or blue represent potently neutralizing mAbs, and blue clone names represent two mAbs tested in detail in subsequent figures. (Figure 33B) Binding strength to live virus isolates or mock virus preparations is shown using EC50 values ​​generated by using an indirect ELISA (Figure 33C) with purified mAb dilutions to fit a sigmoidal dose-response curve. ">" indicates an EC50 value greater than the highest concentration tested, 100 μg / mL, suggesting poor or no binding. [Figure 33-2] See description of Figure 33-1. [Figure 33-3] See description of Figure 33-1. [Figure 34]Figure 34A-C: Comparison of structural features between two immune complexes. (Figure 34A) Radial-color cryo-EM maps of EV-D68:Fab EV68-159 (left) or EV-D68:Fab EV68-228 (right). Each map is projected onto a two-fold symmetry axis. The five-fold, three-fold, and two-fold axes of each asymmetric unit are indicated using triangular outlines labeled with one pentagon, two small triangles, and one ellipse, respectively. (Figure 34B) Comparison of binding positions using asymmetric units. Viral proteins are colored blue (VP1), green (VP2), and red (VP3). Fab molecules are colored gray (EV68-159) or purple (EV68-228), and heavy and light chains are colored the same, depending on their dark or light intensity, respectively. (Figure 34C) Footprint of EV68-159 Fab (left) or EV68-228 Fab (right). Radial colored 2D projections of the viral surface were generated using RIVEM. Viral surface residues facing any atom from the Fab molecule within a distance of 4 Å are shown in light blue (VP1), light green (VP2), and light red (VP3). Canyon regions are shown in yellow. Scale bars in (Figure 34A) and (Figure 34C) indicate radial distance measured in Å. [Figure 35] Figure 35: Close-up of the binding interface of EV68-159 and EV68-228. The viral capsid is shown as the surface, and the Fab is shown in cartoon representation. VP1, VP2, and VP3 are colored white, dark gray, and silver, respectively. The heavy or light chain is colored orange or yellow, respectively. Viral residues that interact are colored based on the heavy and light chain, with color intensity varying based on either VP. The heavy and light chain complementarity-determining regions (HCDRs and LCDRs, respectively) involved in the binding interface are indicated by arrows. [Figure 36] Figures 36A-D: Molecular details of virion-Fab interactions. Representative interactions at the binding interface of EV-D68:Fab EV68-159 (Figures 36A-C) and EV-D68:Fab EV68-228 (Figure 36D). Hydrogen bonds are colored cyan, salt bridges are colored magenta. [Figure 37]Figure 37A-D: MAb EV68-228 protects mice from EV-D68-induced respiratory disease when used as either prophylactic or therapeutic agents. Four-week-old AG129 strain mice (n = 4 / time point) were inoculated intranasally with mouse-adapted B1 clade EV-D68, antibody was administered intraperitoneally, and viral titers in the indicated tissues were measured by CCID50 assay. (Figure 37A-B) Mice were inoculated with the virus 24 hours after the indicated dose of antibody, and viral titers were then measured at the indicated time points. (Figure 37C-D) Mice were inoculated with virus followed by 10 mg / kg (except where indicated) antibody 4, 24, or 48 hours later, and viral titers were then measured. [Figure 38] Figure 38A-F: MAb EV68-228 reduces lung inflammation in EV-D68-infected mice. Four-week-old AG129 mice (n = 4 / time point) were inoculated intranasally with virus 24 hours after the indicated dose of antibody (Figure 38A-C) or with 10 mg / kg (except where indicated) antibody 4, 24, or 48 hours after intranasal inoculation of virus (Figure 38D-F). Cytokines were then measured at the indicated time points. Cytokines were quantified from lung homogenates using ELISA. Values ​​from the treatment groups were compared with the placebo group for each time point using one-way ANOVA with Dunnett's T3 multiple comparison test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). IL, interleukin; MCP, monocyte chemotactic protein. Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 39]Figures 39A-F: MAb EV68-228 protects mice from EV-D68-induced neurological disease when used as either prophylactic or therapeutic agents. Ten-day-old mice were inoculated intraperitoneally with mouse-adapted B1 clade EV-D68, antibody was administered intraperitoneally, and viral titers in the indicated tissues were measured by CCID50 assay. (Figures 39A-C) Mice were inoculated with virus 24 hours after the indicated dose of antibody. (Figure 39A) Viral titers were then measured (n = 3 / time point), (Figure 39B) survival was monitored, and (Figure 39C) neurological scores (n = 6 / time point) were recorded at the indicated time points. Higher scores indicate more severe motor impairment. (Figures 39D-F) Mice were inoculated with virus followed by a 10 mg / kg dose of antibody, then (Figure 39D) viral titers were measured (n=3 / time point), (Figure 39E) survival was monitored, and (Figure 39F) neurological scores (n=6 / time point, except for n=9 at 120 hours) were recorded. Colored vertical arrows indicate the time of treatment. [Figure 40] Figure 40: Western blot data. All mAbs were tested for their ability to stain B1 clade viruses by Western blot. All positive results are shown. [Figure 41-1]Figure 41: Detailed characterization of human mAbs. MAbs were ranked within competitive binding groups by IC50 values ​​in a CCID50 neutralization assay using B1 clade isolates. We also tested the 21 most potently neutralizing mAbs for neutralization of D clade and Fermon (Fer.) isolates. ">" indicates no neutralization detected when tested at concentrations up to 50 μg / mL. Blank cells indicate not tested. Clone numbers in red or blue font are potently neutralizing mAbs, and blue clone names indicate two mAbs tested in detail in subsequent figures. The final IC50 value for each mAb is the average of the logarithm of the EC50 values ​​obtained from three separate experiments performed in duplicate. Binding strength to live virus isolates is shown based on EC50 values ​​generated using an indirect ELISA with purified mAb dilutions to fit a sigmoidal dose-response curve. ">" indicates that the predicted EC50 value exceeds the maximum concentration tested, 100 μg / mL, suggesting poor or no binding. The final EC50 value for each mAb is the mean of the logarithm of the EC50 values ​​obtained from three separate experiments performed in duplicate. IgG subtypes were determined phenotypically. Light chain types were kappa (K) or lambda (L) and were determined genetically. WB indicates Western blot reactivity; all mAbs tested; blank lines indicate no reactivity. [Figure 41-2] See description of Figure 41-1. [Figure 41-3] See description of Figure 41-1. [Figure 42-1] Figure 42: Indirect ELISA data for all mAbs. Binding strength to live virus isolates is shown by EC50 values ​​generated by using indirect ELISA with purified mAb dilutions to fit a sigmoidal dose-response curve. Representative data from one of three experiments is shown, with error bars indicating the standard deviation of duplicate technical replicates. The final EC50 value for each mAb is the average of the logarithm of the EC50 values ​​obtained from all three experiments. Mock virus preparations were obtained from RD cell monolayers that were not inoculated with virus but were prepared in the same manner as the virus-infected cells. [Figure 42-2] See description of Figure 42-1. [Figure 42-3] See description of Figure 42-1. [Figure 42-4] See description of Figure 42-1. [Figure 43] Figure 43: Representative density obtained from the EV-D68:Fab EV68-228 electron density map. Viral proteins are colored blue (VP1), green (VP2), red (VP3) and magenta (VP4). [Figure 44] Figure 44: Estimation of immune complex map resolution. Map resolution is estimated based on a gold standard Fourier shell correlation (FSC) cutoff of 0.143. The final resolution of the EV68-159 (red curve) or EV68-228 (blue curve) complex is 2.9 Å or 3.1 Å, respectively. [Figure 45] Figure 45: Comparison of Fab binding sites. EV68-159 and EV68-228 are colored gold and blue, respectively, and the viral surface is colored cyan. The left panel shows both the variable and constant domains, while the right panel shows only the variable domain, demonstrating that the footprints of the two Fabs do not overlap. [Figure 46] Figure 46: Roadmap showing a close-up of the Fab footprint. A radially colored 2D projection of the virus surface was generated using RIVEM. Viral surface residues facing any atom from the Fab molecule within a distance of 4 Å are shown in light blue (VP1), light green (VP2), and light red (VP3). Canyon regions are shown in yellow. [Figure 47] Figure 47: Bulky side chains of the EV68-228 Fab heavy chain. This figure shows examples of bulky side chains, also seen in the structure of EV68-159 Fab, that form a network of hydrophobic interactions to stabilize the EV68-228 Fab. [Figure 48]Figures 48A-C: Experiment NIA-1930. Lung virus titers in EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours prior to infection. Lung virus titers are shown on days 1 (Figure 48A), 3 (Figure 48B), and 5 (Figure 48C) post-infection. Mice treated with 10 mg / kg EV68-228-TP or EV68-228-CHO had no detectable lung virus titers on days 1, 3, or 5 post-infection. Treatment with 10 mg / kg hIVIg significantly reduced lung virus titers only on day 5 post-infection. (**P<0.01, ****P<0.0001 compared to placebo-treated mice.) [Figure 49] Figures 49A-C: Experiment NIA-1930. Blood virus titers from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours before infection. Blood virus titers are shown on days 1 (Figure 49A), 3 (Figure 49B), and 5 (Figure 49C) post-infection. No virus was detected in the blood of mice treated with 10 mg / kg EV68-228-TP or EV68-228-CHO on days 1, 3, or 5 post-infection. Treatment with 10 mg / kg hIVIg significantly reduced blood virus titers on days 1, 3, and 5 post-infection. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice.) [Figure 50]Figure 50: Experiment NIA-1930. Lung concentrations of IL-1α, IL-1β, IL-2, and IL-3 from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours before infection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced IL-1α concentrations on days 1, 3, and 5 post-infection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO also reduced IL-1β concentrations on day 3 post-infection. Treatment with hIVIg significantly reduced IL-1β lung concentrations only on day 3 post-infection. No significant changes in IL-2 or IL-3 concentrations were observed post-infection. (*P<0.05, ***P<0.001, ****P<0.0001 compared to placebo-treated mice.) Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 51] Figure 51: Experiment NIA-1930. Lung concentrations of IL-4, IL-5, IL-6, and IL-10 from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours before infection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced IL-5 concentrations on day 3 postinfection and IL-6 concentrations on days 3 and 5 postinfection. Treatment with 10 mg / kg hIVIg significantly reduced pulmonary concentrations of IL-5 and IL-6 on day 3 postinfection. No significant changes in IL-4 or IL-10 concentrations were observed postinfection. (*P<0.05, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 52]Figure 52: Experiment NIA-1930. Lung concentrations of IL-12p70, IL-17, MCP-1, and IFN-γ from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours prior to infection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced MCP-1 concentrations on days 3 and 5 post-infection. Treatment with 10 mg / kg hIVIg significantly reduced MCP-1 lung concentrations on days 3 and 5 post-infection. No significant changes in IL-12p70, IL-17, or IFN-γ concentrations were observed post-infection. (**P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 53] Figure 53: Experiment NIA-1930. Lung concentrations of TNFα, MIP-1α, GM-CSF, and RANTES from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours prior to infection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced lung concentrations of MIP-1α on days 1 and 3 post-infection and reduced RANTES concentrations on days 3 and 5 post-infection. A 10 mg / kg dose of hIVIG significantly reduced MIP-1α concentrations on days 1 and 3 post-infection and also reduced RANTES concentrations on day 3 post-infection. No significant changes in TNFα or GM-CSF concentrations were observed following infection with EV-D68. (**P<0.01, ***P<0.001, P<0.0001 compared to placebo-treated mice.) Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 54]Figures 54A-B: Experiment NIA-1930. Lung virus titers in EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours post-infection. Lung virus titers are shown on days 3 (Figure 54A) and 5 (Figure 54B) post-infection. Mice treated with 10 mg / kg EV68-228-TP or EV68-228-CHO had no detectable lung virus titers on days 3 or 5 post-infection. Treatment with 10 mg / kg hIVIg significantly reduced lung virus titers only on day 3 post-infection. (**P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice.) [Figure 55] Figures 55A-B: Experiment NIA-1930. Blood virus titers of EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours post-infection. Blood virus titers are shown on days 3 (Figure 55A) and 5 (Figure 55B) post-infection. No virus was detected in the blood of mice treated with 10 mg / kg EV68-228-TP or EV68-228-CHO on days 3 or 5 post-infection. Treatment with 10 mg / kg hIVIg significantly reduced blood virus titers on days 3 and 5 post-infection. (*P<0.05, ***P<0.001, ****P<0.0001 compared to placebo-treated mice.) [Figure 56] Figure 56: Experiment NIA-1930. Lung concentrations of IL-1α, IL-1β, IL-2, and IL-3 from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours postinfection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced IL-1α and IL-1β concentrations at day 3 postinfection. In mice treated with EV68-228-TP and mice treated with hIVIg, IL-3 concentrations were significantly reduced at day 3 postinfection. No significant changes in IL-2 concentrations were observed postinfection. (*P<0.05, **P<0.01, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 57] Figure 57: Experiment NIA-1930. Lung concentrations of IL-4, IL-5, IL-6, and IL-10 from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours postinfection. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced IL-5 concentrations on day 3 postinfection and IL-6 concentrations on days 3 and 5 postinfection. Treatment with 10 mg / kg hIVIg significantly reduced IL-5 lung concentrations on day 3 postinfection. No significant changes in IL-4 or IL-10 concentrations were observed postinfection. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 58] Figure 58: Experiment NIA-1930. Lung concentrations of IL-12p70, IL-17, MCP-1, and IFN-γ from EV-D68-infected AG129 mice treated 24 hours postinfection with EV68-228-TP or EV68-228-CHO. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced MCP-1 concentrations on days 3 and 5 postinfection. Treatment with 10 mg / kg hIVIg significantly reduced MCP-1 lung concentrations on days 3 and 5 postinfection. No significant changes were observed in IL-12p70, IL-17, or IFN-γ concentrations postinfection. (****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 59]Figure 59: Experiment NIA-1930. Lung concentrations of TNFα, MIP-1α, GM-CSF, and RANTES from EV-D68-infected AG129 mice treated 24 hours postinfection with EV68-228-TP or EV68-228-CHO. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced lung concentrations of MIP-1α on day 3 postinfection and reduced concentrations of RANTES on days 3 and 5 postinfection. No significant changes in TNFα or GM-CSF concentrations were observed after infection with EV-D68. (****P<0.0001 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 60] Figures 60A-B: Experiment NIA-1930. Lung virus titers in EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 48 hours post-infection. Lung virus titers are shown at days 3 (Figure 60A) and 5 (Figure 60B) post-infection. Only EV68-228-CHO significantly reduced lung virus titers at days 3 or 5 post-infection. (****P<0.0001 compared to placebo-treated mice). [Figure 61] Figures 61A-B: Experiment NIA-1930. Blood virus titers from EV-D68-infected AG129 mice treated with EV68-228-TP or EV68-228-CHO 48 hours post-infection. Blood virus titers are shown on days 3 (Figure 61A) and 5 (Figure 61B) post-infection. No virus was detected in the blood of mice treated with 10 mg / kg of EV68-228-TP or EV68-228-CHO on days 3 or 5 post-infection. Treatment with 10 mg / kg of hIVIg significantly reduced blood virus titers on days 3 and 5 post-infection. (****P<0.0001 compared to placebo-treated mice). [Figure 62]Figure 62: Experiment NIA-1930. Lung concentrations of IL-1α, IL-1β, IL-2, and IL-3 from EV-D68-infected AG129 mice treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. Only treatment with 10 mg / kg EV68-228-CHO significantly reduced IL-1α and IL-1β concentrations at day 3 post-infection. No significant changes in IL-2 or IL-3 concentrations were observed post-infection. (**P<0.01 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 63] Figure 63: Experiment NIA-1930. Lung concentrations of IL-4, IL-5, IL-6, and IL-10 from EV-D68-infected AG129 mice treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO significantly reduced IL-6 concentrations at day 3 post-infection. No significant changes were observed in IL-4, IL-5, or IL-10 concentrations post-infection. (**P<0.01 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 64] Figure 64: Experiment NIA-1930. Lung concentrations of IL-12p70, IL-17, MCP-1, and IFN-γ from EV-D68-infected AG129 mice treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. No significant changes in IL-12p70, IL-17, MCP-1, or IFN-γ concentrations were observed post-infection. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 65]Figure 65: Experiment NIA-1930. Lung concentrations of TNFα, MIP-1α, GM-CSF, and RANTES from EV-D68-infected AG129 mice treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. No significant changes in TNFα, MIP-1α, GM-CSF, or RANTES concentrations were observed following infection with EV-D68. Sample names in the graph correspond from left to right with a top-to-bottom vertical description. [Figure 66] Figure 66: Experiment NIA-1931. Survival rate of 10-day-old AG129 mice infected with EV-D68 and treated with EV68-228-TP or EV68-228-CHO 24 hours before infection. (n=10 mice / group). Treatment with EV68-228-TP at a dose of 10 mg / kg completely protected the mice from death. All 10 mice treated with EV68-228-CHO at 10 mg / kg were protected from death. IVIg at a dose of 10 mg / kg protected 8 of 10 mice from death. Nine of 10 placebo-treated mice died from infection (***P<0.001, ****P<0.0001 compared with placebo-treated mice). [Figure 67] Figure 67: Experiment NIA-1931. Percentage of initial body weight of EV-D68-infected 10-day-old AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours prior to infection. No significant difference in weight loss was observed in mice treated with a 10 mg / kg dose of EV68-228-TP or EV68-228-CHO 24 hours prior to infection compared to placebo-treated mice. Treatment with a 10 mg / kg dose of hIVIg did not protect mice from weight loss. [Figure 68]Figures 68A-B: Experiment NIA-1931. Blood virus titers on days 1 and 3 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated 24 hours pre-infection with EV68-228-TP or EV68-228-CHO. Blood virus titers on days 1 (Figure 68A) and 3 (Figure 68B) post-infection are shown. Treatment with 10 mg / kg EV68-228-TP 24 hours pre-infection significantly reduced virus titers on days 1 and 3 post-infection. Treatment with 10 mg / kg EV68-228-CHO 24 hours pre-infection significantly reduced virus titers on days 1 and 3 post-infection. Treatment with a 10 mg / kg dose of IVIg 24 hours pre-infection significantly reduced blood virus titers on day 3 post-infection. (*P<0.05, **P<0.01, ****P<0.0001 compared with placebo-treated mice). [Figure 69] Figures 69A-B: Experiment NIA-1931. Blood virus titers at days 5 and 7 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated with EV68-228-TP or EV68-228-CHO 24 hours prior to infection. Blood virus titers at days 5 (Figure 69A) and 7 (Figure 69B) post-infection are shown. Treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO 24 hours prior to infection reduced virus titers at day 5 post-infection. No virus was detected at day 7 post-infection. [Figure 70] Figures 70A-C: Experiment NIA-1931. Neurological scores on days 2-10 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated with EV68-228-TP or EV68-228-CHO 24 hours before infection. Neurological scores are shown for days 2-4 (Figure 70A), 5-7 (Figure 70B), and 8-10 (Figure 70C) post-infection. Treatment with 10 mg / kg of EV68-228-TP or EV68-228-CHO 24 hours before infection completely prevented clinical signs of paralysis, as measured by neurological score. No neurological scores were observed in mice treated with IVIg at a dose of 10 mg / kg 24 hours before infection. (****P<0.0001 compared to placebo-treated mice). [Figure 71] Figure 71: Experiment NIA-1931. Survival of 10-day-old AG129 mice infected with EV-D68 and treated 24 hours post-infection with EV68-228-TP or EV68-228-CHO (n=10 mice / group). Treatment with EV68-228-TP at a dose of 10 mg / kg completely protected the mice from death. All 10 mice treated with EV68-228-CHO at 10 mg / kg were protected from death. IVIg at a dose of 10 mg / kg protected 10 of 10 mice from death. Eight of 10 placebo-treated mice died from infection (***P<0.001 compared with placebo-treated mice). [Figure 72] Figure 72: Experiment NIA-1931. Percentage of initial body weight of EV-D68-infected 10-day-old AG129 mice treated with EV68-228-TP or EV68-228-CHO 24 hours post-infection. No significant difference in weight loss was observed in mice treated with a 10 mg / kg dose of EV68-228-TP or EV68-228-CHO 24 hours post-infection compared to placebo-treated mice. Treatment with a 10 mg / kg dose of hIVIg did not protect mice from weight loss. [Figure 73] Figures 73A-B: Experiment NIA-1931. Blood virus titers at days 1 and 3 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated 24 hours post-infection with EV68-228-TP or EV68-228-CHO. Blood virus titers at days 1 (Figure 73A) and 3 (Figure 73B) post-infection are shown. Treatment with 10 mg / kg EV68-228-TP 24 hours post-infection significantly reduced virus titers at day 3 post-infection. Treatment with 10 mg / kg EV68-228-CHO 24 hours post-infection significantly reduced virus titers at day 3 post-infection. Treatment with a 10 mg / kg dose of IVIg 24 hours post-infection significantly reduced blood virus titers at day 3 post-infection. (*P<0.05, **P<0.01, ****P<0.0001 compared with placebo-treated mice). [Figure 74]Figures 74A-B: Experiment NIA-1931. Blood virus titers at days 5 and 7 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated with EV68-228-TP or EV68-228-CHO 24 hours post-infection. Blood virus titers at days 5 (Figure 74A) and 7 (Figure 74B) post-infection are shown. After treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO 24 hours post-infection, no virus was detected at day 5 post-infection. No virus was detected in any of the mice at day 7 post-infection. [Figure 75] Figures 75A-B: Experiment NIA-1931. Neurological scores on days 2-9 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated 24 hours post-infection with EV68-228-TP or EV68-228-CHO. Neurological scores are shown on days 2-5 (Figure 75A) and days 6-9 (Figure 75B) post-infection. Treatment with 10 mg / kg of EV68-228-TP or EV68-228-CHO 24 hours post-infection significantly reduced signs of paralysis as measured by neurological scores on days 2-9 post-infection. Treatment with a 10 mg / kg dose of hIVIg 24 hours post-infection reduced neurological scores only on days 2 and 3 post-infection. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to placebo-treated mice.) Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 76]Figures 76A-B: Experiment NIA-1931. Neurological scores on days 10-17 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated with EV68-228-TP or EV68-228-CHO 24 hours post-infection. Neurological scores are shown for days 10-13 (Figure 76A) and days 14-17 (Figure 76B) post-infection. Treatment with 10 mg / kg of EV68-228-TP or EV68-228-CHO 24 hours post-infection significantly reduced signs of paralysis as measured by neurological scores on days 10-16 post-infection. Treatment with a 10 mg / kg dose of hIVIg 24 hours post-infection did not significantly reduce neurological scores. (*P<0.05, **P<0.01 compared to placebo-treated mice). Sample names in the graph correspond from left to right with a vertical description from top to bottom. [Figure 77] Figure 77: Experiment NIA-1931. Survival rate of 10-day-old AG129 mice infected with EV-D68 and treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. (n = 10 mice / group). Treatment with EV68-228-TP at a dose of 10 mg / kg protected 9 of 10 mice from death. Treatment with EV68-228-CHO at 10 mg / kg protected 8 of 10 mice from death. IVIg at a dose of 10 mg / kg protected 6 of 10 mice from death. 9 of 10 placebo-treated mice died from infection (**P<0.01, ***P<0.001 compared with placebo-treated mice). [Figure 78] Figure 78: Experiment NIA-1931. Percentage of initial body weight of EV-D68-infected 10-day-old AG129 mice treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. No significant difference in weight loss was observed in mice treated 24 hours post-infection with a 10 mg / kg dose of EV68-228-TP or EV68-228-CHO compared to placebo-treated mice. Treatment with a 10 mg / kg dose of hIVIg did not protect mice from weight loss. [Figure 79]Figures 79A-B: Experiment NIA-1931. Blood virus titers at days 1 and 3 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. Blood virus titers at days 1 (Figure 79A) and 3 (Figure 79B) post-infection are shown. Treatment with 10 mg / kg of EV68-228-TP or EV68-228-CHO 48 hours post-infection did not significantly reduce blood virus titers. Treatment with IVIg at a dose of 10 mg / kg 48 hours post-infection did not significantly reduce blood virus titers. [Figure 80] Figures 80A-B: Experiment NIA-1931. Blood virus titers at days 5 and 7 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. Blood virus titers at days 5 (Figure 80A) and 7 (Figure 80B) post-infection are shown. After treatment with 10 mg / kg EV68-228-TP or EV68-228-CHO 24 hours post-infection, no virus was detected at day 5 post-infection. No virus was detected in any of the mice at day 7 post-infection. [Figure 81] Figures 81A-B: Experiment NIA-1931. Neurological scores on days 3-10 post-infection in 10-day-old AG129 mice infected with EV-D68 and treated 48 hours post-infection with EV68-228-TP or EV68-228-CHO. Neurological scores on days 3-6 (Figure 81A) and days 7-10 (Figure 81B) post-infection are shown. Treatment with 10 mg / kg EV68-228-TP 48 hours post-infection significantly reduced neurological scores on days 3-5 post-infection. A single administration of a 10 mg / kg dose of EV68-228-CHO 48 hours post-infection significantly reduced signs of paralysis as measured by neurological scores on days 3-6 post-infection. Treatment with a 10 mg / kg dose of hIVIg 48 hours post-infection did not significantly reduce neurological scores post-infection. (*P<0.05, ***P<0.001, ****P<0.0001 compared to placebo-treated mice.) Sample names in the graph correspond from left to right with a vertical description from top to bottom. DETAILED DESCRIPTION OF THE INVENTION

[0026] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS As mentioned above, enterovirus D68 (EV-D68) can cause outbreaks of respiratory disease, primarily in children, worldwide. Clusters of acute flaccid myelitis, a poliomyelitis-like neuromuscular weakness syndrome, often occur simultaneously with EV-D68 respiratory outbreaks. Seroepidemiological studies have found that sera from nearly all individuals over the age of 2–5 contain anti-EV-D68 neutralizing antibodies, suggesting that EV-D68 is a ubiquitous childhood pathogen. However, knowledge of the viral epitopes targeted by the humoral immune response is only inferred from previous studies of related viruses. Herein, we generated 64 human monoclonal antibodies against EV-D68, some of which are potently neutralizing. These are believed to be the first human mAbs against this pathogen, and we propose their use for the detection, treatment, and prevention of EV-D68 infection. These and other aspects of the present disclosure are described in detail below.

[0027] I. Enterovirus D68 Enterovirus 68 (EV68, EV-D68, HEV68) is a member of the enterovirus family, the Picornaviridae. Enterovirus 68 was first isolated in California in 1962 and was once considered rare, but has experienced a rapid increase in global numbers in the 21st century. It is suspected of causing a polio-like disorder called acute flaccid myelitis.

[0028] EV68 is one of over 100 non-enveloped enteroviruses, a group of short-chain RNA viruses that includes poliovirus, coxsackievirus, and echovirus. Unlike all other enteroviruses, EV68 exhibits both acid instability and a relatively low optimal growth temperature, both characteristics of human rhinoviruses. It was previously referred to by some researchers as human rhinovirus 87. Children under the age of 5 and those with asthma appear to be at highest risk for the disease, although disease has also been reported in adults with asthma and immunosuppression.

[0029] Since its discovery, EV68 has been reported mostly sporadically, with isolated cases. Between 2005 and 2011, six clusters (≥10 cases) or outbreaks were reported in the Philippines, Japan, the Netherlands, and the US states of Georgia, Pennsylvania, and Arizona. EV68 was found in two of five children in California during the 2012 / 13 cluster of polio-like illnesses. In the US, the virus caused a cluster of respiratory illnesses in August 2014. By mid-October, 691 cases of respiratory illness caused by EV-D68 had occurred in 46 states and the District of Columbia, resulting in five child deaths. In 2016, 29 cases were reported in Europe (five in France and Scotland, three each in Sweden, Norway, and Spain).

[0030] Since 2000, the original virus strain has diversified, evolving a genetically distinct outbreak strain, clade B1. It is clade B1, not the older strains, that is associated with AFM and is neuropathic in animal models. Cases have been reported to occur late in the enterovirus season, typically in August and September in the Northern Hemisphere (roughly the period between the spring and autumn equinoxes).

[0031] EV68 causes almost exclusively respiratory illness, which can range from mild to severe, but can cause a variety of symptoms, from completely asymptomatic to mild flu-like symptoms, to debilitating respiratory disease, and is suspected of rare involvement in polio-like syndromes. Like all enteroviruses, EV68 can cause a variety of rashes, abdominal pain, and loose stools. Initial symptoms are similar to those of the common cold, including runny nose, sore throat, cough, and fever. As the disease progresses, more severe symptoms can occur, including pneumonia-like difficulty breathing, decreased alertness, decreased urine production, and dehydration, which can lead to respiratory failure.

[0032] The severity of symptoms experienced appears to depend on the demographic group in question. Experts estimate that a large portion of the population is actually exposed to enteroviruses, but healthy adults do not exhibit symptoms. In contrast, EV-D68 is disproportionately debilitating in very young and highly vulnerable children. Although several hundred cases (472), mostly young people, were exposed to the disease, fewer than 100 of these patients were diagnosed with severe symptoms (such as paralysis).

[0033] This virus is suspected as a cause of acute flaccid myelitis, a rare muscle weakness usually attributed to polio, because two California children who tested positive for the virus developed paralysis of one or more limbs that reached peak severity within 48 hours of symptom onset. Recovery of motor function was insufficient at 6 months of follow-up. As of October 2014, the CDC was investigating 10 cases of paralysis and / or cranial dysfunction in Colorado and other reports nationwide, consistent with increased enterovirus D68 activity. As of October 2014, the actual number of cases was believed to be greater than 100. As of 2018, the association between EV-D68 and paralysis was strong, fully meeting six Bradford Hill criteria and partially meeting two. On October 17, 2018, the CDC issued a statement claiming, "We currently know that poliovirus is not the cause of these AFM cases. CDC tests every stool specimen obtained from AFM patients, and none have tested positive for poliovirus." In 2014, real-time PCR tests were developed by the CDC to speed up detection.

[0034] Because there is no specific treatment and no vaccine, the disease will inevitably progress, and treatment is symptomatic (supportive care). While most people recover fully, some require hospitalization, and some die as a result of the virus. Five cases of EV68 paralysis were unsuccessfully treated with steroids, intravenous immunoglobulin, and / or plasma exchange. Treatment was apparently ineffective, as no recovery of motor function was observed. A 2015 trial suggested that the antiviral drug pleconaril may be useful in treating EV-D68.

[0035] Because the virus spreads through saliva, sputum, and feces, hand washing is important. Ill people can attempt to reduce the spread of this virus through basic hygiene measures, such as covering their nose and mouth when sneezing or coughing. Other measures include cleaning surfaces and toys. For hospitalized patients with EV-D68 infection, the CDC recommends transmission-based precautions, i.e., standard and contact precautions, as recommended for all enteroviruses, and droplet precautions. According to the CDC in 2003, surfaces in healthcare settings should be cleaned with a hospital-grade disinfectant with an EPA label claim against any of several non-enveloped viruses (e.g., norovirus, poliovirus, rhinovirus).

[0036] A. Viral epitopes Measurements of serum antibody virus neutralization capacity reflect the activity of the entire polyclonal antibody repertoire. However, to fully understand the humoral response to viruses, it is necessary to define the specific viral epitopes bound by individual antibodies and determine whether antibody binding to specific epitopes confers protection against disease. Previously, testing of murine monoclonal antibodies raised against rhinovirus-B14 identified four neutralizing immune epitopes (Nim) for Enteroviruses (Rossmann et al., 1985). EV-D68-specific monoclonal antibody epitopes have not been tested to date. However, determination of the crystal structure of EV-D68 suggested possible locations of four Nim sites on the EV-D68 virion, based on the observed structural homology with rhinoviruses (Liu et al., 2015a). Studies comparing the amino acid sequences of the surface proteins of recent human isolates of EV-D68 from the United States (Zhang et al., 2015) or Japan (Imamura et al., 2014) with those of the Fermon reference virus strain suggest that mutations in Nim and nearby neighboring residues occur, particularly in the BC and DE loops of the capsid protein VP1, which is disordered in its crystal structure (Liu et al., 2015a). These VP1 polymorphisms may contribute to increased viral pathogenicity by evading pre-existing humoral immunity. Although mouse monoclonal antibodies are commercially available (sources include GeneTex, ThermoFisher, and Sigma), no information is available regarding the epitopes of these antibodies, apart from listing the specific viral surface proteins used as immunogens. Notably, some of these commercially available antibodies were generated from immunogens of other Enteroviruses, suggesting the ability of heterotypic antibodies to bind to EV-D68. Further supporting this possibility, two murine mAbs cross-reacted with 40 different enterovirus species, but interestingly, EV-D68 was the only virus tested to which they did not react ( Miao et al., 2009 ).Heterotypic molecular recognition of EV-D68 by antibodies induced by other types of enteroviruses may contribute to the observed universal EV-D68 seroprevalence during early childhood. Understanding the fine specificity of human antibody epitopes against EV-D68 and the type specificity or breadth of such antibodies will require studies using cloned human monoclonal antibodies induced by natural infection.

[0037] During the 2014 EV-D68 outbreak in the United States, nearly all virus isolates were from the newly emerged B1 clade, with fewer viruses from the closely related B2 clade or the distantly related D clade (Tan et al., 2016). In this study, all but one subject was infected with a B1 clade isolate (Table E). Since 2014, B3 clade viruses have predominated, and B1 clade viruses are no longer circulating (Dyrdak et al., 2019). In 2018, all EV-D68 isolates sequenced by the US Centers for Disease Control and Prevention were from the B3 clade (Kujawski et al., 2019). We first used a B1 clade EV-D68 isolate to measure the 50% cell culture infectious dose (CCID) of 1000 cells / mL. 50 The in vitro neutralization potency of each mAb was measured using a 50% inhibitory concentration (IC) assay (Figure 33A). Twenty-eight mAbs had a median inhibitory concentration (IC) of less than 50 μg / mL. 50 ) and mAb EV68-159 showed neutralization with an IC of 0.32 ng / mL. 50 The 21 mAbs that most potently neutralized against the D clade isolates were further tested, and 11 mAbs neutralized the virus, 7 of which had IC values ​​above 100 against heterologous viruses (Figure 41). 50 We found that the mAbs neutralized the Fermon laboratory reference strain but were not potent enough to inhibit modern B1 clade viruses. The Fermon strain was isolated in 1962 and is sufficiently distant from modern EV-D68 isolates that it does not fit into the clade classification scheme (Tan et al., 2016). Nine mAbs neutralized the Fermon laboratory reference strain but were not potent enough to inhibit modern B1 clade viruses.

[0038] Recognizing that neutralization assays may underestimate cross-reactivity, we used the same indirect ELISA approach described above to determine the 50% effective concentration (EC ) for binding to representative EV-D68 isolates from the B1, B2, or D clades. 50 ) purified mAb was generated (Figures 33B-C and Figure 42). EC 50 Of the mAbs with a β-value, all bound to B2 clade isolates, but approximately half also bound to D clade isolates (Figure 33B and Figure 41). An additional class of mAbs was observed that generally bound weakly but cross-reacted with viruses from all clades tested.

[0039] To date, structural studies of antibody-EV-D68 interactions have been limited to mouse mAbs (Zheng et al., 2019). We selected two potently neutralizing human mAbs, the clade-specific mAb EV68-159 and the highly cross-reactive mAb EV68-228, to generate antigen-binding fragments (Fabs) and immune complexes with B1 clade EV-D68 isolates for cryo-electron microscopy (cryo-EM). The final density maps achieved a resolution of 2.9 Å (EV68-159) or 3.1 Å (EV68-228) (Figure 34A, Figure 43, Figure 44, and Table G). The structures revealed two distinct binding sites: EV68-159 binds around a three-fold symmetry axis, while EV68-228 binds around a five-fold axis between the recesses forming the canyon region (Figures 34A-C, Figure 45). Therefore, a total of 60 copies of each Fab bound to the virus particle. The Fab variable domains that interacted with the virus surface showed strong density similar to that of the virus capsid proteins, and atomic models of each Fab were constructed along with the four virus capsid proteins. In contrast, the Fab constant domains, located far from the virus surface, showed relatively weak density and were excluded from the atomic model construction. The backbone of the polypeptide chain and most of the amino acid side chains were well-ordered in the density map, revealing important features of the binding interface between the virus particle and the Fab molecule.

[0040] B. Animal Models of Neurological Disease Measuring antibody neutralization of viruses in vitro is typically the most rapid and reproducible method for characterizing an antibody's antiviral function. However, this test format does not measure effector functions mediated by the Fc portion of the antibody, such as complement activation or engagement with cell surface Fc receptors, which act only in vivo. As a result, the magnitude of in vitro neutralization potency may not fully correlate with the level of inhibition of viral replication or protection against disease observed in vivo (reviewed in (Lu et al., 2018)). Many of the Fc-mediated effector functions of antibodies require Fc receptor-bearing cells of the innate or adaptive immune system to mediate full protective effects. In preclinical studies, small animal models of infection are necessary to evaluate the effects of humoral immunity.

[0041] Researchers in Colorado (Hixon et al., 2017a; 2017b), Utah (Morrey et al., 2018), and China (Zhang et al., 2018) have reported mouse models of EV-D68 since early 2017. All three reported models involve inoculating mice 10 days of age or younger with the virus. Across these models, EV-D68 has been shown to be capable of causing flaccid limb paralysis and death when administered via the intraperitoneal route (Hixon et al., 2017a; 2017b; Morrey et al., 2018; Zhang et al., 2018), intramuscular route (Hixon et al., 2017a; 2017b), or intracerebral route (Hixon et al., 2017b). Notably, intranasal inoculation caused paralysis in only 2 of 73 outbred NIH Swiss Webster mice tested, with virus detected in the spinal cord (Hixon et al., 2017b), whereas intranasal inoculation of AG129 mice, which lack interferon type I and type II receptors, caused paralysis in 2 of 4 mice, with virus detected in the muscle but not the spinal cord (Morrey et al., 2018). However, all studies evaluating humoral immunity in mice used non-physiological routes of virus inoculation.

[0042] Viral antigens have been visualized in tissues by immunostaining in skeletal muscle (Morrey et al., 2018; Zhang et al., 2018) and spinal cord (Hixon et al., 2017b; Morrey et al., 2018). The involvement of both muscle and spinal cord suggests that EV-D68 can induce paralysis through two mechanisms: 1) direct pathological effects on skeletal muscle and 2) central motor neuron loss. Motor neurons within the spinal cord are infected, which is similar to the pathogenesis of poliomyelitis. This pattern of mouse neuronal involvement in immunostaining studies also correlates with the findings of gray matter changes on spinal cord imaging in patients with AFM (Hixon et al., 2017b; Morrey et al., 2018). In a more sensitive real-time quantitative RT-PCR test, EV-D68 RNA was detected primarily in muscle and spinal cord, but also in the brain, heart, lungs, intestine, liver, spleen, kidney, and blood (Zhang et al., 2018). However, outside the spinal cord and muscle, the significance of the amount of RNA detected is questionable and may well reflect the viral genome copies present in the blood within these tissues rather than true tropism for these tissues.

[0043] Studies in these models of paralysis and lethality have facilitated preliminary testing of antibodies' ability to protect against neurological disease. Passively transferred heat-inactivated serum from immunized mice into naive mice prevented neurological disease, even when EV-D68 was directly inoculated intracerebrally (Hixon et al., 2017b). Furthermore, therapeutic administration of human IVIG reduced motor deficits even up to 6 days after infection in mice (Hixon et al., 2017a), consistent with the finding of high titers of EV-D68-specific antibodies in IVIG (Zhang et al., 2015). This observation offers hope that vaccines or human monoclonal antibodies may mediate therapeutic effects after individual patients are identified as having features of AFM temporally associated with EV-D68. Furthermore, targeted vaccination or immunoprophylaxis in populations such as infants and young children, who are experiencing loss of maternal humoral immunity during known EV-D68 outbreaks, could prevent cases of AFM.

[0044] C. Animal Models of Respiratory Disease Major limitations of current mouse studies of EV-D68 infection include the use of a nonphysiological inoculation route to induce paralysis and death and the lack of respiratory disease, a primary symptom of EV-D68 infection in humans. In both cotton rats (Sigmodon hispidus) (Patel et al., 2016) and ferrets (Mustela putorius furo) (Zheng et al., 2017a), a single study demonstrated that intranasal inoculation of EV-D68 resulted in viral replication in the nose and lungs and induced an increase in proinflammatory innate immune molecule transcripts in the lungs. Approximately 25% of inoculated ferrets also developed clinical symptoms consistent with upper respiratory tract infection (cough, runny nose, and dry nose). Studies did not assess the presence of EV-D68 in muscle or central nervous system tissue, nor did they demonstrate significant limb weakness or paralysis. In findings described further below, vaccination of cotton rats showed that some levels of pre-existing humoral immunity do not fully protect against respiratory infection and may actually be harmful (Patel et al., 2016).

[0045] A theoretical advantage of cotton rats and ferrets as models of human respiratory virus infection is that their respiratory epithelia can relatively closely mimic those of humans, particularly with regard to the sialylation of glycans on the epithelial surface, which is highly α2,6-linked in humans but not in mice (Gagneux et al., 2003). EV-D68 preferentially bound to α2,6-linked sialic acid over α2,3-linked sialic acid in in vitro glycan arrays (Imamura et al., 2014). Staining of the airways with fluorescently labeled lectins showed that cotton rats had only α2,6-linked sialic acid in their lower respiratory tract and a mixture of α2,6- and α2,3-linked sialic acid in their upper respiratory tract (Blanco et al., 2018). Using similar lectin-based methods, ferrets have been shown to have α2,6-linked sialic acids on epithelial glycans in the upper respiratory tract (Leigh et al., 1995), while the lower respiratory tract displays a mixture of α2,6- and α2,3-linked sialic acids (Zheng et al., 2017a). However, more recent advanced glycomic analyses of respiratory tissues from humans (Walther et al., 2013), mice (Bern et al., 2013), and ferrets (Jia et al., 2014) have demonstrated that a more complex array of glycan modifications than simply the density of α2,6-linked sialylation likely determines respiratory virus tropism for various animal species. Nevertheless, the initial success of studies mimicking human EV-D68 respiratory disease in rats and ferrets has encouraged their further development as models of human pathogenesis.

[0046] D. Vaccines Although no experimental vaccine candidates for EV-D68 have yet entered clinical development, early studies of vaccinating mice with either virus-like particle (VLP) vaccines produced in yeast cells (Zhang et al., 2018a) or insect cells (Dai et al., 2018) or β-propiolactone-inactivated EV-D68 (Zhang et al., 2018) have shown promise. The VLP vaccines induced antibody responses that protected mice from subsequent lethal intraperitoneal challenge. For each of these vaccine candidates, passive transfer of vaccine-immune serum into naive mice was sufficient to protect against lethal intraperitoneal challenge. A potential drawback of VLP-based vaccines is uncertainty as to whether these synthetic constructs fully recapitulate all conformationally sensitive structures on the particle's surface. Many potent human virus-neutralizing antibodies against other viruses recognize complex quaternary antigenic sites with strict conformational constraints (Crowe, JE, 2017). The integrity of the human antibody epitopes on VLPs cannot currently be assessed because these epitopes are unknown. Further limiting the promise of these mouse vaccine trials are findings in cotton rats inoculated intramuscularly with either live or UV-inactivated EV-D68 and subsequently challenged intranasally with the homologous live virus, which showed enhanced inflammation in the lungs compared to infection in naive animals (Patel et al., 2016). Specifically, UV-inactivated virus vaccination did not limit viral replication in the lungs or nasal cavity and skewed the resulting cytokine signature toward a Th2 phenotype rather than the balanced Th1 and Th2 phenotypes seen with immunity from live virus vaccination. Enhanced inflammation and disease have been observed with inactivated virus vaccine candidates against respiratory syncytial virus (Karron, R, 2018) and measles (Strebel et al., 2018). Given these contradictory findings, further careful testing of the immune correlates of protection or enhancement elicited by EV-D68 vaccine candidates is warranted.

[0047] II. Monoclonal Antibodies and Their Generation An "isolated antibody" is one that has been separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody is purified (1) to greater than 95% by weight, and most particularly greater than 99% by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0048] The basic four-chain 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 along with an additional polypeptide called the J chain, and therefore contain ten antigen-binding sites, whereas secreted IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units along with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable region (V) at its N-terminus. H ), followed by three constant domains (C H ) followed by four C for μ and isotype H Each L chain has a variable domain (V L ) followed by a constant domain (C L ) followed by V L is V H Align with CL is the first constant domain of the heavy chain (C H1 ) and specific amino acid residues are believed to form an interface between the light-chain variable region and the heavy-chain variable region. H and V L The pairing of these two antibodies together forms a single antigen-binding site. For the structure and properties of various classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

[0049] The L chains from any vertebrate species contain their constant domains (C L Immunoglobulins can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their heavy chains (C H Depending on the amino acid sequence of the constant domain of the immunoglobulin, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called α, δ, ε, γ, and μ, respectively. The γ and α classes are H IgA1 and IgA2 are further divided into subclasses based on relatively minor differences in sequence and function, with humans expressing the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0050] The term "variable" refers to the fact that the sequence of certain segments of the V domain varies significantly among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Naturally occurring heavy and light chain variable regions each contain four FRs that primarily adopt a β-sheet structure, connected by three hypervariable regions, which form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0051] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V, VL, VH ... LApproximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) within V H approximately residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) within the V1 and V2 sequences; and / or residues from the "hypervariable loops" (e.g., V1 and V2 sequences, when numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). L Residues 24–34 (L1), 50–56 (L2), and 89–97 (L3) in V H residues 26-32 (H1), 52-56 (H2), and 95-101 (H3) within the "hypervariable loops" / CDRs (e.g., V when numbered according to the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)) L Residues 27–38 (L1), 56–65 (L2), and 105–120 (L3) in V H Optionally, the antibody comprises the following residues, when numbered according to AHo; Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001): V L 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in V sub They have symmetric insertions within H at one or more of positions 28, 36 (H1), 63, 74-75 (H2) and 123 (H3).

[0052] "Germline nucleic acid residue" refers to a nucleic acid residue that is naturally present in a germline gene encoding a constant or variable region. A "germline gene" is DNA found in germ cells (i.e., cells destined to become eggs or sperm). A "germline mutation" refers to a specific DNA heritable change that occurs in a germ cell or zygote at the single-cell stage; when transmitted to offspring, such a mutation is incorporated into all cells of the body. A germline mutation is in contrast to a somatic mutation, which is acquired in a single somatic cell. In some cases, a nucleotide in a germline DNA sequence encoding a variable region becomes mutated (i.e., a somatic mutation) and is replaced by a different nucleotide.

[0053] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced using recombinant DNA methods directed against bacterial, eukaryotic, or plant cells (see, e.g., U.S. Pat. No. 4,816,567) after single-cell sorting of antigen-specific B cells, antigen-specific plasmablasts in response to infection or immunization, or capture of linked heavy and light chains from single cells within a bulk-sorted antigen-specific collection. "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0054] A. General Method It is understood that monoclonal antibodies that bind to EV-D68 have several uses. These include the production of diagnostic kits for use in detecting and diagnosing EV-D68 infection and treating EV-D68 infection. In these situations, such antibodies may be linked to diagnostic or therapeutic agents, used as capture or competitor agents in competitive assays, or used individually without additional agents attached to them. Antibodies may be mutated or modified, as further described below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Pat. No. 4,196,265).

[0055] Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as methods for preparing polyclonal antibodies. In both of these methods, the first step is the immunization of a suitable host or the identification of a subject who is immune due to a previous natural infection or vaccination with an approved or experimental vaccine. As is well known in the art, a given composition for immunization may vary in its immunogenicity. Therefore, it is often necessary to enhance the host immune system, which can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of nonspecific stimulators of the immune response, known as adjuvants. In animals, exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant; in humans, they include combinations of alum, CpG, MFP59, and immune stimulatory molecules ("adjuvant systems" such as AS01 or AS03). Additional experimental inoculation formats for inducing EV-D68-specific B cells are possible, including nanoparticle vaccines or gene-encoded antigens delivered as DNA or RNA genes in physical delivery systems (such as lipid nanoparticles or gold biolistic beads) and delivered using needles, gene guns, or transcutaneous electroporation devices.The antigen gene can also be carried so as to be encoded by a replication-competent or defective viral vector, such as an adenovirus, adeno-associated virus, poxvirus, herpesvirus or alphavirus replicon, or virus-like particle.

[0056] In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects who have been exposed to the pathogen, for example, subjects who have been diagnosed with the disease, or subjects who have been vaccinated to generate protective immunity against the pathogen or to test the safety or efficacy of an experimental vaccine. Circulating anti-pathogen antibodies can be detected, and antibody-encoded or produced B cells from antibody-positive subjects can then be obtained.

[0057] The amount of immunogen composition used to generate polyclonal antibodies varies depending on the immunogen and the nature of the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored by sampling the immunized animal's blood at various times after immunization. A second booster injection may be administered. The process of boosting and titrating is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled, and the serum can be isolated and stored, and / or the animal can be used to generate MAbs.

[0058] Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in MAb generation protocols. These cells may be obtained from biopsied spleens, lymph nodes, tonsils, or adenoids, bone marrow aspirates or biopsies, tissue biopsies obtained from mucosal organs such as the lungs or gastrointestinal tract, or circulating blood. Antibody-producing B lymphocytes from the immunized animal or immunized human are then fused with cells of an immortal myeloma cell, generally one of the same species as the immunized animal, or human cells or human / mouse chimeric cells. Myeloma cell lines suitable for use in hybridoma-generating fusion procedures are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that render them unable to grow in specific selective media that support the growth of only the desired fused cells (hybridomas). As known to those skilled in the art, any one of a number of myeloma cell types may 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.

[0059] The method for generating hybrids between antibody-producing spleen cells or lymph node cells and myeloma cells typically involves mixing somatic cells and myeloma cells at a 2:1 ratio, but this ratio can vary from approximately 20:1 to approximately 1:1, respectively, in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion. In some cases, transformation of human B cells with Epstein-Barr virus (EBV) as an initial step increases B cell size and enhances fusion with relatively large myeloma cells. The efficiency of EBV transformation is enhanced by using CpG and Chk2 inhibitors in the transformation medium. Alternatively, human B cells can be activated by co-culturing them with a transfected cell line expressing CD40 ligand (CD154) in medium containing additional soluble factors, such as IL-21 and human B cell-activating factor (BAFF), a type II member of the TNF superfamily. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and fusion methods using polyethylene glycol (PEG), e.g., 37% (v / v) PEG, have been described by Gefter et al. (1977). Electrically induced fusion methods are also suitable (Goding, pp. 71-74, 1986), and processes exist to improve efficiency (Yu et al., 2008). Fusion procedures typically involve approximately 1 × 10 -6 ~1×10 -8Although viable hybrids are generated at a low frequency, optimized procedures can achieve fusion efficiencies approaching 1 in 200 (Yu et al., 2008). However, the relatively low efficiency of fusion is not a problem because viable fused hybrids differentiate from the parent injected cells (especially injected myeloma cells, which usually continue to divide indefinitely) by culturing them in selective medium. Selective medium generally contains agents that block de novo synthesis of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium (HAT medium) is supplemented with hypoxanthine and thymidine as a source of nucleotides. When azaserine is used, the medium is supplemented with hypoxanthine. If the source of B cells is an EBV-transformed human B cell line, ouabain is added to eliminate EBV-transformed cells that have not fused to myelomas.

[0060] The preferred selective medium is HAT or HAT containing ouabain. Only cells capable of operating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells lack key enzymes in the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and cannot survive. B cells can operate this pathway, but their lifespan in culture is limited and they generally die within about two weeks. Therefore, the only cells that can survive in selective medium are hybrids formed from myeloma cells and B cells. When the source of B cells used for fusion is an EBV-transformed B cell line, as in this specification, ouabain can also be used for drug selection of the hybrid, because the EBV-transformed B cells are sensitive to drug killing, while the myeloma partner used is selected to be ouabain-resistant.

[0061] Culture provides a population of hybridomas from which specific hybridomas are selected. Hybridoma selection is typically performed by culturing cells by single-clone dilution in microtiter plates, followed (approximately 2–3 weeks later) by testing individual clonal supernatants for the desired reactivity. Assays must be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, or dot immunobinding assays. Selected hybridomas are then serially diluted or single-cell sorted by flow cytometry sorting and cloned into individual antibody-producing cell lines, which can then be propagated indefinitely to produce mAbs. These cell lines can be utilized for MAb generation in two basic ways: A sample of the hybridomas can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animal can be primed with a hydrocarbon, particularly an oil such as pristane (tetramethylpentadecane), prior to injection. When using human hybridomas in this manner, it is best to inject them into immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animals develop tumors secreting the specific monoclonal antibodies produced by the fused cell hybrids. The animal's body fluids, such as serum or ascites, can then be tapped to provide high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where MAbs are naturally secreted into the culture medium, where they can be easily obtained in high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. Cell lines can be adapted for growth in serum-free medium to optimize the ability to recover highly pure human monoclonal immunoglobulins.

[0062] MAbs produced by either means may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0063] It is also contemplated that molecular cloning techniques can be used to generate monoclonal antibodies. Single B cells labeled with the antigen of interest can be physically selected using paramagnetic bead selection or flow cytometry sorting, and then RNA can be isolated from the single cells and antibody genes amplified by RT-PCR. Alternatively, antigen-specific bulk-sorted cell populations can be separated into microvesicles, and matched heavy and light chain variable genes can be recovered from single cells using physical linkage of heavy and light chain amplicons or common barcoding of heavy and light chain genes from vesicles. Matched heavy and light chain genes from single cells can also be obtained from a population of antigen-specific B cells by treating the cells with RT-PCR primers and cell-permeable nanoparticles carrying barcodes to mark transcripts with one barcode per cell. Antibody variable genes can also be isolated by RNA extraction of hybridoma lines, and antibody genes can be obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from cell lines, and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantage of this approach over traditional hybridoma technology is approximately 10 4The advantages of this approach are that twice as many antibodies can be generated and screened at once, and that new specificities can be generated by combining heavy and light chains, further increasing the chances of finding a suitable antibody.

[0064] Other U.S. patents that teach the production of antibodies useful in the present disclosure, each of which is incorporated herein by reference, include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using combinatorial techniques; U.S. Pat. No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0065] B. Antibodies of the Present Disclosure Antibodies according to the present disclosure can be defined, in a first example, by their binding specificity. By evaluating the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art, one skilled in the art can determine whether such an antibody falls within the scope of the present claims. For example, the epitope to which a given antibody binds can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within an antigen molecule (e.g., a linear epitope within a domain). Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within an antigen molecule (e.g., a conformational epitope).

[0066] Various 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 polypeptide or protein. Exemplary techniques include routine 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 by various 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 truncation analysis, high-resolution electron microscopy techniques using single-particle reconstruction, cryo-EM, or tomography, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling a protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit a relatively higher mass than amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing 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.If an antibody neutralizes EV-D68, antibody escape mutant variant organisms can be isolated by growing EV-D68 in vitro or in animal models in the presence of high concentrations of antibody. Sequence analysis of the EV-D68 gene encoding the antigen targeted by the antibody reveals mutations that confer antibody escape, indicating residues within the epitope or that allosterically affect the structure of the epitope.

[0067] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, or at least 8-10 amino acids in a unique spatial conformation.

[0068] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for categorizing multiple monoclonal antibodies (mAbs) directed against the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (see U.S. Patent No. 2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect unique epitopes that are distinct from or partially overlap with epitopes represented by other categories. This technique allows for the rapid filtering of genetically identical antibodies so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. MAP may also be used to sort the antibodies of the present disclosure into groups of antibodies that bind to different epitopes.

[0069] The present disclosure includes antibodies that can bind to the same epitope or a portion of the epitope.Similarly, the present disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof.Whether an antibody binds to the same epitope as a reference antibody or competes for binding with a reference antibody can be easily determined by using routine 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 bound to the target under saturating conditions.Then, the ability of the test antibody to bind to the target molecule is evaluated.If the test antibody can bind to the target molecule after saturating binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope from the reference antibody.On the other hand, if the test antibody cannot bind to the target molecule after saturating binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0070] To determine whether an antibody competes for binding with a reference anti-EV-D68 antibody, the above binding method is performed in two directions: in the first direction, the reference antibody is allowed to bind to the EV-D68 antigen under saturating conditions, followed by assessing the binding of the test antibody to the EV-D68 antigen. In the second direction, the test antibody is allowed to bind to the EV-D68 antigen molecule under saturating conditions, followed by assessing the binding of the reference antibody to the EV-D68 antigen. If only the first (saturating) antibody can bind to EV-D68 in both directions, it is concluded that the test and reference antibodies compete for binding to EV-D68. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody will not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0071] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other by at least 50%, preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially every amino acid mutation in the antigen that reduces or eliminates the binding of one antibody reduces or eliminates the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0072] Additional routine experiments (e.g., peptide mutation analysis and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment 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 by EM or crystallography can also demonstrate whether two antibodies competing for binding recognize the same epitope.

[0073] In another aspect, there are provided monoclonal antibodies having CDR pairings with clones derived from the heavy and light chains shown in Tables 3 and 4, respectively. Such antibodies can be generated using the methods described herein and by the clones illustrated below in the Examples section.

[0074] In another aspect, antibodies can be defined by their variable sequences, including additional "framework" regions. These are provided in Tables 1 and 2, which encode or represent complete variable regions. Furthermore, antibody sequences can be varied from these sequences, optionally using methods described in more detail below. For example, nucleic acid sequences can be prepared such that: (a) the variable regions can be separated from the light and heavy chain constant domains; (b) the nucleic acid can vary from those described above without affecting the residues encoded thereby; (c) the nucleic acid can vary from those described above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; and (d) the nucleic acid can be prepared by annealing in about 0.02 M to about 0.15 M NaCl at a temperature of about 50° C. to about 70° C. (e) the amino acids may vary from those described above by a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or (f) the amino acids may vary from those described above by allowing for conservative substitutions (described below). Each of the above applies to the nucleic acid sequences set forth in Table 1 and the amino acid sequences in Table 2.

[0075] When comparing a polynucleotide sequence with a polypeptide sequence, as described below, if the nucleotide or amino acid sequences in the two sequences are the same when aligned for maximum correspondence, the two sequences are said to be "identical." Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, the term "comparison window" refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, 40 to about 50 consecutive positions, within which the sequence can be compared with a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned.

[0076] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, 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, Suppl. 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, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105;Santou,N.Nes,M.(1987)Mol.Biol.Evol.4:406-425;Sneath,PHAand Sokal,RR(1973)Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy,Freeman Press,San Francisco,Calif.;Wilbur,WJand Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0077] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0078] A specific example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the percent sequence identity of the polynucleotides and polypeptides of the present disclosure. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The rearranged nature of antibody sequences and the variable length of each gene necessitates multiple BLAST searches for a single antibody sequence. Furthermore, manual assembly of different genes is difficult and prone to error. The sequence analysis tool IgBLAST (on the World Wide Web, ncbi.nlm.nih.gov / igblast / ) identifies matches to germline V genes, germline D genes, and germline J genes, details at rearrangement junctions, and delineation of Ig V domain framework regions and complementarity-determining regions. IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches, allowing searches against germline gene databases and other sequence databases simultaneously, minimizing the possibility of potentially missing the best-matching germline V gene.

[0079] In one illustrative example, for nucleotide sequences, the cumulative score may be calculated using the parameters M (reward score value for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score becomes zero or below due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a string length (W) of 11, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, a B of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0080] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0081] In one approach, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain 20 percent or less, typically 5-15 percent, or 10-12 percent additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0082] Yet another way to define an antibody is as a "derivative" of any of the antibodies and antigen-binding fragments thereof described below. The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but contains one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses variants having altered CH1, hinge, CH2, CH3, or CH4 regions to form, for example, antibodies with variant Fc regions that exhibit enhanced or impaired effector or binding properties. The term "derivative" further encompasses non-amino acid modifications, e.g., amino acids that may be glycosylated (e.g., altered in content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytic cleavage, linked to cellular ligands or other proteins, etc. In some embodiments, the altered carbohydrate modification modulates one or more of antibody solubilization, promoting intracellular trafficking and secretion of the antibody, promoting antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, the altered carbohydrate modification enhances antibody-mediated effector function compared to an antibody lacking the carbohydrate modification.Carbohydrate modifications that result in altered antibody-mediated effector functions are well known in the art (see, e.g., Shields, R. Let al. (2002) "Lack of Fucose on Human IgG N-Linked Oligosaccharide Improves Binding to Human Fc gamma RIII and Antibody-Dependent Cellular Toxicity," J. Biol. Chem. 277(30):26733-26740; Davies J. et al. (2001) "Expression of GnTIII in a Recombinant Anti-CD20 CHO Production Cell Line: Expression of Antibodies with Altered Glycoforms Leads to an Increase in ADCC Through Higher Affinity for Fc gamma RIII," Biotechnology & Bioengineering 74(4):288-294).Methods for altering carbohydrate content are known to those skilled in the art, and include, for example, Wallick, SC et al. (1988) "Glycosylation of a VH residue of a monoclonal antibody against alpha(1-6) dextran increases its affinity for antigen," J. Exp. Med. 168(3):1099-1109; Tao, MH et al. (1989) "Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region," J. Immunol. 143(8):2595-2601; Routledge, E. G. et al. (1995) "The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody," Transplantation 60(8):847-53; Elliott, S. et al. (2003) “Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,” Nature Biotechnol. 21:414-21; Shields, RLet al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII 277(30):26733-26740).

[0083] Derivative antibodies or antibody fragments can be generated with engineered sequences or glycosylation states to confer desired levels of activity in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function, as measured by bead- or cell-based assays, or in vivo studies in animal models.

[0084] Derivative antibodies or derivative antibody fragments may be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one aspect, an antibody derivative has similar or identical function as the parent antibody. In another aspect, an antibody derivative exhibits altered activity compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more strongly than the parent antibody or be more resistant to proteolysis than the parent antibody.

[0085] C. Manipulation of antibody sequences In various embodiments, one may choose to manipulate the sequence of an identified antibody for various reasons, such as improving expression, improving cross-reactivity, or reducing off-target binding. Modified antibodies can be produced by any technique known to those skilled in the art, including expression by standard molecular biology techniques or chemical synthesis of polypeptides. Methods for recombinant expression are discussed elsewhere in this document. The following is a general description of relevant targeting techniques for antibody engineering.

[0086] The hybridomas are cultured, and then the cells are lysed and total RNA is extracted. cDNA copies of the RNA can be generated using random hexamers with RT, followed by PCR using a multiplexed mixture of PCR primers predicted to amplify all human variable gene sequences. The PCR products can be cloned into the pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using antibodies recovered from hybridoma supernatants and purified by FPLC using a Protein G column.

[0087] Recombinant full-length IgG antibodies can be produced by subcloning the heavy and light chain Fv DNA from a cloning vector into an IgG plasmid vector and transfecting 293 (e.g., Freestyle) or CHO cells, and the antibody can be recovered and purified from the supernatant of the 293 or CHO cells. Other suitable host cell systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without human-like glycan engineering), algae, or various non-human transgenic contexts, such as mouse, rat, goat, or cow.

[0088] Expression of nucleic acids encoding antibodies is also contemplated, both for subsequent antibody purification and for host immunization. The antibody-encoding sequence can be RNA, such as natural or modified RNA. Modified RNA contemplates specific chemical modifications that confer increased stability and reduced immunogenicity to mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translational potency. In addition to turning off immune / eIF2α phosphorylation-dependent translation inhibition, the incorporated N1mΨ nucleotide dramatically alters the kinetics of the translation process by increasing ribosome pausing and density on mRNA. The increased ribosome loading of modified mRNAs makes them more permissive for initiation by promoting either ribosome recycling or de novo ribosome recruitment on the same mRNA. Such modifications can be used to enhance antibody expression in vivo after RNA inoculation. RNA, whether natural or modified, can be delivered as naked RNA or within a delivery vehicle such as a lipid nanoparticle.

[0089] Alternatively, DNA encoding an antibody can be used for the same purpose. The DNA is contained in an expression cassette containing a promoter active in host cells, and the promoter is designed for the host cell. The expression cassette is advantageously contained in a replicable vector, such as a conventional plasmid or minivector. The vector includes viral vectors, such as poxvirus, adenovirus, herpesvirus, adeno-associated virus, and lentivirus. Replicons encoding antibody genes, such as alphavirus replicons based on VEE virus or Sindbis virus, are also contemplated. Such vectors can be delivered by needle via intramuscular, subcutaneous, or intradermal route, or by transcutaneous electroporation if in vivo expression is desired.

[0090] The rapid availability of antibodies produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to shorten the duration of process development programs. Lonza has developed a general method using pooled transfectants grown in CDACF medium to rapidly produce small amounts (up to 50 g) of antibody in CHO cells. While slightly slower than true transient systems, advantages include relatively high product concentrations and the use of the same host and process as the production cell line. In an example of growth and productivity of a GS-CHO pool expressing a model antibody in a single-use bioreactor, i.e., a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a recovered antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0091] Antibody molecules include, for example, fragments produced by proteolytic cleavage of mAbs (e.g., F(ab'), F(ab')2), or single-chain immunoglobulins, which may be produced, for example, by recombinant means. F(ab')2 antibody derivatives are monovalent, while F(ab')2 antibody derivatives are divalent. In one embodiment, such fragments can be combined with each other, or with other antibody fragments or receptor ligands, to form "chimeric" binding molecules. Importantly, such chimeric molecules can contain substituents capable of binding to different epitopes of the same molecule.

[0092] In a related embodiment, the antibody is a derivative of the disclosed antibody, for example, an antibody comprising the same CDR sequence as the disclosed antibody (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, one may wish to modify the antibody molecule, such as by introducing conservative changes. When making such changes, the hydropathic amino acid index can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, thereby defining the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0093] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, describes that the maximum local average hydrophilicity of a protein, as controlled by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0) and histidine (-0.5); acidic amino acids: aspartate (+3.0±1), glutamate (+3.0±1), asparagine (+0.2) and glutamine (+0.2); hydrophilic non-ionic 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 non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5) and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5) and tyrosine (-2.3).

[0094] It is understood that an amino acid can be substituted for another having a similar hydrophilicity to produce a biologically or immunologically modified protein, with substitution of amino acids whose hydrophilicity values ​​are within ±2 being preferred, those within ±1 being particularly preferred, and those within ±0.5 being even more particularly preferred.

[0095] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0096] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve binding valency.

[0097] Alternatively or additionally, it may be useful to combine the amino acid modification with one or more additional 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. Binding polypeptides of particular interest may be those that bind to C1q and exhibit complement-dependent cytotoxicity. Polypeptides that have existing C1q binding activity and, optionally, the ability to mediate CDC may be modified to enhance one or both of these activities. Amino acid modifications that alter C1q and / or modify its complement-dependent cytotoxicity function are described, for example, in International Publication No. WO 0042072, which is incorporated herein by reference.

[0098] For example, an Fc region of an antibody can be engineered with altered effector functions by modifying C1q binding and / or FcγR binding, thereby altering CDC and / or ADCC activity. An "effector function" serves to activate or reduce a biological activity (e.g., of interest). 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 receptors; BCRs); and the like. Such effector functions may require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0099] For example, a variant Fc region of an antibody can be generated that has improved C1q binding and improved FcγRIII binding (e.g., has both improved ADCC activity and improved CDC activity). Alternatively, if reduced or eliminated effector function is desired, the variant Fc region can be engineered with reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and optionally, the other activity may also be reduced (e.g., generating an Fc region variant with improved ADCC activity but reduced CDC activity, and vice versa).

[0100] FcRn binding. Fc mutations can also be introduced and engineered to alter interactions with the neonatal 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). Several methods are known that can result in extended half-life, including amino acid modifications, through techniques including alanine scanning mutagenesis, random mutagenesis, and screening to evaluate binding to the neonatal Fc receptor (FcRn) and / or in vivo behavior (Kuo and Aveson, (2011)). Computational strategies following mutagenesis can also be used to select one of the amino acid mutations to mutate.

[0101] Thus, the present disclosure provides variants of antigen-binding proteins that are optimized for binding to FcRn. In certain embodiments, the variants of antigen-binding proteins comprise at least one amino acid modification in the Fc region of the antigen-binding protein, wherein the modification is 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 3 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 and 447, wherein the numbering of the amino acids in the Fc region is that of the EU index of Kabat. In a further aspect of the disclosure, the modification is M252Y / S254T / T256E.

[0102] Furthermore, various publications describe methods for obtaining physiologically active molecules with altered half-lives by introducing an FcRn-binding polypeptide into the molecule, or by fusing the molecule to an antibody with preserved FcRn-binding affinity but significantly reduced affinity for other Fc receptors, or by fusing the molecule to the FcRn-binding domain of an antibody, see, for example, Kontermann (2009).

[0103] Derivatized antibodies may be used to alter the half-life (e.g., serum half-life) of a parent antibody in a mammal, particularly a human. Such alterations can result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. In a mammal, preferably a human, an increased half-life of an antibody or antibody fragment thereof of the present disclosure results in a higher serum titer of the antibody or antibody fragment in the mammal, thereby reducing the frequency of administration of the antibody or antibody fragment and / or reducing the concentration of the antibody or antibody fragment administered. Antibodies or fragments thereof with increased in vivo half-lives can be generated by techniques known to those skilled in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified to be involved in the interaction between the Fc domain and the FcRn receptor.

[0104] Beltramello et al. (2010) previously reported the modification of a neutralizing mAb by substituting alanine residues for the leucine residues at positions 1.3 and 1.2 (according to the IMGT unique numbering system for C domains) in the CH2 domain due to its tendency to enhance dengue virus infection. This modification, also known as the "LALA" mutation, abolishes antibody binding to FcγRI, FcγRII, and FcγRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAb were compared for their ability to neutralize and enhance infection by four dengue virus serotypes. The LALA variant retained the same neutralizing activity as the unmodified mAb but completely lacked the enhancing activity. Therefore, LALA mutations of this nature are contemplated in connection with the antibodies of the present disclosure.

[0105] Altered glycosylation. A particular aspect of the present disclosure is an isolated monoclonal antibody or antigen-binding fragment thereof containing substantially uniform glycans that do not contain sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be linked to a heavy chain constant region or a light chain constant region, respectively. The substantially uniform glycans may be covalently linked to the heavy chain constant region.

[0106] Another embodiment of the present disclosure includes a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody or antigen-binding fragment thereof is present in a substantially uniform composition represented by the GNGN glycoform or the G1 / G2 glycoform. Fc glycosylation plays an important role in the antiviral and anticancer properties of therapeutic mAbs. This disclosure is consistent with recent studies showing increased in vitro inhibition of anti-lentiviral cell-mediated virus by fucose-free anti-HIV mAbs. This embodiment of the disclosure, with uniform glycans lacking core fucose, demonstrated a greater than two-fold increase in protection against certain viruses. Removal of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells, but appears to have the opposite effect on ADCC activity of polymorphonuclear cells (PMNs).

[0107] An isolated monoclonal antibody or antigen-binding fragment thereof comprising a substantially homogeneous composition represented by GNGN glycoforms or G1 / G2 glycoforms exhibits increased binding affinity to FcγRI and FcγRIII compared to the same antibody lacking substantially homogeneous GNGN glycoforms and having glycoforms containing G0, G1F, G2F, GNF, GNGNF, or GNGNFX. In one embodiment of the disclosure, the antibody is lysed at a concentration of 1×10 -8 from FcγRI with a Kd of 1 × 10 -7 It dissociates from FcγRIII with a Kd of M or less.

[0108] Glycosylation of the Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequence are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences within a polypeptide creates a potential glycosylation site.

[0109] The glycosylation pattern may be altered, for example, by deleting one or more glycosylation sites found 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 is conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn297 of the heavy chain. Alterations can also be made by adding or substituting one or more serine or threonine residues to the original polypeptide sequence (for O-linked glycosylation sites). Furthermore, altering Asn297 to Ala can eliminate one of the glycosylation sites.

[0110] In certain embodiments, the antibody is expressed in cells expressing β(1,4)-N-acetylglucosaminyltransferase III (GnT III), so that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing antibodies in this manner are provided in International Publication No. 9954342, International Publication No. 03011878, Patent Publication No. 20030003097, 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 specific post-translational modifications, such as glycosylation. For example, CRISPR technology can be used to eliminate genes encoding glycosylation enzymes in 293 cells or CHO cells used to express recombinant monoclonal antibodies.

[0111] Elimination of monoclonal antibody protein sequence liabilities. Antibody variable gene sequences obtained from human B cells can be engineered to increase their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs associated with sites including: 1) unpaired Cys residues, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) SYE cutting, 6) Met oxidation, 7) Trp oxidation, 8) N-terminal glutamate, 9) integrin binding, 10) CD11c / CD18 binding, or 11) Fragmentation

[0112] Such motifs can be eliminated by altering the synthetic gene of the cDNA encoding the recombinant antibody.

[0113] Protein engineering efforts in the field of therapeutic antibody development have clearly revealed that specific 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 indicates 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.

[0114] Stability. Antibodies can be engineered to enhance biophysical properties. High temperatures can be used to unfold antibodies and determine their relative stability using the average apparent melting temperature. Differential scanning calorimetry (DSC) measures the heat capacity, C, of ​​the molecule as a function of temperature. p DSC measures the heat required to warm it (per degree). DSC can be used to test the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it resolves the unfolding of individual domains within the mAb structure, which can produce up to three peaks in the thermogram (Fab domain, C H 2 domain and C H(from the unfolding of three domains). Typically, unfolding of the Fab domain produces the most intense peak. DSC profiles and the relative stability of the Fc portion show distinctive differences among human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). Circular dichroism (CD) performed with a CD spectrometer can also be used to determine the average apparent melting temperature. Far-UV CD spectra are measured for antibodies in the range of 200-260 nm with 0.5 nm increments. The final spectrum can be determined as the average of 20 accumulations. Residue ellipticity values ​​can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg / mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C / min. Dynamic light scattering (DLS) can be used to assess aggregation propensity. DLS is used to characterize the size of various particles, including proteins. When a system is not size-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. DLS essentially measures the fluctuations in scattered light intensity by particles, allowing the particle's diffusion coefficient to be determined. DLS software on commercially available DLS instruments can indicate particle populations of various diameters. Stability testing can be easily performed using DLS. DLS measurements of a sample can indicate whether particles aggregate over time or with temperature fluctuations by determining whether the hydrodynamic radius of the particles increases. When particles aggregate, a relatively large population of particles with a relatively large radius can be observed. Temperature-dependent stability can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include a proven method for characterizing antibody stability.The iCE technique can be used to resolve antibody protein charge variants resulting from deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other alterations to the protein that may result in a change in the protein's pI. Each expressed antibody protein can be evaluated by high-throughput free-solution isoelectric focusing (IEF) in a capillary column (cIEF) using the Protein Simple Maurice instrument. UV absorbance detection across the column can be performed every 30 seconds for real-time monitoring of molecules focusing at their isoelectric point (pI). This technique combines the high resolution of traditional gel IEF with the quantitation and automation advantages of column-based separations while eliminating the need for a mobilization step. This technique provides reproducible quantitative analysis of the identity, purity, and heterogeneity profile of expressed antibodies. Results identify antibody charge heterogeneity and molecular size in both absorbance and native fluorescence detection modes, with a detection sensitivity of as low as 0.7 μg / mL.

[0115] Solubility. The intrinsic solubility score of an antibody sequence can be determined. This can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequence of residues 95-102 (Kabat numbering) within the HCDR3 of each antibody fragment, such as an scFv, can be evaluated via an online program to calculate the solubility score. Laboratory techniques can also be used to determine solubility. Various techniques exist, including adding lyophilized protein to a solution until the solution is saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cutoff. The simplest method is the induction of an amorphous precipitate, which involves protein precipitation using ammonium sulfate to measure protein solubility (Trevino et al., J Mol Biol 366:449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information regarding relative solubility values. Ammonium sulfate precipitation produces a precipitation solution with well-defined aqueous and solid phases and requires a relatively small amount of protein. Solubility measurements performed using ammonium sulfate to induce amorphous precipitation can also be easily performed at various pH values. Protein solubility is highly pH-dependent, and pH is considered the most important extrinsic factor affecting solubility.

[0116] Autoreactivity. While it is generally believed that autoreactive clones should be eliminated by negative selection during ontogeny, it has become clear that many natural human antibodies with autoreactive properties persist in the adult mature repertoire, and that autoreactivity can enhance the antiviral function of many antibodies against pathogens. It has been noted that the HCDR3 loops within antibodies during early B cell development are often rich in positive charges and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). A given antibody can be tested for autoreactivity by assessing its level of binding to cells of human origin using microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometry cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity can also be investigated using assessment of tissue binding in tissue arrays.

[0117] Preferred residues ("human similarity"). Many recent studies have performed extensive deep sequencing of the B cell repertoire of human B cells derived from blood donors. Sequence information for a significant portion of the human antibody repertoire facilitates statistical evaluation of antibody sequence features common in healthy humans. Knowledge of antibody sequence features in the Human Recombinant Antibody Variable Gene Reference Database can be used to estimate the position-specific degree of "human similarity" (HL) of an antibody sequence. HL has been shown to be useful in antibody development for clinical applications, such as therapeutic antibodies or antibodies as vaccines. The goal is to increase the human similarity of antibodies to reduce potential adverse effects and anti-antibody immune responses that can result in a significant reduction in the efficacy of antibody drugs or induce serious health consequences. We were able to evaluate the antibody characteristics of the combined antibody repertoire of three healthy human blood donors, totaling approximately 400 million sequences, and created a novel "relative human similarity" (rHL) score that focuses on the hypervariable regions of antibodies. The rHL score allows for easy discrimination between human sequences (positive scores) and non-human sequences (negative scores). Antibodies can be engineered to eliminate residues that are uncommon in the human repertoire.

[0118] D. Single chain antibody Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains linked together by a short (usually serine or glycine) linker. These chimeric molecules retain the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification usually leaves the specificity unchanged. These molecules were originally generated to facilitate phage display, where it is very convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0119] Flexible linkers are generally composed of helix- and turn-promoting amino acid residues, such as alanine, serine, and glycine, although other residues can function as well. Tang et al. (1996) used phage display as a means to rapidly select tailored linkers for single-chain antibodies (scFv) from protein linker libraries. A random linker library was constructed in which genes for heavy and light chain variable domains were linked by segments encoding 18-amino acid polypeptides of variable composition. The scFv repertoire (approximately 5 × 10 6 Different members of the V-tether were displayed on filamentous phage and subjected to affinity selection with haptens. The population of selected variants showed a marked increase in binding activity while retaining considerable sequence diversity. Screening of 1054 individual variants subsequently yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed the V-tether as the only common feature of the selected tethers. HA conserved proline in the linker two residues after the C-terminus and abundant arginines and prolines at other positions were revealed.

[0120] The recombinant antibodies of the present disclosure may also contain sequences or moieties that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers in conjunction with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin / avidin, which allow the combination of two antibodies.

[0121] In another embodiment, single-chain antibodies can be produced by linking the light and heavy chains of the receptor using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced in separate cells, purified, and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via a suitable chemical bridge).

[0122] Cross-linking reagents, such as stabilizers and coagulants, are used to form molecular bridges that connect the functional groups of two different molecules.However, it is possible to create dimers or multimers of the same analog, or heteromeric complexes composed of different analogs.To link two different compounds stepwise, heterobifunctional cross-linkers can be used, which eliminates the formation of undesired homopolymers.

[0123] An exemplary heterobifunctional crosslinker contains two reactive groups, one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). The crosslinker may react with a lysine residue of one protein (e.g., a selected antibody or fragment) via the primary amine reactive group, while the crosslinker already attached to the first protein reacts with a cysteine ​​residue (free sulfhydryl group) of another protein (e.g., a selected agent) via the thiol reactive group.

[0124] Preferably, the crosslinker used has reasonable stability in blood.Many kinds of disulfide bond-containing linkers are known, which can be used successfully to conjugate targeting agent and therapeutic / prophylactic agent.The linker containing sterically hindered disulfide bond can bring about greater stability in vivo, and can prevent the release of targeting peptide before reaching the site of action.Therefore, these linkers are one group of linking agents.

[0125] Another cross-linking reagent is SMPT, a bifunctional cross-linker containing a disulfide bond "sterically hindered" by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond is thought to protect the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent the conjugate from dissociating before the attached drug can be delivered to the target site.

[0126] The SMPT cross-linking reagent, like many other known cross-linking reagents, confers the ability to cross-link functional groups such as cysteine ​​or primary amine SH (e.g., the ε-amino group of lysine). Another possible type of cross-linker includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenyl azide (upon photolysis) reacts nonselectively with any amino acid residue.

[0127] In addition to hindered crosslinkers, non-hindered linkers can also be used in accordance with the present invention. Other useful crosslinkers that are not thought to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0128] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for generating conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under various mild conditions. This linker is particularly useful in that the active agent of interest can be directly attached to the linker, and cleavage can release the active agent. Specific applications include adding free amino groups or free sulfhydryl groups to proteins, such as antibodies or drugs.

[0129] U.S. Patent No. 5,856,456 provides peptide linkers for use in connecting polypeptide components to create fusion proteins, such as single-chain antibodies. The linkers are up to about 50 amino acids in length and contain at least one charged amino acid (preferably arginine or lysine) followed by proline, and are characterized by relatively high stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immunodiagnostic and separation techniques.

[0130] E. Multispecific antibodies In certain embodiments, the antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies with binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen-binding site with a binding site for a second antigen. Alternatively, an anti-pathogen arm may be combined with an arm that binds to a trigger molecule on leukocytes, such as a T cell receptor molecule (e.g., CD3), or an IgG Fc receptor (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus and localize cellular defense mechanisms to infected cells. Bispecific antibodies can also be used to localize cytotoxic agents to infected cells. These antibodies possess a pathogen-binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate, or radioisotope 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. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-FcγRI antibody. WO 98 / 02463 discloses a bispecific anti-ErbB2 / Fcα antibody. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody.

[0131] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, with the two chains having different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, with only one of the 10 having the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography, but this is quite laborious and results in low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0132] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion comprises the addition of at least one variable domain, such as a hinge region, C H2 Area and C H3 The fusions are with an Ig heavy-chain constant domain containing at least part of the heavy-chain constant region (C) containing the site necessary for light-chain binding, present in at least one of the fusions. H1 ) DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. In embodiments where the optimal yield of the desired bispecific antibody is obtained when the three polypeptide chains used in the construction are not equal in ratio, this provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments. However, where expression of at least two polypeptide chains in equal ratios results in high yields, or where the ratio does not significantly affect the yield of the desired chain combination, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0133] In a particular embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from undesired immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a facile separation method. This approach is disclosed in WO 94 / 04690. For additional details on the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0134] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. A preferred interface is C H3 The antibody comprises at least a portion of a domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain is created on the interface of a second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers.

[0135] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0136] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0137] Technology exists that facilitates the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the generation of humanized bispecific antibody F(ab')2 molecules. To form the bispecific antibody, each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro. The bispecific antibody thus formed was capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, as well as inducing the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0138] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol. 16, 677-681 (1998) doi:10.1038 / nbt0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). Leucine zipper peptides derived from Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. To form antibody heterodimers, antibody homodimers were reduced at the hinge region to form monomers and then reoxidized. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), provides an alternative mechanism for making bispecific antibody fragments. The fragments may be separated by a linker that is too short to allow pairing between the two domains on the same chain. L V connected to H Therefore, one fragment of V H Domains and V L The domain is a complementary V L Domains and V HThe Fv domains are paired together, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0139] In certain embodiments, bispecific or multispecific antibodies can be formed as DOCK-AND-LOCK™ (DNL™) complexes (see, e.g., U.S. Pat. Nos. 7,521,056, 7,527,787, 7,534,866, 7,550,143, and 7,666,400, the Examples section of each of which is incorporated herein by reference). Generally, this technology utilizes the specific, high-affinity binding interaction that occurs between the dimerization and docking domain (DDD) sequence of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA) and the anchor domain (AD) sequence from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005;579:3264; Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004;5:959). The DDD and AD peptides may be attached to any protein, peptide, or other molecule. Because the DDD sequence spontaneously dimerizes and binds to the AD sequence, this technique allows the formation of complexes between any selected molecule that can attach to the DDD or AD sequence.

[0140] Antibodies with more than two binding valencies are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60, 1991; Xu et al., Science, 358(6359):85-90, 2017). Multivalent antibodies may be internalized (and / or catabolized) more quickly by cells expressing the antigen to which the antibody binds than bivalent antibodies. The antibodies of the present disclosure may be multivalent antibodies (e.g., tetravalent antibodies) with three or more antigen-binding sites, which can be easily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies can include a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or hinge region. In this context, the antibody comprises an Fc region and three or more antigen-binding sites amino-terminal to the Fc region. A preferred multivalent antibody herein comprises (or consists of) three to about eight, preferably four, antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), and the polypeptide chain comprises two or more variable regions. For example, the polypeptide chain may comprise VD1-(X1) n -VD2-(X2) n -Fc, where VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (preferably four) light chain variable region polypeptides. The multivalent antibody herein may comprise, for example, about two to about eight light chain variable region polypeptides. The light chain variable region polypeptide contemplated herein comprises a light chain variable region and optionally a C L Further includes domains.

[0141] In the context of multispecific antibodies, charge modifications are particularly useful, as amino acid substitutions in Fab molecules reduce mispairing of light chains with mismatched heavy chains (Bence-Jones type by-products) that can occur during the generation of Fab-based bispecific / multispecific antigen-binding molecules using VH / VL exchange in one (or more, in the case of molecules comprising more than two antigen-binding Fab molecules) of the binding arms of the Fab-based bispecific / multispecific antigen-binding molecule (see also PCT Publication No. WO2015 / 150447, which is incorporated herein by reference in its entirety, and in particular the Examples therein).

[0142] Thus, in certain embodiments, the antibody included in the therapeutic agent is (a) a first Fab molecule that specifically binds to a first antigen; (b) a second Fab molecule that specifically binds to a second antigen, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other; the first antigen is an activating T cell antigen and the second antigen is a target cell antigen, or the first antigen is a target cell antigen and the second antigen is an activating T cell antigen; where: i) in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index), or ii) In the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index).

[0143] The antibody may not comprise both modifications described under i) and ii): the constant domains CL and CH1 of the second Fab molecule are not replaced by each other (i.e., remain unexchanged).

[0144] In another embodiment of the antibody, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in a preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0145] In a further embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (numbering according to Kabat); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (numbering according to Kabat EU index).

[0146] In a particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted (numbering according to Kabat) (in a preferred embodiment independently by lysine (K) or arginine (R)) by lysine (K), arginine (R) or histidine (H), and the amino acid at position 123 is substituted (numbering according to Kabat) (in a preferred embodiment independently by lysine (K) or arginine (R)) by lysine (K), arginine (R) or histidine (H), and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index), and the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0147] In a more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0148] In an even more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by an arginine (R) (numbering according to Kabat); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0149] F. Chimeric Antigen Receptor Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors (CARs)) are engineered receptors that transfer any specificity onto immune effector cells. Typically, these receptors are used to transfer the specificity of monoclonal antibodies into T cells through the transfer of their coding sequences facilitated by retroviral vectors. In this way, large numbers of target-specific T cells can be generated for adoptive cell transfer. Phase I clinical trials of this technique have shown efficacy.

[0150] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the CD3-ζ transmembrane domain and endodomain. Such molecules result in the transduction of the ζ signal in response to recognition of its target by the scFv. An example of such a construct is 14g2a-ζ, a fusion of an scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). Upon expression of this molecule (usually achieved by oncoretroviral vector transduction), T cells recognize and kill target cells (e.g., neuroblastoma cells) that express GD2. To target malignant B cells, researchers have used chimeric immune receptors specific for the B-lineage molecule, CD19, to redirect T cell specificity.

[0151] The variable portions of the immunoglobulin heavy and light chains are fused by a flexible linker to form scFvs. The scFvs are preceded by a signal peptide that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression (which is cleaved). The flexible spacer allows the scFvs to be oriented in different directions, enabling antigen binding. The transmembrane domain is a typical hydrophobic α-helix, usually derived from the original molecule, and the signaling endodomain that protrudes into the cell and transmits the desired signal.

[0152] Type I proteins are actually two protein domains connected by a transmembrane α-helix between them. The cell membrane lipid bilayer through which the transmembrane domain passes acts to isolate the inner part (endodomain) from the outer part (ectodomain). It is not surprising that attaching the ectodomain from one protein to the endodomain of another results in a molecule that combines the recognition of the former with the signal of the latter.

[0153] Ectodomain. The signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential for the receptor to be glycosylated and anchored to the cell membrane. Any eukaryotic signal peptide sequence will usually work well. Generally, the signal peptide naturally attached to the amino-terminal most component is used (e.g., in an scFv with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0154] The antigen recognition domain is usually an scFv, although many alternatives exist. Antigen recognition domains derived from the α and β single chains of natural T cell receptors (TCRs) have been described as having simple ectodomains (e.g., the CD4 ectodomain for recognizing HIV-infected cells) and relatively foreign recognition components, such as linked cytokines (which result in recognition of cells bearing cytokine receptors). In fact, almost anything that binds with high affinity to a given target can be used as an antigen recognition region.

[0155] The spacer region connects the antigen-binding domain to the transmembrane domain. It must be flexible enough to allow the antigen-binding domain to orient in various directions to facilitate antigen recognition. The simplest form is the hinge region 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 must often be determined empirically.

[0156] Transmembrane domain. The transmembrane domain is a hydrophobic α-helix that spans the membrane. Generally, the transmembrane domain from the membrane-most component of the endodomain is used. Interestingly, the use of the CD3-ζ transmembrane domain can result in the integration of an artificial TCR into a native TCR, a factor that depends on the presence of the native CD3-ζ transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stabilities. The CD28 transmembrane domain results in a stable receptor that is vividly expressed.

[0157] The endodomain is the receptor's "end of the line." After antigen recognition, receptors form clusters and transmit signals to the cell. The most commonly used endodomain component is CD3-ζ, which contains three ITAMs. It transmits activation signals to T cells after antigen binding. CD3-ζ may not provide a fully competent activation signal, and additional costimulatory signaling is required.

[0158] "First-generation" CARs typically contained an intracellular domain derived from the CD3 ξ-chain, the primary transmitter of signals from the endogenous TCR. "Second-generation" CARs added intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to T cells. Preclinical studies have shown that second-generation CAR designs improve the antitumor activity of T cells. More recently, "third-generation" CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance efficacy.

[0159] G. ADC Antibody-drug conjugates (ADCs) are a new class of highly potent biopharmaceuticals designed as targeted therapies for treating people with infectious diseases. ADCs are composite molecules composed of an antibody (whole mAb or antibody fragment, e.g., single-chain variable fragment, or scFv) linked to a biologically active cytotoxic / antiviral payload or drug via a stable chemical linker with a labile bond. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0160] By combining the unique targeting capabilities of monoclonal antibodies with the cancer-killing potential of cytotoxic drugs, antibody-drug conjugates enable highly sensitive discrimination between healthy and diseased tissues. This means that, in contrast to traditional systemic approaches, antibody-drug conjugates target and attack infected cells, leaving healthy cells less severely affected.

[0161] In developing ADC-based antitumor therapies, anticancer drugs (e.g., cytotoxins or cytotoxic agents) are coupled to antibodies that specifically target specific cell markers (e.g., ideally, proteins found only in or on infected cells). The antibodies track these proteins throughout the body and attach to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal within the tumor cell, causing the tumor cell to absorb or internalize the antibody along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cell or impairing viral replication. Due to this targeting, the drug ideally has fewer side effects and a wider therapeutic window than other drugs.

[0162] A stable linkage between the antibody and the cytotoxic / antiviral agent is a key aspect of ADCs. Linkers are based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable) or thioethers (non-cleavable), to control the distribution and delivery of the cytotoxic agent to target cells. Preclinical and clinical studies have demonstrated the safety of both cleavable and non-cleavable linkers. Brentuximab vedotin, a potent and highly toxic antimicrotubule agent, contains an enzyme-sensitive cleavable linker that delivers the synthetic anti-neoplastic agent monomethyl auristatin E (MMAE) to human specific CD30-positive malignant cells. Due to its high toxicity, MMAE inhibits cell division by blocking tubulin polymerization, preventing its use as a single-agent chemotherapy drug. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor (TNF) receptor) has been shown to be stable in extracellular fluids, cleavable by cathepsins, and safe for treatment. Another approved ADC, trastuzumab emtansine, is a combination of the microtubule inhibitor mertansine (DM-1), a derivative of maytansine, and the antibody trastuzumab (Herceptin® / Genentech / Roche) attached by a stable, non-cleavable linker.

[0163] The availability of better and more stable linkers has changed the function of chemical bonds. The type of cleavable or non-cleavable linker confers specific properties to cytotoxic (anti-cancer) drugs. For example, a non-cleavable linker retains the drug intracellularly. As a result, the entire antibody, linker, and cytotoxic agent enters the targeted cancer cell, where the antibody is degraded to the amino acid level. The resulting complex (amino acid, linker, and cytotoxic agent) then becomes the active drug. In contrast, a cleavable linker is catalyzed by an enzyme within the host cell, releasing the cytotoxic agent.

[0164] 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 highly flexible ADCs without worrying about changes in cleavage rates. Researchers are also developing new methods for peptide cleavage based on Edman degradation, a method for sequencing amino acids within a peptide. Future directions in ADC development also include the development of site-specific conjugation (TDC) to further improve stability and therapeutic index, as well as alpha-releasing immunoconjugates and antibody-conjugated nanoparticles.

[0165] H. BiTE Bispecific T cell engagers (BiTEs) are a class of engineered bispecific monoclonal antibodies being investigated for use as anti-cancer drugs. They direct the host's immune system, more specifically the cytotoxic activity of T cells, against infected cells. BiTEs is a registered trademark of Micromet AG.

[0166] BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of approximately 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to infected cells via a specific molecule.

[0167] Like other bispecific antibodies, but unlike conventional monoclonal antibodies, BiTEs form a link between T cells and target cells. This allows T cells to exert cytotoxic / antiviral activity against infected cells by producing proteins such as perforin and granzymes, independent of the presence of MHC I or costimulatory molecules. These proteins enter infected cells and initiate cellular apoptosis. This action mimics the physiological process observed during T cell attack against infected cells.

[0168] I. Intrabody In certain embodiments, the antibody is a recombinant antibody suitable for intracellular action; such antibodies are known as "intrabodies." These antibodies can interfere with target function through various mechanisms, for example, by altering intracellular protein trafficking, interfering with enzyme function, and blocking protein-protein or protein-DNA interactions. Their structure in many ways mimics or parallels the structure of the single-chain and single-domain antibodies described above. Indeed, the single transcript / single chain is a key feature that allows intracellular expression within the target cell and makes it more feasible for the protein to cross the cell membrane. However, additional features are required.

[0169] Two major issues affecting the implementation of intrabody therapy are delivery, including cell / tissue targeting, and stability. Regarding delivery, various approaches have been used, such as tissue-directed delivery, the use of cell-type-specific promoters, viral-based delivery, and the use of cell-penetrating / membrane-permeable peptides. Regarding stability, these approaches generally involve brute-force screening, including phage display, sequence maturation or development of consensus sequences, or methods that may include insertion of stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and highly targeted modifications such as disulfide substitutions / modifications.

[0170] An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors have been designed and are commercially available that can target intrabodies (or other proteins) to intracellular regions, such as the cytoplasm, nucleus, mitochondria, and ER (Invitrogen Corp.; Persic et al., 1997).

[0171] Due to their ability to enter cells, intrabodies have additional uses that other types of antibodies cannot achieve. In the case of this antibody, its ability to interact with the MUC1 cytoplasmic domain in living cells can interfere with functions associated with MUC1 CD, such as signal transduction (binding to other molecules) or oligomerization. In particular, it is believed that such antibodies can be used to inhibit MUC1 dimerization.

[0172] J. Purification In certain embodiments, the antibodies of the present disclosure can be purified. As used herein, the term "purified" is intended to refer to a composition that can be isolated from other components, and the protein is purified to any degree compared to its naturally occurring state. Thus, a purified protein also refers to a protein that is free from the environment in which it may naturally occur. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the majority 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.

[0173] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides include ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or by heat denaturation, followed by centrifugation; gel filtration, reverse-phase chromatography, hydroxylapatite chromatography, and affinity chromatography; as well as combinations of such techniques with other techniques.

[0174] In purifying the antibodies of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide can be purified from other cellular components using an affinity column that binds to a tagged portion of the polypeptide. As is generally known in the art, the order in which the various purification steps are performed may be changed or certain steps may be omitted, and still result in a method suitable for preparing a substantially purified protein or peptide.

[0175] Generally, whole antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., Protein A). Alternatively, antigen may be used to simultaneously purify and select suitable antibodies. Such methods often utilize a selection agent bound to a support, such as a column, filter, or beads. The antibody is bound to the support, contaminating components are removed (e.g., washed away), and the antibody is released by applying conditions (salt, heat, etc.).

[0176] Various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. Another way to assess the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thereby calculating the degree of purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen to follow purification and whether the expressed protein or peptide exhibits detectable activity.

[0177] It is known that the migration of polypeptides can vary, sometimes significantly, using different conditions of SDS / PAGE (Capaldi et al., 1977). Therefore, it is understood that the apparent molecular weight of purified or partially purified expression products can vary under different electrophoretic conditions.

[0178] III. Active / Passive Immunization and Treatment / Prevention of Enterovirus D68 Infection A. Formulation and Administration The present disclosure provides pharmaceutical compositions containing anti-EV-D68 antibodies and antigens for generating them. Such compositions comprise a prophylactically or therapeutically effective amount of the antibody or fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be 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, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0179] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceuticals are described in "Remington's Pharmaceutical Sciences." Such compositions contain a prophylactically or therapeutically effective amount of an antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should be compatible with the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, aerosol, bronchial inhalation, rectal, vaginal, topical, or delivered by mechanical ventilation.

[0180] Active vaccines that produce antibodies similar to those disclosed herein in vivo in subjects at risk of EV-D68 infection are also contemplated. Such vaccines can be formulated for parenteral administration, for example, by injection via intradermal, intravenous, intramuscular, subcutaneous, aerosol, or even intraperitoneal routes. Intradermal and intramuscular administration are contemplated. Alternatively, the vaccine can be administered by direct topical routes to mucous membranes, for example, by nasal drops, inhalation, nebulizer, or rectal or vaginal delivery. Pharmaceutically acceptable salts include acid salts and those 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 the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0181] Passive transfer of antibodies, known as artificially acquired passive immunity, generally involves the use of intravenous or intramuscular injections. Antibodies can be in the form of pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, high-titer human IVIG or IG from immunized or disease-recovering donors, or monoclonal antibodies (MAbs), in human or animal plasma or serum. Such immunity is generally short-lived, and there are potential risks of hypersensitivity reactions and serum sickness, particularly due to gamma globulins of non-human origin. However, passive immunization provides immediate protection. Antibodies are formulated in a carrier suitable for injection, i.e., a sterile, injectable carrier.

[0182] Generally, the components of the compositions of the present disclosure are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachette indicating the amount of active ingredient.When the composition is administered by injection, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0183] The compositions of the present disclosure can be formulated as neutral or salt form.Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0184] 2. ADCC Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an Fc region-containing antibody or fragment thereof specifically binds via a protein portion that is generally the N-terminus of the Fc region. An "antibody with increased / decreased antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to an antibody with increased / decreased ADCC as determined by any suitable method known to those skilled in the art.

[0185] As used herein, the term "increased / decreased ADCC" is defined as either an increase / decrease in the number of target cells lysed within a given time period at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or a decrease / increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells within a given time period by the ADCC mechanism. The increase / decrease in ADCC is compared to unengineered ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, the increase in ADCC mediated by an antibody produced by a host cell engineered to have an altered glycosylation pattern (e.g., to express glycosyltransferase, GnTIII, or other glycosyltransferase) by the methods described herein is compared to the ADCC mediated by the same antibody produced by the same type of unengineered host cell.

[0186] 3. CDC Complement-dependent cytotoxicity (CDC) is a function of the complement system. CDC is a process in the immune system that kills pathogens by damaging their membranes without the involvement of antibodies or cells of the immune system. There are three main processes. All three involve inserting one or more membrane attack complexes (MACs) into the pathogen, which causes lethal colloid osmotic swelling, or CDC. This is one mechanism by which antibodies or antibody fragments have antiviral effects.

[0187] IV. Antibody Conjugates The antibodies of the present disclosure can be linked to at least one agent to form an antibody conjugate. To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, it is conventional to link, covalently bond, or conjugate at least one desired molecule or moiety to the antibody molecule. 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. Non-limiting examples of effector molecules attached to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelators, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or colored ligands, such as biotin.

[0188] Antibody conjugates are generally preferred for use as diagnostic agents.Antibody diagnostics are generally divided into two categories: those for use in in vitro diagnosis, such as various immunoassays, and those for use in in vivo diagnostic protocols, generally known as "antibody-directed imaging".Many suitable imaging agents are known in the art, as are the methods for their attachment to antibodies (see, for example, U.S. Patent No. 5,021,236, U.S. Patent No. 4,938,948 and U.S. Patent No. 4,472,509).The imaging moiety used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR detectable substances and X-ray imaging agents.

[0189] In the case of paramagnetic ions, examples include ions such as chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0190] Astatine, in the case of radioisotopes for therapeutic and / or diagnostic use 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 ,indium 111 , 59 iron, 32 Phosphorus, rhenium 186 ,rhenium 188 ,75 selenium, 35 Sulfur, technicium 99m and / or yttrium 90 and in certain embodiments, 125 I is often preferred for use, and technicium 99m and / or indium 111 Radiolabeled monoclonal antibodies of the present disclosure can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium iodide and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. Monoclonal antibodies according to the present disclosure can also be iodinated with technitium by a ligand exchange process, for example, by reducing pertechnate with a stannous solution, chelating the reduced technitium on a Sephadex column, and applying the antibody to the column. 99m Alternatively, direct labeling techniques may be used, for example, by incubating the antibody with a reducing agent such as pertechnate or SNCl2, a buffer such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups often used to attach radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0191] Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine green, rhodamine red, renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0192] Another type of antibody contemplated in the present disclosure is an antibody intended primarily for in vitro use, which is linked to a secondary binding ligand and / or enzyme (enzyme tag) that generates a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin, avidin, and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0193] Yet another known method for site-specific attachment of molecules to antibodies involves reacting the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids in the antigen-binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous because it results in a loss of antigen binding by the antibody conjugate.

[0194] Molecules containing azide groups can also be used to form covalent bonds to proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-specific optical probes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains in purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binders.

[0195] Several methods for attaching or conjugating antibodies to their conjugate moieties are known in the art. Some attachment methods include, for example, the use of organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA) attached to antibodies; ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or metal chelate complexes using tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patent Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, and a detectable imaging moiety is attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0196] In another embodiment, immunoglobulins are derivatized by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce antibodies with diagnostic and therapeutic potential that are currently undergoing clinical evaluation.

[0197] V. Immunodetection Methods In yet a further aspect, the present disclosure relates to immunodetection methods for binding, purifying, removing, quantifying, and generally detecting EV-D68 and its associated antigens. While such methods can be applied in a conventional sense, another application is in the quality control and monitoring of vaccines and other viral stocks, where antibodies according to the present disclosure can be used to assess the quantity or integrity of antigens within the virus (i.e., long-term stability). Alternatively, the methods can be used to screen various antibodies for appropriate / desired reactivity profiles.

[0198] Other immunodetection methods include specific assays for determining the presence of EV-D68 in a subject. A wide variety of assay formats are contemplated, particularly those used to detect EV-D68 in fluids obtained from a subject, such as saliva, blood, plasma, sputum, semen, urine, respiratory droplets, or aerosols. Semen, in particular, has been shown to be a viable sample for detecting EV-D68 (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). These assays can be conveniently adapted for non-healthcare (home) use, including lateral flow assays (see below) similar to home pregnancy tests. These assays may be packaged in kit form with appropriate reagents and instructions to allow for use by the family member subject.

[0199] Some immunodetection methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescence assay, bioluminescence assay, and Western blot, to name a few. Competitive assays for detecting and quantifying EV-D68 antibodies specifically directed against specific parasite epitopes in a sample are also provided. The steps of various useful immunodetection methods are 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 EV-D68 and, optionally, contacting the sample with a first antibody according to the present disclosure under conditions effective to allow the formation of an immune complex.

[0200] These methods include purifying EV-D68 or related antigens from a sample. The antibody is preferably attached to a solid support, e.g., in the form of a column matrix, and a sample suspected of containing EV-D68 or antigenic components is applied to the immobilized antibody. Undesired components are washed from the column, leaving the EV-D68 antigen immunocomplexed to the immobilized antibody, which is then recovered by removing the organism or antigen from the column.

[0201] Immunobinding methods also include methods for detecting and quantifying the amount of EV-D68 or related components in a sample, as well as detecting and quantifying any immune complexes formed during the binding process. In this method, a sample suspected of containing EV-D68 or its antigen is obtained, contacted with an antibody that binds to EV-D68 or its component, and then the amount of immune complexes formed under specific conditions is detected and quantified. For antigen detection, the biological sample analyzed can be any sample suspected of containing EV-D68 or an EV-D68 antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid including blood and serum, or a secretion such as feces or urine.

[0202] Contacting a selected biological sample with an antibody under conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibody to form immune complexes with, i.e., bind to, EV-D68 or any antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove nonspecifically bound antibody species and allow detection of only specifically bound antibodies within the primary immune complexes.

[0203] Generally, the detection of immune complex formation is well known in the art and can be achieved by applying a number of techniques.These methods are generally based on the detection of labels or markers, such as radioactive tags, fluorescent tags, biological tags and enzymatic tags.The patents relating to the use of such labels include U.S. Patent No. 3,817,837, U.S. Patent No. 3,850,752, U.S. Patent No. 3,939,350, U.S. Patent No. 3,996,345, U.S. Patent No. 4,277,437, U.S. Patent No. 4,275,149 and U.S. Patent No. 4,366,241.Of course, as known in the art, additional advantages can be found through the use of secondary binding ligands, such as second antibodies and / or biotin / avidin ligand binding sequences.

[0204] The antibody used for detection may itself be linked to a detectable label, in which case the amount of primary immune complexes in the composition can be determined simply by detecting the label. Alternatively, the first antibody that becomes bound within the primary immune complex may be detected by a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody and may therefore be referred to as a "secondary" antibody. The primary immune complexes are contacted with a labeled secondary binding ligand or antibody under conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove nonspecifically bound labeled secondary antibodies or secondary ligands, and the remaining label in the secondary immune complexes is detected.

[0205] Another method involves detecting primary immune complexes using a two-step approach. As described above, a second binding ligand, such as an antibody, having binding affinity for the antibody is used to form secondary immune complexes. After washing, the secondary immune complexes are again contacted with a third binding ligand or antibody having binding affinity for the second antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing the detection of the tertiary immune complexes thus formed. This system can provide signal amplification, if desired.

[0206] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect the biotin attached to the complexed biotin. 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, a portion of the antibody binds to the antigen, forming a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding an additional biotin moiety to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second-step antibody directed against biotin. This second-step antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymology using a chromogenic substrate. Suitable amplification can produce macroscopically visible conjugates.

[0207] Another known method of immunodetection utilizes immunopolymerase chain reaction (PCR). PCR is similar to the Cantor method up to the incubation with biotinylated DNA. However, instead of using multiple incubations of streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed away using a low-pH or high-salt buffer, which releases the antibody. The resulting wash solution is then used to perform a PCR reaction using suitable primers with appropriate controls. At least theoretically, the enormous amplification power and specificity of PCR can be exploited to detect single antigen molecules.

[0208] A. ELISA Immunoassay, in its simplest and most direct sense, is binding assay.Particular preferred immunoassays are various types of enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) known in the art.Immunohistochemical detection using tissue sections is also particularly useful.However, it is easy to understand that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, etc. can also be used.

[0209] In one exemplary ELISA, the antibodies of the present disclosure are immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected of containing EV-D68 or an EV-D68 antigen is then added to the well. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-EV-D68 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-EV-D68 antibody, followed by a third antibody with binding affinity for the second antibody, where the third antibody is linked to a detectable label.

[0210] In another exemplary ELISA, a sample suspected of containing EV-D68 or an EV-D68 antigen is immobilized on a well surface and then contacted with the anti-EV-D68 antibody of the present disclosure. After binding and washing to remove nonspecifically bound immune complexes, the bound anti-EV-D68 antibody is detected. If the first anti-EV-D68 antibody is linked to a detectable label, the immune complex can be detected directly. Again, the immune complex can be detected using a second antibody that has binding affinity for the first anti-EV-D68 antibody, and the second antibody is linked to a detectable label.

[0211] Regardless of the format used, ELISAs have certain features in common, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes, which are described below.

[0212] When coating a plate with an antigen or antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody overnight or for a specified period of time. The wells are then washed to remove incompletely adsorbed material. The remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, or milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thereby reducing the background caused by nonspecific binding of the antiserum to the surface.

[0213] In ELISA, it may be more conventional to use secondary or tertiary detection means rather than a direct procedure. Thus, after binding the protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilized surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or secondary antibody, and the secondary binding ligand or secondary antibody in combination with a labeled tertiary antibody or third binding ligand.

[0214] By "under conditions effective to allow immune complex (antigen / antibody) formation" is meant that the conditions preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to help reduce nonspecific background.

[0215] "Suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. The incubation step is typically performed at a temperature of preferably about 25°C to 27°C for about 1 to 2 to 4 hours, or overnight at about 4°C.

[0216] In ELISA, after all incubation steps, the contacted surface is washed to remove uncomplexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.

[0217] To provide a detection means, the second or third antibody has an associated label that allows detection.Preferably, this is an enzyme that generates color when incubated with an appropriate chromogenic substrate.Thus, for example, it is desirable to contact or incubate the first and second immune complexes with a urease-conjugated antibody, a glucose oxidase-conjugated antibody, an alkaline phosphatase-conjugated antibody, or a hydrogen peroxidase-conjugated antibody for a period and under conditions that promote the development of additional immune complex formation (e.g., incubate in a PBS-containing solution, such as PBS-Tween, at room temperature for 2 hours).

[0218] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified by incubation with, for example, a chromogenic substrate such as urea or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) or HO in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0219] In another aspect, the present disclosure contemplates the use of a competitive format, which is particularly useful for detecting EV-D68 antibodies in a sample. In a competitive-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 is an indication of the amount of unknown antibody or analyte in the sample.

[0220] Here, we propose the use of a labeled EV-D68 monoclonal antibody to determine the amount of EV-D68 antibody in a sample. The basic format involves contacting a known amount of EV-D68 monoclonal antibody (linked to a detectable label) with an EV-D68 antigen or particle. The EV-D68 antigen or particle 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 displace, the labeled monoclonal antibody. By measuring either the lost or remaining label (and subtracting it from the original amount of bound label), we can determine how much unlabeled antibody is bound to the support and, therefore, how much antibody was present in the sample.

[0221] B. Western Blot Western blot (or protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or tissue extract. It uses gel electrophoresis to separate native or denatured proteins by polypeptide 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) where they are probed (detected) using antibodies specific for the target protein.

[0222] Samples can be taken from whole tissues or cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (for large sample volumes), a homogenizer (for small volumes), or sonication. Cells may also be disrupted by one of the above mechanical methods. However, it should be noted that Western blotting is not limited to cellular testing, as bacterial, viral, or environmental samples may also be sources of proteins. Various detergents, salts, and buffers may be used to promote cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0223] Proteins in a sample are separated using gel electrophoresis. Protein separation can be by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample processing and the properties of the gel. This is a very useful method for determining proteins. It is also possible to use two-dimensional (2-D) gels, which spread proteins from a single sample across two dimensions. Proteins are separated according to their isoelectric point (pH with a neutral net charge) in the first dimension and according to molecular weight in the second dimension.

[0224] To make proteins accessible for antibody detection, they are transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). A stack of filter paper is placed on top of the membrane, which is then placed on top of the gel. The entire stack is then placed in a buffer solution, which rises up the paper by capillary action, carrying the proteins along with it. Another method of protein transfer, called electroblotting, uses an electric current to draw proteins from the gel onto a PVDF or nitrocellulose membrane. The proteins migrate from the gel onto the membrane while maintaining the configuration they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both types of membranes are chosen for their nonspecific protein-binding properties (i.e., they bind all proteins equally well). Protein binding is based on hydrophobic interactions and charged interactions between the membrane and the protein. Nitrocellulose membranes are less expensive than PVDF but are much more fragile and do not withstand repeated probing well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be confirmed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. After transfer, proteins are detected using labeled or unlabeled primary antibodies, followed by indirect detection using labeled Protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0225] C. Lateral Flow Assay Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in a sample (matrix) without the need for dedicated, expensive equipment, although many laboratory-based applications exist that require the aid of a reader. Typically, these tests are used as low-resource medical diagnostics for either home testing, point-of-care testing, or laboratory use. One popular and well-known application is home pregnancy testing.

[0226] This technology is based on a series of capillary beds, such as strips of porous paper or sintered polymer. Each of these elements has the ability to spontaneously transport fluid (e.g., urine). The first element (sample pad) acts as a sponge, retaining excess sample fluid. Upon immersion, the fluid migrates to the second element (conjugate pad), which contains, according to the manufacturer, so-called conjugates, a dried format of bioactive particles (see below), stored in a salt-sugar matrix that contains everything needed to ensure an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., an antibody) immobilized on the particle's surface. The sample fluid dissolves the salt-sugar matrix, but also the particles, and in a combined transport process, the sample and conjugate mix as they flow through the porous structure. In this way, the analyte binds to the particles as it travels further through the third capillary bed. This material has one or more regions (often called stripes) where the manufacturer has immobilized a third molecule. By the time the sample-conjugate mixture reaches these strips, the analyte has bound to the particles, and a third "capture" molecule binds to the complex. After a while, as more and more fluid passes through the stripes, particles accumulate, causing the striped region to change color. Typically, there are at least two stripes: one (control) captures any particles, thereby indicating that the reaction conditions and technology worked well, and the second stripe contains a specific capture molecule and captures only particles with immobilized analyte molecules. After passing through these reaction zones, the fluid enters a final porous material (wick) that simply acts as a waste container. Lateral flow tests can function as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Patent No. 6,485,982.

[0227] D. Immunohistochemistry The antibodies of the present disclosure can also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for testing by immunohistochemistry (IHC). Methods for preparing tissue blocks from these particulate specimens have been successfully used in previous IHC tests 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).

[0228] In summary, frozen sections may be prepared by rehydrating 50 ng of frozen "ground" tissue at room temperature in phosphate-buffered saline (PBS) in small plastic capsules, pelleting the particles by centrifugation, resuspending them in viscous embedding medium (OCT), inverting the capsule and / or pellet by centrifugation, snap-freezing in -70 °C isopentane, cutting the plastic capsule and / or removing the frozen cylindrical tissue, fixing the cylindrical tissue on a cryostat microtome chuck, and / or cutting 25-50 serial sections from the capsule. Alternatively, the entire frozen tissue sample may be used for serial sectioning.

[0229] Permanent sections may be prepared by a similar method involving rehydrating a 50 mg sample in a plastic microcentrifuge tube, pelleting, resuspending in 10% formalin and fixing for 4 hours, washing / pelleting, resuspending in warm 2.5% agar, pelleting, chilling in ice water to harden the agar, removing the tissue / agar block from the tube, infiltrating and / or embedding the block in paraffin, and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be substituted.

[0230] E. Immunodetection Kits In yet a further aspect, the present disclosure relates to an immunodetection kit for use with the above-described immunodetection method. Because the antibody can be used to detect EV-D68 or an EV-D68 antigen, the antibody can be included in the kit. Thus, the immunodetection kit includes, in suitable container means, a first antibody that binds to EV-D68 or an EV-D68 antigen, and optionally, an immunodetection reagent.

[0231] In certain embodiments, the EV-D68 antibody may be pre-bound to a solid support, such as the column matrix and / or wells of a microtiter plate. The immunodetection reagent of the kit may take any one of a variety of forms, including a detectable label associated with or linked to a given antibody. Detectable labels associated with or bound to a secondary binding ligand are also contemplated. An exemplary secondary ligand is a secondary antibody that has binding affinity for the first antibody.

[0232] Further suitable immunodetection reagents for use in the present kits include two-component reagents comprising a second antibody having binding affinity for the first antibody, along with a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, numerous exemplary labels are known in the art, and any such label may be used in connection with the present disclosure.

[0233] The kit may further include a suitably dispensed composition of EV-D68 or an EV-D68 antigen, whether labeled or unlabeled, that can be used to generate a standard curve for the detection assay. The kit may include the antibody-label conjugate either in fully conjugated form, in the form of an intermediate, or as a separate moiety to be conjugated by the user of the kit. The components of the kit may be packaged in either aqueous medium or lyophilized form.

[0234] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, or preferably suitably aliquoted. The kits of the present disclosure also typically will include means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials may be retained.

[0235] F. Vaccine and Antigen Quality Control Assays The present disclosure also contemplates the use of the antibodies and antibody fragments described herein for use in assessing the antigenic integrity of viral antigens in samples.Biological pharmaceuticals, such as vaccines, differ from chemical drugs in that they cannot usually be molecularly characterized.Antibodies are large, fairly complex molecules, and their capabilities vary greatly from preparation to preparation.Because they are administered to healthy individuals, including children at the start of life, strong emphasis must be placed on their quality to ensure that they are as effective as possible in preventing or treating life-threatening diseases without causing harm themselves.

[0236] The increasing globalization of vaccine production and distribution has opened up new possibilities for better managing public health concerns, but it has also raised questions regarding the equivalence and interchangeability of vaccines sourced from various sources. Therefore, setting high expectations for international standardization of starting materials, production and quality control testing, and regulatory oversight of how these products are manufactured and used is fundamental for continued success. However, this is a constantly changing field, and while continued technological advances in this field offer the promise of developing powerful new weapons against the most ancient public health threats, as well as new ones—malaria, pandemic influenza, and HIV, to name a few—they also place significant pressure on manufacturers, regulators, and the broader medical community to ensure that products continue to meet the highest achievable quality standards.

[0237] Therefore, antigen or vaccine can be obtained from any source or at any time during the manufacturing process.Therefore, the quality control process can start with preparing a sample for immunoassay to identify the binding of the antibody or fragment disclosed herein to viral antigen.Such immunoassays are disclosed elsewhere herein, and any of them can be used to evaluate the structural integrity / antigen integrity of antigen.The criteria for finding a sample that contains an acceptable amount of antigenically accurate and intact antigen can be established by regulatory authorities.

[0238] Another important aspect of evaluating the integrity of antigen is to determine shelf life and storage stability.Most pharmaceuticals, including vaccines, can deteriorate over time.Therefore, it is important to determine the extent to which, for example, the antigen in vaccines degrades or becomes unstable over time, so that it is no longer antigenic when administered to a subject and / or cannot produce an immune response.Again, the standard for finding a sample that contains an acceptable amount of antigenically intact antigen can be established by regulatory authorities.

[0239] In certain embodiments, a viral antigen may contain multiple protective epitopes. In these cases, it may prove useful to use an assay that examines the binding of multiple antibodies, for example, two, three, four, five, or more antibodies. These antibodies may bind to closely related epitopes, so that they are adjacent to or overlap each other. Alternatively, they may represent epitopes that are distinct from heterogeneous portions of the antigen. By examining the integrity of multiple epitopes, a more complete picture of the overall integrity of the antigen and, therefore, its ability to generate a protective immune response can be determined.

[0240] The antibodies and fragments thereof described in this disclosure can also be used in kits for monitoring the effectiveness of a vaccination procedure by detecting the presence of protective EV-D68 antibodies. The antibodies, antibody fragments, or variants and derivatives thereof described in this disclosure can also be used in kits for monitoring the production of a vaccine with desired immunogenicity. [Example]

[0241] VI. Working Examples The following examples are included to demonstrate preferred embodiments. Those of skill in the art should understand that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in carrying out the embodiments, and as such, can be considered to constitute preferred modes for their practice. However, those of skill in the art should understand in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0242] Example 1 - Materials and Methods Animals. Four-week-old male and female AG129 mice from a specific pathogen-free colony were housed in the Utah Science Technology and Research (USTAR) building at Utah State University. Mice were bred and maintained on irradiated Teklad Rodent Diet (Harlan Teklad) and autoclaved tap water at the USTAR building at Utah State University.

[0243] Antibodies and Compounds. The monoclonal antibody (mAb) EV-D68-228 was provided by James Crowe at Vanderbilt University Medical Center. EV-D68-228 was provided in solution at a concentration of 1.134 mg / ml and diluted with sterile saline to achieve doses of 10, 3, and 1 mg / kg for treatment. rRSV-90 was provided in solution at a concentration of 5 mg / ml and used as a negative control antibody at a dose of 10 mg / kg. Intravenous immunoglobulin (IVIg, Carimune, CSL Behring, King of Prussia, PA) was purchased from a local pharmacy and used as a comparative control for EV-D68-228 mAb. Guanidine HCl (guanidine) was obtained from Sigma-Aldrich (St. Louis, MO) and served as a positive control.

[0244] Virus. Enterovirus D68 was obtained from BEI Resources, NIAID, NIH: Enterovirus D68, US / MO / 14-18949, NR-49130. The virus was serially passaged 30 times in the lungs of 4-week-old AG129 mice and then plaque-purified three times in rhabdomyosarcoma (RD) cells obtained from the American Type Culture Collection (Manassas, VA). The resulting virus stock was amplified twice in RD cells to generate a working stock. The virus used for infection was designated EV-D68 MP30 PP.

[0245] Experimental Design. As shown in Table I, a total of 78 mice were randomly assigned to six groups of 12 mice each, with a group of 6 mice used as normal controls. Mice were treated by intraperitoneal (IP) administration of EV-D68-228 mAb, IVIg, or placebo mAb 24 hours before infection. 1 × 10 mAb in a volume of 90 μl of MEM was administered. 4.5 CCID 50Mice were infected by intranasal (IN) instillation of EV-D68 MP30 PP. Treatment with guanidine was initiated 4 hours postinfection and continued twice daily for 5 days. Mice were weighed before treatment and daily thereafter. Four mice from each treatment group were euthanized on days 1, 3, and 5 postinfection for assessment of lung virus titers, blood virus titers, and lung cytokine concentrations.

[0246] Assessment of pulmonary cytokines / chemokines. Each sample of lung homogenate was tested for cytokines and chemokines using a chemiluminescent ELISA-based assay according to the manufacturer's instructions (Quansys Biosciences Q-Plex™ Array, Logan, UT). The Quansys multiplex ELISA is a quantitative test in which 16 different capture antibodies are applied to each well of a 96-well plate in a defined array. Each sample supernatant was tested for IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MCP-1, IFN-γ, TNFα, MIP-1α, GM-CSF, and RANTES. Abbreviations are defined as follows: IL - interleukin; MCP - monocyte chemotactic protein; IFN - interferon; TNF - tumor necrosis factor; MIP - macrophage inflammatory protein; GM-CSF - granulocyte / macrophage colony-stimulating factor; and RANTES - regulated upon activation, normal T cell expressed and secreted.

[0247] Statistical Analysis. All numerical and statistical analyses were completed using Prism 8.0.2 (GraphPad Software Inc.). For each day post-infection, lung and blood viral titers obtained from treatment groups were compared with those obtained from placebo-treated mice using one-way analysis of variance (ANOVA). For each cytokine / chemokine, concentrations obtained from treated mice were compared with those obtained from placebo-treated mice using two-way ANOVA.

[0248] Ethics Statement Regarding Laboratory Animals. This study was conducted in accordance with approval by the Institutional Animal Care and Use Committee at Utah State University, dated March 2, 2019 (expiring March 1, 2022). This research was conducted at the Laboratory Animal Research Center at Utah State University, which is accredited by AAALAC. U.S. government (National Institutes of Health) approval was updated March 9, 2018 (PHS Assurance No. D16-00468 [A3801-01]) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (revised; 2011).

[0249] Table I: Experimental design for the text efficacy of EV-D68-228 for the treatment of EV-D68 respiratory infection in mice. TIFF0007644087000001.tif79149

[0250] Example 2 - Results The purpose of this study was to determine the effect of treatment with EV-D68-228 on enterovirus D68 (EV-D68) respiratory infection in 4-week-old AG129 mice. This study determined the effect of EV-D68-228 mAb on the treatment of EV-D68 respiratory infection in 4-week-old AG129 mice.

[0251] Figures 6A–C show lung viral titers in EV-D68-infected AG129 mice treated with EV-D68-228. In mice treated with EV-D68-228 at doses of 10, 3, or 1 mg / kg, lung viral titers were not detected on days 1, 3, or 5 postinfection. Treatment with IVIg at a dose of 10 mg / kg significantly reduced lung viral titers on day 1 postinfection, but not on days 3 or 5 postinfection. Guanidine treatment at a dose of 100 mg / kg / day significantly reduced lung viral titers on days 1 and 3 postinfection, but not on day 5 postinfection.

[0252] Figures 7A-C show blood viral titers in EV-D68-infected AG129 mice treated with EV-D68-228. In mice treated with EV-D68-228 at doses of 10, 3, or 1 mg / kg, blood viral titers were not detected on days 1, 3, or 5 postinfection. Treatment with IVIg at a dose of 10 mg / kg also reduced blood viral titers on days 1, 3, and 5 postinfection. Guanidine significantly reduced blood viral titers on days 1 and 5 postinfection, but not on day 3.

[0253] Figure 8 shows lung concentrations of IL-1α and IL-1β from EV-D68-infected AG129 mice treated with EV-D68-228. Treatment with EV-D68-228 significantly reduced IL-1α and IL-1β concentrations on day 3 postinfection. Treatment with IVIg or guanidine did not significantly reduce lung concentrations of IL-1α or IL-1β.

[0254] Lung concentrations of IL-6 and MCP-1 from EV-D68-infected AG129 mice treated with EV-D68-228 are shown in Figure 9. Treatment with EV-D68-228 significantly reduced IL-6 and MCP-1 concentrations on day 3 postinfection. Treatment with IVIg or guanidine significantly reduced lung concentrations of MCP-1, but not IL-6, on day 3 postinfection.

[0255] Lung concentrations of RANTES from EV-D68-infected AG129 mice treated with EV-D68-228 are shown in Figure 10. Treatment with EV-D68-228 significantly reduced RANTES concentrations on days 3 and 5 postinfection compared with placebo-treated mice. Treatment with IVIg or guanidine did not significantly reduce lung concentrations of RANTES on days 1, 3, or 5 postinfection.

[0256] Figure 11 shows lung concentrations of IL-2, IL-3, and IL-4 from EV-D68-infected AG129 mice treated with EV-D68-228. No significant changes in IL-2, IL-3, or IL-4 concentrations were observed after infection with EV-D68.

[0257] Figure 12 shows lung concentrations of IL-5, IL-10, and IL-12p70 from EV-D68-infected AG129 mice treated with EV-D68-228. No significant changes in IL-5, IL-10, or IL-12p70 concentrations were observed after infection with EV-D68.

[0258] Lung concentrations of IL-17, IFNγ, and TNFα from EV-D68-infected AG129 mice treated with EV-D68-228 are shown in Figure 13. No significant changes in the concentrations of IL-17, IFNγ, or TNFα were observed after infection with EV-D68.

[0259] Lung concentrations of MIP-1α and GM-CSF from EV-D68-infected AG129 mice treated with EV-D68-228 are shown in Figure 14. No significant changes in the concentrations of MIP-1α or GM-CSF were observed after infection with EV-D68.

[0260] Example 3 - Discussion In this study, the efficacy of EV-D68-228, a mAb against EV-D68, for treating respiratory infection caused by EV-D68 in 4-week-old AG129 mice was determined.

[0261] EV-D68-228 at doses of 10, 3, and 1 mg / kg was highly effective in reducing lung and blood viral titers and proinflammatory cytokines caused by EV-D68 infection. The lung concentrations of IL-1α, IL-1β, IL-6, MCP-1, and RANTES were significantly reduced by treatment with EV-D68-228.

[0262] EV-D68-228 was highly effective in preventing infection with EV-D68.

[0263] Example 4 - Materials and Methods Animals. 10-day-old AG129 mice from a specific pathogen-free colony housed in the Utah Science Technology and Research (USTAR) building at Utah State University. Mice were bred and maintained on irradiated Teklad Rodent Diet (Harlan Teklad) and autoclaved tap water at the USTAR building at Utah State University.

[0264] Antibodies and Compounds. The monoclonal antibody (mAb) EV-D68-228 was provided by James Crowe at Vanderbilt University Medical Center. EV-D68-228 was provided in solution at a concentration of 1.134 mg / ml and diluted with sterile saline to achieve doses of 10, 3, and 1 mg / kg for treatment. rRSV-90 was provided in solution at a concentration of 5 mg / ml and used as a negative control antibody at a dose of 10 mg / kg. Intravenous immunoglobulin (IVIg, Carimune, CSL Behring, King of Prussia, PA) was purchased from a local pharmacy and used as a comparative control for EV-D68-228 mAb. Guanidine HCl (guanidine) was obtained from Sigma-Aldrich (St. Louis, MO) and served as a positive control.

[0265] Virus. Enterovirus D68 was obtained from BEI Resources, NIAID, NIH: Enterovirus D68, US / MO / 14-18949, NR-49130. The virus was serially passaged 30 times in the lungs of 4-week-old AG129 mice and then plaque-purified three times in rhabdomyosarcoma (RD) cells obtained from the American Type Culture Collection (Manassas, VA). The resulting virus stock was amplified twice in RD cells to generate a working stock. The virus used for infection was designated EV-D68 MP30 PP.

[0266] Experimental Design. A total of 42 mice were randomized into seven groups of six mice each, as shown in Table II. Mice were treated 24 hours before infection by intraperitoneal (IP) administration of EV-D68-228 mAb, IVIg, or placebo mAb. 1 × 10 mAb in a 100 μl volume of MEM was administered. 6.5 CCID 50 Mice were infected by intraperitoneal (IP) administration of EV-D68 MP30 PP. Guanidine treatment was initiated 4 hours postinfection and continued twice daily for 5 days. Mice were weighed before treatment and daily thereafter. All mice were observed daily for morbidity, mortality, and neurological score until day 21. Neurological scores (NS) were recorded as follows: NS0 - no observable paralysis; NS1 - abnormally splayed hind limbs, but normal or slightly slowed gait; NS2 - partial hind limb collapse and limp while used for forward movement; NS3 - rigid paralysis of the hind limbs, with no use of the hind limbs for forward movement; and NS4 - rigid paralysis of the hind limbs, with no forward movement. Any animals with an NS4 score were humanely euthanized.

[0267] Statistical analysis. Kaplan-Meier survival curves were generated using Prism 8.0.2 (GraphPad Software Inc.). Survival curves were compared using the Log-rank (Mantel-Cox) test followed by the Gehan-Breslow-Wilcoxon test. For each day postinfection, blood virus titers from treatment groups were compared with lung and blood titers from placebo-treated mice using one-way analysis of variance (ANOVA). Mean body weights were compared using one-way analysis of variance. Neurological scores were compared using the Kruskal-Wallis test followed by Dunn's multiple comparison test.

[0268] Ethics Statement Regarding Laboratory Animals. This study was conducted in accordance with approval by the Institutional Animal Care and Use Committee at Utah State University, dated March 2, 2019 (expiring March 1, 2022). This research was conducted at the Laboratory Animal Research Center at Utah State University, which is accredited by AAALAC. U.S. government (National Institutes of Health) approval was updated March 9, 2018 (PHS Assurance No. D16-00468 [A3801-01]) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (revised; 2011).

[0269] Table II: Experimental design for the text efficacy of EV-D68-228 for the treatment of EV-D68 neurological infection in mice. TIFF0007644087000002.tif72149

[0270] Example 5 - Results The purpose of this study was to determine the effect of treatment with EV-D68-228 on enterovirus D68 (EV-D68) neurological infection in 10-day-old AG129 mice. This study determined the effect of EV-D68-228 mAb on the treatment of EV-D68 neurological infection in 10-day-old AG129 mice.

[0271] Figure 15 shows Kaplan-Meier survival curves for 10-day-old AG129 mice infected with EV-D68 and treated with EV-D68-228, IVIg, or guanidine. In each group of six mice infected with EV-D68, EV-D68-228 at doses of 10, 3, or 1 mg / kg provided 100% protection from death. Two of the six mice treated with IVIg at a dose of 10 mg / kg did not survive infection. Treatment with guanidine at a dose of 100 mg / kg / day protected all six mice from death.

[0272] Figure 16 shows the average body weight of 10-day-old AG129 mice infected with EV-D68 and treated with EV-D68-228, IVIg, or guanidine. Doses of 10, 3, or 1 mg / kg protected mice from infection-associated weight loss. IVIg at a dose of 10 mg / kg also protected mice from weight loss. Furthermore, guanidine at a dose of 100 mg / kg / day protected mice from weight loss.

[0273] Figures 17A-C show blood viral titers in EV-D68-infected AG129 mice treated with EV-D68-228. Mice treated with EV-D68-228 at doses of 10, 3, or 1 mg / kg had undetectable blood viral titers on days 1, 3, or 5 postinfection. Treatment with IVIg at a dose of 10 mg / kg also reduced blood viral titers on days 1, 3, and 5 postinfection. Guanidine treatment at a dose of 100 mg / kg / day significantly reduced blood viral titers on days 1, 3, and 5 postinfection.

[0274] The neurological scores of 10-day-old AG129 mice infected with EV-D68 and treated with EV-D68-228, IVIg, or guanidine are shown in Figure 18. No neurological scores were observed in mice treated with EV-D68-228 Ab at doses of 10, 3, or 1 mg / kg. Neurological scores were observed in mice treated with IVIg at a dose of 10 mg / kg on days 4, 5, and 6 postinfection. However, the neurological scores of mice treated with IVIg were significantly reduced compared with placebo-treated mice. No neurological scores were observed in mice treated with guanidine at a dose of 100 mg / kg / day.

[0275] Example 6 - Discussion In this study, the efficacy of EV-D68-228, a mAb against EV-D68, for treating neurological infection caused by EV-D68 in 10-day-old AG129 mice was determined.

[0276] In EV-D68-infected mice, EV-D68-228 at doses of 10, 3, and 1 mg / kg provided protection from death and weight loss. Furthermore, no blood viral titers were detected in mice treated with EV-D68-228 on days 1, 3, or 5 postinfection. No paralysis, as measured by neurological score, was observed in mice treated with EV-D68-228 at doses of 10, 3, or 1 mg / kg.

[0277] EV-D68-228 appeared to provide protection from EV-D68 infection compared with commercial IVIg, as indicated by the survival rates, blood viral titers, and neurological scores observed in mice treated with a 10 mg / kg dose of IVIg.

[0278] EV-D68-228 provided equivalent protection from death and paralysis compared with treatment with the positive control, guanidine. However, whereas blood virus titers were still detectable in guanidine-treated mice on day 3 postinfection, virus was not detectable in the blood of mice treated with EV-D68-228 at doses of 10, 3, or 1 mg / kg on days 1, 3, or 5 postinfection.

[0279] Example 7 - Effect of EV-D68-228 on respiratory infection in mice material and method Animals. Four-week-old male and female AG129 mice from a specific pathogen-free colony were housed in the Utah Science Technology and Research (USTAR) building at Utah State University. Mice were bred and maintained on irradiated Teklad Rodent Diet (Harlan Teklad) and autoclaved tap water at the USTAR building at Utah State University.

[0280] Antibodies and Compounds. The monoclonal antibody (mAb) EV-D68-228 was provided by James Crowe at Vanderbilt University Medical Center. EV-D68-228 was provided in solution at a concentration of 1.134 mg / ml and diluted with sterile saline to achieve doses of 10 or 1 mg / kg for treatment. rRSV-90 was provided in solution at a concentration of 5 mg / ml and used as a negative control antibody at a dose of 10 mg / kg. Intravenous immunoglobulin (IVIg, Carimune, CSL Behring, King of Prussia, PA) was purchased from a local pharmacy and used as a comparative control for EV-D68-228 mAb.

[0281] Virus. Enterovirus D68 was obtained from BEI Resources, NIAID, NIH: Enterovirus D68, US / MO / 14-18949, NR-49130. The virus was serially passaged 30 times in the lungs of 4-week-old AG129 mice and then plaque-purified three times in rhabdomyosarcoma (RD) cells obtained from the American Type Culture Collection (Manassas, VA). The resulting virus stock was amplified twice in RD cells to generate a working stock. The virus used for infection was designated EV-D68 MP30 PP.

[0282] Experimental Design. As shown in Table C, a total of 52 mice were randomly assigned to six groups of eight mice each, with a group of four mice used as normal controls. Mice were treated by intraperitoneal (IP) administration of EV-D68-228 mAb, IVIg, or placebo mAb 4, 24, or 48 hours postinfection. Mice were infected by intranasal (IN) instillation of 1 × 10 CCID50 of EV-D68 MP30 PP in 90 μl of MEM. Mice were weighed before treatment and daily thereafter. Four mice from each treatment group were euthanized on days 3 and 5 postinfection for assessment of lung virus titers, blood virus titers, and lung c...

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that binds to enterovirus D68, (i) heavy chain CDR1, CDR2 and CDR3 sequences corresponding to SEQ ID NOs: 314, 315 and 316, respectively, and light chain CDR1, CDR2 and CDR3 sequences corresponding to SEQ ID NOs: 506, 507 and 508, respectively; or (ii) heavy chain CDR1, CDR2 and CDR3 sequences corresponding to SEQ ID NOs: 413, 414 and 415, respectively, and light chain CDR1, CDR2 and CDR3 sequences corresponding to SEQ ID NOs: 605, 606 and 607, respectively. The monoclonal antibody or antigen-binding fragment thereof,

2. the antibody or antigen-binding fragment thereof (i) encoded by light chain variable sequences and heavy chain variable sequences corresponding to SEQ ID NOs: 40 and 39, respectively, or SEQ ID NOs: 106 and 105, respectively; or (ii) comprising a light chain variable sequence and a heavy chain variable sequence corresponding to SEQ ID NOs: 168 and 167, respectively, or SEQ ID NOs: 234 and 233, respectively; The monoclonal antibody or antigen-binding fragment thereof described in claim 1.

3. The antigen-binding fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, which is an Fv fragment or an Fv fragment.

4. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is a chimeric antibody or a bispecific antibody.

5. The antibody, IgG or a recombinant IgG antibody or antigen-binding fragment thereof, comprising an Fc portion mutated to alter (eliminate or enhance) FcR interaction, such as LALA, N297, GASD / ALIE, YTE, DHS or LS mutations to increase half-life and / or to increase therapeutic effect, or glycans modified to alter (eliminate or enhance) FcR interaction, such as enzymatic or chemical addition or removal of glycans, or expression in an engineered cell line with a defined glycosylation pattern; A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof further comprises a cell-penetrating peptide and / or is an intrabody.

7. A pharmaceutical composition for treating a subject infected with Enterovirus D68 (EV-D68) or reducing the likelihood of infection in a subject at risk of contracting EV-D68, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a polynucleotide comprising an RNA or DNA sequence encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a vector comprising said polynucleotide.

8. (i) the pharmaceutical composition is used in combination with a second therapy; (ii) the pharmaceutical composition is administered before or after infection; (iii) the treatment treats or reduces a pulmonary and / or neurological infection; and / or (iv) the antibody or antigen-binding fragment thereof is delivered to the subject by administration or by genetic delivery by a polynucleotide comprising an RNA or DNA sequence encoding the monoclonal antibody or antigen-binding fragment thereof, or a vector comprising the polynucleotide; 8. The pharmaceutical composition of claim 7.

9. 9. The pharmaceutical composition of claim 8, wherein the second therapy is an anti-viral therapy or an anti-inflammatory therapy.

10. 1. An in vitro method for detecting Enterovirus D68 (EV-D68) infection in a subject, comprising the steps of: (a) contacting a sample obtained from the subject with the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and (b) detecting EV-D68 in the sample by binding the antibody or antigen-binding fragment thereof to an EV-D68 antigen in the sample.

11. (i) the sample is a body fluid, blood, sputum, tears, saliva, mucous membrane or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, filtrate, respiratory droplets or aerosol, tissue scraping, or feces; (ii) detection comprises ELISA, RIA, lateral flow assay or Western blot; and / or the method further comprising performing steps (a) and (b) again and determining a change in EV-D68 antigen levels compared to the first assay; 11. The method of claim 10.

12. 7. An engineered cell expressing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

13. (a) one or more monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1 to 6; or (b) one or more expression vectors comprising a nucleic acid encoding the first monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

13. A vaccine formulation comprising:

14. (i) the expression vector is a Sindbis virus vector or a VEE vector; (ii) the vaccine formulation is formulated for delivery by needle injection, jet injection or electroporation; (iii) the expression vector is a Sindbis virus vector or a VEE vector and the vaccine formulation is formulated for delivery by needle injection, jet injection or electroporation; or (iv) the vaccine formulation further comprises one or more expression vectors encoding a second monoclonal antibody or antigen-binding fragment thereof; 14. The vaccine formulation of claim 13.

15. The vaccine formulation of claim 14, wherein the second monoclonal antibody or antigen-binding fragment thereof is a distinct antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

16. A pharmaceutical composition for protecting the health of the placenta and / or fetus of a pregnant subject infected with or at risk of being infected with Enterovirus D68, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, or a polynucleotide comprising an RNA or DNA sequence encoding the monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, or a vector comprising said polynucleotide.

17. (i) the pharmaceutical composition is administered before or after infection; (ii) the subject is a pregnant woman, a sexually active woman, or a woman undergoing fertility treatment; and / or (iii) the antibody or antigen-binding fragment thereof is delivered to the subject by administration or by genetic delivery by a polynucleotide comprising an RNA or DNA sequence encoding the antibody or antigen-binding fragment thereof, or a vector comprising the polynucleotide; or (iv) the monoclonal antibody or antigen-binding fragment thereof increases placental size compared to an untreated control; or (v) the monoclonal antibody or antigen-binding fragment thereof reduces fetal viral load and / or pathology compared to an untreated control.

17. The pharmaceutical composition of claim 16.

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