Protective antibodies against respiratory viral infections

Abstract

Protective antibodies against respiratory viral infections, including human parainfluenza virus (HPIV), respiratory syncytial virus (RSV) and human metapneumovirus (HMPV), are described. Certain antibodies disclosed herein neutralize two or more viruses, e.g., RSV and HMPV. The antibody binding fragments can be engineered into a variety of formats, including formats that allow for simultaneous protection against multiple viruses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 154,166, filed February 26, 2021, the contents of which are incorporated by reference in their entirety as if set forth herein.

[0002] Sequence Listing Reference The sequence listing accompanying this application is provided in text format rather than hardcopy, and is incorporated herein by reference. The text file containing the sequence listing is named F053-0125PCT_ST25.txt. The text file is 68.4kb in size, was created on February 24, 2022, and was submitted electronically via EFS-Web.

[0003] The present disclosure provides protective antibodies against respiratory viral infections, including human parainfluenza virus (HPIV), respiratory syncytial virus (RSV) and human metapneumovirus (HMPV). Certain antibodies disclosed herein neutralize two or more viruses, such as RSV and HMPV. The antibody binding fragments can be recombined into a variety of formats, including formats that can provide simultaneous protection against multiple viruses. [Background technology]

[0004] Respiratory infections caused by paramyxoviruses and pneumoviruses cause tens of thousands of deaths in the United States each year. Patients undergoing hematopoietic stem cell transplantation (HCT) are particularly susceptible to infections while their immune systems are repopulating. After HCT, infections caused by human parainfluenza virus (HPIV), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV) are the most lethal. Unfortunately, there is no prevention or treatment for RSV, HMPV, or HPIV infections in elderly individuals or HCT recipients. The antibody drug palivizumab is given to premature infants to prevent RSV infection, but there is no prevention for HMPV or HPIV infections. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides novel antibodies that protect against one or more of human parainfluenza viruses (HPIV1 and / or HPIV3), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV). Particular antibodies disclosed herein are protective antibodies against HPIV3. Other antibodies disclosed herein are protective antibodies against HPIV3 and HPIV1. Other antibodies disclosed herein are protective antibodies against RSV and HMPV. The novel antibodies disclosed herein can be used to protect against various forms of respiratory viruses.

[0006] In certain embodiments, various binding fragments derived from these newly disclosed antibodies and other antibodies can be recombined to provide multispecific formats capable of simultaneously protecting against multiple types of viruses, including a variety of additional respiratory viruses. In certain embodiments, these antibodies and their recombinant formats provide simultaneous protection against multiple medically important respiratory viruses affecting children, the elderly, and immunosuppressed patients.

[0007] The PI3 antibody disclosed herein binds to and neutralizes HPIV3, the "3x1" antibody disclosed herein binds to and neutralizes HPIV3 and HPIV1, and the MxR antibody disclosed herein binds to and neutralizes RSV and HMPV. [Brief description of the drawings]

[0008] Many of the drawings submitted in this application are believed to be more easily understood in color, and applicants hereby contemplate color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0009] [Figure 1] An example of an embodiment in which the antibodies disclosed herein are used to bridge the compromised period of hematopoietic stem cell transplant recipients is shown below.

[0010] [Fig. 2A-2I]Screening of human PBMCs, human tonsils, and human spleens for HPIV3-specific B cells is shown. Figure 2A) Biotinylated HPIV3 fusion (F) protein was tetramerized with APC-conjugated streptavidin, and the tetramer was used to label HPIV3-specific B cells, which were then magnetically enriched with anti-APC microbeads. Representative flow cytometry plots of enriched HPIV3-specific B cells after gating on live cells (negative with fixable viability dye), CD3 / CD14 / CD16-, and CD19+ B cells are shown. Cells in the enriched fraction, outlined in gray, are B cells that bound the pre-fusion structure of HPIV3 F protein (preF) but not the post-fusion structure of HPIV3 F protein (postF). Percentages shown on the flow cytometry plots represent the percentage of total cells shown on each flow cytometry plot. (B) Frequency of HPIV3 preF-specific B cells in human PBMCs (N = 2 donors), human tonsils (N = 2 donors) and human spleens (N = 2 donors). (C, D) Representative flow cytometry plots of class-switched (IgM- / IgD-) B cells as a percentage of total B cells in PBMCs (N = 2 donors), tonsils (N = 2 donors) and spleens (N = 2 donors). (E, F) Frequency of class-switched (IgM- / IgD-) HPIV3 preF-specific B cells as a percentage of total B cells in PBMCs (N = 2 donors), tonsils (N = 2 donors) and spleens (N = 2 donors). Figure 2G) Biolayer interferometry assay of binding and dissociation between 25 monoclonal antibodies (mAbs) cloned directly from selected individual HPIV3 preF-specific B cells and HPIV3 preF protein. Figure 2H) Biolayer interferometry assay of binding kinetics between PI3-E12 or PI3-C9 monoclonal antibodies and HPIV3 preF protein. Figure 2I) Plaque reduction neutralization assay of PI3-E12 monoclonal antibody. Asterisk indicates P<0.05 by t-test compared to PBMC.

[0011] [Figure 3A-3F] High-throughput screening of B cells capable of producing neutralizing antibodies against HPIV3 in human PBMC, human tonsils, and human spleen is shown. (Fig. 3A) IgG was detected by ELISA in the supernatants of randomly selected B cells from individually sorted B cells (N=2, independent experiments) and grown on feeder cells. (Fig. 3B) Plaque reduction neutralization assay of supernatants of HPIV3 preF-specific B cells individually sorted and then grown on feeder cells (PBMC-derived B cells: N=120, tonsil-derived B cells: N=120, spleen-derived B cells: N=1,235). (Fig. 3C) Neutralization titers of HPIV3-specific monoclonal antibodies were measured by 60% plaque reduction neutralization assay on Vero cells with GFP-labeled HPIV3. The preF (Fig. 3D) or postF (Fig. 3E) structure of HPIV3 F protein was immobilized on the Penta-HIS probe. The binding of each monoclonal antibody was then measured by biolayer interferometry. All measurements were corrected for a negative control antibody. The positive control antibody was a human monoclonal antibody known to bind to the HPIV3 postF structure. (Figure 3F) Antinuclear antibody assay in HEp-2 cells using a monoclonal antibody targeting the HPIV3 preF structure. Binding was detected using an Alexa Fluor 647 (AF647)-labeled goat anti-human antibody as the secondary antibody. As a negative control for the autoreactivity assay, the monoclonal antibody palivizumab was used, and as a positive control, the 2F5 antibody. The mean fluorescence intensity was calculated from two independent experiments.

[0012] [Figure 4A-4D]Structural analysis of anti-HPIV3 preF monoclonal antibodies. (Fig. 4A) 2D classification of negative stain electron microscopy (EM) of HPIV3 preF complexed with PI3-E12 Fab. (Fig. 4B) 3D reconstruction of negative stain electron microscopy. Coordinates of HPIV3 preF trimer (label: PDB ID 6MJZ), GCN4 trimer domain (label: PDB ID 4DME), and crystal structure of PI3-E12 Fab (label: this study) were fitted to the 3D map. (Fig. 4C) Structure of PI3-E12 Fab with CDRs marked. Left: cartoon model. Right: surface model. (Fig. 4E) Structural alignment of PIA174 (indicated by star). Dots in the CDR sequence indicate gaps based on IMGT unique numbering.

[0013] [Figure 5A-5E] Efficacy of prophylactic and therapeutic administration of HPIV3 monoclonal neutralizing antibodies in vivo. (FIG. 5A) Schematic diagram of prophylactic experiment performed in cotton rats (N=4 per group). (FIG. 5B) Lung histopathology of cotton rats 4 days after infection. Arrows indicate areas of peribronchiolitis. Peribronchiolitis was scored as severity (%). (FIG. 5C) Viral titers by plaque assay in nasal and lung homogenates 4 days after infection. (FIG. 5D) Schematic diagram of therapeutic experiment performed in immunodeficient cotton rats (N=5 per group). (FIG. 5E) Viral titers by plaque assay in nasal (upper) and lung (lower) homogenates 4 days after infection. Asterisks indicate P<0.05 by t-test compared with PBS control.

[0014] [Figure 6A-6B]Dual binding and dual neutralization by MxR monoclonal antibodies are shown. (Figure 6A) The results of detecting the binding of MxR-01 monoclonal antibody to RSV preF (upper row) and HMPV preF (lower row) by biolayer interference method are shown. MxR-01 antibody is also called MxR-B11. (Figure 6B) The neutralization titer of MxR-01 antibody or palivizumab against RSV or HMPV was measured by plaque reduction neutralization test. Measurement of the neutralization titer of MxR-01 antibody showed a low value of 0.07 μg / mL, indicating that MxR-01 antibody exhibits strong protective effect against infection by HMPV or RSV in vitro. This assay also showed that MxR-01 antibody has a superior neutralization effect against RSV, which is almost 10 times higher than palivizumab.

[0015] [Figure 7] The formats and representative uses of recombinant antibodies are presented.

[0016] [Figure 8] Taking advantage of different bispecific formats, we show the design of a quadruple defense antibody that combines the antigen-binding sites of the MxR-01 antibody and the 3x1 antibody in a single monoclonal antibody. (Top) A configuration obtained using a knobs-into-holes approach is shown, which promotes heterodimerization to obtain an antibody that contains one antigen-binding fragment (Fab) from the MxR-01 antibody and one antigen-binding fragment (Fab) from the 3x1 antibody, linked by their constant (Fc) regions. As an alternative approach (bottom), an IgG-scFv fusion antibody is shown, in which the scFv of one antibody is linked to the constant region of another antibody.

[0017] [Figure 9A-9C]Isolation of HMPV / RSV (MxR) and HPIV3 / HPIV1 (3x1) cross-neutralizing monoclonal antibodies (Figure 9A) RSV-binding B cells and HMPV-binding B cells were isolated from human blood, and RSV-binding B cells were labeled with APC-labeled streptavidin tetramer of biotinylated RSV prefusion protein (preF), and separately, HMPV-binding B cells were labeled with PE-labeled streptavidin tetramer of biotinylated HMPV preF. Flow cytometry plots of B cells binding RSV preF and B cells binding HMPV preF after gating on live cells, CD3-CD14-CD16- (excluding non-B cells), CD19+CD20+ (B cells), IgD- / IgM- (isotype switched), and APC / Dylight755-HMPV postF-PE / DL650-RSV postF- (decoy to exclude cells binding RSV / HMPV postF, APC, PE, or streptavidin). Grey boxes indicate B cells from which MxR antibodies were obtained. (Figure 9B) HPIV3-binding B cells were isolated from human spleens and labeled with APC-labeled streptavidin tetramer of biotinylated HPIV3 preF. Flow cytometry plots of B cells binding to HPIV3 after gating on live cells, CD3-CD14-CD16-, CD19+CD20+, and IgD- / IgM- are shown. Black boxed cells in the bound fraction are B cells that bind HPIV3 preF but not HPIV3 postF. Grey boxes indicate B cells from which 3x1 antibodies were obtained. (Figure 9A, Figure 9B) The bound fraction represents cells magnetically enriched using anti-allophycocyanin (APC) or anti-phycoerythrin (PE) microbeads. The numbers in each gate are the percentage of total cells in that flow cytometry plot. (Figure 9C) Schematic of the "bait-and-switch" method to identify cross-neutralizing B cells using a single antigen. Using this method, two lead candidate antibodies were identified: the MxR antibody, which neutralizes RSV and HMPV, and the 3x1 antibody, which neutralizes HPIV3 and HPIV1.

[0018] [Figure 10A-10C] The phenotype and potency of MxR and 3x1 cross-neutralizing monoclonal antibodies in B cells are shown. (FIG. 10A) The source, phenotype, and alleles involved of B cells producing MxR and 3x1 antibodies are shown. (FIG. 10B) Vero cells were infected with RSV in the presence of serial dilutions of palivizumab (Pali.) or MxR antibodies. The upper dotted line indicates PRNT60. (FIG. 10C) PRNT60 results of palivizumab against RSV; PRNT60 results of MxR antibodies against RSV-A, RSV-B, or HMPV; and PRNT60 results of 3x1 antibodies against HPIV1 or HPIV3. Each data point is from three independent experiments, with two technical replicates in each experiment. The asterisk indicates a P value <0.01 compared to palivizumab using an unpaired two-tailed t-test with Welch's correction. In vitro, the MxR and 3x1 antibodies have stronger neutralizing ability against each virus than palivizumab.

[0019] [Figures 11A-11C]Epitope binning and structural analysis are shown. (FIG. 11A) A Penta-HIS probe was coated with His-tagged HPIV3 preF. The monoclonal antibodies shown on the left side of the diagram were immobilized on the coated probe and reacted with the monoclonal antibodies shown on the top line of the diagram. The legend indicates the degree of competition between the antibodies, expressed as the percentage of reduction in the maximum signal of the monoclonal antibody shown on the top line in the presence of the monoclonal antibody shown on the left side compared to the maximum signal with the monoclonal antibody shown on the top line alone. Site 0 is located at the apical end of HPIV3 preF36. Site X is a newly discovered antigenic site. Structures of the 3x1 antibody complexed with HPIV3 preF at 3.7 Å resolution (FIG. 11B) and the MxR antibody complexed with RSV preF at 2.3 Å resolution (FIG. 11C) are shown by cryo-electron microscopy. The top view is shown on the left. The side view is shown on the right. The 3x1 antibody binds to a novel antigenic site on HPIV3 preF, and the MxR antibody binds to the equatorial region of RSV preF.

[0020] [Figure 12] In vitro resistance analysis. Vero cells were inoculated with HPIV3 or RSV in the presence of increasing concentrations of 3x1 antibody, MxR antibody, or palivizumab (Pali.). At each subculture, the well with the highest antibody concentration in which viral replication was detectable by measuring cytopathic effect (CPE) was selected 5 days after infection, and the supernatant was harvested from this well and inoculated onto fresh Vero cells in the presence of increasing concentrations of each monoclonal antibody. MxR and HPIV3 appeared to have a higher resistance barrier than palivizumab.

[0021] [Figure 13]Measurement of the binding kinetics of MxR antibody and RSV preF by biolayer interferometry (BLI). BLI assays were performed at room temperature with shaking at 500 rpm using an Octet.Red instrument (ForteBio). A Penta-His capture sensor (ForteBio, Cat. No. 18-5120) was placed in kinetics buffer containing 1 μM His-tagged F protein, and His-tagged F protein was immobilized for 300 s. After His-tagged F protein was immobilized on the capture sensor, a baseline signal was recorded in kinetics buffer for 60 s. The capture sensor was then immersed in kinetics buffer containing purified MxR antibody to perform a binding step for 300 s, followed by immersion in kinetics buffer without antibody to measure the dissociation phase for 1200 s. Curve fitting was performed with the ForteBio data analysis software using a 1:1 binding model. The MxR antibody bound to the RSV preF protein with very high affinity, exceeding the measurement limit.

[0022] [Figure 14]Binding kinetics of MxR antibody and HMPV preF measured by biolayer interferometry (BLI). BLI assays were performed at room temperature with shaking at 500 rpm using an Octet.Red instrument (ForteBio). A Penta-His capture sensor (ForteBio, Cat. No. 18-5120) was placed in kinetics buffer containing 1 μM His-tagged F protein, and His-tagged F protein was immobilized for 300 s. After His-tagged F protein was immobilized on the capture sensor, a baseline signal was recorded for 60 s in kinetics buffer. The capture sensor was then immersed in kinetics buffer containing purified MxR antibody for a 300 s binding step, followed by immersion in kinetics buffer without antibody for a 600 s dissociation phase measurement. Curve fitting was performed with ForteBio data analysis software using a 1:1 binding model. MxR antibody bound to HMPV preF protein with nanomolar affinity.

[0023] [Figure 15]Measurement of the binding kinetics of 3x1 antibody and HPIV3 preF by biolayer interferometry (BLI). BLI assays were performed at room temperature with shaking at 500 rpm using an Octet.Red instrument (ForteBio). A Penta-His capture sensor (ForteBio, Cat. No. 18-5120) was placed in kinetics buffer containing 1 μM His-tagged F protein, and His-tagged F protein was immobilized for 300 s. After His-tagged F protein was immobilized on the capture sensor, a baseline signal was recorded in kinetics buffer for 60 s. The capture sensor was then immersed in kinetics buffer containing purified 3x1 antibody for a 300 s binding step, followed by immersion in kinetics buffer without antibody for a 1200 s dissociation phase measurement. Curve fitting was performed with ForteBio data analysis software using a 1:1 binding model. The 3x1 antibody bound to HPIV3 preF protein with a very high affinity that exceeded the measurement limit.

[0024] [Figure 16] We show that prophylactic administration of MxR antibody inhibits RSV replication in hamster lungs and nasal turbinates. Hamster challenge experiments were performed with N=4–5 per group. Hamsters were infected by intranasal administration of 100 μL of 105 pfu of virus. Two days prior to this infection, MxR monoclonal antibody (5 mg / kg) or PBS control was administered intramuscularly. Five days after infection, nasal turbinates and lungs were harvested and virus titers were measured by plaque assay. Lung and nasal homogenates were clarified by centrifugation. Confluent monolayers of Vero cells were seeded in duplicate onto each diluted homogenate in a 24-well plate. After 1 h of incubation at 37°C, each well was overlaid with methylcellulose. Four days later, the number of plaques was counted and titers were measured as pfu per gram of tissue. Prophylactic administration of MxR antibodies can suppress RSV replication in the nasal passages and lungs of hamsters.

[0025] [Figure 17] Figure 1 shows the inhibition of HPIV3 replication in hamster lungs and nasal turbinates by prophylactic administration of 3x1 antibody. Hamster challenge experiments were performed with N=4-5 per group. Hamsters were infected by intranasal administration of 100 μL of 105 pfu of virus. Two days prior to this infection, hamsters were administered intramuscularly with 3x1 monoclonal antibody (5 mg / kg) or PBS control. Five days after infection, nasal turbinates and lungs were harvested and virus titers were measured by plaque assay. Lung and nasal homogenates were clarified by centrifugation. Confluent monolayers of Vero cells were seeded in duplicate on each diluted homogenate in a 24-well plate. After 1 h of incubation at 37°C, each well was overlaid with methylcellulose. Four days later, the number of plaques was counted and titers were measured as pfu per gram of tissue. Prophylactic administration of 3x1 antibody can suppress HPIV3 replication in hamster lungs.

[0026] [Figure 18] Prophylactic administration of MxR and 3x1 antibodies (5 mg / kg each) suppressed RSV and HPIV3 co-infection in hamster lungs and nasal turbinates. Hamster challenge experiments were performed with N=4–5 animals per group. Hamsters were infected by intranasal administration of 105 pfu of each virus in 100 μL. Two days prior to this infection, each monoclonal antibody or PBS control was administered intramuscularly. Five days after infection, nasal turbinates and lungs were harvested and virus titers were measured by plaque assay. Lung and nasal homogenates were clarified by centrifugation. Confluent monolayers of Vero cells were seeded in duplicate on each diluted homogenate in 24-well plates. After 1 h of incubation at 37°C, each well was overlaid with methylcellulose. Four days later, the number of plaques was counted and titers were measured as pfu per gram of tissue. Prophylactic administration of a cocktail containing MxR and 3x1 antibodies suppresses the replication of RSV and HPIV3 in a hamster co-infection model.

[0027] [Figures 19A-19E](Figure 19A) Schematic diagram of an experiment in which prophylactic administration of MxR and 3x1 antibodies (5 mg / kg each) suppressed RSV and HPIV3 replication in the lungs and nasal turbinates of hamsters superinfected with RSV and HPIV3. Hamster challenge experiments were performed with N = 6 to 10 animals per group. Hamsters were infected by intranasal administration of 100 μL of each virus at 105 pfu. Two days prior to this infection, each monoclonal antibody or PBS control was administered intramuscularly. Five days after infection, nasal turbinates and lungs were harvested and virus quantification was performed by real-time PCR. Lung and nasal homogenates were clarified by centrifugation. Viral RNA was extracted from each sample homogenate using the QIAamp vRNA Mini Kit. Reverse transcription was performed using a custom reverse transcription primer for RSV (TCCAGCAAATACACCATCCAAC (SEQ ID NO: 187)) and a custom reverse transcription primer for HPIV3 (CTAGAAGGTCAAGAAAAGGGAACTC (SEQ ID NO: 188)). Virus-specific custom Taqman probes for real-time PCR were generated. 20x Gene Expression Assays for RSV (forward primer: TGACTCTCCTGATTGTGGGATGATA (SEQ ID NO: 189), reverse primer: CGGCTGAAGACCAGATCTGT (SEQ ID NO: 190) and reporter: CCCCTGCTGCTAATTT (SEQ ID NO: 191)) and 20x Gene Expression Assays for HPIV3 (forward primer: CGGTGACACAGTGGATCAGATT (SEQ ID NO: 192), reverse primer: TGTTTCAACCATAAGAGTTACCAAGCT (SEQ ID NO: 193) and reporter: ACCGCATGATTGACCC (SEQ ID NO: 194)) were used with Taqman Universal mastermix II with UNG. Samples were measured using a QuantStudio 7 Flex Real-Time PCR System and quantified based on standard curves generated from vRNA extracted from RSV and HPIV3 viral stocks of known titers.Viral titers were calculated in pfu / g by interpolation of cycle thresholds. Virus quantification following administration of PBS or the monoclonal antibody cocktail is shown for (Figure 19B) RSV in the lungs, (Figure 19C) RSV in the nasal turbinates, (Figure 19D) HPIV3 in the lungs, and (Figure 19E) HPIV3 in the nasal turbinates. Asterisks indicate P<0.01 by Mann-Whitney test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Paramyxovirus and pneumovirus respiratory infections cause tens of thousands of deaths each year in the United States and hundreds of thousands of deaths worldwide. In addition to representing significant healthcare costs, these respiratory infections are particularly virulent and deadly in immunosuppressed patients and high-risk adults and infants.

[0029] Hematopoietic stem cell transplant recipients: Patients undergoing hematopoietic stem cell transplantation (HCT) are particularly susceptible to infections during the six months it takes for their immune system to repopulate. Over 50,000 hematopoietic stem cell transplants have been performed worldwide, with 20,000 in the United States. Up to one-third of hematopoietic stem cell transplant recipients will develop a respiratory viral infection within six months of transplant. In up to one-third of these patients, the viral infection progresses from the upper to the lower respiratory tract. Once the virus gains a foothold in the lower respiratory tract, most viruses are unable to provide more than supportive care. As a result, up to 40% of patients with lower respiratory tract disease will die within three months. RSV, HMPV, and HPIV account for half of all serious respiratory viral infections after hematopoietic stem cell transplantation. Of patients who recover from acute infection, over 25% develop fixed airway obstruction, a debilitating condition that accelerates the loss of lung function and increases mortality.

[0030] Lung transplant recipients: Lung transplant recipients are another immunosuppressed population at particular risk for morbidity and mortality. Over 3,000 lung transplants are performed annually worldwide, with 2,000 lung transplants performed annually in the United States. Up to 25% of lung transplant recipients become infected with RSV, HMPV, or HPIV, with the majority of these infections occurring in the first year after transplant, when patients have resumed social activities. Infections with these viruses can lead to bacterial superinfections, tissue rejection, and chronic allograft dysfunction.

[0031] adult: People over 65, living on the street, and those with chronic lung disease are at risk for severe lung disease. The U.S. population over 65 is approaching 50 million, or 15% of the total population. In addition, 550,000 people in the U.S. live on the street, and an estimated 24 million adults in the U.S. have chronic obstructive pulmonary disease (COPD).

[0032] infant: Prematurity is a well-known risk factor for developing severe lung disease after RSV infection. Premature births before 37 weeks of gestation account for 11% of premature births in the United States. Infections occurring before the age of one year can have long-term effects on the lungs and reduce lung function. RSV accounts for the majority of hospitalizations due to lower respiratory tract viral infections, followed by HMPV and then HPIV.

[0033] Currently, there are no clinically available prophylaxis or treatments for RSV, HMPV, or HPIV infections in adults or transplant recipients. In premature infants, the antibody palivizumab is administered as a defense against RSV infection, but there is no defense against HMPV or HPIV infection. Palivizumab has a short half-life, which requires monthly reinjections, limiting its use.

[0034] The present disclosure provides novel antibodies that protect against one or more of human parainfluenza virus (HPIV), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV). A particular antibody disclosed herein is a protective antibody against HPIV3. Another antibody disclosed herein is a protective antibody against HPIV3 and HPIV1. Also disclosed herein is a protective antibody against RSV and HMPV. The novel antibodies disclosed herein can be used to protect against various forms of respiratory viruses.

[0035] In certain embodiments, various binding fragments derived from these newly disclosed antibodies and other antibodies can be recombined to obtain a multispecific format capable of simultaneously protecting against multiple types of viruses. That is, cross-neutralizing antibodies can be recombined into a single compound capable of protecting against multiple medically important respiratory viruses. An exemplary antibody, referred to herein as MRP3 / 1, comprises an extended half-life quadruple protection bispecific antibody created by linking together two monoclonal cross-neutralizing antibodies (see, e.g., Figures 7 and 8). MRP3 / 1 is a quad-protection bispecific antibody that binds to H. M P.V., R S.V., H. P IV 3 and HPIV 1 The antibody has high potency and broad applicability in vitro and is expected to have a half-life 4-6 times longer than palivizumab. Thus, MRP3 / 1 could replace palivizumab as the standard of care for RSV prophylaxis in premature infants, with the added benefit of providing protection against three viruses in addition to RSV. Furthermore, such recombinant constructs could be used as immune protection in hematopoietic stem cell transplant recipients during the immunocompromised period of 6 months post-transplant, or as treatment for patients hospitalized with any of the aforementioned infections.

[0036] Various aspects of the disclosure are described in further detail below under the following headings: (i) antibodies and recombinant binding molecules; (ii) formulations; (iii) methods of use; (iv) representative embodiments; (v) experimental examples; and (vi) conclusion. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the disclosure.

[0037] (i) Antibodies and recombinant binding molecules The structural unit of a natural antibody comprises a tetramer. Each tetramer comprises two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each light chain and each heavy chain comprises a variable region responsible for antigen recognition and binding to an epitope. The variable regions share a common general structure consisting of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity determining regions (CDRs). The CDRs of the two chains of each pair are aligned across the framework regions, allowing binding to a specific epitope. The light chain variable region and the heavy chain variable region comprise, from the N-terminus to the C-terminus, the FR1 domain, the CDR1 domain, the FR2 domain, the CDR2 domain, the FR3 domain, the CDR3 domain and the FR4 domain.

[0038] The amino acid assignments for each domain are based on Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al. (1997) JMB 273:927-948 (“Chothia” numbering scheme)); Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”); Gelfand; Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.(Contact numbering scheme));IMGT(Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77(“IMGT” numbering scheme));AHo(Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 June.8; 309(3):657-70 (AHo numbering scheme); North (North et al., J Mol Biol. 406(2):228-256 (2011), "A new clustering of antibody CDR loop conformations"); or other numbering schemes.

[0039] Software programs and bioinformatics tools such as ABodyBuilder and Paratome can be used to determine the CDR sequences. Additionally, X-ray crystallography can be used to determine the CDRs.

[0040] At the carboxy-terminal end of each light chain and each heavy chain is a constant region that is responsible, particularly for the heavy chain (Fc), for effector functions such as binding to C1q and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., the B cell receptor), and activation of B cells.

[0041] In full-length light chains and full-length heavy chains, the variable and constant regions are linked by a "J" region amino acid sequence, and the heavy chain further includes a "D" region amino acid sequence. See, e.g., Chapter 7 of Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0042] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α or ε chains, which define the antibody isotype: IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including IgM1 and IgM2. IgA is also divided into subclasses, including IgA1 and IgA2.

[0043] As described herein, an antibody binds to an epitope on an antigen. An "antigen" refers to a molecule or a portion thereof that can undergo binding by an antibody. An epitope is an area of ​​an antigen that undergoes binding by the variable region of an antibody. Epitope determinants may include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, sulfonyl groups, etc., and may have specific three-dimensional structural characteristics and / or specific charge characteristics. When an antigen is a protein or peptide, an epitope includes specific amino acids in the protein or peptide that make contact with the variable region of an antibody.

[0044] In certain embodiments, epitopes refer to binding sites on viral peptides that are bound by the corresponding variable regions of antibodies. Variable regions may bind to linear epitopes (e.g., epitopes that include a sequence of 5-12 consecutive amino acids) or may bind to three-dimensional structures formed by the spatial arrangement of several short sequences of a target protein. For example, a three-dimensional epitope recognized by a variable region, such as an epitope recognition site or paratope of an antibody or antibody fragment, can be considered as a three-dimensional surface feature of the epitope molecule. Such three-dimensional surface features of the epitope molecule fit (fit) into a corresponding variable region binding site, thereby facilitating binding of the variable region to its target protein (more generally, an antigen). In certain embodiments, epitopes can be considered to be of two types: (i) "covered portions," which can be considered as a shadow cast by the variable region of an antibody on the antigen to which it binds; and (ii) individual side chains and backbone residues that are involved in facilitating binding. Binding then occurs through ionic interactions, hydrogen bonding and hydrophobic interactions.

[0045] The epitopes of the antibodies of this disclosure (i.e., the epitopes to which the antibodies of this disclosure bind) are present on a virus selected from HPIV3, HPIV1, RSV, and / or HMPV. In certain embodiments, the epitopes of the antibodies of this disclosure are present on the F protein of the virus, e.g., the F protein of the virus in the prefusion state.

[0046] In certain embodiments, "binding" refers to a binding sequence in which the variable region is -8 This means that the molecule binds to its target epitope with a dissociation constant (Kd or KD) of 10 or less. In certain embodiments, -5 M~10 -13 This means that the molecule binds to its target epitope with a dissociation constant (Kd or KD) of 10 M, and in certain embodiments, -5 M~10 -10 This means that the molecule binds to its target epitope with a dissociation constant (Kd or KD) of 10 M, and in certain embodiments, -5 M~10 -7 This means that the molecule binds to its target epitope with a dissociation constant (Kd or KD) of 10 M, and in certain embodiments, -8 M~10 -13 This means that the molecule binds to its target epitope with a dissociation constant (Kd or KD) of 10 M or, in certain embodiments, 10 -9 M~10 -13 This means that the variable region binds to its target epitope with a dissociation constant (Kd or KD) of 10 M. Furthermore, the term "binding" can be used to indicate that the variable region does not bind to other biomolecules in the surrounding area (e.g., the variable region binds to the target epitope with a dissociation constant (Kd or KD) of 10 M. -4 M or greater, and in certain embodiments, can be used to indicate that the molecule binds to another biomolecule with a dissociation constant (Kd) of 10 -4 It can be used to indicate that a molecule binds to other biomolecules with a dissociation constant (Kd) of M~1M.

[0047] In certain embodiments, Kd can be assessed using BIAcore. For example, in certain embodiments, Kd can be measured by surface plasmon resonance assay using BIACORE® 2000 or BIACORE 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using a CM5 chip with antigen immobilized at a concentration of 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) can be activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen protein can be bound to the biosensor chip at a concentration of 10 response units (RU) by diluting the antigen to 5 μg / ml (0.2 μM) in 10 mM sodium acetate (pH 4.8) and injecting at a flow rate of 5 μl / min. After antigen injection, unreacted groups can be blocked by injecting 1M ethanolamine. For kinetic measurements, 0.05% polysorbate 20 (TWEEN-20) was used. TM ) 2-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected into PBS containing a surfactant (PBST) at 25°C and a flow rate of 25 μl / min. The binding rate constant (k on ) and the dissociation rate constant (k off The equilibrium dissociation constant (Kd) can be calculated by simultaneously curve fitting the association and dissociation sensorgrams with a simple 1:1 Langmuir binding model (BIACORE evaluation software, version 3.2). The equilibrium dissociation constant (Kd) is k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881, 1999. In this surface plasmon resonance assay, the binding rate constant is 10 6 M -1 s -1If the binding rate exceeds 100%, then the binding rate constant can be measured using a fluorescence quenching technique, which can be performed using a spectrophotometer equipped with a stopped-flow system (Aviv Instruments) or an 8000 series SLM-AMINCO equipped with a stirrable cuvette. TM A spectrometer such as a spectrophotometer (ThermoSpectronic) is used to measure the increase or decrease in fluorescence emission intensity of 20 nM anti-antigen antibody (in the form of Fab) in PBS (pH 7.2) at 25° C. in the presence of increasing concentrations of antigen (excitation=295 nm; emission=340 nm, bandwidth: 16 nm).

[0048] Unless otherwise stated, "antibody" includes variants, derivatives and fragments thereof (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above). Examples of these are also described below. Furthermore, unless expressly excluded, antibodies may include monoclonal antibodies, human or humanized antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multispecific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, fusion antibodies and fragments thereof. In certain embodiments, antibodies may include oligomeric or multiplexed antibodies.

[0049] "Monoclonal antibody" refers to an antibody obtained from a substantially homogenous antibody population, i.e., the individual antibodies in the antibody population are identical and / or bind to the same epitope, except for possible variant antibodies, such as those containing natural mutations or those that arise during the preparation of the monoclonal antibody, which are usually present in only minor amounts. In contrast to a polyclonal antibody preparation, which contains different antibodies targeting different epitopes, each monoclonal antibody in a monoclonal antibody preparation targets a single epitope on an antigen. Thus, the modifier "monoclonal" indicates the characterization of the antibody as being obtained from a substantially homogenous antibody population and should not be construed as requiring the antibody to be produced by a particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including hybridoma, recombinant DNA, phage display, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin locus.

[0050] A "human antibody" is an antibody that contains an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or that contains an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from a human antibody repertoire or other non-human source that utilizes coding sequences for human antibodies.

[0051] The "human consensus framework" is a set of human immunoglobulin V L or V H The framework sequence of human immunoglobulins is usually selected from the V L Array or V HThe sequence is selected from a subgroup of variable domain sequences, which may be a subgroup of variable domain sequences set forth in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In certain embodiments, the V L The subgroup of V is subgroup κI as reported by Kabat et al. (supra). H This subgroup is subgroup III reported by Kabat et al. (supra).

[0052] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one variable domain (usually two variable domains), with all or substantially all of the CDRs corresponding to the CDRs of a non-human antibody, and all or substantially all of the FRs corresponding to the FRs of a human antibody. A humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0053] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008, and further described, for example, in Riechmann et al., Nature 332:323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033, 1989; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34, 2005 (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498, 1991 (discussing "resurfacing"); Dall'Acqua et al., Methods 36:43-60,2005 (discussing "framework region shuffling"); and Osbourn et al., Methods 36:61-68, 2005 and Klimka et al., Br. J. Cancer, 83:252-260, 2000 (discussing "guided selection" methods of framework region shuffling). European Patent Publication No. 0239400(B) provides a detailed description of "CDR grafting methods," in which one or more CDR sequences of a first antibody are placed within the framework regions of the sequence of an antibody other than the first antibody (e.g., another antibody).

[0054] Human framework regions that may be used for humanization include framework regions selected by the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); framework regions derived from consensus sequences of human antibodies within a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Nati. Acad. Sci. USA, 89:4285, 1992; and Presta et al., J. Immunol., 151:2623, 1993); human mature framework regions (somatically mutated framework regions) or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008); and framework regions obtained by screening framework region libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684, 1997; and Rosok et al., J. Biol. Chem. 271:22611-22618, 1996).

[0055] In certain embodiments, the monoclonal antibody PI3-E12 disclosed herein has a CDRH1 comprising GFTFSDHY (SEQ ID NO: 1); a CDRH2 comprising ISSSGSNT (SEQ ID NO: 2); a CDRH3 comprising ARAKWGTMGRGAPPTIYDH (SEQ ID NO: 3); a CDRL1 comprising QSLLQSNGNNY (SEQ ID NO: 4); a CDRL2 comprising LGS; and a CDRL3 comprising MQALQTPLT (SEQ ID NO: 5).

[0056] In certain embodiments, PI3-E12 has a heavy chain sequence comprising QVQLLESGGKLVKPGGSLRLSCAASGFTFSDHYMIWIRQAPGKGLEWISYISSSGSNTIYADSLMGRFTISRDNAKNSLYLQMNSLRTEDTAVYYCARAKWGTMGRGAPPTIYDHWGQGTLVTVSS (SEQ ID NO: 166) and a light chain sequence comprising DIVMTQSPLSLPVTPGEPASISCRSSQSLLQSNGNNYLEWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDAGVYYCMQALQTPLTFGGGTKVEIK (SEQ ID NO: 167).

[0057] In certain embodiments, the PI3-E12 antibody is CAGGTGCAGCTGTTGGAGTCTGGGGGAAAGTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACCACTACATGATCTGGATCCGCCAGGCTCCCGGGAAGGGGCTGGAGTGGATTTCATACATAAGTAGTAGTGGTAGTAACACAAT CTACGCAGACTCTTTGATGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCTCTGTATCTACAAATGAACAGCCTGAGGACCGAGGACACGGCCGTTTATTACTGTGCGAGAGCAAAGTGGGGTACTATGGGTCGGGGAGCACCCCCGACAATTTATGACCACTGGGCCAGGGAACCCTGGTCACCGTCTCC and a kappa light chain variable region sequence encoded by GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGTAGGTCTAGTCAGAGCCTCCTGCAAAGTAATGGAAACAATTATTTGGAGTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCCAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAGATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC (SEQ ID NO:7).

[0058] In certain embodiments, the monoclonal antibody PI3-A3 disclosed herein has a CDRH1 comprising GFTFSNYW (sequence number 8); a CDRH2 comprising VKEEGSEK (sequence number 9); a CDRH3 comprising AGEVKSGWFGRYFDS (sequence number 10); a CDRL1 comprising QSVGSW (sequence number 11); a CDRL2 comprising KTS; and a CDRL3 comprising QQYSSFPYT (sequence number 12).

[0059] In a specific embodiment, the monoclonal antibody, PI3-A3, has a heavy chain sequence comprising EVQLVESGGGLVQPGGSLRLSCTASGFTFSNYWMSWVRQAPGKGLEWVANVKEEGSEKHYVDSVKGRFTISRDNAKNSVYLQMSSLRAEDTAVYYCAGEVKSGWFGRYFDSWGQGTLVTVSS (SEQ ID NO: 168) and a light chain sequence comprising DIQMTQSPSTLSASVGDRVTINCRASQSVGSWLAWYQQKPGKAPKLLMYKTSTLQRGVPSRFSGSGSGTEFTLTISSLQPDDFAAYYCQQYSSFPYTFGQGTKLEIK (SEQ ID NO: 169).

[0060] In certain embodiments, the PI3-A3 antibody is GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCTTTAGCAATTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAATGTGAAGGAAGAAGGAAGTGA GAAACACTATGTAGACTCTGTGAAGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAGTGTATCTGCAGATGAGCAGCCTGAGAGCCGAGGACACGGCTGTCTATTACTGTGCGGGAGAGGTTAAGAGTGGCTGGTTCGGTCGGTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCC and a kappa light chain variable region sequence encoded by GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTGGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATGTATAAGACATCTACTTTACAAAGAGGGGTCCCATCAAGGTTCAGCGGCGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAGCTTATTACTGCCAACAGTATAGTAGTTTTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC (SEQ ID NO: 14).

[0061] In certain embodiments, the monoclonal antibody PI3-B5 disclosed herein has a CDRH1 comprising GYNFTNYW (sequence number 15); a CDRH2 comprising IYPADSDT (sequence number 16); a CDRH3 comprising ARPSTRWFVPGGMDV (sequence number 17); a CDRL1 comprising QSIGAW (sequence number 18); a CDRL2 comprising KAS; and a CDRL3 comprising QQHSSYPST (sequence number 19).

[0062] In a specific embodiment, the monoclonal antibody, PI3-B5, has a heavy chain sequence comprising: EVQLVQSGAEVKKPGESLRISCKGSGYNFTNYWIAWVRQMPGKGLEWMGIIYPADSDTRYSPSFQGQVTISADKSITTAYLQWSSLKASDTAIYYCARPSTRWFVPGGMDVWGQGTTVIVSS (SEQ ID NO: 170) and a light chain sequence comprising: DIQMTQSPSTLSASVGDRVTISCRATQSIGAWLAWYQQKPGEPPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDSATYYCQQHSSYPSTFGGGTKVEIK (SEQ ID NO: 171).

[0063] In certain embodiments, the PI3-B5 antibody is GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATACAACTTTACCAACTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGCTGACTCGGA TACCAGATACAGCCCGTCCTTCCAAGGCCAAGTCACCATCTCAGCCGACAAGTCCATCACCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATATACTACTGTGCGAGACCGAGTACTAGGTGGTTCGTCCCTGGCGGTATGGACGTCTGGGGCCAAGGCACCACGGTCATCGTCTCC and a kappa light chain variable region sequence encoded by GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCTTCTGTAGGAGACAGAGTCACCATTTCTTGCCGGGGCCACTCAGAGTATTGGTGCCTGGTTGGCCTGGTATCAGCAGAAACCAGGGGAACCCCCTAAGCTCCTGATCTATAAGGCGTCTACTTTAGAGAGTGGGGTCCCATCAAGGTTCAGCGGCGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTCTGCAACTTATTACTGCCAACAGCATAGTAGTTATCCTTCTACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC (SEQ ID NO:21).

[0064] In certain embodiments, the monoclonal antibody PI3-A10 disclosed herein has a CDRH1 comprising GFNFNNYG (SEQ ID NO: 22); a CDRH2 comprising VSFDGSNR (SEQ ID NO: 23); a CDRH3 comprising SKSKYSDFWSEI (SEQ ID NO: 24); a CDRL1 comprising QNVMRY (SEQ ID NO: 25); a CDRL2 comprising DAS; and a CDRL3 comprising QQRTNHRFS (SEQ ID NO: 26).

[0065] In a specific embodiment, the monoclonal antibody PI3-A10 has a heavy chain sequence comprising QVQLVESGGGVVRPGRSLRLSCVASGFNFNNYGLQWIRQAPGKGLEWVAGVSFDGSNRYYADSVKGRVTISRDDSKNTLYLEMNSLRAEDTGIYYCSKSKYSDFWSEIWGQGTLVTVSS (SEQ ID NO: 172) and a light chain sequence comprising EIVLTQSPATLSLSPGERATLSCRASQNVMRYLAWYQQRPGQAPRLLFYDASSRATGIPARFTASGSGTDFTLTISGLEPGDFAVYYCQQRTNHRFSFGPGTKVDIK (SEQ ID NO: 173).

[0066] In certain embodiments, the PI3-A10 antibody is CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCAACTTCAATAACTATGGGCTGCAGTGGATCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGGTGTCTCGTTTGATGGGAG TAATAGATATTATGCAGACTCCGTGAAGGGCCGAGTCACCATATCCAGAGACGATTCCAAGAACACCCTGTATCTAGAAATGAACAGCCTGAGAGCTGAGGACACAGGAATATATTACTGTTCGAAGTCCAAGTACTCCGACTTTTGGAGCGAAATATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO:27), and a kappa light chain variable region sequence encoded by GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAATGTTATGAGGTACTTAGCCTGGTACCAACAGAGACCTGGCCAGGCTCCCAGACTCCTCTTCTATGATGCATCCAGCCGGGCCACTGGCATCCCAGCCCGGTTCACTGCCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCGGCCTCGAGCCTGGGAGATTTTGCAGTTTATTACTGTCAGCAGCGTACCAACCATAGATTCTCTTTCGGCCCTGGGACCAAGGTGGATATCAAAC (SEQ ID NO:28).

[0067] In certain embodiments, the monoclonal antibody PI3-A12 disclosed herein has a CDRH1 comprising GDSVKSDDFY (sequence number 29); a CDRH2 comprising IYYGGRT (sequence number 30); a CDRH3 comprising VRVEGLLWFGELFDY (sequence number 31); a CDRL1 comprising NSNIGNNF (sequence number 32); a CDRL2 comprising KDY; and a CDRL3 comprising AAWQDGLSGPL (sequence number 33).

[0068] In a particular embodiment, the monoclonal antibody PI3-A12 has a heavy chain sequence comprising QVQLQESGPGLVKPSETLSLTCTVSGDSVKSDDFYWSWIRQPPGKGLEWIGFIYYGGRTYYNPSLSGRGTISVDTSKNHFFLELTSVTAADTAVYYCVRVEGLLWFGELFDYWGQGTLVTVSS (SEQ ID NO: 174) and a light chain sequence comprising QSVLTQPPSASGTPGQRVTISCSGSNSNIGNNFVYWYQQVPGSAPKVVIYKDYQRPSGVPDRFSASKSGTSASLTISGLRSDDEAHYYCAAWQDGLSGPLFGGGTKLTVL (SEQ ID NO: 175).

[0069] In certain embodiments, the PI3-A12 antibody is CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCAGAGACCCTGTCCCTCACTTGCACTGTCTCTGGTGACTCCGTCAAGAGTGATGATTTCTACTGGAGTTGGATCCGGCAGCCCCCAGGGAAGGGCCTGGAGTGGATTGGCTTCATCTATTACGGTGGCAG AACTTACTACAACCCGTCCCTCAGTGGTCGAGGAACCATTTCAGTGACACGTCCAAGAACCACTTCTTCCTGGAGCTGACCTCTGTGACTGCCGCAGACACGGCCGTATACTACTGTGTCAGGGTCGAAGGATTACTGTGGTTCGGGGAGTTATTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA and a lambda light chain variable region sequence encoded by CAGTCTGTGCTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAACTCCAACATCGGAAATAATTTTGTCTACTGGTACCAACAAGTCCCAGGATCGGCCCCCAAAGTCGTCATTTACAAGGATTATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGACCATCAGCGGGCTCCGGTCCGACGATGAGGCCCATTATTACTGTGCAGCATGGCAGGACGGTCTGAGTGGGCCGTTATTTGGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO:35).

[0070] In certain embodiments, the monoclonal antibody 3x1 disclosed herein has a CDRH1 comprising GFTFSSFG (SEQ ID NO: 36); a CDRH2 comprising ISHSAGFL (SEQ ID NO: 37); a CDRH3 comprising AKRLAGLPDLEWLLYPNFLDH (SEQ ID NO: 38); a CDRL1 comprising ILRTYY (SEQ ID NO: 39); a CDRL2 comprising GKN; and a CDRL3 comprising SSRDRSGNHVL (SEQ ID NO: 40).

[0071] In a specific embodiment, the monoclonal antibody 3x1 has a heavy chain sequence comprising EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQSPGKGLEWVADISHSAGFLNYADSVKGRFTVSRDNSKSTLHLQMKSLRAEDTAVYYCAKRLAGLPDLEWLLYPNFLDHWGQGTLVTVSS (SEQ ID NO: 176) and a light chain sequence comprising SSELTQDPAVSVALGQTVRITCQGDILRTYYVSWYQQKPGQAPLLVIYGKNNRPSVIPDRFSGSTSGDTASLTITGAQAEDEAEYYCSSRDRSGNHVLFGGGTKLTVL (SEQ ID NO: 177).

[0072] In certain embodiments, the monoclonal antibody 3x1 is GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGTGGGTCCCTGAGACTGTCTTGTGCGGCCTCAGGATTCACCTTTAGCAGCTTTGGCATGAGCTGGGTCCGCCAATCTCCAGGAAAGGGGCTGGAGTGGGTCGCAGATATAAGCCATAGTGCTGGCTT CTTAAACTACGCAGACTCCGTGAAGGCCGGTTCACCGTCTCCAGAGACAATTCTAAGAGCACGCTGCATCTCCAAATGAAGAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGACTTGCCGGATTACCAGATTTGGAGTGGTTACTTTACCCTAACTTCTTAGACCACTGGGGCCAGGGAACCCTGG and a lambda light chain variable region sequence encoded by TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACATACTGAGAACCTATTATGTAAGCTGGTACCAGCAGAAACCAGGACAGGCCCCGCTACTTGTCATCTATGGTAAAAACAACCGACCCTCAGTGATCCCAGACCGATTCTCTGGCTCCACCTCAGGAGACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAGTATTATTGTAGCTCTCGGGACAGGAGTGGAAACCATGTGCTATTCGGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO:42).

[0073] In certain embodiments, the monoclonal antibody MxR-B11 (also referred to as MxR-01) disclosed herein has a CDRH1 that comprises GFPFSSYK (sequence number 43); a CDRH2 that comprises ISASGSYI (sequence number 44); a CDRH3 that comprises ARDGGRELSPFEK (sequence number 45); a CDRL1 that comprises NSNIGTGYD (sequence number 46); a CDRL2 that comprises DNN; and a CDRL3 that comprises QSYDKSLGGWV (sequence number 47).

[0074] In a specific embodiment, MxR-B11 (MxR-01) comprises a heavy chain variable region having the sequence set forth in EVQVVESGGGLVKPGGSLRLSCAASGFPFSSYKMDWVRQAPGKGLEWVSSISASGSYINYADSVKGRFTISRDNAKNSLYLQMKSLRADDTAVYFCARDGGRELSPFEKWGQGILVTVSS (SEQ ID NO: 178) and a light chain variable region having the sequence set forth in QSVLTQPPSVSGAPGQRVTISCTGTNSNIGTGYDVHWYQQLPGTAPKVVLFDNNNRPSGVPDRFSGSKSGTSAALAITGLQAEDEAVYYCQSYDKSLGGWVFGGGTKLTVL (SEQ ID NO: 179).

[0075] In a specific embodiment, the MxR-B11 antibody has the structure: GAGGTGCAGGTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCCCCTTCAGTTCTTATAAGATGGACTGGGTCCGCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCGTCCATCAGTGCTAGTGGAAGTTACATAAACTATGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAAAGCCTGAGAGCCGACGACACGGCTGTATATTTTTGTGCGAGAGACGGCGGAAGAGAACTGAGCCCCTTTGAAAAGTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 48). and a light chain variable region encoded by the sequence shown in CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGACAGAGGGTCACCATCTCCTGCACTGGGACCAACTCCAACATCGGGACAGGTTATGATGTACACTGGTACCAGCAGCTTCCGGGAACAGCCCCCCAAAGTCGTCCTCTTTGATAACAACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACTTCAGCCGCCCTGGCCATCACTGGCCTCCAGGCTGAGGATGAGGCTGTTTATTACTGCCAGTCCTATGACAAGAGCCTGGGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO: 49).

[0076] In certain embodiments, the monoclonal antibody disclosed herein, MxR-D10 (also referred to as MxR-02), has a CDRH1 that comprises GFIFSNYD (SEQ ID NO: 50); a CDRH2 that comprises ITGGSSFI (SEQ ID NO: 51); a CDRH3 that comprises ARDGGRQLSPCEH (SEQ ID NO: 52); a CDRL1 that comprises SSNIGAGYD (SEQ ID NO: 53); a CDRL2 that comprises DNN; and a CDRL3 that comprises QSYDRGLSGWA (SEQ ID NO: 54).

[0077] In a specific embodiment, the monoclonal antibody MxR-D10 (MxR-02) comprises a heavy chain variable region having the sequence set forth in EVQVVESGGGLVKPGGSLRLSCTASGFIFSNYDMNWVRQAPGKGLEWVASITGGSSFINYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARDGGRQLSPCEHWGQGTLVTVSS (SEQ ID NO: 180) and a light chain variable region having the sequence set forth in QSVLTQPPSVSGSPGQRVTISCTGGSSNIGAGYDVHWYQQLPGSAPKLLMYDSNNRPSGVPDRFSGSSKSGTSASLAITGLQAEDEADYYCQSYDRGLSGWAFGGGTKLTVL (SEQ ID NO: 181).

[0078] In a specific embodiment, the MxR-D10 antibody has the structure: GAGGTGCAGGTGGTGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCATATTCAGTAATTATGACATGAACTGGGTCCGCAGGCTCCAGGGAAGGGCCTGGAGTGGGTCGCCTCCATTACTGGTGGTAGTAGTTTCATAAATTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCACTGTATCTGCAAATGAACAGCCTCAGAGCCGAGGACACGGCTGTCTATTACTGTGCGAGAGATGGCGGGAGACAGTTGAGTCCGTGTGAACATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 55). and a light chain variable region encoded by the sequence shown in CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGTCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGGGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAACAACTTCCAGGATCAGCCCCCAAACTCCTCATGTATGATAGTAATAATCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGGGGCCTGAGTGGGTGGGCGTTCGGCGGAGGGACCAAGCTGACCGTCCTGG (SEQ ID NO:56).

[0079] As described herein, the antibodies disclosed herein can be used to prepare binding fragment molecules related to the invention in a variety of forms. For example, certain embodiments may include binding fragments of antibodies, such as Fv, Fab, Fab', F(ab')2 and single chain Fv fragments (scFv), or biologically effective fragments of immunoglobulins that specifically bind to an epitope described herein.

[0080] In certain embodiments, antibody fragments are used. An "antibody fragment" refers to an intact antibody or a portion of a full-length antibody that retains the ability to bind to an epitope. Antibody fragments can be generated by a variety of techniques, including proteolysis of a full-length antibody, or production by recombinant host cells (e.g., mammalian suspension cell lines, E. coli, or phage), as described herein. Antibody fragments can be screened for binding properties in the same manner as full-length antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; and linear antibodies.

[0081] A "single-chain variable fragment (scFv)" is a fusion protein consisting of the heavy and light variable chains of an immunoglobulin linked by a short linker peptide. Fv fragments are the V fragments derived from one arm of an antibody. L Domain and V H The Fv fragment contains two domains, namely, the V domain and the V domain, but lacks the constant region. L Domain and V H Although the domains are encoded by separate genes, a pair of V domains can be synthesized, for example, by recombinant methods, by linking the domains with a synthetic linker that allows the domains to be assembled into a single protein chain. L Area and V HMonovalent molecules consisting of the Fv and scFv domains (single chain Fv (scFv)) can be formed. For more information on Fv and scFv, see, for example, Bird, et al., Science 242:423-426, 1988; Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, (1994) 269-315; WO1993 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0082] scFv V L and V H The linker sequence used to link V is usually 5 to 35 amino acids in length. L -V H The linker may be 5 to 35 amino acids long, 10 to 30 amino acids long, or 15 to 25 amino acids long. Varying the length of the linker may maintain activity or may enhance activity, which may provide superior efficacy in activity studies. The linker sequence of the scFv is typically a Gly-Ser linker, which is described in detail elsewhere herein.

[0083] Other examples of antibody-based binding fragment formats include grababodies generated from scFvs and soluble VH domain antibodies, which use only the heavy chain variable region to form the binding region. See, e.g., Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0084] The Fab fragment is V L Domain, V H Domain, C L Domain and C H A diabody is a monovalent antibody fragment that contains one or more Fab fragments linked by a disulfide bridge at the hinge region. A F(ab')2 fragment is a bivalent fragment that contains two Fab fragments linked by a disulfide bridge at the hinge region. For a discussion of Fab and F(ab')2 fragments with extended half-life in vivo, see U.S. Pat. No. 5,869,046. A diabody contains two epitope binding sites that may be bivalent. See, e.g., European Patent Publication No. 0404097; WO1993 / 01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Dual affinity retargeting antibody (DART) TM based on the diabody format, but characterized by a disulfide bridge at the C-terminus for further stabilization (Moore et al., Blood 117:4542-51, 2011) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9:129-134, 2003.

[0085] Variants of the antibodies described herein are also included in the present invention. Antibody variants may include those with one or more conservative amino acid substitutions, or those with one or more non-conservative substitutions that do not adversely affect protein binding.

[0086] In certain embodiments, conservative amino acid substitutions may not substantially alter the structural characteristics of the reference sequence (e.g., the substituting amino acid should not tend to disrupt helices found in the reference sequence or other types of secondary structure characteristic of the reference sequence). Examples of polypeptide secondary and tertiary structures known in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature, 354:105 (1991).

[0087] In certain embodiments, V L The region is V L or may be derived from the V L and V of the present disclosure. L In one embodiment, each CDR contains no changes or at most one, two or three changes, and the recombined V LThe insertion, deletion, or substitution may be made without affecting the V L V including the amino or carboxy termini or both of the regions L It may be located anywhere in the region.

[0088] In certain embodiments, V H The region is V H or may be derived from the V H and V of the present disclosure. H In one embodiment, each CDR may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of these changes, compared to the recombined V. H The insertion, deletion, or substitution may be made without affecting the V H V including the amino or carboxy termini or both of the regions H It may be located anywhere in the region.

[0089] In certain embodiments, the variant comprises or is a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the antibody sequences disclosed herein. L ) and / or the heavy chain variable region (V H), or both, and which contain no, or at most one, two or three changes in each CDR compared to a reference antibody or fragment or derivative thereof disclosed herein that specifically binds to a target viral epitope.

[0090] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, human IgG2, human IgG3 or human IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0091] In certain embodiments, variants are modified from the reference sequence to provide administration advantages. Exemplary administration advantages include: (1) reduced susceptibility to hydrolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity to form protein complexes, (4) altered binding affinity, (5) reduced immunogenicity; and / or (6) increased half-life. In the following disclosure, these modifications are described as being applied to antibodies, but may also be applied to other specific binding fragment formats (e.g., scFvs and bispecific antibodies) where applicable.

[0092] In certain embodiments, a mutation can be introduced into an antibody to increase affinity to an Fc receptor. A representative mutation that increases affinity to an Fc receptor is G236A / S239D / A330L / I332E (GASDALIE) (Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6181-6186, 2012). In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, and the one or more amino acid substitutions that improve ADCC include, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (residue positions according to EU numbering). In certain embodiments, the Fc region can be modified to modify binding to C1q and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551 and WO 99 / 51642 and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.

[0093] In certain embodiments, it may be desirable to generate antibodies that incorporate cysteines, e.g., "thioMAbs" in which one or more residues on an antibody are replaced with cysteine ​​residues. In certain embodiments, such residue replacements are introduced into accessible sites on the antibody. By replacing residues at these sites with cysteine, reactive thiol groups can be placed at accessible sites on the antibody that may be used to conjugate the antibody to another moiety, e.g., to conjugate the antibody with a drug moiety or a linker-drug moiety to generate an immunoconjugate, as described below. In certain embodiments, the 5400th residue (EU numbering) of the heavy chain Fc region is selected. Antibodies that incorporate cysteines may be generated, e.g., as described in U.S. Pat. No. 7,521,541.

[0094] Further provided are antibody variants having a glycan structure lacking fucose linked (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose can be measured by calculating the percentage of the average amount of fucose in the glycan linked to Asn297 relative to the total amount of all glycan structures (e.g., complex, hybrid and high mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to the 297th asparagine residue of the Fc region (residue position of the Fc region according to EU numbering), although Asn297 may be located ±3 residues upstream or downstream from 297, i.e., between 294th and 300th, depending on rare antibody changes. Such fucosylation variants may have improved ADCC activity. See, for example, WO2000 / 61739; WO2001 / 29246; WO2002 / 031140; U.S. Patent Publication No. 2002 / 0164328; WO2003 / 085119; WO2003 / 084570; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent Publication No. 2003 / 0157108; U.S. Patent Publication No. 2003 / 0157208; U.S. Patent Publication No. 2003 / 0157320; See U.S. Patent Publication No. 2004 / 0093621; U.S. Patent Publication No. 2004 / 0110704; U.S. Patent Publication No. 2004 / 0132140; U.S. Patent Publication No. 2004 / 0110282; U.S. Patent Publication No. 2004 / 0109865; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and WO2003 / 085107).

[0095] In certain embodiments, the modified antibodies include antibodies in which one or more amino acids have been replaced with non-amino acid moieties, antibodies in which functional groups have been attached to amino acids, or antibodies in which functional groups unrelated to amino acids have been attached to amino acids. The modified amino acids may be, for example, glycosylated, PEGylated, farnesylated, acetylated, biotinylated, attached to lipid moieties, or attached to organic derivatizing agents. For example, amino acids may be modified co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation in NXS / T motifs when expressed in mammalian cells) or by synthetic methods. The modified amino acids may be present within the sequence or at the end of the sequence. Modifications also include nitritation constructs.

[0096] In certain embodiments, variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the reference sequence. In certain embodiments, glycosylation variants have more or fewer N-linked glycosylation sites than the reference sequence. N-linked glycosylation sites are characterized by a sequence designated Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated X can be any amino acid residue except proline. Substitution of amino acid residues to obtain this sequence provides a new site to which an N-linked glycan can be added. Alternatively, substitution can be made to remove this sequence, thereby removing existing N-linked glycans. N-linked glycans can also be rearranged such that one or more (e.g., native) N-linked glycosylation sites are removed and one or more new N-linked sites are formed. Another antibody variant includes a cysteine ​​variant in which one or more cysteine ​​residues are removed or replaced with another amino acid, such as serine, compared to the reference sequence. Such cysteine ​​variants may be useful when the antibody needs to be refolded into a biologically active structure, such as after isolating insoluble inclusion bodies. Such cysteine ​​variants usually contain fewer cysteine ​​residues than the reference sequence, and the number of cysteine ​​residues is usually an even number to minimize interactions with cysteines that do not have a binding partner.

[0097] Specifically, PEGylation is the process by which polyethylene glycol (PEG) polymer chains are covalently attached to another molecule, such as a protein. Several methods for PEGylation of proteins have been reported in the literature. For example, N-hydroxysuccinimide (NHS)-PEG has been used to PEGylate the free amine groups of lysine residues and the N-terminus of proteins; PEG with aldehyde groups has been used to PEGylate the amino terminus of proteins in the presence of reducing agents; PEG with maleimide functional groups has been used to selectively PEGylate the free thiol groups of cysteine ​​residues of proteins; site-specific PEGylation of acetylphenylalanine residues is also possible.

[0098] Covalent attachment of PEG to proteins has proven to be a useful method to extend the half-life of proteins in vivo (Abuchowski, A. et al., Cancer Biochem. Biophys.,1984, 7:175-186; Hershfield, MS et al., N. Engl. J. Medicine,1987, 316:589-596; and Meyers, FJ et al., Clin. Pharmacol. Ther.,49:307-313,1991). Attachment of PEG to proteins not only protects the molecule from enzymatic degradation but also reduces the rate of clearance from the body. The size of the PEG attached to a protein has a large effect on the half-life of the protein. The reduction in clearance due to PEGylation is usually independent of the number of PEG groups attached to the protein and depends on the total molecular weight of the PEGylated protein. Generally, the larger the PEG, the longer the in vivo half-life of the PEG-added protein. Additionally, PEGylation can reduce protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter the immunogenicity of a protein (Abuchowski et al., J. Biol. Chem. 252: 3582, 1977), and increase the solubility of a protein, as described, for example, in PCT Publication No. WO 92 / 16221.

[0099] PEGs of various sizes suitable for producing proteins with desired blood half-lives are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalog Ver 7.1). Various types of activated PEGs have been used, such as mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes (e.g., mPEG-propionaldehyde).

[0100] In certain embodiments, antibodies may be fused or linked to an Fc polypeptide containing amino acid changes that can increase the half-life of the antibody in vivo, such that the antibody containing the altered Fc polypeptide has an increased half-life in vivo compared to the half-life of a similar antibody that contains the same Fc polypeptide but does not contain the amino acid changes.

[0101] In certain embodiments, the amino acid changes in the Fc polypeptide include M252Y, S254T, T256E, M428L and / or N434S, which can be used simultaneously, separately, or in any combination. For example, the introduction of "YTE" mutations (M252Y, S254T, and T256E) into the Fc region of IgG can extend the half-life of the antibody in non-human primates by 4 times, from 3 weeks to more than 3 months, allowing the antibody to be effective with a single dose throughout the entire respiratory virus season. More information on YTE mutations can be found in Acqua et al., J. Immunol. 169(9) 5171-5180 (2002). The M428L / N434S pair is a mutation that can increase the half-life of antibodies in serum and is described in Zalevsky et al., Nature Biotechnology 28, 157-159, 2010. Other potentially useful changes are described in U.S. Patent No. 7,083,784, U.S. Patent No. 7,670,600, U.S. Patent Publication No. 2010 / 0234575, PCT U.S. Patent Publication No. 2012 / 070146, and Zwolak, Scientific Reports 7: 15521, 2017. In certain embodiments, half-life may be increased by substituting amino acid positions in the Fc polypeptide at: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, or 436. Each of these changes, or a combination of these changes, may be used to increase the half-life of the bispecific antibodies described herein.

[0102] In certain embodiments, the antibodies disclosed herein are generated using the Daedalus expression system reported by Pechman et al. (Am J Physiol 294: R1234-R1239 (2008)). The Daedalus expression system suppresses or prevents genome silencing by introducing minimal ubiquitous chromatin opening elements into the transduction vector, and maintains stable expression at decigram levels. The Daedalus expression system utilizes the secretion pathway of human suspension cell lines suitable for serum-free media, such as the 293 Freestyle line, thereby eliminating laborious and time-consuming steps of other protein production methods. By using optimized lentiviral vectors, it is possible to obtain correctly folded, post-translationally modified, endotoxin-free proteins of up to 70 kDa in size at yields of 20-100 mg / l in conventional small-scale cultures (100 ml). With such yields, the majority of the protein can be purified in a single size-exclusion chromatography step and is immediately suitable for use in structural, biophysical or therapeutic applications (Bandaranayake et al., Nucleic Acids Res., 39(21) 2011). In some cases, the purity of the production according to the methods described herein may not require chromatographic purification.

[0103] Antiviral bispecific antibodies of the invention bind to at least two epitopes, at least one of which is present in HPIV3, HPIV1, RSV or HMPV. Antiviral trispecific antibodies of the invention bind to at least three epitopes, at least one of which is present in HPIV3, HPIV1, RSV, HMPV, etc. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). For example, WO 1996 / 016673 describes bispecific anti-ErbB2 / anti-FcγRIII antibodies; U.S. Patent No. 5,837,234 describes bispecific anti-ErbB2 / anti-FcγRI antibodies; WO 1998 / 002463 describes bispecific anti-ErbB2 / Fcα antibodies; and U.S. Patent Publication No. 5,821,337 describes bispecific anti-ErbB2 / anti-CD3 antibodies. In certain embodiments, the bispecific antibody may be in the form of a bispecific T cell engager (BiTE®) antibody.

[0104] Some other representative bispecific antibodies have two heavy chains (each heavy chain having three heavy chain CDRs followed (from N- to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain) and two immunoglobulin light chains that confer antigen-binding specificity through binding to each heavy chain. However, as noted herein, other configurations are also contemplated, such as bispecific antibodies in which one or more light chains are associated with each heavy chain but do not (or only minimally) contribute to antigen-binding specificity; bispecific antibodies in which one or more light chains can bind to one or more epitopes bound by the antigen-binding region of the heavy chain; or bispecific antibodies in which one or more light chains can bind to each heavy chain, allowing one or both of the heavy chains to bind one or two epitopes.

[0105] Instead of full-length antibodies, scFv dimers or diabodies may be used. Diabodies and scFvs can be constructed without the Fc region, using only variable domains (usually containing variable domain portions derived from the light and heavy chains of the original antibody), to reduce the effects of anti-idiotypic reactions. Another form of bispecific antibody is the single-chain antibody "Janusin" reported by Traunecker et al. (Embo Journal, 10, 3655-3659, 1991).

[0106] Bispecific antibodies with extended half-lives are described, for example, in U.S. Pat. No. 8,921,528 and U.S. Patent Publication No. 2014 / 0308285.

[0107] The method of producing bispecific antibodies is known in the art.For example, the conventional method of producing full-length bispecific antibodies is based on co-expressing two immunoglobulin heavy and light chain pairs with different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983).For example, similar techniques are disclosed in WO1993 / 008829, Traunecker et al., EMBO J. 10:3655-3659, 1991 and Holliger & Winter, Current Opinion Biotechnol. 4, 446-449 (1993).

[0108] In certain embodiments, bispecific antibodies can be prepared using chemical linkage. For example, in the procedure reported by Brennan et al. (Science 229: 81, 1985), full-length antibodies are proteolytically cleaved to obtain F(ab')2 fragments. The F(ab')2 fragments are then reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab'-TNB derivatives is then reduced with mercaptoethylamine to convert it to a Fab'-thiol and mixed with the other Fab'-TNB derivative in an equimolar amount to form the bispecific antibody.

[0109] In certain embodiments, bispecific antibodies can be prepared using the knobs-into-holes technique. The knobs-into-holes technique refers to a technique in which different mutations are introduced into the CH3 domains of two antibody heavy chains to modify the contact interface of each CH3 domain, forcing these antibody heavy chains to pair with each other. A bulky amino acid in one heavy chain is replaced with an amino acid with a short side chain to form a "hole". An amino acid with a large side chain is introduced into the CH3 domain of the other heavy chain to form a "knob". By co-expressing these two heavy chains (and two identical light chains, which must be appropriate for both heavy chains), heterodimers ("knob-hole") are formed in higher yields than homodimers ("hole-hole" or "knob-knob") (Ridgway, JB, Protein Eng. 9 (1996) 617-621; and WO96 / 027011).

[0110] In certain embodiments, the "knob" and / or "hole" may be native to the polypeptide or may be synthetically introduced (e.g., by modifying a nucleic acid encoding the polypeptide). The knob and / or hole are synthetically introduced by replacing the nucleic acid encoding the original amino acid residue (or other non-amino acid group, e.g., a hydrocarbon group) present at the contact interface of the polypeptide with DNA encoding at least one inserted amino acid residue. The contact interface refers to one or more amino acid residues (or other non-amino acid groups) that are in contact between the first heavy chain constant region and the second heavy chain constant region. The inserted residue to form the hole is preferably an amino acid whose side chain volume is smaller than that of the original amino acid residue, such as alanine (A), serine (S), threonine (T), valine (V), glycine (G), etc. The inserted residue to form the knob is preferably an amino acid whose side chain volume is larger than that of the original amino acid residue, such as tyrosine (Y), arginine (R), phenylalanine (F), tryptophan (W), etc. The proportion of heterodimers can also be increased by remodeling the interaction surfaces of the two CH3 domains using phage display techniques and the introduction of disulfide bridges that stabilize the heterodimers (Merchant A. M, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35).

[0111] In certain embodiments, the binding fragments disclosed herein can be used to generate bispecific, trispecific or tetraspecific antibody constructs (or specific antibody constructs with more specificities) that bind to a second virus. In the present specification, the second virus is a virus other than HPIV3, HPIV1, RSV, HMPV. In certain embodiments, the second virus is a respiratory virus. In certain embodiments, the second respiratory virus is selected from adenovirus, bocavirus, coronavirus, enterovirus, influenza virus, metapneumovirus, parainfluenza virus and / or rhinovirus. In certain embodiments, respiratory viruses include human adenovirus, human bocavirus (HBoV), human coronavirus (HCoV) (including SARS-CoV, MERS-CoV, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, coronavirus NL, coronavirus NH), influenza virus (types A and B), human parainfluenza virus (HPIV2 or HPIV4), and / or human rhinovirus (HRVA-HRVC).

[0112] Representative binding fragments that can be used in the recombinant format to bind to a second virus include 8C4, 5Hx-l, 5Hx-2, 5Hx-3, 5Hx-4, 5Hx-5, 5.100K-1, 5PB-1, 5Fb-l, and 1E11, which bind adenovirus; EPR23305-44, which binds coxsackievirus and adenovirus; 47D11 antibody, which binds SARS-CoV and SARS-CoV-2; CR3022, which binds SARS-CoV-2; CDC2-A2, G2, 5F9, FIB-H1, and JC57-13, which bind MERS-CoV; 32D6, which binds H1N1 influenza virus; CH65, which binds H1 influenza virus; CR9114, which binds influenza A virus, MAb 22 / 1, MAb70 / l, MAb 110 / 1, MAb 264 / 2, MAb W18 / 1, MAb 14 / 3, MAb 24 / 4, MAb 47 / 8, MAb 198 / 2, MAb 215 / 2, H2 / 6A5, H3 / 4C4, H2 / 6C4, H2 / 4B3, H9 / B20, H2 / 4B1, CA6261, 6F12, CR9114 and PEG-1; CR8033, CR8071, 113 / 2, 124 / 4, 128 / 2, 134 / 1, 146 / 1, 152 / 2, 160 / 1, 162 / 1, 195 / 3, 206 / 2, 238 / 4 and 280 / 2 which bind influenza B virus; PAR2 (boca231 / 9F) which binds HPIV2; or TCN-711 which binds rhinovirus.

[0113] Exemplary binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence set forth in EFTFNTYG (SEQ ID NO:57), ISYDGTKK (SEQ ID NO:58) and ARSGDSDAFDI (SEQ ID NO:59), and / or a light chain having three CDRs each comprising the amino acid sequence set forth in ELGDKF (SEQ ID NO:60), QDS and QAWDSNSYV (SEQ ID NO:61).

[0114] Exemplary binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence set forth in GGTFGSYA (SEQ ID NO: 62), the amino acid sequence set forth in IDAANGNT (SEQ ID NO: 63) and the amino acid sequence set forth in ARDRWMTTRAFDI (SEQ ID NO: 64), and / or a light chain having three CDRs each comprising the amino acid sequence set forth in SSNIGSNY (SEQ ID NO: 65), the amino acid sequence set forth in RNN and the amino acid sequence set forth in AAWDDSLRGPV (SEQ ID NO: 66).

[0115] Exemplary binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence set forth in GGTFSSYA (SEQ ID NO: 135), the amino acid sequence set forth in IIPIFGKA (SEQ ID NO: 136) and the amino acid sequence set forth in ARDQGISANFKDAFDI (SEQ ID NO: 137), and / or a light chain having three CDRs each comprising the amino acid sequence set forth in ESVGSN (SEQ ID NO: 138), the amino acid sequence set forth in GAS and the amino acid sequence set forth in QQYNNWPLT (SEQ ID NO: 139).

[0116] Exemplary binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence represented by GGTFSSYA (SEQ ID NO: 135), the amino acid sequence represented by IIPIFGTA (SEQ ID NO: 141) and the amino acid sequence represented by ARVGYCSSTSCHIGAFDI (SEQ ID NO: 142), and / or a light chain having three CDRs each comprising the amino acid sequence represented by QSVSSS (SEQ ID NO: 143), the amino acid sequence represented by DSS and the amino acid sequence represented by QQYSSSPYT (SEQ ID NO: 144), respectively.

[0117] Exemplary binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence represented by GGTFSSYA (SEQ ID NO: 135), the amino acid sequence represented by IIPIFGTA (SEQ ID NO: 141) and the amino acid sequence represented by ARASYCSTTSCASGAFDI (SEQ ID NO: 147), and / or a light chain having three CDRs each comprising the amino acid sequence represented by QSVLYSSNNNY (SEQ ID NO: 148), the amino acid sequence represented by WAS and the amino acid sequence represented by QQYYSVPFT (SEQ ID NO: 149), respectively.

[0118] Exemplary binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence set forth in GYTFNVYA (SEQ ID NO: 150), the amino acid sequence set forth in IIPILGIA (SEQ ID NO: 151) and ARDYYGSGARGFDY (SEQ ID NO: 152), and / or a light chain having three CDRs each comprising the amino acid sequence set forth in SNNVGNQG (SEQ ID NO: 153), the amino acid sequence set forth in TNN and the amino acid sequence set forth in ASWDSSLSVWV (SEQ ID NO: 154), respectively.

[0119] Representative binding fragments that can be used in a recombinant format to bind to a second virus, such as MERS-CoV, include a heavy chain having three CDRs each comprising the amino acid sequence GGTFSSYA (SEQ ID NO: 135), the amino acid sequence IIPIFGIA (SEQ ID NO: 156) and the amino acid sequence ASSNYYGSGSYYPRSAFDI (SEQ ID NO: 157), and / or a light chain having three CDRs each comprising the amino acid sequence QSISND (SEQ ID NO: 158), the amino acid sequence GAS and the amino acid sequence QQYGVSPLT (SEQ ID NO: 159). Further examples are described in U.S. Patent No. 9,718,872.

[0120] Exemplary heavy and light chain variable regions that can be used in a recombinant format that binds to a second virus, such as SARS-CoV, include a heavy chain variable region sequence that includes: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTNTAYMELRSLRSDDTAVYYCAVGRYLDYWGQGTLVTV (SEQ ID NO: 160), and a light chain variable region sequence that includes: DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSYPLTFGGGTKVEIK (SEQ ID NO: 161).

[0121] Exemplary heavy and light chain variable regions that can be used in a recombinant format that binds to a second virus, such as SARS-CoV, include a heavy chain variable region sequence that includes: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAVGRYLDYWGQGTLVTVSS (SEQ ID NO: 162) and a light chain variable region sequence that includes: DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSYPLTFGGGTKVEIK (SEQ ID NO: 161).

[0122] Exemplary heavy and light chain variable regions that can be used in a recombinant format that binds to a second virus, such as SARS-CoV, include a heavy chain variable region sequence that includes: QVHLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYFCAGGRYLDYWGQGTLVTVSS (SEQ ID NO: 164) and a light chain variable region sequence that includes: DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKSGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNSYPITFGQGTRLEIK (SEQ ID NO: 165). Further examples are described in U.S. Patent No. 7,728,110.

[0123] In certain embodiments, the binding fragments disclosed herein can be used to generate bispecific or trispecific antibody constructs (or specific antibody constructs with more specificities) that function as immune cell engagers. Examples of multispecific antibody constructs that function as immune cell engagers include multispecific antibody constructs that bind to viral epitopes on HPIV3, HPIV1, RSV, or HMPV and epitopes that activate immune cells (e.g., T cells or NK cells) with the goal of bringing immune cells into contact with virus-infected cells that present the viral epitopes of HPIV3, HPIV1, RSV, or HMPV to which the antibodies disclosed herein bind. See, for example, U.S. Patent Publication No. 2008 / 0145362. Such constructs are referred to herein as immune-activating multi-specifics, or I-AMS. BiTE® (Amgen, Thousand Oaks, Calif.) is a form of I-AMS. In the present disclosure, immune cells that can be targeted for local activation by I-AMS include, for example, T cells, natural killer (NK) cells, and macrophages, which are described in detail below.

[0124] T cell activation can be mediated by two types of signals. One signal initiates antigen-dependent primary activation and provides a T cell receptor-like signal (primary intracellular signaling sequence), and the other signal provides an antigen-independent secondary or costimulatory signal (secondary intracellular signaling sequence). The I-AMS disclosed herein can target T cell activation epitopes that can induce T cell activation upon binding of the I-AMS. Examples of such T cell activation epitopes include epitopes on T cell markers, including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB (CD 137), OX40, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C, and B7-H3.

[0125] In certain embodiments, the CD3 binding fragment (e.g., scFv) is derived from the OKT3 antibody (the same antibody utilized in Blinatumomab). The OKT3 antibody is described in detail in U.S. Pat. No. 5,929,212. The OKT3 antibody comprises a light chain variable region comprising a CDRL1 sequence comprising SASSSVSYMN (SEQ ID NO: 67), a CDRL2 sequence comprising RWIYDTSKLAS (SEQ ID NO: 68), and a CDRL3 sequence comprising QQWSSNPFT (SEQ ID NO: 69). In certain embodiments, the epitope binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) comprising a heavy chain variable region comprising a CDRH1 sequence comprising KASGYTFTRYTMH (SEQ ID NO: 70), a CDRH2 sequence comprising INPSRGYTNYNQKFKD (SEQ ID NO: 71), and a CDRH3 sequence comprising YYDDHYCLDY (SEQ ID NO: 72).

[0126] The sequence represented by QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 73) is an scFv derived from OKT3 and retains CD3 binding ability. This sequence may be used as a CD3 binding fragment.

[0127] In certain embodiments, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a light chain variable region that comprises a CDRL1 sequence that comprises QSLVHNNGNTY (SEQ ID NO: 74), a CDRL2 sequence that comprises KVS, and a CDRL3 sequence that comprises GQGTQYPFT (SEQ ID NO: 75). In certain embodiments, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a heavy chain variable region that comprises a CDRH1 sequence that comprises GFTFTKAW (SEQ ID NO: 76), a CDRH2 sequence that comprises IKDKSNSYAT (SEQ ID NO: 77), and a CDRH3 sequence that comprises RGVYYALSPFDY (SEQ ID NO: 78). These CDR sequences constitute the CDR sequences of the 20G6-F3 antibody.

[0128] In certain embodiments, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a light chain variable region that comprises a CDRL1 sequence that comprises QSLVHDNGNTY (SEQ ID NO: 79), a CDRL2 sequence that comprises KVS, and a CDRL3 sequence that comprises GQGTQYPFT (SEQ ID NO: 75). In certain embodiments, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a heavy chain variable region that comprises a CDRH1 sequence that comprises GFTFSNAW (SEQ ID NO: 80), a CDRH2 sequence that comprises IKARSNNYAT (SEQ ID NO: 81), and a CDRH3 sequence that comprises RGTYYASKPFDY (SEQ ID NO: 82). These CDR sequences constitute the CDR sequences of the 4B4-D7 antibody.

[0129] In a particular embodiment, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a light chain variable region that comprises a CDRL1 sequence that comprises QSLEHNNGNTY (SEQ ID NO: 83), a CDRL2 sequence (not shown in the sequence listing) that comprises KVS, and a CDRL3 sequence that comprises GQGTQYPFT (SEQ ID NO: 75). In a particular embodiment, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a heavy chain variable region that comprises a CDRH1 sequence that comprises GFTFSNAW (SEQ ID NO: 80), a CDRH2 sequence that comprises IKDKSNNYAT (SEQ ID NO: 84), and a CDRH3 sequence that comprises RYVHYGIGYAMDA (SEQ ID NO: 85). These CDR sequences constitute the CDR sequences of the 4E7-C9 antibody.

[0130] In certain embodiments, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a light chain variable region that comprises a CDRL1 sequence that comprises QSLVHTNGNTY (SEQ ID NO: 86), a CDRL2 sequence that comprises KVS, and a CDRL3 sequence that comprises GQGTHYPFT (SEQ ID NO: 87). In certain embodiments, the epitope-binding fragment that activates CD3 T cells is a human or humanized binding fragment (e.g., scFv) that comprises a heavy chain variable region that comprises a CDRH1 sequence that comprises GFTFTNAW (SEQ ID NO: 88), a CDRH2 sequence that comprises KDKSNNYAT (SEQ ID NO: 89), and a CDRH3 sequence that comprises RYVHYRFAYALDA (SEQ ID NO: 90). These CDR sequences constitute the CDR sequences of the 18F5-H10 antibody.

[0131] Further examples of anti-CD3 antibodies, binding fragments and CDRs are described in WO2016 / 116626. TR66 may also be used.

[0132] CD28 is a surface glycoprotein present on 80% of human peripheral T cells, both resting and activated. CD28 binds to B7-1 (CD80) and B7-2 (CD86), the most potent of the known costimulatory molecules (June et al., Immunol. Today 15:321, 1994; Linsley et al., Ann. Rev. Immunol. 11:191, 1993). In certain embodiments, the CD28 binding fragment (e.g., scFv) is derived from the CD80, CD86 or 9D7 antibody. Other antibodies that bind to CD28 include 9.3, KOLT-2, 15E8, 248.23.2 and EX5.3D10. Additionally, there is 1YJD, a crystal structure of human CD28 complexed with the Fab fragment of a mitogenic antibody (5.11A1).

[0133] In certain embodiments, the CD28 binding fragment is derived from TGN1412. In certain embodiments, the heavy chain variable region of TGN1412 comprises QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS (SEQ ID NO: 91) and the light chain variable region of TGN1412 comprises DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO: 92).

[0134] In a specific embodiment, the CD28 binding fragment comprises a light chain variable region comprising a CDRL1 sequence comprising HASQNIYVWLN (SEQ ID NO: 93), a CDRL2 sequence comprising KASNLHT (SEQ ID NO: 94), and a CDRL3 sequence comprising QQGQTYPYT (SEQ ID NO: 95), and a heavy chain variable region comprising a CDRH1 sequence comprising GYTFTSYYIH (SEQ ID NO: 96), a CDRH2 sequence comprising CIYPGNVNTNYNEK (SEQ ID NO: 97), and a CDRH3 sequence comprising SHYGLDWNFDV (SEQ ID NO: 98).

[0135] In a specific embodiment, the CD28 binding fragment comprises a light chain variable region comprising a CDRL1 sequence comprising HASQNIYVWLN (SEQ ID NO: 93), a CDRL2 sequence comprising KASNLHT (SEQ ID NO: 94), and a CDRL3 sequence comprising QQGQTYPYT (SEQ ID NO: 95), and a heavy chain variable region comprising a CDRH1 sequence comprising SYYIH (SEQ ID NO: 99), a CDRH2 sequence comprising CIYPGNVNTNYNEKFKD (SEQ ID NO: 100), and a CDRH3 sequence comprising SHYGLDWNFDV (SEQ ID NO: 98).

[0136] Activated T cells express 4-1BB (CD137). In a specific embodiment, the 4-1BB binding fragment comprises a light chain variable region comprising a CDRL1 sequence comprising RASQSVS (SEQ ID NO: 101), a CDRL2 sequence comprising ASNRAT (SEQ ID NO: 102), and a CDRL3 sequence comprising QRSNWPPALT (SEQ ID NO: 103), and a heavy chain variable region comprising a CDRH1 sequence comprising YYWS (SEQ ID NO: 104), a CDRH2 sequence comprising INH, and a CDRH3 sequence comprising YGPGNYDWYFDL (SEQ ID NO: 105).

[0137] In a specific embodiment, the 4-1BB binding fragment comprises a light chain variable region comprising a CDRL1 sequence comprising SGDNIGDQYAH (SEQ ID NO: 106), a CDRL2 sequence comprising QDKNRPS (SEQ ID NO: 107), and a CDRL3 sequence comprising ATYTGFGSLAV (SEQ ID NO: 108), and a heavy chain variable region comprising a CDRH1 sequence comprising GYSFSTYWIS (SEQ ID NO: 109), a CDRH2 sequence comprising KIYPGDSYTNYSPS (SEQ ID NO: 110), and a CDRH3 sequence comprising GYGIFDY (SEQ ID NO: 111).

[0138] Certain embodiments disclosed herein include binding fragments that bind to an epitope on CD8. In certain embodiments, the CD8 binding fragment (e.g., scFv) is derived from the OKT8 antibody. For example, in certain embodiments, the epitope-binding fragment that activates CD8 T cells is a human or humanized binding fragment (e.g., scFv) that includes a light chain variable region that includes a CDRL1 sequence that includes RTSRSISQYLA (SEQ ID NO: 112), a CDRL2 sequence that includes SGSTLQS (SEQ ID NO: 113), and a CDRL3 sequence that includes QQHNENPLT (SEQ ID NO: 114). In certain embodiments, the epitope-binding fragment that activates CD8 T cells is a human or humanized binding fragment (e.g., scFv) that includes a heavy chain variable region that includes a CDRH1 sequence that includes GFNIKD (SEQ ID NO: 115), a CDRH2 sequence that includes RIDPANDNT (SEQ ID NO: 116), and a CDRH3 sequence that includes GYGYYVFDH (SEQ ID NO: 117). These CDR sequences constitute the CDR sequences of the OKT8 antibody.

[0139] In certain embodiments, natural killer cells (also known as NK cells, K cells and killer cells) are targeted for local activation by I-AMS. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cell membranes and can secrete cytokines to recruit other immune cells.

[0140] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that can activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0141] Examples of commercially available antibodies that bind to NK cell receptors and induce and / or enhance NK cell activation include 5C6 and 1D11 (available from BioLegend®, San Diego, Calif.), which bind and activate NKG2D; mAb 33 (available from BioLegend), which binds and activates KIR2DL4; P44-8 (available from BioLegend), which binds and activates NKp44; SK1, which binds and activates CD8; and 3G8, which binds and activates CD16.

[0142] In certain embodiments, I-AMS can enhance NK cell activation by binding to and blocking NK cell inhibitory receptors. Examples of NK cell inhibitory receptors that can be blocked by I-AMS binding include KIR2DL1, KIR2DL2 / 3, KIR3DL1, NKG2A, and KLRG1. In certain embodiments, the binding fragment that binds to and blocks the NK cell inhibitory receptors KIR2DL1 and KIR2DL2 / 3 comprises a light chain variable region consisting of the sequence set forth in EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT (SEQ ID NO: 118) and a heavy chain variable region consisting of the sequence set forth in QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS (SEQ ID NO: 119). Further examples of antibodies that activate NK cells are described in WO / 2005 / 0003172 and U.S. Patent No. 9,415,104.

[0143] In certain embodiments, the target of local activation by I-AMS is a macrophage, a type of white blood cell that can engulf and digest cells, cellular debris, and / or contaminants through a process known as phagocytosis.

[0144] I-AMS can be designed to bind to proteins expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursor cells, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors (TLR) 1-9, IL-4Rα, and MARCO. Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70 (available from BioLegend), which binds and activates CD11b; KP1 (available from ABCAM®, Cambridge, UK), which binds and activates CD68; and ab87099 (available from ABCAM), which binds and activates CD163.

[0145] In certain embodiments, I-AMS can target pathogen recognition receptors (PRRs). PRRs are proteins or protein complexes that recognize danger signals and activate and / or enhance innate immune responses. Examples of PRRs include the TLR4 / MD-2 complex, which recognizes gram-negative bacteria; dectin-1 and dectin-2, which recognize mannose moieties of fungi and other pathogens; TLR2 / TLR6 or TLR2 / TLR1 heterodimers, which recognize gram-positive bacteria; TLR5, which recognizes flagellin; and TLR9 (CD289), which recognizes CpG motifs in DNA. In certain embodiments, I-AMS can bind to and activate TLR4 / MD-2, dectin-1, dectin-2, TRL2 / TLR6, TLR2 / TLR1, TLR5, and / or TLR9.

[0146] In certain embodiments, I-AMS can target the complement system, an immune pathway induced by antigen-bound antibodies and involved in complement protein signaling responsible for immune recognition and elimination of antibody-coated antigens.

[0147] The binding fragments in the I-AMS and other recombinant formats described herein may be linked via a linker. The linker is an amino acid sequence that can provide flexibility and space for conformational movement between the binding fragments in the I-AMS. Any suitable linker may be used.

[0148] Examples of linkers are described in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. The linker may be flexible, rigid, or semi-rigid, depending on the desired functional domain to be presented to the target.

[0149] A commonly used flexible linker is a linker sequence consisting of the amino acids glycine and serine (Gly-Ser linker). In a particular embodiment, the linker sequence includes a repeat sequence of glycine and serine, for example, 1 to 10 (Gly x Ser y ) n (SEQ ID NO: 120), where x and y are independently integers from 0 to 10, excluding the case where both x and y are 0, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. A specific example is a repeat sequence consisting of (Gly4Ser) n (SEQ ID NO: 121), (Gly3Ser) n (Gly4Ser) n (SEQ ID NO: 122), (Gly3Ser) n (Gly2Ser) n (SEQ ID NO: 123), and (Gly3Ser) nIn certain embodiments, the Gly-Ser linker is (Gly4Ser)4 (SEQ ID NO: 125), (Gly4Ser)3 (SEQ ID NO: 126), (Gly4Ser)2 (SEQ ID NO: 127), (Gly4Ser)1 (SEQ ID NO: 128), (Gly3Ser)2 (SEQ ID NO: 129), (Gly3Ser)1 (SEQ ID NO: 130), (Gly2Ser)2 (SEQ ID NO: 131), (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 132), GGSGGGSGSG (SEQ ID NO: 133), or GGSGGGSG (SEQ ID NO: 134).

[0150] Also, linkers comprising one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, for example, a linker consisting of only CH3 or a linker consisting of a CH2CH3 sequence, can be used.

[0151] In some cases, flexible linkers may not be able to maintain the distance or position of the binding fragments required for a particular application. In such cases, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In certain embodiments, proline-rich linkers are peptide sequences that have more proline residues than would be expected to be included in a sequence by chance alone. In certain embodiments, proline-rich linkers are linkers whose sequence is at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% full proline residues. Particular examples of proline-rich linkers include fragments of salivary proline-rich proteins (PRPs).

[0152] The cytolytic properties of I-AMS molecules can be confirmed by comparative in vitro assays. Briefly, in experiments with cell lines, target virus-infected cells can be seeded at a density of 5–10,000 cells / well in 96-well round-bottom plates with increasing concentrations of various I-AMS antibodies (e.g., viral epitopes can be incubated with CD3 I-AMS, such as viral epitope-CD3 bispecific antibodies (BiAbs)) in the presence or absence of T cells from healthy donors. After 48 hours, cell counts and drug-induced cytotoxicity can be measured by flow cytometry using 4',6-diamidino-2-phenylindole (DAPI) for dead cell detection. In experiments with healthy donor T cells, virus-infected cells can be identified by forward / side scatter characteristics and negative staining with CellVue Burgundy dye. Experiments can have two technical replicates.

[0153] In certain embodiments involving I-AMS constructs, the epitope-binding fragment that activates T cells is the V of a known TCR. α , V β , C α or C β The recombined V includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of these changes, compared to the CDRs. Each CDR includes no changes or at most one, two or three changes, and the recombined V α area, V β area, C α Area or C β As long as a binding fragment containing the region is capable of specifically binding to a target with an affinity similar to that of the wild type, the insertion, deletion, or substitution may be α area, Vβ area, C α Area or C β V including the amino or carboxy termini or both of the regions α area, V β area, C α Area or C β It may be located anywhere in the region.

[0154] In certain embodiments, bispecific molecules can be constructed in a standard BiTE antibody format by synthesizing each scFv as a DNA fragment with overlapping ends compatible with Gibson assembly. For example, a prototypical intervening region such as a (Gly4Ser)3 linker (SEQ ID NO: 126) can be used between the paired variable domains, and a short Gly4Ser linker (SEQ ID NO: 128) can be used between the two scFvs.

[0155] Antiviral trispecific antibodies are artificial proteins that simultaneously bind to three antigens, at least one of which is a viral epitope on HPIV1, HPIV3, RSV or HMPV that is bound by the antibodies disclosed herein. Trispecific antibodies are described, for example, in WO2016 / 105450, WO2010 / 028796; WO2009 / 007124; WO2002 / 083738; U.S. Patent Publication No. 2002 / 0051780; and WO 2000 / 018806.

[0156] (ii) Preparation The various antibodies described herein in representative formats can be used alone to prepare a composition for administration to a subject, or several of these antibodies can be combined to prepare a composition for administration to a subject. Salts and / or prodrugs of these antibodies can also be used.

[0157] Pharmaceutically acceptable salts include salts that retain the activity of the antibodies described herein and are acceptable for pharmaceutical use. Pharmaceutically acceptable salts also refer to salts that may be formed in vivo by administering an acid, another salt, or a prodrug that is converted into an acid or salt.

[0158] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids.Examples of such inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid and phosphoric acid.Suitable organic acids can be selected from aliphatic organic acids, alicyclic organic acids, aromatic organic acids, aromatic aliphatic organic acids, heterocyclic organic acids, carboxylic acids and sulfonic acids.

[0159] Suitable pharma- ceutically acceptable base addition salts include metallic salts prepared from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc; and organic salts prepared from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine, and procaine.

[0160] Prodrugs include active ingredients that are converted into therapeutically active compounds after administration, such as by cleavage or hydrolysis of biologically labile groups.

[0161] In certain embodiments, the compositions described herein comprise at least 0.1% w / v of the antibody relative to the total composition; at least 1% w / v of the antibody relative to the total composition; at least 10% w / v of the antibody relative to the total composition; at least 20% w / v of the antibody relative to the total composition; at least 30% w / v of the antibody relative to the total composition; at least 40% w / v of the antibody relative to the total composition; at least 50% w / v of the antibody; at least 60% w / v of the total composition; at least 70% w / v of the antibody; at least 80% w / v of the total composition; at least 90% w / v of the antibody; at least 95% w / v of the total composition; or at least 99% w / v of the antibody.

[0162] Representative pharma- ceutically acceptable carriers in common use include any absorption retarders, antioxidants, binders, buffers, bulking or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonicity agents, lubricants, preservatives, salts, solvents or cosolvents, stabilizers, surfactants, and / or delivery vehicles.

[0163] Representative antioxidants include ascorbic acid, methionine and vitamin E.

[0164] Representative buffers include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and / or trimethylamine salts.

[0165] A typical chelating agent is EDTA (ethylenediaminetetraacetic acid).

[0166] Representative tonicity agents include polyhydric sugar alcohols including trihydric sugar alcohols or sugar alcohols having a higher valence than trihydric sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0167] Representative preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkylparabens (e.g., methylparaben and propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0168] Stabilizers refer to a wide variety of excipients that can have various functions, from additives that can solubilize antibodies, prevent denaturation of antibodies, and prevent antibodies from adhering to the walls of containers to bulking agents. Typical stabilizers include polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, and cyclitols (e.g., inositol); PEG; amino acid polymers; urea, glutathione, thioctic acid, thioglycerol, and thioglycerol; and thioglycerol. Sulfur-containing reducing agents such as sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., polypeptides with less than 10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, and sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer is usually included in the range of 0.1 to 10,000 parts by weight based on the total weight of the therapeutic agent.

[0169] The compositions disclosed herein can be formulated for administration by injection, inhalation, infusion, irrigation, or oral ingestion.Furthermore, the compositions disclosed herein can be formulated for intravenous, intradermal, intraarterial, intralymphatic, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, oral, sublingual, and / or subcutaneous administration.

[0170] The composition for injection can be formulated as an aqueous solution in a buffer solution such as Hank's solution, Ringer's solution, or physiological saline. Hank's solution refers to an isotonic buffer solution containing inorganic salts and sugars. Ringer's solution contains sodium chloride, potassium chloride, and calcium chloride at physiological concentrations, and the pH is adjusted with sodium bicarbonate (or sodium lactate). The aqueous solution may contain formulation additives such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the formulation may be in lyophilized and / or powder form, which is reconstituted with a suitable solvent such as, for example, sterile water that does not contain pyrogens.

[0171] Compositions for oral administration can be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Suitable excipients for oral solid preparations such as powders, capsules, tablets, etc. include binders such as gum tragacanth, gum arabic, corn starch, gelatin, etc.; fillers such as sugars, e.g., lactose, sucrose, mannitol, sorbitol, etc.; dicalcium phosphate, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP); granulating agents; and binders. If desired, disintegrants can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof (e.g., sodium alginate). If desired, solid dosage forms may be sugar-coated or enteric-coated by standard techniques. Flavorings, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like, may also be used.

[0172] The compositions described herein can be formulated as aerosols. In certain embodiments, the aerosols are provided as part of anhydrous inhalers, liquid inhalers or dry powder inhalers. The aerosol sprays that are delivered from pressurized packs or nebulizers can be used in combination with suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. The compositions can also be formulated into gelatin capsules and cartridges that are used in inhalers or nebulizers, and these gelatin capsules and cartridges contain a powder mix of the compositions described herein and a suitable powder base, such as lactose or starch.

[0173] The compositions described herein can also be formulated as depot preparations. Depot preparations can be formulated using suitable polymeric or hydrophobic materials (e.g., as an emulsion in a pharma-ceutically acceptable oil), can be formulated using ion exchange resins, or can be formulated as sparingly soluble derivatives (e.g., sparingly soluble salts).

[0174] Furthermore, the compositions described herein can be formulated as sustained release systems comprising at least one antibody using semipermeable matrices of solid polymers. A variety of sustained release materials have been developed and are well known to those skilled in the art. Depending on their chemical nature, sustained release systems may release one or more antibodies for several weeks up to more than 100 days after administration. Depot formulations can be administered by injection; parenteral injection; infusion; or implantation into soft tissue or body cavity, or optionally into blood vessels by injection using a fine needle.

[0175] The depot formulations may contain a variety of biodegradable polymers, including poly(lactide), poly(glycolide), poly(caprolactone), and poly(lactide)-co-(glycolide) (PLG), with the desired lactide to glycolide ratio, average molecular weight, polydispersity, and end group chemical structure. Different grades of different types of polymers can be mixed in different ratios to obtain the characteristics derived from each constituent polymer.

[0176] The size and structure of the microparticles can be modified to control the release characteristics by using a variety of solvents (e.g., dichloromethane, chloroform, ethyl acetate, triacetin, N-methylpyrrolidone, tetrahydrofuran, phenol, or combinations thereof). Other useful solvents include water, ethanol, dimethylsulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0177] Representative release modifying agents include surfactants, detergents, internal phase viscosity enhancing agents, complexing agents, surface active molecules, co-solvents, chelating agents, stabilizers, cellulose derivatives, (hydroxypropyl)methylcellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Del.), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Del.), sucrose acetate isobutyrate (SAIB), salts and buffers.

[0178] By adding excipients such as surfactants, such as polysorbates, dioctyl sulfosuccinate, poloxamer, PVA, etc., to the external phase interface of the microparticles, properties such as microparticle stability, erosion rate, hydration, flow channel structure, interfacial transport, and pharmacokinetics can be advantageously modified.

[0179] Further processing of the sustained release depot formulations of the present disclosure can utilize stabilizing additives including mannitol, sucrose, trehalose and glycine, as well as other ingredients such as polysorbates, PVA, dioctyl sulfosuccinate, etc., in buffers such as Tris, citrate, histidine, etc. Additionally, a lyophilization cycle can be utilized to produce a powder with very low moisture content that can be reconstituted to a volume and performance characteristics similar to the original suspension.

[0180] The compositions disclosed herein may advantageously include other pharma- ceutically acceptable carriers, such as carriers that do not cause significant adverse, allergic or other untoward reactions that outweigh the benefits of administration. Representative pharma-ceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, the formulations can be prepared to meet the standards of sterility, pyrogenicity, general safety and purity as required by the Office of Biologics Standards of the U.S. Food and Drug Administration and / or other relevant regulatory authorities in other countries.

[0181] (iii) How to use The methods disclosed herein include treating a subject (e.g., humans, companion animals (dogs, cats, reptiles, birds), livestock (e.g., horses, cows, goats, pigs, chickens), or research animals (e.g., monkeys, rats, mice, fish)) with a composition disclosed herein. Treating a subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes an amount that can provide an effective amount, prophylactic treatment, and / or therapeutic treatment.

[0182] "Effective amount" is the amount of composition required to cause a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amount disclosed herein is an amount that can cause a statistically significant effect in animal models or in vitro assays related to the evaluation of the onset, progression and / or resolution of infectious diseases.

[0183] "Prophylactic treatment" includes treatment administered to a subject who does not exhibit signs or symptoms of an infection or who exhibits only early signs or symptoms of an infection with the intent of reducing or decreasing the risk of the infection developing further. Thus, prophylactic treatment serves as a treatment to prevent an infection. In certain embodiments, prophylactic treatment inhibits, slows, or prevents the progression of an infection.

[0184] "Therapeutic treatment" includes treatment administered to a subject exhibiting a symptom or sign of an infectious disease with the intent of reducing or eliminating the sign or symptom of the infectious disease. Therapeutic treatment may prevent, control or eliminate the presence or activity of an infectious disease and / or may prevent, control or eliminate the side effects of an infectious disease.

[0185] The terms effective amount, functioning as a prophylactic treatment or a therapeutic treatment are not mutually exclusive, and in certain embodiments, an administered dose may accomplish more than one treatment.

[0186] In certain embodiments, the therapeutically effective amount can provide an anti-infectious effect. Anti-infectious effect includes suppression or prevention of cell infection by a virus, reduction in the number of infected cells, reduction in the volume of infected tissue, extension of life span, extension of life expectancy, and reduction or elimination of the symptoms associated with infection (e.g., reduced lung capacity). In certain embodiments, the therapeutically effective amount induces an immune response. This immune response may be against a virus.

[0187] When administered, the therapeutically effective amount (also referred to herein as "dosage") can be estimated initially based on the results of in vitro assays and / or animal model studies. Such information can be used to more accurately determine the dosage that is useful for the intended subject. The actual dosage administered to a particular subject can be determined by a physician, veterinarian or researcher, taking into account parameters such as physical and physiological factors, such as target, body weight, disease severity, type of infection, stage of infection, previous or concurrent therapeutic interventions, subject's idiopathic disease and route of administration.

[0188] Useful doses may range from 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In another example, doses include 1 μg / kg, 15 μg / kg, 30 μg / kg, 50 μg / kg, 55 μg / kg, 70 μg / kg, 90 μg / kg, 150 μg / kg, 350 μg / kg, 500 μg / kg, 750 μg / kg, 1000 μg / kg, 0.1 to 5 mg / kg or 0.5 to 1 mg / kg. In another example, doses include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0189] The therapeutically effective amount can be achieved by one or multiple administrations during the course of the treatment regimen (e.g., daily, every other day, every 3rd day, every 4th day, every 5th day, every 6th day, once a week, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or once a year). In certain embodiments, the treatment protocol may be determined according to a clinical trial protocol or an FDA-approved treatment protocol.

[0190] As described herein, certain methods include use in immunocompromised individuals, including those who have undergone hematopoietic stem cell transplants, lung transplant recipients, premature infants, adults over 65 years of age, or individuals with other health problems that increase their susceptibility to infection with respiratory viruses.

[0191] The pharmaceutical compositions described herein can be administered, for example, by injection, inhalation, infusion, irrigation, or oral ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intralymphatic, intravesical, intrathecal, intraperitoneal, parenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and / or sublingual administration.

[0192] The following representative embodiments and examples are provided to illustrate specific embodiments of the present disclosure. Those skilled in the art having reference to this disclosure will appreciate that various changes can be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit and scope of the present disclosure.

[0193] (iv) Representative embodiments 1. An antibody or binding fragment thereof comprising the complementarity determining regions (CDRs) of PI3-E12, PI3-A3, PI3-B5, PI3-A10, PI3-A12, 3x1, MxR-B11 or MxR-D10. 2. The antibody or binding fragment of embodiment 1, wherein the CDRs are numbered according to the Kabat, Chothia, Martin, Contact, IMGT, AHo or North numbering systems. 3. The antibody or binding fragment thereof CDRH1 contains GFTFSDHY (SEQ ID NO:1); CDRH2 contains ISSSGSNT (SEQ ID NO:2); CDRH3 contains ARAKWGTMGRGAPPTIYDH (SEQ ID NO:3); CDRL1 contains QSLLQSNGNNY (SEQ ID NO:4); CDRL2 contains LGS; and CDRL3 contains MQALQTPLT (SEQ ID NO:5); CDRH1 comprises GFTFSNYW (SEQ ID NO:8); CDRH2 comprises VKEEGSEK (SEQ ID NO:9); CDRH3 comprises AGEVKSGWFGRYFDS (SEQ ID NO:10); CDRL1 comprises QSVGSW (SEQ ID NO:11); CDRL2 comprises KTS; and CDRL3 comprises QQYSSFPYT (SEQ ID NO:12); CDRH1 comprises GYNFTNYW (SEQ ID NO: 15); CDRH2 comprises IYPADSDT (SEQ ID NO: 16); CDRH3 comprises ARPSTRWFVPGGMDV (SEQ ID NO: 17); CDRL1 comprises QSIGAW (SEQ ID NO: 18); CDRL2 comprises KAS; and CDRL3 comprises QQHSSYPST (SEQ ID NO: 19); CDRH1 comprises GFNFNNYG (SEQ ID NO:22); CDRH2 comprises VSFDGSNR (SEQ ID NO:23); CDRH3 comprises SKSKYSDFWSEI (SEQ ID NO:24); CDRL1 comprises QNVMRY (SEQ ID NO:25); CDRL2 comprises DAS; and CDRL3 comprises QQRTNHRFS (SEQ ID NO:26); CDRH1 comprises GDSVKSDDFY (SEQ ID NO:29); CDRH2 comprises IYYGGRT (SEQ ID NO:30); CDRH3 comprises VRVEGLLWFGELFDY (SEQ ID NO:31); CDRL1 comprises NSNIGNNF (SEQ ID NO:32); CDRL2 comprises KDY; and CDRL3 comprises AAWQDGLSGPL (SEQ ID NO:33); CDRH1 comprises GFTFSSFG (SEQ ID NO:36); CDRH2 comprises ISHSAGFL (SEQ ID NO:37); CDRH3 comprises AKRLAGLPDLEWLLYPNFLDH (SEQ ID NO:38); CDRL1 comprises ILRTYY (SEQ ID NO:39); CDRL2 comprises GKN; and CDRL3 comprises SSRDRSGNHVL (SEQ ID NO:40); CDRH1 comprises GFPFSSYK (SEQ ID NO: 43); CDRH2 comprises ISASGSYI (SEQ ID NO: 44); CDRH3 comprises ARDGGRELSPFEK (SEQ ID NO: 45); CDRL1 comprises NSNIGTGYD (SEQ ID NO: 46); CDRL2 comprises DNN; and CDRL3 comprises QSYDKSLGGWV (SEQ ID NO: 47); or CDRH1 comprises GFIFSNYD (SEQ ID NO:50); CDRH2 comprises ITGGSSFI (SEQ ID NO:51); CDRH3 comprises ARDGGRQLSPCEH (SEQ ID NO:52); CDRL1 comprises SSNIGAGYD (SEQ ID NO:53); CDRL2 comprises DNN; and CDRL3 comprises QSYDRGLSGWA (SEQ ID NO:54). 2. The antibody or binding fragment of embodiment 1, comprising: 4. An antibody or binding fragment thereof, a heavy chain variable region having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 166 and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 167; a heavy chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 168 and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 169; a heavy chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 170 and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 171; a heavy chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 172 and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 173; a heavy chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 174 and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 175; a heavy chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 176 and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 177; a heavy chain variable region having a sequence that has at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 178 and a light chain variable region having a sequence that has at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 179; or A heavy chain variable region having a sequence having at least 90% or at least 95% sequence identity with the sequence set forth in SEQ ID NO: 180, and a light chain variable region having a sequence having at least 90% or at least 95% sequence identity with the sequence set forth in SEQ ID NO: 181. An antibody or binding fragment thereof comprising: 5. An antibody or binding fragment thereof, a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 166 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 167; a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 168 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 169; a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 170 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 171; a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 172 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 173; a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 174 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 175; a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 176 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 177; a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 178 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 179; or a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 180 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 181; An antibody or binding fragment thereof, comprising: 6. An antibody or binding fragment thereof, a sequence having at least 90% or at least 95% sequence identity with the sequence shown in SEQ ID NO:6 and the sequence shown in SEQ ID NO:7; a sequence having at least 90% or at least 95% sequence identity with the sequence shown in SEQ ID NO: 13 and the sequence shown in SEQ ID NO: 14; a sequence having at least 90% or at least 95% sequence identity with the sequence shown in SEQ ID NO: 27 and the sequence shown in SEQ ID NO: 28; a sequence having at least 90% or at least 95% sequence identity to the sequence shown in SEQ ID NO: 34 and the sequence shown in SEQ ID NO: 35; a sequence having at least 90% or at least 95% sequence identity with the sequence shown in SEQ ID NO: 41 and the sequence shown in SEQ ID NO: 42; A sequence having at least 90% or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 48 and the sequence set forth in SEQ ID NO: 49; or A sequence having at least 90% or at least 95% sequence identity with the sequence shown in SEQ ID NO: 55 and the sequence shown in SEQ ID NO: 56 An antibody or binding fragment thereof, encoded by 7. An antibody or binding fragment thereof, encoded by a sequence comprising the sequence set forth in SEQ ID NO:6 and the sequence set forth in SEQ ID NO:7; a sequence comprising the sequence set forth in SEQ ID NO:13 and the sequence set forth in SEQ ID NO:14; a sequence comprising the sequence set forth in SEQ ID NO:27 and the sequence set forth in SEQ ID NO:28; a sequence comprising the sequence set forth in SEQ ID NO:34 and the sequence set forth in SEQ ID NO:35; a sequence comprising the sequence set forth in SEQ ID NO:41 and the sequence set forth in SEQ ID NO:42; a sequence comprising the sequence set forth in SEQ ID NO:48 and the sequence set forth in SEQ ID NO:49; or a sequence comprising the sequence set forth in SEQ ID NO:55 and the sequence set forth in SEQ ID NO:56. 8. An antibody or binding fragment thereof according to any one of embodiments 1 to 7, comprising an IgG having an M252Y mutation, an S254T mutation and a T256E mutation in the Fc region. 9. A binding molecule comprising binding fragments of at least two antibodies according to any of embodiments 1 to 8. 10. The binding molecule of embodiment 9, wherein the binding fragments of at least two antibodies of any of embodiments 1 to 3 comprise Fab or single chain variable fragments (scFv). 11. The binding molecule according to embodiment 9 or 10, wherein said binding fragments comprise Fabs linked via inter-heavy chain bonds by knobs-into-holes technology (Figure 8, top row). 12. The binding molecule according to any of embodiments 9 to 11, wherein the binding fragment comprises an scFv linked to a constant region of an antibody which has one binding fragment of the scFv linked to another binding fragment different from the scFv (Figure 8, bottom). 13. The binding molecule according to embodiment 12, comprising a linker comprising a Gly-Ser linker. 14. The binding molecule according to any of embodiments 9 to 13, wherein the binding fragments of at least two antibodies according to any of embodiments 1 to 8 comprise scFvs. 15. Contains a binding fragment of a PI3 antibody and a binding fragment of an MxR antibody; a binding fragment of a PI3 antibody and a binding fragment of a 3x1 antibody; or comprising a binding fragment of the 3x1 antibody and a binding fragment of the MxR antibody, A binding molecule according to any one of embodiments 9 to 14. 16. A binding molecule according to any of embodiments 9 to 15, comprising a binding fragment of a PI3 antibody, a binding fragment of a 3x1 antibody and a binding fragment of an MxR antibody. 17. The binding molecule of embodiment 15, wherein the binding fragment comprises a Fab and / or a scFv. 18. A binding molecule according to any of embodiments 9 to 17, comprising MxR-B11 Fab and 3x1 Fab; MxR-B11 scFv and 3x1 Fab; MxR-B11 Fab and 3x1 scFv; or MxR-B11 scFv and 3x1 scFv. 19. A binding molecule according to any of embodiments 9 to 18, comprising MxR-D10 Fab and 3x1 Fab; MxR-D10 scFv and 3x1 Fab; MxR-D10 Fab and 3x1 scFv; or MxR-D10 scFv and 3x1 scFv. 20. A binding molecule comprising a binding fragment according to any one of embodiments 1 to 8 and a binding fragment of an antibody that binds to a second virus. 21. The binding molecule of embodiment 20, wherein the second virus is selected from adenovirus, bocavirus, coronavirus, enterovirus, influenza virus, metapneumovirus, parainfluenza virus and / or rhinovirus. 22. The binding molecule of embodiment 20 or 21, wherein the second virus is selected from human adenovirus, human bocavirus (HBoV) and / or human coronavirus (HCoV). 23. The binding molecule according to any of embodiments 20 to 22, wherein the second virus is a coronavirus selected from SARS-CoV, MERS-CoV, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, coronavirus NL and / or coronavirus NH. 24. The binding molecule according to any of embodiments 20 to 23, wherein the second virus is selected from influenza virus A, influenza virus B, human parainfluenza virus 2 (HPIV2), HPIV4, human rhinovirus (HRV) A, HRVB and / or HRVC. 25. The binding fragment is 8C4, 5Hx-l, 5Hx-2, 5Hx-3, 5Hx-4, 5Hx-5, 5.100K-1, 5PB-1, 5Fb-l, 1E11, EP R23305-44, 47D11, CR3022, CDC2-A2, G2, 5F9, FIB-H1, JC57-13, 32D6, CH65, CR9114, MAb 22 / 1, MAb70 / l, MAb 110 / 1, MAb 264 / 2, MAb W18 / 1, MAb 14 / 3, MAb 24 / 4, MAb 47 / 8, MAb 198 / 2, MAb 35. The binding molecule according to any of embodiments 20 to 34, comprising the CDRs of an antibody selected from 215 / 2, H2 / 6A5, H3 / 4C4, H2 / 6C4, H2 / 4B3, H9 / B20, H2 / 4B1, CA6261, 6F12, CR9114, PEG-1, CR8033, CR8071, 113 / 2, 124 / 4, 128 / 2, 134 / 1, 146 / 1, 152 / 2, 160 / 1, 162 / 1, 195 / 3, 206 / 2, 238 / 4, 280 / 2, PAR2 (boca231 / 9F) or TCN-711. 26. A binding molecule comprising a binding fragment according to any one of embodiments 1 to 8 and a binding fragment that activates an immune cell. 27. The binding molecule according to embodiment 26, wherein the binding fragment that activates an immune cell binds to CD3, CD28 or 4-1BB. 28. The binding molecule of embodiment 26 or 27, wherein the binding fragment that activates an immune cell comprises the CDRs of OKT3 or TGN1412. 29. A composition comprising an antibody or binding fragment thereof according to any one of embodiments 1 to 28 and a pharma- ceutically acceptable carrier. 30. The composition of embodiment 29, wherein the pharma- ceutically acceptable carrier comprises an aqueous solution. 31. The composition of embodiment 29 or 30, wherein the pharma- ceutically acceptable carrier comprises saline. 32. The composition of any of embodiments 29-31, wherein the pharma-ceutically acceptable carrier comprises sodium chloride, potassium chloride, and calcium chloride. 33. The composition of any of embodiments 29-32, wherein the pharma-ceutically acceptable carrier comprises sodium bicarbonate or sodium lactate. 34. The composition of any of embodiments 29-33, wherein the pharma- ceutically acceptable carrier comprises an inorganic salt. 35. The composition of any of embodiments 29-34, wherein the pharma- ceutically acceptable carrier comprises a sugar. 36. The composition of any of embodiments 29-35, wherein the pharma- ceutically acceptable carrier comprises an antioxidant, a buffering agent, a chelating agent, an isotonicity agent, a preservative and / or a stabilizer. 37. The composition of any of embodiments 29-36, wherein the pharma- ceutically acceptable carrier comprises a release modifier. 38. A composition according to any of embodiments 29 to 37, comprising a therapeutically effective amount of the antibody or binding fragment thereof for administration to a subject. 39. A method for providing an antiviral effect to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition described in any one of embodiments 29-38, thereby providing an antiviral effect. 40. The method of embodiment 39, wherein the antiviral effect comprises an anti-HPIV3 effect, an anti-HPIV1 effect, an anti-respiratory syncytial virus (RSV) effect, and / or an anti-human metapneumovirus (HMPV) effect. 41. The method of embodiment 39 or 40, wherein the antiviral effect comprises an anti-HPIV3 effect and an anti-HPIV1 effect. 42. The method of any one of embodiments 39 to 32, wherein the antiviral effect includes an anti-RSV effect and an anti-HMPV effect. 43. The method of any one of embodiments 39 to 42, wherein the antiviral effect includes an anti-HPIV effect, an anti-RSV effect, and an anti-HMPV effect. 44. The method of any of embodiments 39-43, wherein the subject in need of an antiviral effect is a hematopoietic stem cell transplant (HCT) recipient, a lung transplant recipient, a premature infant, a person over 65 years of age, a homeless person, or a person with a lung disease. 45. The method of any of embodiments 39-44, wherein the subject has chronic obstructive pulmonary disease (COPD). 46. ​​The method of any one of embodiments 39 to 45, wherein the antiviral effect includes an antiviral effect against a second virus. 47. The method of embodiment 46, wherein the second virus is selected from adenovirus, bocavirus, coronavirus, enterovirus, influenza virus, metapneumovirus, parainfluenza virus and / or rhinovirus. 48. The method of embodiment 46 or 47, wherein the second virus is selected from human adenovirus, human bocavirus (HBoV) and / or human coronavirus (HCoV). 49. The method of any of embodiments 46-48, wherein the second virus is a coronavirus selected from SARS-CoV, MERS-CoV, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, coronavirus NL and / or coronavirus NH. 50. The method of any of embodiments 46-49, wherein the second virus is selected from influenza A virus, influenza B virus, human parainfluenza virus 2 (HPIV2), HPIV4, human rhinovirus (HRV) A, HRVB and / or HRVC.

[0194] (v) Experimental Examples Introduction HPIV3 is a common cause of respiratory disease in infants and children. More than 11,000 hospitalizations due to fever or acute respiratory illness caused by HPIV3 occur annually in the United States. Like respiratory syncytial virus (RSV), HPIV3 often causes severe bronchiolitis and pneumonia in infants under 6 months of age, who are infected during early childhood and are unable to mount a strong antibody response. HPIV3 is also important as a factor influencing mortality, morbidity, and health care costs in other immunocompromised populations, particularly hematopoietic stem cell transplant (HCT) recipients who are immunosuppressed. Up to one-third of hematopoietic stem cell transplant recipients will develop a respiratory viral infection within six months of transplant. In up to one-third of these patients, the viral infection progresses from the upper to the lower respiratory tract. Once the virus gains a foothold in the lower respiratory tract, most viruses are unable to provide more than supportive care, and up to 40% of patients with lower respiratory tract disease will die within three months. HPIV3 is an important cause of serious respiratory viral infections after hematopoietic stem cell transplantation, with post-transplant incidence rates as high as 18% in some centers. The lack of a vaccine or treatment highlights the need for preventive and therapeutic interventions against HPIV3.

[0195] Monoclonal neutralizing antibodies correlate with protection against several respiratory viruses, including RSV and influenza. The monoclonal antibody palivizumab is a humanized antibody that targets the fusion (F) protein of RSV and is licensed for use as immunoprophylaxis to prevent severe disease in high-risk newborns. The RSV F protein is an essential surface glycoprotein and is therefore often targeted by neutralizing antibodies. As a class I fusion protein, the F protein mediates viral entry by transitioning between a metastable pre-fusion (preF) and a stable post-fusion (postF) conformation. Because the preF conformation is a common conformation in infectious viruses, anti-preF antibodies have potent virus neutralization, whereas antibodies targeting the postF conformation do not typically exert such potent virus neutralization (Ngwuta, et al. Sci Transl Med 7, 309ra162, (2015); Gilman, et al. Sci Immunol 1, (2016)). Similar to RSV, the HPIV3 F protein also has a preF and postF structure. Recently, it has been reported that the prefusion structure of the HPIV3 F protein has been successfully stabilized, and that F protein immunogens stabilized with the preF structure can be used to induce higher neutralizing antibody titers than the same F protein immunogen with the postF structure (Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018)). Furthermore, a high-throughput screening method was developed that allows rapid selection and testing of human B cells specific for the HPIV3 preF protein for their neutralizing ability against HPIV3, with the aim of isolating potential monoclonal antibodies for prophylactic and therapeutic use. Using this method, we isolated several potent monoclonal neutralizing antibodies, assessed their binding, and tested one of these antibody candidates in an in vivo challenge model.

[0196] result Identification of HPIV3-specific B cells and their isotypes in the human B cell repertoire The HPIV3 F protein of the prefusion structure (preF) and the HPIV3 F protein of the postfusion structure (postF) were separately biotinylated and mixed with streptavidin labeled with a fluorescent dye. Then, HPIV3 preF-specific B cells and HPIV3 postF-specific B cells were enriched using magnetic microbeads bound to antibodies targeting the fluorescent dye. It has been previously reported that animals inoculated with the HPIV3 F protein of the preF structure induced significantly higher neutralization titers than animals inoculated with the HPIV3 F protein of the postF structure (Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018)).

[0197] Using this approach, B cells that bound to the preF structure but not to the postF structure were identified by flow cytometry using fluorochrome-labeled streptavidin (Figure 2A). Because the seroprevalence of HPIV3 in humans is nearly 100% (Henrickson, Clin Microbiol Rev 16, 242-264, (2003)), there was no need to prescreen donors for seropositivity to HPIV3. Because secondary lymphoid organs such as peripheral blood, tonsils, and spleen are thought to be rich in B cells that have undergone affinity maturation for binding to HPIV3, the approach was evaluated in peripheral blood, tonsil, and spleen samples from unqualified donors. Overall, 0.20-1.75% of total B cells in these tissue samples bound the HPVI3 preF protein (Figure 2B). IgM - IgD - The overall frequency of isotype-switched B cells was high in the spleen and tonsils (Fig. 2C and 2D), with HPIV3 preF detected in the tonsils and spleen. + The increase in population showed statistical significance (Figures 2E and 2F).

[0198] Binding and neutralization of HPIV3 in human B cell repertoires We next assessed how many of the HPIV3 preF-specific B cells identified by flow cytometry produced antibodies capable of binding and neutralizing HPIV3. To do this, 92 HPIV3 preF-specific B cells sorted from PBMC from two donors were plated into individual wells of a 96-well plate, resulting in 25 pairs of heavy and light chain sequences (Table 1).

[0199] [Table 1]

[0200] These 25 pairs of heavy and light chain sequences were cloned as monoclonal antibodies and tested for binding by biolayer interferometry (BLI), confirming that 100% of them (25 of 25) bound to the purified HPIV3 preF protein (Fig. 2G). Two monoclonal antibodies from this group, designated PI3-E12 and PI3-C9, each showed a binding titer of 1 × 10 -12 M and 2.5×10 -9 High affinity (K D ) bound to the HPIV3 preF protein (Fig. 2H). However, binding kinetics analysis revealed that the dissociation rates of PI3-E12 and PI3-C9 were 1 × 10 -7 / s and 2.7 × 10 -4 / s and a slower dissociation rate for PI3-E12 than for PI3-C9, indicating that PI3-E12 has a higher affinity for the HPIV3 preF protein than PI3-C9 (Fig. 2H). Accordingly, PI3-E12, but not PI3-C9, was able to neutralize live virus in vitro (Fig. 2I). These data indicate that B cells that bind HPIV3 preF tetramers are found at low frequencies but express high-affinity antibodies that can neutralize HPIV3.

[0201] To focus on neutralizing antibody-producing B cells, we modified the assay by sorting individual B cells and growing them on irradiated CD40L / IL2 / IL21-expressing 3T3 feeder cells. We performed high-throughput screening of neutralizing culture supernatants by stimulating antibody secretion and then cloning the antibodies. Overall, more than half of the sorted B cells produced antibodies detectable by ELISA (Figure 3A). In this assay, we stimulated single B cells specific for HPIV3 preF and excluded IgD-expressing cells from the assay, which were considered to be the least likely to undergo somatic hypermutation and affinity maturation required for potent neutralization. This method allowed us to obtain HPIV3 pre-F-specific IgD ... - HPIV3 neutralizing antibodies were produced by 14% of B cells, whereas HPIV3 pre-F specific IgD was not produced by spleen-selected B cells. - HPIV3 neutralizing antibodies were produced by 5% of B cells, and HPIV3 pre-F specific IgD was selected from peripheral blood. - HPIV3-neutralizing antibodies were produced by 2% of B cells (2%) ( Fig. 3B ).

[0202] By the above-mentioned novel screening method, four new monoclonal antibodies that neutralize HPIV3 were found, named PI3-A3, PI3-B5, PI3-A10 and PI3-A12, respectively, and were sequenced, cloned and produced (Fig. 3C). The neutralizing potency of these antibodies ranged from 7.0 to 61.4 ng / mL (Fig. 3C). Each monoclonal neutralizing antibody had a different immunoglobulin heavy chain allele (Table 2). [Table 2]

[0203] All of the immunoglobulin light chain alleles were different, except for PI3-A3 and PI3-B5, whose κ chain gene alleles were 1-5*03. None of the neutralizing antibodies matched the alleles of the previously reported HPIV3 antibody PIA174 (Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018)). The similarity of the germline sequences of the variable region genes to these neutralizing antibodies ranged from 90 to 97% (Table 2). All of the neutralizing antibodies newly discovered in this study bound strongly to the preF structure, but no detectable binding was observed to the postF structure (Figures 3D and 3E). The previously reported antibody PIA174 also bound strongly to the HPIV3 preF structure, but also weakly to the postF structure (Figures 3D and 3E). Taking into account the administration of these antibodies in vivo, we performed experiments following common methods for evaluating autoreactivity (Steach, et al. J Immunol 204, 498-509, (2020); Bancroft, et al. J Exp Med 216, 2331-2347, (2019)), and found no antibody binding to permeabilized HEp-2 cells (Figure 3F).

[0204] Next, cross-competitive binding experiments were performed to evaluate the antigenic site on HPIV3 preF that allows neutralization. Two of the four novel monoclonal neutralizing antibodies (PI3-A3 and PI3-B5) fully competed with each other and with the previously reported antibody PIA174 (Figure 12A). PI3-A10 also competed with this group, but only partially with PI3-E12 (Figure 12A). Based on the known binding site of PIA174, this antigenic site was likely located at the apical end of HPIV3 preF (Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018)).

[0205] TIFF2024507948000004.tif19158

[0206] TIFF2024507948000005.tif19154

[0207] TIFF2024507948000006.tif20154Negative staining electron microscopy (nsEM) of PI3-E12 F complexed with HPIV3 preF. ab As predicted, the PI3-E12 F ab is a 1:1 ratio (F ab PI3-E12 F bound to the apical end of HPIV3 preF in the form of a PI3-E12 trimer. ab PI3-E12 F appears to bind to HPIV3 preF at a different bond angle than PIA174 (Figures 4A and 4B). ab The structure was obtained (Figures 4B and 4C and Table 3). [Table 3]

[0208] PI3-E12 F ab When the structure of and PIA174 were superimposed (root-mean-square deviation: 1.4 A2), the main differences in structural organization were found to be in CDRL1 and CDRH3, with CDRL1 being longer in PI3-E12 (Fig. 4D).

[0209] TIFF2024507948000008.tif13152

[0210] Protection from HPIV3 infection in vivo Next, we investigated the clinical utility of PI3-E12 in an animal challenge model of HPIV3 infection. Although human parainfluenza viruses do not replicate in mice, lower respiratory tract lesions and viral replication can be observed in cotton rats intranasally infected with HPIV3 (Henrickson, Clin Microbiol Rev 16, 242-264, (2003); Ottolini, et al. J Gen Virol 77 (Pt 8), 1739-1743, (1996)).

[0211] The cotton rat model has been used in previous experiments to predict the efficacy of antibody-based immunoprophylaxis as well as the efficacy of palivizumab at a dose of 15 mg / kg, which is thought to be effective against RSV in human infants (Johnson, et al. J Infect Dis 176, 1215-1224, (1997)). Based on this, a similar experimental design was adopted. 5 Cotton rats were infected intranasally with pfu HPIV3 and then injected intramuscularly with 0.625–5 mg / kg PI3-E12 the following day (Fig. 5A). None of the rats receiving PI3-E12 developed peribronchiolitis, whereas peribronchiolitis was detected in rats receiving PBS 4 days after infection (Fig. 5B). Consistent with the suppression of peribronchiolitis, even this low dose of PI3-E12 reduced the amount of HPIV3 detected in the lungs by 6-fold, falling below the limit of detection in 8 of 9 rats injected with doses of 2.5 mg / kg or higher (Fig. 5C). A mild reduction in HPIV3 replication was also detected in the nasal cavity (Fig. 5C), which was expected considering the relative lack of penetration of IgG antibodies into the nasal cavity (Kirkeby, et al. Clin Diagn Lab Immunol 7, 31-39, (2000); Fisher, et al. J Infect Dis 180, 1324-1327, (1999)). These data suggest that the EC 50 The EC 99was shown to be 1.80 mg / kg.

[0212] Because patients undergoing cytotoxic therapy for cancer or autoimmune disease and other immunosuppressed populations are at highest risk for severe disease and mortality via HPIV3 infection, we tested the efficacy of PI3-E12 as a treatment in immunosuppressed animals. To do so, we employed an experimental design similar to that previously described to model RSV in immunodeficient cotton rats (Boukhvalova, et al. Bone Marrow Transplant 51, 119-126, (2016); Johnson, et al. Infect Immun 37, 369-373 (1982); Ottolini, et al. Bone Marrow Transplant 29, 117-120, (2002)).

[0213] Cotton rats were treated with 5 mg / kg of Cytoxan every 3 days for 3 weeks, and then 5 pfu of HPIV3 was intranasally infected (Fig. 5D). Five days after infection, 10 4 pfu / g of HPIV3 were detected (Fig. 5E). In contrast, injection of 5 mg / kg PI3-E12 1 day after infection reduced lung and nasal virus titers 28-fold and 2-fold, respectively (Fig. 5E).

[0214] These data indicate that PI3-E12 can prevent and treat HPIV3 infection.

[0215] Materials and Methods Study design Experimental group sizes are stated in the figure legends. Peripheral blood was collected by venipuncture from HIV-seronegative healthy adult volunteers enrolled in the Seattle Area Control Study, which was approved by the Institutional Review Board overseeing the conduct of the study. PBMCs were isolated from whole blood using Accuspin System Histopaque-1077 (Sigma-Aldrich).

[0216] Institutional review board approval for studies with human tonsils was obtained from Seattle Children's Hospital. Studies with human spleens were considered non-human subjects research because spleen tissue was de-identified, discarded, or obtained from deceased individuals. Tissue pieces were passed through a basket sieve, centrifuged at 1400 rpm for 7 min, incubated with ACK lysis buffer (Thermo Fisher) for 3.5 min, resuspended in RPMI medium (Gibco), and passed through a stack of 500 μm and 70 μm cell strainers. Cells were resuspended in heat-inactivated fetal bovine serum with 10% dimethyl sulfoxide (Gibco) and stored frozen in liquid nitrogen until use.

[0217] cell line 293F cells (Thermo Fisher) were cultured in Freestyle 293 medium (Thermo Fisher). Vero cells (ATCC CCL-81), LLC-MK2 cells (ATCC CCL-7.1), and HEp-2 (ATCC CCL-23) were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / mL streptomycin (Gibco). 3T3-CD40L / IL2 / IL21 feeder cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and 0.4 mg / mL geneticin. Irradiation was performed at 5,000 rad.

[0218] virus Wild-type rHPIV3 was a recombinant of the JS strain (GenBank accession number: Z11575) and was modified to enhance GFP expression as previously reported (Liu, et al. PLoS One 15, e0228572, (2020)). Virus was grown in LLC-MK2 cells and purified by centrifugation at 120,000 × g for 90 min at 4 °C on a 30% / 60% discontinuous sucrose density gradient (Sigma-Aldrich) containing 0.05 M HEPES and 0.1 M MgSO4. Viral titers were measured by infecting Vero cell monolayers seeded in 24-well plates with 10-fold serial dilutions of virus, overlaying them with DMEM containing 4% methylcellulose (Sigma-Aldrich), and counting fluorescent plaques using a Typhoon scanner (GE Life Sciences) 5 days after infection.

[0219] Antigen expression and purification The expression plasmid for expressing the His-tagged HPIV3 preF antigen and the expression plasmid for expressing the His-tagged postF antigen are described in Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018). 6Each expression plasmid was transfected into 293F cells at a density of 1000 / mL. The transfected cells were cultured for 7 days at 37°C with gentle shaking. The supernatant was collected by centrifugation of the culture at 2,500 × g for 30 min and filtered through a 0.2 μM filter. The clarified supernatant was incubated overnight at 4°C with Ni-Sepharose beads and washed with a wash buffer containing 50 mM Tris, 300 mM NaCl and 8 mM imidazole. The His-tagged proteins were then eluted with an elution buffer containing 25 mM Tris, 150 mM NaCl and 500 mM imidazole. The purified proteins were run on a 10 / 300 Superose 6 size-exclusion column (GE Life Sciences). Fractions containing the trimer HPIV3 F protein were pooled and concentrated by centrifugation into an Amicon ultrafiltration unit with a molecular weight cutoff of 50 kDa (Millipore). The tags were cleaved by mixing 1 mg of each protein with 2 units of biotinylated thrombin (Millipore) and incubating overnight, after which streptavidin agarose (Millipore) was added and incubated for an additional hour to remove the thrombin and cleaved tags, and the resulting mixture was passed through a PVDF filter (Millipore) and centrifuged to remove the streptavidin agarose. Concentrated samples were placed in 50% glycerol and stored at -20°C.

[0220] Antigen tetramerization Purified HPIV3 F protein was biotinylated using an EZ-link Sulfo-NHS-LC-Biotinylation kit (Thermo Fisher) at a molar ratio of biotin to HPIV3 F protein of 1:1.3. Unbound biotin was removed by centrifugation using a 50 kDa Amicon Ultra size-exclusion column (Millipore). To determine the average number of biotin molecules bound to each molecule of F protein, a constant amount of biotinylated F protein was titrated with increasing concentrations of streptavidin-PE (ProZyme) and incubated for 30 min at room temperature. Each sample was run on an SDS-PAGE gel (Invitrogen), transferred to nitrocellulose, and incubated with 1:10,000 diluted streptavidin-Alexa Fluor 680 (Thermo Fisher) to determine the time point when there was an excess of biotin available for binding to the streptavidin-Alexa Fluor 680 reagent. Biotinylated F protein and streptavidin-APC were mixed at the ratio determined above to completely saturate streptavidin and incubated at room temperature for 30 min. Unbound F protein was removed by centrifugation using a 300K Nanosep centrifuge (Pall Corporation). APC / DyLight 755 tetramers were made by mixing F protein prelabeled with streptavidin-APC and DyLight 755 (Thermo Fisher) according to the manufacturer's instructions. APC / DyLight 755 contained an average of 4–8 DyLight molecules per APC. The concentration of each F protein tetramer was determined based on the absorbance of APC (650 nm, extinction coefficient = 0.7 μM -1 cm -1 ) was measured.

[0221] Tetramer enrichment 100–200 × 10 6 Frozen PBMCs, 20-50 x 10 610 frozen tonsil cells or 40-80 x 10 6 Frozen splenocytes were thawed. The cells were centrifuged and resuspended in 50 μL of ice-cold FACS buffer consisting of 1% newborn calf serum (Thermo Fisher) and PBS. In the presence of 2% rat and mouse serum (Thermo Fisher), APC / DyLight 755-labeled tetramers were added at a final concentration of 25 nM and incubated at room temperature for 10 min. APC tetramers were then added at a final concentration of 5 nM, incubated on ice for 25 min, and washed with 10 mL of ice-cold FACS buffer. 50 μL of anti-APC-labeled microbeads (Miltenyi Biotec) that recognize each APC were added and incubated on ice for 30 min. Next, 3 mL of FACS buffer was added and passed through an LS column (Miltenyi Biotec) with a magnetic field applied. The column was washed once with 5 mL of ice-cold FACS buffer and then removed from the magnetic field. 5 mL of ice-cold FACS buffer was passed twice through the column with the magnetic field removed using a plunger to elute the cell fraction that had been bound to the column.

[0222] Flow cytometry Cells were incubated for 30 min on ice in 50 μL of FACS buffer containing an antibody cocktail that included anti-IgM FITC (G20-127, BD), anti-CD19 BUV395 (SJ25C1, BD), anti-CD3 BV711 (UCHT1, BD), anti-CD14 BV711 (M0P-9, BD), anti-CD16 BV711 (3G8, BD), anti-CD20 BUV737 (2H7, BD), anti-IgD BV605 (IA6-2, BD), and a fixable viability dye (FV). The absolute number of cells in each sample was calculated by adding a known amount of AccuCheck Counting Beads (Thermo Fisher). Individual B cells were sorted by 1) seeding into empty 96-well PCR plates and immediately freezing, or 2) seeding the B cells into 96-well flat-bottom plates that had been pre-seeded with feeder cells at a density of 28,600 cells / well in 100 μL of IMDM medium (Gibco) containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2.5 μg / mL amphotericin the day before seeding the B cells. Sorted B cells on feeder cells were cultured at 37° C. for 13 days.

[0223] ELISA Nunc Maxisorp 96-well plates (Thermo Fisher) were pre-filled with 100 ng of goat anti-human F ab(Jackson ImmunoResearch) for 90 min at 4°C. Each well was washed three times with PBS and blocked with PBS containing 1% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. The antigen-coated plate was incubated with culture supernatant for 90 min at 4°C. A standard curve was prepared using a two-fold serial dilution series of palivizumab. Each well was washed three times with PBS and incubated with horseradish peroxidase-conjugated goat anti-human full-length immunoglobulin (Invitrogen) diluted 1:6000 for 1 h. Next, each well was washed four times with PBS and incubated with TMB substrate (SeraCare) for 5–15 min. The absorbance was measured at 405 nm using a Softmax Pro plate reader (Molecular Devices). The concentration of the antibody in each sample was measured by referring to the standard curve and dilution factor. Neutralization assay To screen the culture supernatant for neutralization, Vero cells were seeded in 96-well flat-bottom plates and cultured for 48 hours. After 13 days of culture, 25 μL of virus solution containing GFP-HPIV3 purified on a sucrose density gradient and diluted to 2,000 plaque-forming units (pfu) / mL was mixed with 40 μL of culture supernatant and incubated at 37°C for 1 hour. 50 μL of the mixture of culture supernatant and virus was added to Vero cells and incubated at 37°C for 1 hour to allow the virus to adsorb. Next, 100 μL of DMEM containing 4% methylcellulose was overlaid on each well. Five days after infection, fluorescent plaques were counted using a Typhoon imaging device. The titers of HPIV3-specific monoclonal antibodies were determined by a 60% plaque reduction neutralization test (PRNT). 60) was measured. Vero cells were seeded in 24-well plates and cultured for 48 hours. Monoclonal antibodies serially diluted 1:4 were placed in 120 μL of DMEM and mixed with 120 μL of sucrose gradient-purified HPIV3 diluted to 2,000 pfu / mL and incubated at 37°C for 1 hour. 100 μL of the antibody-virus mixture was added to Vero cells and incubated at 37°C for 1 hour to allow virus adsorption. Each well was then overlaid with 500 μL of DMEM containing 4% methylcellulose. Five days after infection, fluorescent plaques were counted using a Typhoon imager. PRNT 60 The titers measured in were calculated by linear regression analysis.

[0224] Sequencing and cloning of the B cell receptor Sorted individual B cells were cryopreserved in empty 96-well PCR plates, and reverse transcription (RT) was performed directly after thawing the plates containing B cells using SuperScript IV (Thermo Fisher) as previously reported (Wu, et al. Science 329, 856-861, (2010); Tiller, et al. J Immunol Methods 329, 112-124, (2008)).

[0225] Briefly, 3 μL of RT reaction mix consisting of 3 μL 50 μM random hexamers (Thermo Fisher), 0.8 μL 25 mM deoxyribonucleotide triphosphates (dNTPs; Thermo Fisher), 1 μL (20 U) SuperScript IV RT, 0.5 μL (20 U) RNaseOUT (Thermo Fisher), 0.6 μL 10% Igepal (Sigma-Aldrich) and 15 μL RNase-free water was added to each well containing sorted single B cells and incubated at 50 °C for 1 h. Individual B cells sorted on feeder cells were cultured for 13 days, after which the supernatant was removed and the plate was immediately frozen on dry ice and stored at -80 °C, and after thawing, RNA was extracted using an RNeasy Micro Kit (Qiagen). The total eluate obtained by RNA extraction was used instead of water to perform the reverse transcription (RT) reaction. After the RT reaction, 2 μL of the resulting cDNA was added to 19 μL of PCR reaction mix to obtain a reaction containing 0.2 μL (0.5 U) HotStarTaq polymerase (Qiagen), 0.075 μL 50 μM 3' reverse primer, 0.115 μL 50 μM 5' forward primer, 0.24 μL 25 mM dNTPs, 1.9 μL 10× buffer (Qiagen) and 16.5 μL water. The PCR program for the heavy and kappa light chains was 94°C for 30 s, 57°C for 30 s and 72°C for 55 s for 50 cycles, with a final run at 72°C for 10 min. The PCR program for the lambda light chain was 94°C for 30 s, 60°C for 30 s and 72°C for 55 s for 50 cycles, with a final run at 72°C for 10 min. After the first PCR, 2 μL of the resulting PCR product was added to 19 μL of the second PCR reaction mix, resulting in a reaction containing 0.2 μL (0.5 U) HotStarTaq polymerase, 0.075 μL 50 μM 3' reverse primer, 0.075 μL 50 μM 5' forward primer, 0.24 μL 25 mM dNTPs, 1.9 μL 10× buffer, and 16.5 μL water. The same PCR program as in the first PCR was used. 4 μL of the PCR product was run on an agarose gel to confirm the presence of a 500 bp heavy chain band or a 450 bp light chain band.Five microliters of PCR reaction showing the presence of heavy or light chain amplicons were mixed with 2 μL of ExoSAP-IT (Thermo Fisher) and incubated at 37°C for 15 min, then incubated at 80°C for 15 min to hydrolyze excess primers and nucleotides. The hydrolyzed second PCR products were sequenced by Genewiz using the respective reverse primers used in the second PCR, and the sequences were analyzed by IMGT / V-Quest to identify the V, D and J gene segments. Pairs of heavy chain VDJ and light chain VJ sequences were cloned into pTT3-derived expression vectors containing human IgG1, human IgK or human IgL constant regions using In-Fusion cloning (Clontech) as described by McGuire, et al. Nat Commun 7, 10618, (2016).

[0226] Monoclonal antibody production The heavy and light chain expression plasmid pairs were incubated at 1:1 ratio in Freestyle 293 medium at 10 6 Secreted IgG was produced by co-transfecting 293F cells at a density of 100 / mL. The transfected cells were cultured for 7 days at 37°C with gentle shaking. The supernatant was harvested by centrifuging the culture at 2,500 × g for 15 min and filtered through a 0.2 μM filter. The clarified supernatant was then incubated with Protein A agarose (Thermo Scientific) and washed with IgG binding buffer (Thermo Scientific). Antibodies were eluted in 1 M Tris base-containing neutralization buffer (pH 9.0) using IgG elution buffer (Thermo Scientific). The purified antibodies were concentrated and buffer exchanged into DBPS using an Amicon ultrafiltration unit with a molecular weight cutoff of 50 kDa.

[0227] Biolayer Interferometry (BLI) BLI assays were performed using an Octet.Red instrument (ForteBio). Binding assays were performed at room temperature with shaking at 500 rpm. First, an anti-human IgG capture sensor (ForteBio) was placed in kinetics buffer (PBS (pH 7.4) containing 0.01% BSA, 0.02% Tween 20, and 0.005% NaN3) containing 40 μg / mL of purified monoclonal antibody, and the purified monoclonal antibody was immobilized on the capture sensor for 150 seconds. After the purified monoclonal antibody was immobilized on the capture sensor, a baseline signal was recorded in kinetics buffer for 60 seconds. Next, the capture sensor was immersed in kinetics buffer containing 1 μM purified HPIV3 F protein to perform a binding step for 300 seconds, and then immersed in kinetics buffer without antibody to measure the dissociation phase for 300 seconds. The maximum response was determined by averaging the shift (nm) during the last 5 s of the binding step after subtracting the background signal from each well at each time point containing analyte using a negative control monoclonal antibody. Curve fitting was performed with ForteBio data analysis software using a 1:1 binding model. For competitive binding assays, Penta-His capture sensors (ForteBio) were placed in kinetics buffer containing 1 μM His-tagged HPIV3 F protein and His-tagged HPIV3 F protein was immobilized for 300 s. After His-tagged HPIV3 F protein was immobilized on the capture sensor, a baseline signal was recorded in kinetics buffer for 30 s. The capture sensor was then immersed in kinetics buffer containing 40 μg / mL of the first antibody for 300 s, followed by immersion in kinetics buffer containing 40 μg / mL of the second antibody for 300 s. The competitive ratio was determined by dividing the maximum increase in signal of the second antibody in the presence of the first antibody by the maximum signal with the second antibody alone.

[0228] Autoreactivity assay HEp-2 cells were seeded at a density of 50,000 cells / well in 96-well plates and fixed the following day with 50% acetone and 50% methanol at -20°C for 10 min. Cells were then permeabilized and blocked by incubation with 1% Triton (Sigma-Aldrich) and 1% bovine serum albumin in PBS for 30 min at room temperature. 100 μL of each monoclonal antibody at 0.1 mg / mL was added and incubated for 30 min at room temperature. 2F5 positive control was obtained from the AIDS Reagent Program of the National Institutes of Health. Each well was then washed four times with PBS and incubated with goat anti-human IgG Alexa Fluor 594 (Thermo Fisher) diluted 1:200 in PBS for 30 min at room temperature in the dark. After washing four times with 1× PBS, images were acquired using an EVOS Cell Imaging System (Thermo Fisher).

[0229] structural analysis PI3-E12 F was prepared by adding 10 μg of LysC (New England Biolabs) to 10 mg of each IgG and incubating overnight at 37 °C, followed by addition of protein A and incubation at room temperature for 1 h. ab The resulting mixture was passed through a PVDF filter and centrifuged, then passed through a 30 kDa Amicon Ultra size-exclusion column and concentrated in PBS, and further purified by size-exclusion chromatography using Superdex 200 (GE Healthcare Life Sciences) eluted with 5 mM Hepes and 150 mM NaCl. PI3-E12 F was screened by mixing 0.1 μL of protein and 0.1 μL of reservoir solution by vapor diffusion using an NT8 dispensing robot (Formulatrix) and commercial screening kits (Rigaku Wizard Precipitant Synergy block #2, Molecular Dimensions Proplex screen HT-96, Hampton Research Crystal Screen HT). abCrystals of 1000 nm were obtained. The crystals used to acquire the diffraction data were grown in a solution containing 0.2 M monoammonium phosphate, 0.1 M Tris (pH 8.5) and 50% (±)-2-methyl-2,4-pentanediol. The crystals were cryoprotected in Parabar oil (Hampton). Diffraction analysis of the crystals was performed at 2.1 Å resolution (Table 3). Data were collected using an in-house X-ray generator at the Fred Hutchinson Cancer Research Center and analyzed using an HKL2000 55 Data processing was performed using Phaser included in CCP4 (Collaborative Computational Project, Number 4). The structure was solved by molecular replacement using HPIV3 preF (PDB accession number 6MJZ) as a search model (Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018); Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-763, (1994)). Iterative structure building and refinement were performed using COOT (Emsley & Cowtan, Acta Crystallogr D Biol Crystallogr 60, 2126-2132, (2004)) and Phenix (Adams, et al. Acta Crystallogr D Biol Crystallogr 66, 213-221, (2010)). Structure maps were generated using Pymol (DeLano, Curr Opin Struct Biol 12, 14-20, (2002)) and Chimera (Pettersen, et al. J Comput Chem 25, 1605-1612, (2004)).

[0230] PI3-E12 F abPI3-E12 F was prepared by mixing HPIV3 preF and PI3-E12 F at a 1:1 molar ratio and incubating overnight at 4°C. ab +HPIV3 preF complexes were formed. PI3-E12 F was purified by size exclusion chromatography using Superdex 200 with elution solution consisting of 5 mM Hepes and 150 mM NaCl (pH 7.4). ab +HPIV3 preF complex was purified. Negative staining electron microscopy was performed as described by Kong, et al. Nat Struct Mol Biol 20, 796-803, (2013). - / Å 2 Data were collected by imaging at 52,000x with an electron dose of 1000 nm. This magnification corresponds to a pixel size of 1.6 Å in the plane of the sample. Images were acquired with a Tietz TemCam-F416 CMOS camera using a Leginon interface with a nominal defocus of 800-1,000 nm. Image processing was performed using a cisTEM (Grant, et al. Elife 7, doi:10.7554 / eLife.35383 (2018)). The final reconstruction was calculated as C1 symmetry with no binning using 12,000 pixels and refined 20 times. ab (this disclosure), HPIV3 (PDB ID 6MJZ (Stewart-Jones, et al. Proc Natl Acad Sci USA 115, 12265-12270, (2018)), and GNC4 (PDB ID 4DME (Oshaben, et al. Biochemistry 51, 9581-9591, (2012)). Fitting was performed using the Fit function in Chimera (Pettersen, et al. J Comput Chem 25, 1605-1612, (2004)).

[0231] Animals and HPIV3 challenge The cotton rat challenge experiment was conducted with the approval of the Animal Care and Use Committee. N = 4-5 rats per group were administered 10 5 Mice were infected intranasally with 100 μL of pfu HPIV3. Monoclonal antibodies were administered intramuscularly the day before or the day after infection. Cyclophosphamide (50 mg / kg) was administered intramuscularly 21 days before infection and re-administered every 3 days for the duration of the immunosuppressive experiment. Four days after infection, nasal turbinates were harvested for virus titer determination by plaque assay. Also, four days after infection, lungs were harvested for virus titer determination by plaque assay and histopathological examination. Lung and nasal homogenates were clarified by centrifugation in EMEM medium (Gibco). Confluent monolayers of HEp-2 cells were seeded in duplicate on each diluted homogenate in a 24-well plate. After 2 h of incubation at 37°C, each well was overlaid with 0.75% methylcellulose. After 4 days, cells were fixed and stained with 0.1% crystal violet for 1 h, and plaques were counted and titered as pfu per gram of tissue. Histopathology was performed by inflating excised lungs with 10% formalin, immersing in 10% formalin, embedding in paraffin, sectioning, and staining with hematoxylin and eosin. Each slide was blindly scored on a 0-4 scale of severity. Each score was converted to a histopathology scale of 0-100% as previously reported. See Boukhvalova, et al. Bone Marrow Transplant 51, 119-126, (2016); Porter, et al. J Virol 65, 103-111 (1991).

[0232] statistical analysis Statistical analysis was performed using GraphPad Prism 7. Pairwise statistical comparisons were performed using unpaired two-tailed t-tests. Differences were considered statistically significant when P<0.05. Data points from individual samples are shown.

[0233] Hypothetical Example 1 Golden Syrian hamsters or cotton rats are used as small animal models. For preventive treatment, hamsters are administered 5 mg / kg of 3x1 antibody or control antibody, and the next day, infected with HPIV1 and HPIV3, and viral titers are measured in the nasal cavity and lungs 4 days after infection, when viral replication in the hamsters reaches its peak. For therapeutic treatment, 5 mg / kg of 3x1 antibody or control antibody is administered 1 day after infection. Groups of 5 animals are included for each condition. Treatment with 3x1 antibody can confer an antiviral effect.

[0234] Hypothetical Example 2 Golden Syrian hamsters or cotton rats are used as small animal models. For preventive treatment, hamsters are administered 5 mg / kg of MxR-01 antibody or control antibody, and the next day, infected with RSV or HMPV, and viral titers are measured in the nasal cavity and lungs 4 days after infection, when viral replication in the hamsters reaches its peak. For therapeutic treatment, 5 mg / kg of MxR-01 antibody or control antibody is administered 1 day after infection. Groups of 5 animals are included for each condition. Treatment with MxR-01 antibody can confer an antiviral effect.

[0235] (vi) Conclusion Variants of the sequences disclosed and referenced herein are also included herein. Guidelines for determining which amino acid residues can be substituted, inserted or deleted without losing biological activity can be determined using computer programs well known in the art, such as DNASTAR. TM Software (Madison, WI, USA) can be used to find the amino acid changes in the protein variants disclosed herein. The amino acid changes are preferably conservative amino acid changes, i.e., substitutions of similarly charged amino acids with each other or of uncharged amino acids with each other. Conservative amino acid changes include substitutions with members of a family of amino acids whose side chains are related.

[0236] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can be made without generally altering the biological activity of the resulting molecule.Those skilled in the art will be familiar with the fact that generally, a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter the biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu), and ketone (K). Group 7 (polar uncharged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp) and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu and Ile; Group 10 (small aliphatic residues that are non-polar or slightly polar): Ala, Ser, Thr, Pro and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0237] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is widely understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity index of each amino acid is Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamic acid (-3.5); Gln (-3.5); aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0238] It is well known in the art that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity degree can also result in a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such changes, substitution of amino acids with hydrophobicity indices within ±2 is preferred, substitution of amino acids with hydrophobicity indices within ±1 is particularly preferred, and substitution of amino acids with hydrophobicity indices within ±0.5 is even more particularly preferred. Furthermore, it is well known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity.

[0239] As detailed in U.S. Patent Publication No. 4,554,101, each amino acid residue is assigned a hydrophilicity value as follows: Arg (+3.0); Lys (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is well known that a particular amino acid can be substituted with another amino acid having a similar hydrophilicity value, and that such substitutions will result in biologically equivalent, and in particular immunologically equivalent, proteins. When making such changes, substitutions of amino acids with hydrophilicity values ​​within ±2 are preferred, substitutions of amino acids with hydrophilicity values ​​within ±1 are particularly preferred, and substitutions of amino acids with hydrophilicity values ​​within ±0.5 are even more particularly preferred.

[0240] As outlined above, amino acid substitutions may be made on the basis of the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Also, as described elsewhere herein, variants of a gene sequence include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that have no statistically significant effect on the function of the encoded product.

[0241] Variants of the protein, nucleic acid and gene sequences disclosed herein also include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to the protein, nucleic acid or gene sequences disclosed herein.

[0242] "Percent sequence identity" refers to the relatedness of two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein, nucleic acid or gene sequences, as determined by the matching between strings of protein, nucleic acid or gene sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods for determining identity are preferably designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal format alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)). Related programs further include the GCG suite of programs (Wisconsin package version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the "default values" that are the basis of the program. In this specification, "default values" refers to a set of numerical values ​​or parameters that are preregistered in the software at the time of initialization of the software.

[0243] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20μg / ml denatured salmon sperm DNA that has been fragmented, followed by washing the filter at 50°C with 0.1xSSC. Hybridization stringency and signal detection are altered primarily by adjusting the concentration of formamide (lower percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately stringent conditions include overnight incubation at 37° C. in 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml blocking salmon sperm DNA, followed by washing with 1×SSPE and 0.1% SDS at 50° C. Even lower stringency is achieved by performing stringent post-hybridization washes at high salt concentrations (e.g., 5×SSC). The above conditions can be varied by adding and / or substituting other blocking reagents used to reduce the background of hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary preparations. The addition of certain blocking reagents may require some modification of the hybridization conditions described above due to compatibility issues.

[0244] "Specifically binds" means that it does not bind significantly to other molecules or components in the relevant environmental sample, but does bind 10 5 M -1"High affinity" refers to binding between a binding fragment (e.g., a specific binding fragment) and its cognate binding molecule with an affinity equal to or greater than Ka (i.e., the equilibrium binding constant for a particular binding interaction, expressed in units of 1 / M). As used herein, "specifically binds" is also referred to as "binding." Binding fragments may be classified as "high affinity" or "low affinity." In certain embodiments, a "high affinity" binding fragment has an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 Or at least 10 13 M -1 In certain embodiments, a "low affinity" binding fragment refers to a binding fragment having a Ka of 10 7 M -1 Below, 10 6 M -1 Less than or equal to 10 5 M -1 Alternatively, affinity refers to the equilibrium dissociation constant (Kd) (units: M) of a particular binding interaction (e.g., 10 -5 M~10 -13M) for the recognition binding molecule. In certain embodiments, a binding fragment may have an "enhanced affinity," which refers to a selected or engineered binding fragment exhibiting stronger binding to its recognition binding molecule than the wild-type (or parent) binding fragment. For example, the enhanced affinity may be due to a higher Ka (equilibrium binding constant) for the recognition binding molecule than the reference binding fragment, a lower Kd (dissociation constant) for the recognition binding molecule than the reference binding fragment, or a lower dissociation rate (Koff) for the recognition binding molecule than the reference binding fragment. A variety of assays for detecting binding fragments that specifically bind to a particular recognition binding molecule and for measuring binding affinity are known, including Western blots, ELISAs, and BIACORE analyses (see also, e.g., Scatchard, et al., 1949, Ann. NY Acad. Sci. 51:660; and U.S. Patent Publication Nos. 5,283,173, 5,468,614, or similar publications).

[0245] Unless otherwise stated, the present disclosure can be carried out using conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); FM Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and RI Freshney, ed. Animal Cell Culture (1987).

[0246] As will be appreciated by those of skill in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the particular components, steps, materials, or ingredients described. Thus, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprise" means that an unrecited component, step, material, or ingredient is included, even if in large amounts. The transitional phrase "consisting of" excludes all unrecited components, steps, materials, or ingredients. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the recited components, steps, materials, or ingredients and those components, steps, materials, or ingredients that do not materially affect the embodiment. A significant effect refers to an effect that statistically significantly reduces binding between an antibody disclosed herein and an epitope of a virus.

[0247] Unless otherwise indicated, all numerical values ​​expressing quantities or properties of materials, such as molecular weight and reaction conditions, in the specification and claims are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding procedures. For clarity, the term "about," when used in conjunction with a stated value or range, has a meaning that would be reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0248] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation associated with their respective testing measurements.

[0249] In the description of the present invention (particularly in the description of the claims below), the terms "a", "an", "the" and similar modifiers are intended to include both the singular and the plural unless otherwise indicated or the context clearly indicates otherwise. Numerical ranges described herein are intended to be a shorthand way of referring to each numerical value falling within the range individually. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any method described herein can be performed in any suitable order unless otherwise indicated or the context clearly indicates otherwise. The use of any examples or language of examples (e.g., "etc.") provided herein is intended to be for the purpose of illustrating the invention only and does not limit the scope of the invention as described in the claims. No term described herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0250] Groupings of other elements of the invention disclosed herein or of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0251] Specific embodiments of the present invention are described herein, including those embodiments known to the inventors to be the best mode for carrying out the invention. Of course, those skilled in the art will readily appreciate that the embodiments described herein may be modified in various ways upon review of the above detailed description. The inventors anticipate that such modifications may be adopted by those skilled in the art, and intend that the present invention may be practiced in other ways than as specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter of the present invention to the extent permitted within the scope of applicable law. Moreover, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless otherwise indicated or the context clearly dictates otherwise.

[0252] Additionally, throughout this specification, various patents, publications, journal articles and other documents are cited (references herein). Each reference cited herein is individually incorporated herein by reference for the teachings thereof as if it were a part of this specification.

[0253] Finally, the embodiments of the invention disclosed herein are to be considered as illustrative of the principles of the invention. Other modifications may be adopted within the scope of the invention. Thus, by way of example, but not by way of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not to be limited to what has been precisely shown and described herein.

[0254] The details described herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the present invention, to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no structural details of the present invention are described in more detail than is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by reading the description of the present invention in conjunction with the drawings and / or examples.

[0255] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clear and unambiguous changes are made in the examples, or the meaning of the terms makes the interpretation meaningless or substantially meaningless. If the definition of a term does not make sense or substantially meaningless from the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. An antibody or binding fragment thereof comprising the complementarity determining region (CDR) of MxR-B11, MxR-D10, 3x1, PI3-E12, PI3-A3, PI3-B5, PI3-A10 or PI3-A12.

2. The antibody or binding fragment thereof of claim 1 , wherein the CDRs are numbered according to the Kabat, Chothia, Martin, Contact, IMGT, AHo or North numbering systems.

3. The antibody or binding fragment thereof according to claim 1, comprising any one of the following (a) to (c): (a) CDRH1 comprises GFPFSSYK (SEQ ID NO: 43), CDRH2 comprises ISASGSYI (SEQ ID NO: 44), CDRH3 comprises ARDGGRELSPFEK (SEQ ID NO: 45), CDRL1 comprises NSNIGTGYD (SEQ ID NO: 46), CDRL2 comprises DNN, and CDRL3 comprises QSYDKSLGGWV (SEQ ID NO: 47); CDRH1 containing GFTFSDHY (SEQ ID NO:1), CDRH2 containing ISSSGSNT (SEQ ID NO:2), CDRH3 containing ARAKWGTMGRGAPPTIYDH (SEQ ID NO:3), CDRL1 containing QSLLQSNGNNY (SEQ ID NO:4), CDRL2 containing LGS, and CDRL3 containing MQALQTPLT (SEQ ID NO:5); CDRH1 comprises GFTFSNYW (SEQ ID NO:8), CDRH2 comprises VKEEGSEK (SEQ ID NO:9), CDRH3 comprises AGEVKSGWFGRYFDS (SEQ ID NO:10), CDRL1 comprises QSVGSW (SEQ ID NO:11), CDRL2 comprises KTS, and CDRL3 comprises QQYSSFPYT (SEQ ID NO:12); CDRH1 comprising GYNFTNYW (SEQ ID NO:15), CDRH2 comprising IYPADSDT (SEQ ID NO:16), CDRH3 comprising ARPSTRWFVPGGMDV (SEQ ID NO:17), CDRL1 comprising QSIGAW (SEQ ID NO:18), CDRL2 comprising KAS, and CDRL3 comprising QQHSSYPST (SEQ ID NO:19); CDRH1 comprising GFNFNNYG (SEQ ID NO:22), CDRH2 comprising VSFDGSNR (SEQ ID NO:23), CDRH3 comprising SKSKYSDFWSEI (SEQ ID NO:24), CDRL1 comprising QNVMRY (SEQ ID NO:25), CDRL2 comprising DAS, and CDRL3 comprising QQRTNHRFS (SEQ ID NO:26); CDRH1 comprises GDSVKSDDFY (SEQ ID NO:29), CDRH2 comprises IYYGGRT (SEQ ID NO:30), CDRH3 comprises VRVEGLLWFGELFDY (SEQ ID NO:31), CDRL1 comprises NSNIGNNF (SEQ ID NO:32), CDRL2 comprises KDY, and CDRL3 comprises AAWQDGLSGPL (SEQ ID NO:33); CDRH1 comprises GFTFSSFG (SEQ ID NO:36), CDRH2 comprises ISHSAGFL (SEQ ID NO:37), CDRH3 comprises AKRLAGLPDLEWLLYPNFLDH (SEQ ID NO:38), CDRL1 comprises ILRTYY (SEQ ID NO:39), CDRL2 comprises GKN, and CDRL3 comprises SSRDRSGNHVL (SEQ ID NO:40); or CDRH1 comprising GFIFSNYD (SEQ ID NO:50), CDRH2 comprising ITGGSSFI (SEQ ID NO:51), CDRH3 comprising ARDGGRQLSPCEH (SEQ ID NO:52), CDRL1 comprising SSNIGAGYD (SEQ ID NO:53), CDRL2 comprising DNN, and CDRL3 comprising QSYDRGLSGWA (SEQ ID NO:54), (b) a heavy chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 178, and a light chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 179; a heavy chain variable region having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 166 and a light chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 167; a heavy chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 168 and a light chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 169; a heavy chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 170 and a light chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 171; a heavy chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 172 and a light chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 173; a heavy chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 174 and a light chain variable region having a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 175; a heavy chain variable region having a sequence that has at least 95% sequence identity to the sequence set forth in SEQ ID NO: 176 and a light chain variable region having a sequence that has at least 95% sequence identity to the sequence set forth in SEQ ID NO: 177; or a heavy chain variable region having a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 180; and a light chain variable region having a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 181; (c) a heavy chain variable region having the sequence set forth in SEQ ID NO: 178 and a light chain variable region having the sequence set forth in SEQ ID NO: 179; a heavy chain variable region having the sequence set forth in SEQ ID NO: 166 and a light chain variable region having the sequence set forth in SEQ ID NO: 167; a heavy chain variable region having the sequence set forth in SEQ ID NO: 168 and a light chain variable region having the sequence set forth in SEQ ID NO: 169; a heavy chain variable region having the sequence set forth in SEQ ID NO: 170 and a light chain variable region having the sequence set forth in SEQ ID NO: 171; a heavy chain variable region having the sequence set forth in SEQ ID NO: 172 and a light chain variable region having the sequence set forth in SEQ ID NO: 173; a heavy chain variable region having the sequence set forth in SEQ ID NO: 174 and a light chain variable region having the sequence set forth in SEQ ID NO: 175; a heavy chain variable region having the sequence set forth in SEQ ID NO: 176 and a light chain variable region having the sequence set forth in SEQ ID NO: 177; or A heavy chain variable region having the sequence set forth in SEQ ID NO:180 and a light chain variable region having the sequence set forth in SEQ ID NO:

181. (a) a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:48 and a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:49; a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:6 and a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:7; a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 13 and a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 14; a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:27 and a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:28; a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 34 and a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 35; A sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 41 and a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 42; or A sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO: 55 and a sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO: 56; or (b) An antibody or binding fragment thereof encoded by a sequence comprising the sequence set forth in SEQ ID NO:48 and the sequence set forth in SEQ ID NO:49; a sequence comprising the sequence set forth in SEQ ID NO:6 and the sequence set forth in SEQ ID NO:7; a sequence comprising the sequence set forth in SEQ ID NO:13 and the sequence set forth in SEQ ID NO:14; a sequence comprising the sequence set forth in SEQ ID NO:27 and the sequence set forth in SEQ ID NO:28; a sequence comprising the sequence set forth in SEQ ID NO:34 and the sequence set forth in SEQ ID NO:35; a sequence comprising the sequence set forth in SEQ ID NO:41 and the sequence set forth in SEQ ID NO:42; or a sequence comprising the sequence set forth in SEQ ID NO:55 and the sequence set forth in SEQ ID NO:

56.

5. The antibody according to any one of claims 1 to 4, which is an IgG antibody having an M252Y mutation, an S254T mutation, a T256E mutation, an M428L mutation and an N434S mutation in the Fc region.

6. A binding molecule comprising binding fragments of at least two antibodies according to any one of claims 1 to 5.

7. The binding molecule of claim 6, characterized in any one of the following (a) to (h): (a) the binding fragments of at least two antibodies according to any one of claims 1 to 5 comprise Fab or single chain variable fragments (scFv); (b) the binding fragments comprise Fabs linked via inter-heavy chain linkages by knobs-into-holes technology; (c) the binding fragments of at least two antibodies according to any one of claims 1 to 5 comprise scFv; (d) the binding molecule comprises a binding fragment of a PI3 antibody and a binding fragment of an MxR antibody; a binding fragment of a PI3 antibody and a binding fragment of a 3x1 antibody; or comprising a binding fragment of the 3x1 antibody and a binding fragment of the MxR antibody, (e) the binding molecule comprises a binding fragment of a PI3 antibody, a binding fragment of a 3x1 antibody, and a binding fragment of an MxR antibody; (f) the binding fragment comprises a Fab and / or scFv; (g) the binding molecule comprises MxR-B11 Fab and 3x1 Fab; MxR-B11 scFv and 3x1 Fab; MxR-B11 Fab and 3x1 scFv; or MxR-B11 scFv and 3x1 scFv; (h) the binding molecule comprises MxR-D10 Fab and 3x1 Fab; MxR-D10 scFv and 3x1 Fab; MxR-D10 Fab and 3x1 scFv; or MxR-D10 scFv and 3x1 scFv.

8. The binding molecule of claim 6 , wherein the binding fragment comprises an scFv linked to a constant region of an antibody in which one binding fragment of the scFv has another binding fragment that is different from the other binding fragment.

9. The binding molecule of claim 8 having a linker comprising a Gly-Ser linker.

10. A binding molecule comprising a binding fragment according to any one of claims 1 to 4 and a binding fragment of an antibody that binds to a second virus.

11. The binding molecule of claim 10, characterized in any one of the following (a) to (e): (a) the second virus is selected from adenovirus, bocavirus, coronavirus, enterovirus, influenza virus, metapneumovirus, parainfluenza virus, and rhinovirus; (b) the second virus is selected from human adenovirus, human bocavirus (HBoV) and human coronavirus (HCoV); (c) the second virus is a coronavirus selected from SARS-CoV, MERS-CoV, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, coronavirus NL, and coronavirus NH; (d) the second virus is selected from influenza A virus, influenza B virus, human parainfluenza virus 2 (HPIV2), HPIV4, human rhinovirus (HRV) A, HRVB, and HRVC; (e) the binding fragment is 8C4, 5Hx-l, 5Hx-2, 5Hx-3, 5Hx-4, 5Hx-5, 5.100K-1, 5PB-1, 5Fb-l, 1E11, EP R23305-44, 47D11, CR3022, CDC2-A2, G2, 5F9, FIB-H1, JC57-13, 32D6, CH65, CR9114, MAb 22 / 1, MAb70 / l, MAb 110 / 1, MAb 264 / 2, MAb W18 / 1, MAb 14 / 3, MAb 24 / 4, MAb 47 / 8, MAb 198 / 2, MAb The CDRs include those of an antibody selected from 215 / 2, H2 / 6A5, H3 / 4C4, H2 / 6C4, H2 / 4B3, H9 / B20, H2 / 4B1, CA6261, 6F12, CR9114, PEG-1, CR8033, CR8071, 113 / 2, 124 / 4, 128 / 2, 134 / 1, 146 / 1, 152 / 2, 160 / 1, 162 / 1, 195 / 3, 206 / 2, 238 / 4, 280 / 2, PAR2 (boca231 / 9F), and TCN-711.

12. A binding molecule comprising a binding fragment according to any one of claims 1 to 4 and a binding fragment that activates an immune cell.

13. The binding molecule of claim 12, wherein the binding fragment that activates an immune cell (a) binds to CD3, CD28 or 4-1BB; or (b) comprises the CDRs of OKT3 or TGN1412.

14. A composition comprising the antibody or binding fragment thereof according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

15. The composition of claim 14, characterized in any one of the following (a) to (f): (a) the pharma- ceutically acceptable carrier comprises an aqueous solution; (b) the pharma- ceutically acceptable carrier comprises saline; (c) the pharma- ceutically acceptable carrier comprises sodium chloride, potassium chloride, and calcium chloride; (d) the pharma- ceutically acceptable carrier comprises an inorganic salt; (e) the pharma- ceutically acceptable carrier comprises an antioxidant, a buffer, a chelating agent, an isotonicity agent, a preservative and / or a stabilizer; (f) the pharma- ceutically acceptable carrier comprises a release modifier.

16. The pharma- ceutically acceptable carrier is (a) containing sodium chloride, potassium chloride and calcium chloride, and further containing sodium bicarbonate or sodium lactate; or The composition of claim 14, comprising (b) an inorganic salt and a sugar.

17. The composition of claim 14, comprising a therapeutically effective amount of the antibody or binding fragment thereof for administration to a subject.

18. The composition of claim 14 for use in a method comprising providing an antiviral effect to a subject in need of such effect by administering a therapeutically effective amount of the composition to the subject.

19. A composition for use according to claim 18, characterized in that it is any one of the following (a) to (f): (a) the antiviral effect includes an anti-HPIV3 effect, an anti-HPIV1 effect, an anti-respiratory syncytial virus (RSV) effect, and / or an anti-human metapneumovirus (HMPV) effect; (b) the antiviral effect includes an anti-HPIV3 effect and an anti-HPIV1 effect; (c) the antiviral effect includes an anti-RSV effect and an anti-HMPV effect; (d) the antiviral effect includes an anti-HPIV effect, an anti-RSV effect, and an anti-HMPV effect; (e) the subject in need of an antiviral effect is a hematopoietic stem cell transplant (HCT) recipient, a lung transplant recipient, a premature infant, a person over 65 years of age, a homeless person, or a person with a pulmonary disease; (f) the subject has chronic obstructive pulmonary disease (COPD).

20. 20. The composition for use according to claim 18, wherein the antiviral effect comprises an antiviral effect against a second virus.

21. A composition for use according to claim 20, characterized in that it is any one of the following (a) to (d): (a) the second virus is selected from adenovirus, bocavirus, coronavirus, enterovirus, influenza virus, metapneumovirus, parainfluenza virus, and rhinovirus; (b) the second virus is selected from human adenovirus, human bocavirus (HBoV) and human coronavirus (HCoV); (c) the second virus is a coronavirus selected from SARS-CoV, MERS-CoV, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, coronavirus NL, and coronavirus NH; (d) the second virus is selected from influenza A virus, influenza B virus, human parainfluenza virus 2 (HPIV2), HPIV4, human rhinovirus (HRV) A, HRVB, and HRVC.

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