Antibodies and vaccines having VH3-21 and VL1-40 binding domains and uses thereof

Abstract

An antibody against respiratory syncytial virus (RSV) and / or human metapneumovirus (HMPV) is described herein. The antibody includes binding domains from anti-idiotypic monoclonal antibodies (ai-mAbs) that bind an RSV neutralizing antibody VH3-21 / VL1-40 presented as B cell receptors (BCRs). The antibodies can be used as a vaccine to selectively elicit antibodies capable of neutralizing RSV and / or HMPV without a need for somatic mutation. The vaccines are particularly useful to treat and / or reduce the risk of RSV and / or HMPV infection in infants.

Description

ANTIBODIES AND VACCINES HAVING VH3-21 AND VL1-40 BINDING DOMAINS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 635,785 filed April 18, 2024, which is incorporated herein by reference in its entirety as if fully set forth herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Al 156063 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3EX8084.xml. The file is 147,508 bytes, was created on April 18, 2025, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0004] The current disclosure describes binding domains that bind VH3-21 and VL1-40 and vaccines against respiratory syncytial virus (RSV) and / or human metapneumovirus (HMPV) based on the same. The binding domains are based on anti-idiotypic monoclonal antibodies (ai-mAbs) that bind VH3-21 / VL1-40 presented as B cell receptors (BCRs). The vaccines can be used to selectively elicit antibodies capable of neutralizing RSV and / or HMPV without a need for somatic mutation. The vaccines are particularly useful to treat and / or reduce the risk of RSV and / or HMPV infection in infants.BACKGROUND OF THE DISCLOSURE

[0005] Respiratory syncytial virus (RSV) is a common seasonal pathogen and infection generally causes mild respiratory symptoms in adults but can cause serious lower respiratory infection in infants and older adults. RSV infection is responsible for 60,000 recorded in-hospital deaths annually in children under 5 and accounts for a substantial hospitalization burden in infants as well as aged adults (Shi et al., Lancet 390, 946-958, 2017; Lozano et al., Lancet 380, 2095-2128, 201; Widmer eta / ., Influenza Other Respir Viruses 8, 347-352, 2014; Hall eta / ., The New England journal of medicine 360, 588-598, 2009). Human metapneumovirus (HMPV) is in the same Pneumoviridae family as RSV and cause similar respiratory illnesses. Like RSV, HMPV hospitalizations are a significant concern in young children and older adults. In the US, the annual hospitalization rate in children under 5 is 1 per 1,000. In older adults (age 65 and up), the rate is 231 per 100,000 individuals.

[0006] In 2023, two RSV vaccines, AREXVY™ (GSK, United Kingdom) and ABRYSVO™ (Pfizer, New York, NY), were approved for the use in adults over 60 years old. However, the current treatments available for infant protection are prophylactics based on the transfer of neutralizing antibodies. Historically, the monoclonal antibody (mAb) palivizumab (SYNAGIS™ (Sobi, Sweden)), has been available for infants at high risk of infection, such as those born prematurely (Pediatrics 102, 531-537, 1998 (“Pediatrics”); Homaira et al., Int J Pediatr 2014, 571609-571609 (“Homaira”)). Morerecently, the extended half-life mAb nirsevimab (BEYFORTUS™ (Sanofi, France)), has been approved for all newborns and babies under 1 year of age entering their first RSV season (Hammitt et al., N Engl J Med 386, 837-846, 2022; Muller et al., N Engl J Med 388, 1533-1534, 2023). These mAbs target the virally encoded fusion protein, F. As an alternative prophylactic strategy, ABRYSVO™ (Pfizer) was recently approved for use in pregnant individuals in the final month of gestation to protect infants from birth through the first 6 months of age (ed FDA Office of Media Affairs, FDA, 2023). This strategy relies on the transplacental transfer of protective antibodies from the mother to the fetus (Kampmann et al., N Engl J Med 388, 1451-1464, 2023). These prophylactic strategies afford transient protection to the infant for the duration serum half-life of the antibodies, but this protection wanes as these antibodies are cleared from the infant’s system. Furthermore, the high-risk infant population where a vaccine is most needed, faces unique immunologic challenges. These include immunological immaturity resulting in reduced levels of T cell help which limits somatic mutation, affinity maturation and antibody class switching, and the suppression of B cell responses by maternal antibody.SUMMARY OF THE DISCLOSURE

[0007] The present disclosure describes anti-idiotypic monoclonal antibodies (ai-mAbs) that have a high affinity and specificity for unmutated and mutated VH3-21 / L1 -40 B cell receptors (BCRs). Binding domains based on the ai-mAbs can be used in a vaccine that rapidly and selectively elicits neutralizing antibodies against respiratory syncytial virus (RSV) and / or human metapneumovirus (HMPV).

[0008] In particular embodiments, the ai-mAbs includes a first binding domain and a second binding domain wherein the first binding domain binds VH3-21 and the second binding domain binds VL1 -40.

[0009] In particular embodiments, the binding domain that binds VH3-21 includes a heavy chain variable region including a complementarity determining region (CDR) heavy (H)1 including the sequence: GYWIE (SEQ ID NO: 13), the CDRH2 including the sequence: EILSGRGTTNYNEKFKG (SEQ ID NO: 14), and the CDRH3 including the sequence: GGFLYGDGPFDY (SEQ ID NO: 15) and a light chain variable region including a CDR light (L)1 including the sequence: RASQNIDTHIH (SEQ ID NO: 16), the CDRL2 including the sequence: YASESIS (SEQ ID NO: 17), and the CDRL3 including the sequence: QQSNSWPFT (SEQ ID NO: 18).

[0010] In particular embodiments, the binding domain that binds VL1-40 includes a heavy chain variable region including aCDRHI including the sequence: DYEIH (SEQ ID NO: 19), the CDRH2 including the sequence: TSDPETGGFAYTQKFKG (SEQ ID NO: 20), and the CDRH3 including the sequence: RPYYGSGAWFAY (SEQ ID NO: 21) and a light chain variable region including a CDR light (L)1 including the sequence: KANHDVNIAVA (SEQ ID NO: 22), the CDRL2 including the sequence: SASYRYT (SEQ ID NO: 23), and the CDRL3 including the sequence: QQHYDTPRT (SEQ ID NO: 24).

[0011] In particular embodiments, the binding domain that binds VH3-21 includes a variable heavy chain including the sequence of SEQ ID NO: 1 and a variable light chain including the sequence of SEQ ID NO: 2.

[0012] In particular embodiments, the binding domain that binds VL1-40 includes a variable heavy chain including the sequence of SEQ ID NO: 3 and light chain variable region including the sequence of SEQ ID NO: 4.

[0013] In particular embodiments, the ai-mAb further includes Fc antibody regions. The presence of an Fc allows for extended half-life and recirculation through FcRn. When included, Fc regions can include one or more of silencingmutations, mutations that lower an isoelectric point, and heterodimer mutations. In particular embodiments, Fey interactions are reduced or eliminated.BRIEF DESCRIPTION OF THE FIGURES

[0014] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0015] FIG. 1. Isolation of anti-idiotypic antibodies specific for antibodies encoded by VH3-21 and VL1-40 genes. Immunoglobulin G (IgG)s purified from hybridoma supernatants 2C2, 2C1, 2F1, and 1D3 were evaluated for their ability to bind to a panel of recombinant monoclonal antibodies (mAbs) by enzyme-linked immunosorbent assay (ELISA). The panel included unmutated site Ill-directed VH3-21 / VL-140 mAbs, chimeric mAbs with VH3-21 derived heavy chains paired with non-VL1-40 encoded light chains, chimeric mAbs VL1-40 light chains paired with non-VH3-21 encoded heavy chains, and control mAbs derived from non-VH3-21 encoded heavy chains paired with non-VL1-40 encoded light chains (control mAbs) as indicated. The scale indicates the A280 signal as measured by ELISA. Data shown is average of duplicate wells.

[0016] FIGs. 2A-2D. Anti-idiotypic monoclonal antibody (ai-mAb)-specific B cell sorting and sequencing. (2A) ai-mAbs were fluorescently labeled with Allophycocyanin (APC) conjugated to a unique oligonucleotide barcode and used to stain naive B cells (CD3-, CD14-, CD20+, CD 19+, lgM+, lgD+, CD27 ) from human peripheral blood mononuclear cells (PBMCs) as indicated. Representative test stain shown above. Naive B cells stained with oligonucleotide barcoded APC were used as a control for non-specific staining (upper left panel). The frequency of APC positive B cells is shown below the gate. Gating strategy for sort shown in FIG. 3. (2B-2D) APC+naive B cells were sorted and the paired B cell receptor transcripts were amplified and indexed using the chromium platform from 10X genomics and then subjected to next-generation sequencing. (2B) The percentage of VH3-21 / L1-40 pairs among all B-cell receptor (BCR) sequences sorted by each ai- mAb bait is shown. (2C) The percentage of BCRs expressing a heavy chain derived from the VH3-21 gene sorted by each ai-mAb bait is shown. (2D) The percentage of BCRs expressing a light chain derived from the VL1-40 gene sorted by each ai-mAb bait is shown. The number of B cells analyzed is shown at the bottom of FIGs. 2B-2D. The frequency of naive B cells expressing these genetic features identified by high-throughput unbiased sorting is included for comparison (Dekosky et al., PNAS, 113(19), E2636-E2645, 2016) in FIGs. 2B-2D.

[0017] FIG. 3. Gating strategy to identify and bulk sort ai-mAb specific naive B cells (related to FIG. 2). Gating as follows: Single cells -> Lymphocytes -> Live cells - CD3’ CD14- - CD19+CD20+^ lgD+lgM+— >■ ai-mAb-APC+B cells. Cell percentages of parent gate are shown, representative staining plots from 1 group of sorted cells (See Methods in Experimental Example 1).

[0018] FIGs. 4A, 4B. Assigning ai-mAb-specificity for BCR using feature barcodes (related to FIG. 2). Ai-mAbs were labeled with APC conjugated to a unique oligonucleotide feature barcode and used to stain naive PBMCs in two cocktails containing iv2, 2C1, 1D3 (4A) and iv2, 2F1, and 2C2 (4B). Individual B cell libraries were generated using the chromium platform from 10X genomics. Each panel shows the number of times the indicated feature barcode was observed for a given cell plotted from highest to lowest where each dot represents a single B cell. The thresholds, represented by thehorizontal dotted line in each panel, were estimated by segmented generalized linear models using the chngpt R package. BCR sequences from B cells above the threshold were considered positive for a given ai-mAb. B cells above the threshold from the Iv2 isotype control in each sort were discarded from subsequent analysis.

[0019] FIGs. 5A, 5B. Binding kinetics of 2C1 and 1D3 ai-mAbs to ADI-19425 Fab. Binding kinetics of recombinant 2C1 (5A) or 1D3 (5B) IgG to ADI-19425 Fab at the indicated concentrations. The average association rates (ka), kaerror, dissociation rates (kd), kd error and average apparent affinity (KD) values are shown below each plot.

[0020] FIG. 6. Data collection and refinement statistics for crystal structures.

[0021] FIGs. 7A-7D. Crystal structures of parental ai-mAbs in complex with ADI-19425. (7 A, 7C) Surface representation of the epitopes on the ADI-19425 Fab bound by (7 A) 1D3 Fab (7C) and 2C1 Fab. 1D3 and 2C1. Views are looking down on the ADI-19425 paratope. Residues within 5A of (7 A) 1 D3 or (7C) 2C1 are outlined in white. Complementarity determining regions (CDRs) are labeled as indicated. (7B, 7D) Buried surface area (BSA) plots shown as stacked bar graphs of ADI- 19425 HC (top, SEQ ID NO: 33) and ADI-19425 LC (bottom, SEQ ID NO: 34) bound by (7B) 1D3 Fab or (7D) 2C1 Fab. BSA contributed by the HC of either 1 D3 or 2C1 have solid-colored bars while BSA contributed by the LC of either 1D3 or 2C1 have empty bars. Residues participating in hydrogen-bond interactions are labeled with an “H”. The ADI-19425 sequences encoded by the V-gene are boxed. The ADI-19425 LC (GenBank Accession: MG524528) differs by one amino acid from germline V gene VL1-40. It contains a proline at position 2, while VL1-40 contains a serine at this position.

[0022] FIGs. 8A-8C. Fab-Fab structures of 2C1 and 1D3 bound to ADI-19425 (related to FIG. 7). Full view of complexes between (8A) 2C1 Fab (heavy chain (HC), light chain (LC)) and (8B) 1D3 Fab with ADI-19425 Fab. (8C) Overlays of both complexes are shown aligned by the VHA / L region of ADI-19425 Fab and rotated on the y-axis by 190°.

[0023] FIGs. 9A-9F. Engineering a VH3-21 / VL1-40 BCR-targeting bispecific ai-mAb. (9A) Schematic of the bispecific platform used. The 1D3 Arm is a conventional heavy chain / light chain (HC / LC) pair with mutations introduced into the Fc (constant fragment) to lower the isoelectric point indicated by white stars. The 2C1 arm is a single chain fragment variable (scFv)-Fc fusion. Both Fc regions contain mutations that ablate Fc binding (black stars). (9B) Anion exchange (AEX) chromatography of protein A-affinity purified material from 293 cells co-transfected with expression plasmids encoding the three chains depicted in FIG. 9A. A280 is shown on the left y-axis, and conductivity is shown on the right y-axis. (9C-9F) Binding of ADI-19425 Fab and the indicated chimeric Fabs to recombinant 2C1 -IgG (9C) 1 D3-lgG (9D), or IgG from peak 1 (9E) or peak 2 (9F) from FIG. 9A was measured by Bio-Layer Interferometry (BLI).

[0024] FIG. 10. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of the 2 bispecific ai-mAb peaks eluted off AEX column (related to FIG. 9). Both bispecific antibody peaks eluted off the anion exchange column were run on an SDS-PAGE gel along with an IgG standard under reduced (left) and non-reduced (right) conditions.

[0025] FIGs. 11A-11D. Bispecific ai-mAb-specific B cell sorting and sequencing. (11A) Fluorescently-labeled 1D3 / 2C1 bispecific ai-mAb was used to stain and sort naive human B cells. Representative sorting plot from 1 of 3 independent sorting experiments. See FIG. 12 for gating strategy. (11 B) From the sorted cells, the percentage of productive VH3-21 HCs, VL1-40 LCs, and VH3-21 / L1 -40 pairs are shown. Each data point represents the frequency of gene usage from 3 independent sorting experiments. Colored bars represent the mean and the error bars represent the standard deviation. Naive BCRs obtained from unbiased high-throughput sequencing of 3 unique donors from Dekosky et al. (Proc Natl AcadSci USA 113, E2636-2645, 2016 ("Dekosky") are shown for comparison. Significant differences were determined using Student two-tailed t tests. **p = 0.001-0.01, ***p = 0.0001-0.001, ****p < 0.0001. (11C) VH3-21 / VL1-40 BCRs from FIG. 11 B were expressed as recombinant mAbs and tested for binding to respiratory synctial virus (RSV) pre-fusion F protein (preF) and post-fusion F protein (postF) by BLI. Data shown is average from 2 duplicate experiments. (11D) mAbs from FIG. 11C were tested for their ability to compete for binding with ADI-14337 to preF by BLI. % binding was was calculated by Rmax of binding in presence of ADI-143371 Rmax binding in the presence of an irrelevant mAb x 100%. Each data point represents an independent experiment and the bars represent the average. Horizontal dotted line represents the average ADI-14337 competition against itself. D25 is an RSV preF site 0 mAb (Kwakkenbos et al., Nature Medicine 16, 123-128, 2010), and included as a negative control.

[0026] FIG. 12. Gating strategy to identify and single cell sort bispecific ai-mAb specific naive B cells (related to FIG. 11). Gating as follows: Lymphocytes -> Single cells -> Live cells -> CD3-, CD 14- - CD19+CD20+— > lgD+lgM+— > CD27- - lgM+Bispecific ai-mAb+. Percentages of parent gate indicated.

[0027] FIGs. 13A, 13B. High throughput VH3-21 / VL1-40 mAb expression and screening (related to FIG. 11). (13A) Schematic of high throughput mAb cloning and expression. Briefly, fragments 1 and 2 are PCR-amplified from pTT3 IgG expression plasmids encoding HC and LCs. HC and LC variable regions from sequences recovered through single cell sorting experiments with 5’ and 3’ homology arms are synthesized (sDNA, See FIG. 11). Fragments 1, 2, and the corresponding HC / LC sDNA are assembled by In-Fusion cloning, and then PCR amplified with the flanking forward / reverse (F / R) primers. Linearized amplicons are then transfected into 2mL 293E cultures in 24-deep well plates, and incubated for 5-6 days. Supernatants are harvested from cultures and screened by BLI. Created with BioRender.com (13B) Representative BLI plots from production of mAbs recovered from sort #2. Screened for binding to 2C1 Fab, RSV-A preF and postF. Grey curves represent mAbs that bound preF and were further evaluated, black curves represent mAbs that failed to bind preF in the initial screen.

[0028] FIGs. 14A-14D. Neutralization screen of VH3-21 / VL1 -40 sorted mAbs against RSV-A / B and Steady State Affinity Measurements (related to FIG. 15). All recombinant VH3-2WL1 -40 preF-binding mAbs sorted with the bispecific ai-mAb were screened for neutralization activity against RSV-A (14A) or RSV-B (14B). All mAbs were used at 500 g / mL final dilution in triplicate wells. % Infectivity is the infectivity in the presence of mAb infectivity in the absence of mAb X100%. Colored mAbs indicate those followed up for IC50 determination. (14C) Steady-state analysis of VH3-21 / VL1-40 mAb binding response to a dilution series of RSV-A preF measured by BLI. E1D1 is an irrelevant EBV mAb (Sathiyamoorthy et al., Nature Communications 7.1, 13557, 2016) included as a negative control. Each data point represents the average response from two independent experiments. (14D) Dissociation rates for VH3-21 / VL1-40 mAbs averaged over the dilution series in FIG. 14C. Each data points is from an individual experiment, and bars represent the average.

[0029] FIGs. 15A-15D. VH3-21 / VL1 -40 mAb RSV neutralization and affinity (15A) Representative neutralization curves of selected VH3-21 / VL1-40 mAbs against RSV-A (left) and RSV-B (right) measured using a plaque reduction assay. Points shown are average of 2 technical replicates from a single experiment. (15B) Average IC50 values of selected mAbs against RSV-A and RSV-B. Each dot represents an independent experimental replicate, and error bars represent standard deviation. Dotted line represents maximum tested concentration. (15C) Steady-state kinetic analysis of neutralizing VH3-21 / VL1-40 mAbs binding to a dilution series of RSV-A (solid lines) and RSV-B (dashed lines) preF measured by BLI . Each data point represents the average response from two independent experiments. (15D) Apparent affinity of neutralizing VH3-21 / VL1 -40 mAbs to RSV-A / RSV-B preF determined from dilution series experiments in FIG. 15C.

[0030] FIGs. 16A-16I. Activation of on and off-target BCR-expressing cell lines by calcium flux assays (16A-16C) Surface staining of DG75 B cell lines transduced to express the indicated BCRs. Staining shown as histograms, with signals normalized to mode. (16A) Staining with phycoerythrin (PE)-conjugated 1D3 ai-mAb. (16B) Staining with APC-conjugated 2C1 mAb. (16C) Staining with fluorescein isothiocyanate (FITC)-conjugated bispecific ai-mAb. (16D-16E) Calcium flux assay in response to the addition of the 1D3 ai-mAb (16D), 2C1 ai-mAb (16E), or the 1D3 / 2C1 bispecific ai-mAb (16F) as immunogen. Signals shown are representative of 2 independent experiments. (16G-16I) Calcium flux assays in nonneutralizing RSV preF specific BCR-expressing cell lines as indicated. Cell lines were stimulated with RSV-A preF (16G), the bispecific ai-mAb (16H), and as a positive control an a-IgG Fey F(ab’)2 (161). Signals shown are representative of 2 independent experiments.

[0031] FIG. 17. Bi-specific ai-mAb-specific B cell sorting and sequencing. Fluorescently-labeled 1D3 / 2C1 bi-specific ai- mAb was used to stain naive B cells.

[0032] FIGs. 18A-18C. Schematic of bi-specific antibody binding to representative B cells expressing VH3-21 heavy chains but with non VL1-40 light chains (18A), non-VH3-21 heavy chains paired with VL1-40 light chain (18B) or B cells expressing VH3-2WL1-40 BCR pairs (18C).

[0033] FIGs. 19A-19D. CRISPR Cas9 was used to edit the rearranged AD114337 VDJ into the murine Ig locus (19A), and the rearranged AD114337 VLJL into the murine IgK locus (19B). Peripheral B cells from a wildtype mouse (19C), or mouse heterozygous for the knocked in AD114337 heavy and light chains (19D) were stained with the VH3-21 -specific mAb 1D3 conjugated to APC and the VL1 -40-specific mAb 2C1 conjugated to phycoerythrin (PE).

[0034] FIGs. 20A-20C. Schematic of bi-specific ai-mAb / CpG conjugate. (20B) Non-reducing SDS-PAGE of bispecific and bispecific monovalent streptavidin (mSA) fusion (20C) binding of bispecific-mSA fusion to the indicated Fabs.

[0035] FIG. 21. Table of expected outcomes of immunization studies.

[0036] FIG. 22. Schema for adoptive transfer-challenge experiments.

[0037] FIG. 23. Schema for modeling the effect of maternally transferred antibodies on the VH3-21 / L1 -40 B cell response to RSV vaccine.

[0038] FIGs. 24A-24C. (24A) Binding of the ai-mAb to RSV-human metapneumovirus (HMPV) cross neutralizing VH3- 21A / L1-40 mAbs. (24B) Binding of ai-mAb-sorted antibodies (denoted by MLR_*) to HMPV preF. (24C) Venn diagram indicating binding properties of ai-mAb-sorted VH3-21 / VL1-40 mAbs. The numbers indicate the mAbs belonging to each category and the HMPV-binding mAbs from FIG. 24B are indicated.

[0039] FIGs. 25A-25D. Knock-in strategy and characterization of ADI-19425 BCR+ mice. (25A-25B) CRISPR-Cas9 was used to edit rearranged ADI-19425 variable diversity joing (VDJ) into the murine Ig locus 26A and the rearranged ADI- 19425 VJA into the murine IgK locus 25B. (25C) Peripheral B cells from a wildtype (WT) mouse or a KI ADI-19425 BCR+ mouse were stained with RSV preF conjugated to APC or PE. (25D) 0.5x106B cells from knock-in AD119425 mice were transferred into MD4 (anti-HEL) transgenic mice and immunized with the ai-mAb (n=3) or an isotype control (n=3). Serumcollected before and at 5 days after immunization was tested for binding to DS-Cav1 by ELISA.

[0040] FIGs. 26A-26C. Development of an adoptive transfer mouse model to recapitulate the human frequency of naive VH3-21 / VL1 -40 B cells. (26A) Schematic of the adoptive transfer system and evaluation. (26B) Enumeration of transferred B cells by differential CD45.1 / CD45.2 staining (gate in bottom right). (26C) Frequency of CD45.2 B cells 24 hours posttransfer (n = 5 mice).

[0041] FIGs. 27A-27F. Immunization and serum neutralization in adoptive transfer mouse model. (27A) Schematic of experimental timeline. (27B-27E) In vitro RSV-A serum neutralization from (27B) day (d)7, (27C) d17, (27D) d21, and (27E) d28 of immunized mice by plaque reduction assay in Vero cells. (27F) Reciprocal I D50 RSV-A neutralizing titers of bispecific ai-mAb immunized mice at all timepoints.

[0042] FIGs. 28A-28E. Immunization and RSV challenge in adoptive transfer mouse model. (28A) Schematic of experimental timeline. (28B & 28C) In vitro RSV-A serum neutralization from (28B) d 12 or (28C) d 17 of immunized mice by plaque reduction assay in Vero cells. (28D) Reciprocal ID50 RSV-A neutralizing titers of bispecific ai-mAb immunized mice at d12 and d17. One mouse that did not show serum neutralization was excluded from analysis (#4 from 28B and 28C). (28E) RSV-A lung titers from immunized mice harvested at d17, 5 days post RSV infection.

[0043] FIG. 29. Sequences supporting the disclosure including AD1 19425 variable heavy chain coding (SEQ ID NO: 91); AD1 19425 variable light chain coding (SEQ ID NO: 92); 1D3 variable heavy chain coding sequence (SEQ ID NO: 93); 1D3 variable light chain coding sequence (SEQ ID NO: 94); 2C1 variable heavy chain coding sequence (SEQ ID NO: 95); 2C1 variable light chain coding sequence (SEQ ID NO: 96); scFv linker (SEQ ID NO: 100); 1D3 scFv (SEQ ID NO: 46 and SEQ ID NO: 47); 2C1 scFv (SEQ ID NO: 44 and SEQ ID NO: 45); 2C1 scFv-Fc coding sequence (SEQ ID NO: 97); 1D3 scFv- Fc (SEQ ID NO: 9 and SEQ ID NO: 10); 2C1 scFv-Fc (SEQ ID NO: 5 and SEQ ID NO: 8); 1D3 heavy chain coding sequence (SEQ ID NO: 98); 1D3 light chain coding sequence (SEQ ID NO: 99); 1D3 heavy chain (SEQ ID NO: 6); 1D3 light chain (SEQ ID NO: 7); 2C1 heavy chain (SEQ ID NO: 11); and 2C1 light chain (SEQ ID NO: 12).DETAILED DESCRIPTION

[0044] Respiratory syncytial virus (RSV) is a common seasonal pathogen and infection generally causes mild respiratory symptoms in adults but can cause serious lower respiratory infection in infants and older adults. RSV infection is responsible for 60,000 recorded in-hospital deaths annually in children under 5 and accounts for a substantial hospitalization burden in infants as well as aged adults (Shi et a / ., Lancet 390, 946-958, 2017; Lozano ef a / ., Lancet 380, 2095-2128, 2012; Widmer ef a / ., Influenza Other Respir Viruses 8, 347-352, 2014; Hall ef a / ., The New England journal of medicine 360, 588-598, 2009). In 2023, two RSV vaccines, AREXVY™ (GlaxoSmithKline Biological SA, United Kingdom) and ABRYSVO® (Pfizer Inc., New York, NY) were approved for use in adults over 60 and pregnant individuals, respectively. Both are protein subunit vaccines including the viral fusion protein (F), which has been stabilized in the prefusion conformation (PreF), the relevant target of most neutralizing antibodies (Margo et al., PNAS 109: 3089-3094, 2012). During infection, F irreversibly converts to an antigenically distinct post-fusion state (PostF). Previously unsuccessful infant vaccine formulations contained PostF and elicited non-neutralizing antibodies that enhanced respiratory disease and, in some cases, led to death. RSV vaccine development has proceeded slowly and cautiously since. In the absenceof a vaccine, currently the prophylaxis for infant RSV infection is passive transfer of neutralizing monoclonal antibodies (mAbs). This treatment is cost-prohibitive and limited to use in high-risk infants in developed countries. As an alternative, ABRYSVO® (Pfizer Inc.) was approved for use in pregnant individuals in the final month of gestation to protect infants against lower respiratory tract disease from birth through 6 months of age (ed FDA Office of Media Affairs, FDA, 2023). This vaccination strategy relies on transplacental transfer of protective antibodies from the mother to the fetus and consequently the protection afforded to infants wanes in step with maternal antibody decay (Kampmann et al., N Engl J Med. 2023;388(16): 1451-64). This approach will be less effective in pre-term infants, as placental antibody transfer is directly related to gestational age (Palmeira etal., Clin Dev Immunol. 2012, 2012:985646; Pitcher-Wilmott et al., Clin Exp Immunol. 1980, 41(2):303-8; Malek et al., Am J Reprod Immunol. 1994, 32(1):8-14; and Wilkins ef al., Nat Med. 2023, 29(5):1172-9). A vaccine that can be directly delivered to infants to elicit neutralizing antibodies that protect beyond 6 months and establish long-term humoral memory remains an unmet need.

[0045] Maternal transfer of high-titers of RSV antibodies may in fact be detrimental to infant vaccination as they can negatively impact the infant’s ability to mount de novo antibody responses (Siegrist, Vaccine. 2003, 21 (24): 3406- 12 ("Siegrist”); Vono et al., Cell reports. 2019, 28(7):1773-84.e5; Murphy ef al., J Clin Microbiol. 1986, 23(6):1009-14 ("Murphy”); and Shinoff et al., The Journal of Infectious Diseases. 2008, 198(7):1007-15). Furthermore, the infant population faces additional unique immunologic challenges, including reduced levels of T cell help which limits somatic mutation, affinity maturation and antibody class switching; processes involved in humoral responses (Siegrist, Murphy, and Siegrist and Aspinall, Nature reviews Immunology. 2009;9(3): 185-94).

[0046] Most vaccine efforts have aimed to elicit neutralizing antibodies against the viral fusion (F) protein. F exists in a metastable pre-fusion (Pre F) state and a highly stable post-fusion (PostF) state, the former of which is the relevant target of most neutralizing antibodies. In previously unsuccessful vaccine formulations, F preferentially adopted the PostF conformation. In fact, the earliest RSV vaccine trial resulted in enhanced respiratory disease that has been attributed in part due to the elicitation of non-neutralizing antibodies directed at PostF.

[0047] A class of F-specific mAbs have recently been isolated from RSV-infected infants that could potently neutralize RSV. These mAbs are derived from a VH3-21 variable heavy (VH) chain gene and a VL1-40 variable light chain (VL) gene, and bind an epitope present on PreF. Neutralizing PreF-specific VH3-21 / VL1 -40 mAbs were isolated from cord blood and naive B cells from adults, demonstrating that these antibodies represent a reproducible class that is pre-configured to neutralize RSV and is therefore targetable through vaccination.

[0048] Human metapneumovirus (HMPV) is in the same Pneumoviridae family as RSV and cause similar respiratory illnesses. Like RSV, HMPV hospitalizations are a significant concern in young children and older adults. In the US, the annual hospitalization rate in children under 5 is 1 per 1,000. In older adults (age 65 and up), the rate is 231 per 100,000 individuals.

[0049] The present disclosure describes anti-idiotypic monoclonal antibodies (ai-mAbs) that have a high affinity and specificity for unmutated VH3-21 / VL1-40 B cell receptors (BCRs) as a vaccine that will rapidly and selectively elicit antibodies capable of cross-reacting with and potently neutralizing RSV and / or HMPV. This approach has several advantages over RSV F-derived vaccines: i) the target antibodies are pre-configured to bind to RSV F, so a faithful mimicof PreF is not required to present the correct epitope, overcoming potential stability issues; ii) since ai-mAbs are antigenically distinct from RSV F, they will not present off-target epitopes that might distract from the development of a VH3-21 / VL1-40-dominated antibody response thereby reducing the risk of vaccine-enhanced disease mediated by nonneutralizing antibodies; ill) the antigenic disparity between RSV F and ai-mAbs will avoid interference of infant humoral response by maternal antibodies; and iv) VH3-21 / VL1 -40 antibodies do not require affinity maturation to achieve potent neutralization so they can be rapidly induced by vaccination in infants who are immunologically immature.

[0050] In particular embodiments, a vaccine against RSV and / or HMPV includes an ai-mAb. In particular embodiments, the ai-mAb binds VH3-21 / VL1 -40. In particular embodiments, the ai-mAb includes a multi-specific ai-mAb. In particular embodiments, the multi-specific ai-mAb is a bispecific ai-mAb.

[0051] In particular embodiments, the ai-mAb includes a first binding domain and a second binding domain. In particular embodiments, the ai-mAb includes a binding domain that binds VH3-21 and a binding domain that binds VL1-40.

[0052] In particular embodiments, the binding domain that binds VH3-21 includes a heavy chain variable region including a complementarity determining region (CDR) heavy (H)1 including the sequence: GYWIE (SEQ ID NO: 13), the CDRH2 including the sequence: EILSGRGTTNYNEKFKG (SEQ ID NO: 14), and the CDRH3 including the sequence: GGFLYGDGPFDY (SEQ ID NO: 15) and a light chain variable region including a CDR light (L)1 including the sequence: RASQNIDTHIH (SEQ ID NO: 16), the CDRL2 including the sequence: YASESIS (SEQ ID NO: 17), and the CDRL3 including the sequence: QQSNSWPFT (SEQ ID NO: 18), according to Kabat.

[0053] In particular embodiments, the binding domain that binds VL1-40 includes a heavy chain variable region including aCDRHI including the sequence: DYEIH (SEQ ID NO: 19), the CDRH2 including the sequence: TSDPETGGFAYTQKFKG (SEQ ID NO: 20), and the CDRH3 including the sequence: RPYYGSGAWFAY (SEQ ID NO: 21) and a light chain variable region including a CDR light (L)1 including the sequence: KANHDVNIAVA (SEQ ID NO: 22), the CDRL2 including the sequence: SASYRYT (SEQ ID NO: 23), and the CDRL3 including the sequence: QQHYDTPRT (SEQ ID NO: 24), according to Kabat.

[0054] In particular embodiments, the binding domain that binds VH3-21 includes a variable heavy chain including the sequence of SEQ ID NO: 1 and a variable light chain including the sequence of SEQ ID NO: 2.

[0055] In particular embodiments, the binding domain that binds VL1-40 includes a variable heavy chain including the sequence of SEQ ID NO: 3 and light chain variable region including the sequence of SEQ ID NO: 4.

[0056] In particular embodiments, the ai-mAb further includes a first Fc region and a second Fc region. In particular embodiments, the ai-mAb is an IgG antibody. In particular embodiments, the ai-mAb includes a first arm and a second arm, wherein the first arm includes the first Fc region and the second arm includes the second Fc region. In particular embodiments, the first Fc region includes mutations that lower the isoelectric point. In particular embodiments, the second Fc region includes mutations that lower the isoelectric point. In particular embodiments, the first and / or second Fc region include Fc silencing mutations. In particular embodiments, Fc silencing mutations are mutations that reduce or eliminate Fc interactions. In particular embodiments, Fc interactions include binding to Fey receptors I, Ila, lib, and Illa. In particular embodiments, the ai-mAb is depicted in FIG. 9A.

[0057] In particular embodiments, the ai-mAb is a multi-domain binding molecule. In particular embodiments, the ai-mAb is a multi-specific antibody. In particular embodiments, the ai-mAb includes a first binding domain and a second binding domain. In particular embodiments, the first binding domain binds VH3-21 and the second binding domain binds VL1-40. In particular embodiments, the first binding domain binds VL1-40 and the second binding domain binds VH3-21.

[0058] In particular embodiments, to generate a ai-mAb, a first binding domain is produced as an scFv and fused to an Fc, while the variable light (VL) chain of the second binding domain is cloned in frame with a kappa light chain constant region and the variable heavy (VH) chain of the second binding domain is cloned into an IgG heavy chain expression vector.

[0059] In particular embodiments, to generate the ai-mAb, the binding domain that binds VL1-40 is produced as an scFv and fused to an Fc, while the variable light (VL) chain of the binding domain that binds VH3-21 is cloned in frame with a kappa light chain constant region and the variable heavy (VH) chain of the binding domain that binds VH3-21 is cloned into an IgG heavy chain expression vector.

[0060] In particular embodiments, the presence of the Fc allows for an extended half-life and recirculation through the FcRn. In particular embodiments, Fey interactions are reduced or eliminated. In alternative embodiments, the ai-mAb is produced by constructing a binding domain that binds VH3-21 as an scFv and fusing it to an Fc, while VL of the binding domain that binds VL1-40 is cloned in frame with a kappa light chain constant region and the VH of the binding domain that binds VL1-40 is cloned into an IgG heavy chain expression vector.

[0061] Aspects of the current disclosure are now described in more supporting detail as follows: (I) Antibodies; (II) MultiDomain Binding Molecules; (III) Recombinant Production; (IV) Compositions for Administration; (V) Methods of Use; (VI) Kits; (VII) Exemplary Embodiments; (VIII) Examples; and (IX) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0062] (I) Antibodies. Traditionally, an antibody includes a tetramer structure with two full-length heavy chains and two full- length light chains. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (PR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0063] The assignment of amino acids to each domain can be in accordance with 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 etal., J. Mol. Biol. 262:732-745 (1996), “Antibodyantigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc M P 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 automaticmodeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309 (3): 657-70, (AHo numbering scheme)), North (North eta / ., J Mol Biol. 406 (2): 228-256 (2011), "A new clustering of antibody CDR loop conformations"), or other numbering schemes.

[0064] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs, such as ABody Builder can also be used.

[0065] An antibody disclosed herein can include an anti-idiotypic monoclonal antibody (ai-mAb) that binds VH3-21 / VL1 - 40 B cell receptors (BCRs). Upon binding, ai-mAbs will selectively engage and stimulate B cells to rapidly and selectively elicit antibodies capable of cross-reacting with and potently neutralizing RSV and / or HMPV. RSV includes at least two antigenic subgroups (A and B). This antigenic dimorphism is due primarily to differences in the surface G glycoproteins. Two surface glycoproteins, G and F, are present in the envelope and mediate attachment and fusion with cells of the respiratory epithelium. The F proteins also mediate coalescence of neighboring cells to form the characteristic syncytial cells for which the virus receives its name. HMPV has 4 antigenic subgroups: A1, A2, B1, and B2. These subgroups circulate globally with varying patterns and have distinct geographical distributions. While there's some antigenic crossreactivity between subgroups, research suggests that the F protein, a major viral surface glycoprotein, is a key determinant of cross-lineage neutralization and protection.

[0066] The RSV genome is 15,000 nucleotides in length and is composed of a single strand of RNAwith negative polarity. It has 10 genes encoding 11 proteins; there are 2 open reading frames of M2. The genome is transcribed sequentially from NS 1 to L with reduction in expression levels along its length. N encodes nucleocapsid protein that associates with the genomic RNA forming the nucleocapsid. M encodes the Matrix protein used for viral assembly. Nucleolin at the cell surface is the receptor for the RSV fusion protein. Interference with the nucleolin-RSV fusion protein interaction has been shown to be therapeutic against RSV infection in cell cultures and animal models. L encodes the RNA polymerase. The phosphoprotein P is a cofactor for the L protein.

[0067] SH, G and F form the viral coat. The G protein is a surface protein that is heavily glycosylated and functions as the attachment protein. Unlike the RSV G protein, the F protein is conserved among RSV strains, making it an attractive vaccine candidate capable of inducing broadly neutralizing antibodies. The F protein is a transmembrane protein that is incorporated into the virion membrane from the cell membrane during viral budding. The RSV F protein facilitates infection by fusing the viral and host cell membranes. During the fusion process, the F protein irreversibly refolds from an unstable pre-fusion conformation to a stable post-fusion conformation. The protein precursor F0 needs to be cleaved by furin-like proteases during intracellular maturation. There are two Furin sites, cleavage of which removes the p27 peptide, forming two domains, the N-terminal F2 domain and the C-terminal F1 domain. The F2 and F1 domains are joined by two cystine bridges. Antibodies directed against the fusion protein can prevent viral uptake into cells and thus have a neutralizing effect. In addition to being a target for neutralizing antibodies, RSV F contains cytotoxic T cell epitopes (Pemberton etal, 1987, J. Gen. Virol. 68:2177-2182).

[0068] In particular embodiments, RSV fusion protein includes the sequence: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRA RRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAWSLSNGVSVL TFKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYWQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFP QAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTF SNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKS (SEQ ID NO: 32).

[0069] In particular embodiments, RSV neutralizing antibodies are derived from an antibody including a VH3-21 variable heavy (VH) and a VL1-40 variable light (VL) chain. These VH3-21 / VL1-40 mAbs map to an epitope on antigenic site III present on prefusion F proteins (preF).

[0070] In particular embodiments, the VH3-21 variable heavy chain includes the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARLGYCSGGSCHFDYWGQGTLVTVSS (SEQ ID NO: 33).

[0071] In particular embodiments, the VL1-40 variable light chain includes the sequence: QPVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGL QAEDEADYYCQSYDSSLSGFYVFGTGTKLTV (SEQ ID NO: 34).

[0072] In particular embodiments, RSV F site Ill-directed mAbs derived from unmutated VH3-21 and VL1-40 genes include ADI-19425, ADI-25532, and ADI-14337.

[0073] In particular embodiments, the ADI-19425 antibody includes a variable heavy chain including the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARLGYCSGGSCHFDYWGQGTLVTVSS (GenBank Accession MG524063.1 , SEQ ID NO: 33) and a variable light chain including the sequence: QPVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGL QAEDEADYYCQSYDSSLSGFYVFGTGTKLTV (GenBank Accession MG524528.1, SEQ ID NO: 34).

[0074] In particular embodiments, the ADI-19425 antibody includes a heavy chain variable region including a complementarity determining region (CDR) heavy (H)1 including the sequence: GFTFSSY (SEQ ID NO: 35), the CDRH2 including the sequence: SSSSSY (SEQ ID NO: 36), and the CDRH3 including the sequence: LGYCSGGSCHFDY (SEQ ID NO: 37) and a light chain variable region including a CDR light (L)1 including the sequence: TGSSSNIGAGYDVH (SEQ ID NO: 38), the CDRL2 including the sequence: GNSNRPS (SEQ ID NO: 39), and the CDRL3 including the sequence: QSYDSSLSGFYV (SEQ ID NO: 40).

[0075] In particular embodiments, the binding domain that binds VH3-21 includes the binding domain of 1 D3. In particular embodiments, the binding domain that binds VL1-40 includes the binding domain of 2C1, 2C2, or 2F1. In particular embodiments, the binding domain that binds VL1-40 includes the binding domain of 2C1.

[0076] In particular embodiments, the binding domain of 1 D3 includes a variable heavy chain including the sequence: QVQIQQSGAELMRPGASVKLSCKATGYTFTGYWIEWVKQRPGHGLEWIGEILSGRGTTNYNEKFKGKATFTADTYSNTAYMQLSSLTTGDSAIYYCARGGFLYGDGPFDYWGQGTTLTVSS (SEQ ID NO: 1) and a variable light chain including the sequence:DILLTQSPAILSVSPGERVSFSCRASQNIDTHIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDI ADYYCQQSNSWPFTFGSGTKLEIK (SEQ ID NO: 2).

[0077] In particular embodiments, the binding domain of 2C1 includes a variable heavy chain including the sequence: QVQLQQSGAELVRPGASVTLSCKASGYIFTDYEIHWLKQTPVHGLEWIGTSDPETGGFAYTQKFKGKAILTADKSSSTAY MELRSLTSEDSAVYYCTRRPYYGSGAWFAYWGQGTLVTVSA (SEQ ID NO: 3) and a variable light chain including the sequence:DIVMTQSHKFMSTSVGDRVSITCKANHDVNIAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQA EDLAVYYCQQHYDTPRTFGGGTKLEIK (SEQ ID NO: 4).

[0078] In particular embodiments, CDRs for the 1D3 binding domain are in Table 1. In particular embodiments, CDRs for the 2C1 binding domain are in Table 1.

[0079] Table 1. CDR sequences.

[0080] The carboxy-terminal portion of each chain of a naturally occurring antibody defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation. A portion of an Fc region is a fragment of an Fc region. The fragment can include 10% of an Fc region, 20% of an Fc region, 30% of an Fc region, 40% of an Fc region, 50% of an Fc region, 60% of an Fc region, 70% of an Fc region, 80% of an Fc region, 90% of an Fc region, or 95% of an Fc region. A portion of an Fc region can also include a characterized segment of an Fc region, such as a CH2 region or a CH3 region.

[0081] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains. In particular embodiments, a human IgK Fc region includes the sequence:RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25). In particular embodiments, a human IgA Fc region includes the sequence:SVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVIYEDNRRPSGIPERFSGSSSRTLATLTISGAQVEDEADYYCY STDSSGNHWFGGGTKLTVLRQPKAAPSVTSVPT (SEQ ID NO: 26).

[0082] Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, lgG1, lgG2, lgG3, and lgG4. IgM has subclasses including lgM1 and lgM2. IgA is similarly subdivided into subclasses including lgA1 and lgA2. IgG causes opsonization and cellular cytotoxicity and crosses the placenta, IgA functions on the mucosal surface, IgM is most effective in complement fixation, and IgE mediates degranulation of mast cells and basophils. The function of IgD is still not well understood. Resting B cells, which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies these B cells can express any of the five isotypes. The heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 6, and £ chains.

[0083] The constant region of the antibody with multiple binding domains may be of any suitable immunoglobulin subtype. In particular embodiments the subtype of the antibody may be of the class IgG, IgD, IgE, IgA, or IgM. Such an antibody may further belong to any subclass, e.g., lgG1, lgG2a, lgG2b, lgG3 and lgG4. In particular embodiments, a constant region includes a light chain constant region and a heavy chain constant region. A "functional constant heavy chain” or "functional CH” activates an aspect of the immune response.

[0084] In particular embodiments, a human lgG1 Fc region includes the sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 27).

[0085] In particular embodiments, a human lgG1 Fc region includes the sequence: THTCPPCPAPEFFGGPSVFFFPPKPKDTFMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVETVFHQDWENGKEYKCKVSNKAFPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[0086] In particular embodiments, a human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTW HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29)

[0087] In particular embodiments, a human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPE NNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 30).

[0088] In particular embodiments, a human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 31).

[0089] The human IgD constant region typically includes the amino acid sequence: APTKAPDVFPIISGCRHPKDNSPWLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQ GEYKCWQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKT PECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLP RSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVN TSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVSYVTDHGPMK (SEQ ID NO: 101).

[0090] The human IgE constant region typically includes the amino acid sequence: ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSGHYATISLLTVSGAWAK QMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDL STASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCL WDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSG PRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWE QKDEFICRAVHEAASPSQTVQRAVSVNPGK (SEQ ID NO: 102).

[0091] Within full-length light and heavy chains, the variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g. , Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0092] Antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody when in the non-blocked presence of the antibody. In particular embodiments, an antigen includes an antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.

[0093] Unless otherwise indicated, the term "antibody” includes (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies can include monoclonal antibodies (mAb), human or humanized antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi-specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, single chain variable fragments (scFvs), ai-Ab, polyclonal antibodies, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of antibodies.

[0094] A mAb refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a mAb preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each mAb of a mAb preparation is directed against a single epitope on an antigen. Thus, themodifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, mAb can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0095] An "anti-idiotypic antibody” or "anti-idiotype antibody” refers to an antibody, e.g., an antibody molecule that binds to an antigen binding site or variable region of an antibody. In certain embodiments, the anti-idiotype antibody molecule binds to an epitope of an antibody that comes into contact with an antigen, e.g., an antigen described herein (e.g., RSV or HMPV). Vaccination with an anti-idiotype antibody actively induces a polyclonal antibody response. As a result, such antiidiotype antibody-based vaccines have several advantages over passive immunization with standard mAb. There is no antibody response to ai-mAb that limits its effectiveness. In addition, due to the fact that anti-idiotypic treatment is active immunization, the drug need only be injected from time to time to promote the antibody response that the patient produces and maintain a sustained titer of specific antibodies.

[0096] A “human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences.

[0097] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In particular embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al. (supra). In particular embodiments, for the VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0098] (II) Multi-Domain Binding Molecules. The ai-mAb disclosed herein includes multiple binding domains and is a multidomain binding molecule. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes an anti-VH3-21 binding domain and at least one binding domain includes an anti VL1-40 binding domain. In particular embodiments, a multi-domain binding molecule includes at least one, at least two, at least, three, at least four binding domains that bind an epitope on VH3-21; and at least one, at least two, at least, three, at least four binding domains that bind an epitope on VL1-40. In particular embodiments, multi-domain binding molecules include bispecific antibodies, trispecific antibodies, and so on.

[0099] In particular embodiments, a multi-domain binding molecule disclosed herein binds VH3-21 / L1 -40. In particular embodiments, the multi-domain binding molecule disclosed herein includes an ai-mAb that binds VH3-21 / L1-40. In particular embodiments, the ai-mAb is a multi-specific ai-mAb. In particular embodiments, the ai-mAb is a bispecific ai- mAb. In particular embodiments, the ai-mAb has cross reactivity with RSV and / or HMPV reactive B cell receptors.

[0100] In particular embodiments, the ai-mAb includes a binding domain that binds the VH3-21 heavy chain and a binding domain that binds the VL1-40 light chain. In particular embodiments, the binding domain that binds VH3-21 includes the binding domain of 1D3. In particular embodiments, the binding domain that binds VL1-40 includes the binding domain of 2C1, 2C2, or 2F1. In particular embodiments, the binding domain that binds VL1-40 includes the binding domain of 2C1.In particular embodiments, the ai-mAb includes a first binding domain including the binding domain of 1 D3 and a second binding domain including the binding domain of 2C1.

[0101] The ai-mAb can be in any form known in the art such that it binds both VH3-21 and VL1-40. In particular embodiments, the ai-mAb includes 2, 3, 4, 5, 6, 7, 8, 9, or more binding domains. In particular embodiments, the ai-mAb is an IgG, IgA, or IgM antibody. In particular embodiments, the ai-mAb is any antibody or series of antibodies with at least two binding domains.

[0102] In particular embodiments, the ai-mAb includes a first arm and a second arm. An "arm” as it refers to an antibody describes a half of an antibody that includes a heavy chain and a light chain. An example of a 1D3 arm and 2C1 arm are shown in FIG. 9A.

[0103] In particular embodiments, a first binding domain is on the first arm and a second binding domain is on the second arm. In particular embodiments, the first arm includes a variable light chain of a first binding domain, a constant light chain, a variable heavy chain of the first binding domain, and a constant heavy chain; and wherein the second arm includes an scFv of the second binding domain fused to a portion of an Fc region. In particular embodiments, the variable light chain and variable heavy chain of the first arm include the binding domain that binds VH3-21; and the scFv of the second arm includes the binding domain that binds VL1 -40. In particular embodiments, the variable light chain and variable heavy chain of the first arm include the binding domain that binds VL1-40; and the scFv of the second arm includes the binding domain that binds VH3-21.

[0104] The constant heavy chain of the first arm can include any antibody heavy chain including an IgG heavy chain. In particular embodiments, the IgG heavy chain includes an lgG1 antibody, lgG2 antibody, lgG3 antibody, or lgG4 antibody. In particular embodiments, the IgG heavy chain includes an lgG1 antibody.

[0105] The constant light chain of the first arm can include a kappa light chain or lambda light chain. In particular embodiments, the constant light chain of the first arm includes a kappa light chain.

[0106] In particular embodiments, a constant heavy chain is derived from an lgG1 constant heavy domain. In particular embodiments, the constant heavy chain includes the sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSDTKVDKRVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVKHEDPEVKFNWY VDGVEVHNAKTKPREEEYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCDVSGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWDQGDVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 42).

[0107] In particular embodiments, a constant heavy chain is derived from an lgG1 constant heavy domain. In particular embodiments, the constant heavy chain includes the sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSDTKVDKRVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVKHEDPEVKFNWY VDGVEVHNAKTKPREEEYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCDVSGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWEQGDVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 142).

[0108] In particular embodiments, a constant light chain is derived from a kappa light chain. In particular embodiments, the constant light chain includes the sequence:RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41).

[0109] The portion of the Fc region of the second arm can include a fragment of the constant heavy chain of any type of antibody including a fragment of an lgG1 antibody, fragment of an lgG2 antibody, fragment of an lgG3 antibody, or fragment of an lgG4 antibody. In particular embodiments, the portion of the Fc region of the second arm includes a fragment of an lgG1 antibody. In particular embodiments, the portion of the Fc region can include the entire constant heavy chain, 99% of the constant heavy chain, at least 95% of the constant heavy chain, at least 90% of the constant heavy chain, at least 85% of the constant heavy chain, at least 80% of the constant heavy chain, at least 70% of the constant heavy chain, at least 60% of the constant heavy chain, at least 50% of the constant heavy chain, at least 40% of the constant heavy chain, at least 30% of the constant heavy chain, at least 20% of the constant heavy chain, at least 10% of the constant heavy chain, or at least 1 % of the constant heavy chain. In particular embodiments, the portion of the Fc region can include at least 50 residues, at least 100 residues, at least 200 residues, at least 231 residues, at least 232 residues, at least 250 residues, at least 300 residues, at least 400 residues, or at least 500 residues of a constant heavy chain sequence.

[0110] In particular embodiments, the fragment of the lgG1 antibody includes variations of the native lgG1 Fc sequence. In particular embodiments, the fragment of the native lgG1 antibody with variations includes the sequence: EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVKHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDQLTKNQVKLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 43).

[0111] In particular embodiments, the fragment of the lgG1 antibody includes residues 216 through 447 the native lgG1 Fc sequence.

[0112] In particular embodiments, residues 216 through 447 of the native lgG1 Fc sequence include:EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103).

[0113] In particular embodiments, the first arm includes mutations that lower the isoelectric point. In particular embodiments, the second arm includes mutations that lower the isoelectric point. In particular embodiments, the first and / or second arm include Fc silencing mutations (mutations that reduce or eliminate Fc interactions). In particular embodiments, the first arm includes mutations that lower the isoelectric point, heterodimer mutations, and Fc silencing mutations. In particular embodiments, the second arm includes mutations that lower the isoelectric point, heterodimer mutations, and Fc silencing mutations. In particular embodiments, the mutations that lower the isoelectric point include N208D, Q295E, N384D, Q418E, and / or N421D (These mutation positions are in reference to a longer sequence of the lgG1 Fc region. These mutations reference SEQ ID NO: 27 if the position numbering starts at 118 in SEQ ID NO: 27). In particularembodiments, the heterodimer mutations include L368D and / or K370S (These mutation positions are in reference to a longer sequence of the lgG1 Fc region. These mutations reference SEQ ID NO: 27 if the position numbering starts at 118 in SEQ ID NO: 27). In particular embodiments, the Fc silencing mutations include E233P, L234V, L235A, G236del, and S267K (These mutation positions are in reference to a longer sequence of the lgG1 Fc. The mutations are made to reference sequence SEQ ID NO: 103 if the position numbering starts at 216 in SEQ ID NO: 103. The mutations result in SEQ ID NO: 43.). Fc silencing mutations can reduce or eliminate interactions with the Fc receptors. In particular embodiments, the second arm includes mutations, wherein these mutations include C220S, E357Q, and / or S364K (These mutation positions are in reference to a longer sequence of the lgG1 Fc. The mutations are made to reference sequence SEQ ID NO: 103 if the position numbering starts at 216 in SEQ ID NO: 103. The mutations result in SEQ ID NO: 43.). In particular embodiments, the first arm includes mutations, wherein these mutations include C220S, E357Q, and / or S364K (These mutation positions are in reference to a longer sequence of the lgG1 Fc. The mutations are made to reference sequence SEQ ID NO: 103 if the position numbering starts at 216 in SEQ ID NO: 103. The mutations result in SEQ ID NO: 43.). In particular embodiments, a C220S mutation makes the chain more adaptable to the heterodimer / scFv-Fc format. In particular embodiments, an E357Q mutation and S364K mutation increase the isoelectric point (pl). In particular embodiments, increasing the isoelectric point allows for more efficient heterodimer isolation.

[0114] In particular embodiments, to generate a bispecific, the binding domain that binds VL1-40 is produced as an scFv and fused to an Fc region or fragment thereof, while the variable light (VL) chain of the binding domain that binds VH3-21 is cloned in frame with a kappa light chain constant region and the variable heavy (VH) of the binding domain that binds VH3-21 is cloned into an IgG heavy chain expression vector. In particular embodiments, the presence of the Fc region or fragment thereof allows for an extended half-life and recirculation through the FcRn. In particular embodiments, Fey interactions are silenced. In alternative embodiments, the ai-mAb is produced by producing the binding domain that binds VH3-21 as an scFv and fusing it to an Fc region or fragment thereof, while VL of the binding domain that binds VL1-40 is cloned in frame with a kappa light chain constant region and the VH or the binding domain that binds VL1-40 is cloned into an IgG heavy chain expression vector.

[0115] In particular embodiments, a multi-domain binding molecule includes a first arm and a second arm, wherein the first arm includes a first binding domain and the second arm includes a second binding domain. In particular embodiments, the first arm includes a variable heavy chain of the first binding domain, a constant heavy chain, a variable light chain of the first binding domain, and a constant light chain. In particular embodiments, the second arm includes the second binding domain linked to a fragment of an Fc region. In particular embodiments, the second binding domain includes an scFv.

[0116] In particular embodiments, the first binding domain includes a 1 D3 binding domain and the second binding domain includes a 2C1 binding domain. In particular embodiments, the variable heavy chain of the first binding domain includes the sequence of SEQ ID NO: 1, the constant heavy chain includes the sequence of SEQ ID NO: 42, the variable light chain of the first binding domain includes the sequence of SEQ ID NO: 2, and the constant light chain includes the sequence of SEQ ID NO: 41. In particular embodiments, the second binding domain includes an scFv including the sequence of SEQ ID NO: 44 or SEQ ID NO: 45. In particular embodiments, the fragment of the Fc region of the second arm includes the sequence of SEQ ID NO: 43.

[0117] In particular embodiments, the first binding domain includes a 1 D3 binding domain and the second binding domain includes a 201 binding domain. In particular embodiments, the first arm includes a heavy chain and a light chain, wherein the heavy chain includes the sequence of SEQ ID NO: 6 and the light chain includes the sequence of SEQ ID NO: 7. In particular embodiments, the second arm includes an scFv and fragment of an Fc region including the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.

[0118] In particular embodiments, the first binding domain includes a 2C1 binding domain and the second binding domain includes a 1D3 binding domain. In particular embodiments, the variable heavy chain of the first binding domain includes the sequence of SEQ ID NO: 3, the constant heavy chain includes the sequence of SEQ ID NO: 42, the variable light chain of the first binding domain includes the sequence of SEQ ID NO: 4, and the constant light chain includes the sequence of SEQ ID NO: 41. In particular embodiments, the second binding domain includes an scFv having the sequence of SEQ ID NO: 46 or SEQ ID NO: 47. In particular embodiments, the fragment of the Fc region of the second arm includes the sequence of SEQ ID NO: 43.

[0119] In particular embodiments, the first binding domain includes a 201 binding domain and the second binding domain includes a 1 D3 binding domain. In particular embodiments, the first arm includes a heavy chain and a light chain, wherein the heavy chain includes the sequence of SEQ ID NO: 11 and the light chain includes the sequence of SEQ ID NO: 12. In particular embodiments, the second arm includes an scFv and fragment of an Fc region including the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0120] In particular embodiments, to generate the multi-domain binding molecule, 2C1 is produced as an scFv and fused to an Fc region or fragment thereof, while the 1D3 VL is cloned in frame with a kappa light chain constant region and the 1D3 VH is cloned into an IgG heavy chain expression vector. In particular embodiments, the presence of the Fc allows for an extended half-life and recirculation through the FcRn. In particular embodiments, Fey interactions are silenced. In alternative embodiments, the ai-mAb is produced by producing 1D3 as an scFv and fusing it to an Fc region or fragment thereof, while 2C1 VL is cloned in frame with a kappa light chain constant region and the 2C1 VH is cloned into an IgG heavy chain expression vector.

[0121] In particular embodiments, linkers are used to connect portions of the multi-domain binding molecule. Linkers are described elsewhere herein. In particular embodiments, a linker includes a GlySer linker. In particular embodiments, a GlySer linker includes (Gly4Ser)2 (SEQ ID NO: 111). In particular embodiments, a linker includes a Whitlow linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 104).

[0122] In particular embodiments, other methods of generating multi-domain binding molecules can be used.

[0123] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, F(ab')2 bispecific antibodies). For example, WO 1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma Rill antibody; US Pat. No. 5,837,234 describes a bispecific anti-ErbB2 / anti-Fc gamma Rl antibody; WO 1998 / 002463 describes a bispecific anti- ErbB2 / Fc alpha antibody; and US 5,821,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody. In particular embodiments, a bispecific antibody can be in the form of a Bispecific T-cell Engaging (BiTE®) antibody.

[0124] Some additional exemplary bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) aCH1 domain, a hinge, aCH2 domain, and a CH3 domain), and two immunoglobulinlight chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including bi-specific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.

[0125] scFv dimers or diabodies may be used, rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains (usually including the variable domain components from both light and heavy chains of the source antibody), potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins” described in Traunecker ef al. (Embo Journal, 10, 3655-3659, 1991).

[0126] Bispecific antibodies with extended half-lives are described in, for example, US Patent No. 8,921,528 and US Patent Publication No. 2014 / 0308285.

[0127] Methods for making antibodies with two binding domains are known in the art. For example, traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al. Nature 305:37-39, 1983). Similar procedures are disclosed in, for example, WO 1993 / 008829, Traunecker ef a / ., EMBO J. 10:3655-3659, 1991 and Holliger & Winter, Current Opinion Biotechnol. 4, 446-449 (1993).

[0128] In particular embodiments, antibodies with two binding domains can be prepared using chemical linkage. For example, Brennan ef al. (Science 229: 81, 1985) describes a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated then are converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives then is reconverted to the Fab'- thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the antibody having two binding domains.

[0129] In particular embodiments, bispecific antibodies (or antibodies with two binding domains) can be prepared using knobs-into holes techniques. Knobs-into-holes refers to forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids are replaced by amino acids with short side chains to create a 'hole'. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a 'knob'. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (‘knob-hole’) versus homodimer formation ('hole-hole' or 'knob-knob') is observed (Ridgway, J. B., Protein Eng. 9 (1996) 617-621; and WO 96 / 027011).

[0130] In particular embodiments, the 'knob' and / or the 'hole' may exist in the original polypeptide or may be introduced synthetically (e.g., by altering nucleic acid encoding the polypeptide). To synthetically introduce a knob and / or hole, the nucleic acid encoding the original amino acid residue (or other non-amino acid groups such as, for example carbohydrate groups) in the interface of the polypeptide is replaced with DNA encoding at least one import amino acid residue, wherein the interface refers to amino acid residues in contact between a first heavy chain constant region and one or more amino acid residues (or other non-amino acid groups) in a second heavy chain constant region.. The preferred import residuesfor the formation of a hole are amino acids with smaller side chain volumes than the original amino acid residue such as alanine (A), serine (S), threonine (T), valine (V), or glycine (G). The preferred import residues for the formation of a knob are amino acids with larger side chain volumes than the original amino acid residue such as tyrosine (Y), arginine (R), phenylalanine (F), or tryptophan (W). The percentage of heterodimer can be increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A. M, eta / ., Nature Biotech 16 (1998) 677-681; Atwell, S.,J. Mol. Biol. 270 (1997) 26-35).

[0131] Two or more binding domains can be linked through a linker to form a multi-domain binding molecule. Examples of linkers can be found in Chen eta / ., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0132] Commonly used flexible linkers include a linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 105), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 106), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 107), and (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 108). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 109), (Gly4Ser)3(SEQ ID NO: 110), (Gly4Ser)2(SEQ ID NO: 111), (Gly4Ser)i (SEQ ID NO: 112), (Gly3Ser)2(SEQ ID NO: 113), (Gly3Ser)i (SEQ ID NO: 114), (Gly2Ser)2(SEQ ID NO: 115) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 116), GGSGGGSGSG (SEQ ID NO: 117), or GGSGGGSG (SEQ ID NO: 118).

[0133] Linkers that include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used. Additional examples of linkers can be found in Chen eta / ., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0134] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having 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% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0135] T-cell activation can be mediated by two distinct signals: those that initiate antigen-dependent primary activation and provide a T-cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Immune cell activating multispecific (l-AMS) disclosed herein can target any T-cell activating epitope that upon binding induces T-cell activation. Examples of such T-cell activating epitopes are on T-cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD 137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3. Binding domains that bind T-cell markers are known in the art. B cell activation can be initiated by binding of an antigen to the B cell receptor (BCR) (e.g., IgM or IgD). Exemplary immune cell activating factors for NK cells include IL-15 and CD137.

[0136] In particular embodiments macrophages are targeted for localized activation by l-AMS. Macrophages are a type of leukocyte (or white blood cell) that can engulf and digest cells, cellular debris, and / or foreign substances in a process known as phagocytosis.

[0137] The l-AMS can be designed to bind to a protein expressed on the surface of macrophages. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, and MARCO. Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70, which binds and activates CD11b (available from BioLegend®); KP1, which binds and activates CD68 (available from ABCAM®, Cambridge, United Kingdom); and ab87099, which binds and activates CD163 (available from ABCAM®).

[0138] In particular embodiments, l-AMS can target a pathogen recognition receptor (PRR). PRRs are proteins or protein complexes that recognize a danger signal and activate and / or enhance the innate immune response. Examples of PRRs include the TLR4 / MD-2 complex, which recognizes gram negative bacteria; Dectin-1 and Dectin-2, which recognize mannose moieties on fungus 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 particular embodiments, l-AMS can bind and activate TLR4 / MD-2, Dectin-1, Dectin-2, TRL2 / TLR6, TLR2 / TLR1, TLR5, and / or TLR9.

[0139] In particular embodiments, l-AMS can target the complement system. The complement system refers to an immune pathway that is induced by antigen-bound antibodies and involves signaling of complement proteins, resulting in immune recognition and clearance of the antibody-coated antigens.

[0140] Binding domains of l-AMS and other engineered formats described herein may be joined through a linker. A linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of a l-AM. Any appropriate linker may be used.

[0141] Examples of linkers can be found in Chen et al. (Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369) and described elsewhere herein. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0142] Cytolytic properties of l-AMS molecules can be confirmed in comparative in vitro assays. Briefly, for cell line experiments, target cells can be incubated in 96-well round bottom plates at 5-10,000 cells / well containing increasing concentrations of the various l-AMS antibodies with / without healthy donor T-cells (used at an E:T cell ratio of 1:1 and 3:1). After 48 hours, cell numbers and drug-induced cytotoxicity, using 4', 6-diamidino-2-phenyli ndole (DAPI) to detect non-viable cells, can be determined by flow cytometry. In experiments where healthy donor T-cells are added, cells can be identified by forward / side scatter properties and negativity for CellVue Burgundy dye. Experiments can include technical duplicates.

[0143] In particular embodiments, T-cell activating epitope binding domains including l-AMS constructs include 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 amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, Vp, Ca, or Cp of a known TOR. An insertion, deletion or substitution may be anywhere in a Va, Vp, Ca, or Cp region, including at the amino- or carboxyterminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or threechanges and provided a binding domain including a modified Va, Vp, Ca, or Cp region can still specifically bind its target with an affinity similar to wild type.

[0144] Tri-specific antibodies are artificial proteins that simultaneously bind to three different types of antigens. Tri-specific antibodies are described in, for example, WO2016 / 105450, WO 2010 / 028796; WO 2009 / 007124; WO 2002 / 083738; US 2002 / 0051780; and WO 2000 / 018806.

[0145] In some embodiments, a multi-domain binding molecule includes a basic immunoglobulin structure such as an IgA domain or an IgM domain. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow eta / ., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0146] In particular embodiments, multi-domain binding molecules are multimers of an antibody disclosed herein. Multimerization strategies include formation of a fusion protein using protein linkers or use of IgA or IgM constant regions as a multimerization scaffold. In certain aspects, multimerization is achieved by linking antibodies or binding domains of antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0147] Multimerized antibodies and antibody-like molecules such as IgA and IgM antibodies have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1, US20180118814A1, US20180118816A1 , US20190185570A1, and US20180265596A1, and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, and WO 2019 / 165340.

[0148] Particular embodiments include using IgA and IgM constant region domains to allow the binding portion of molecules provided herein to readily multimerize into dimers, pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0149] Immunoglobulin A (IgA), as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcaR, are key mediators of IgA effector function. Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains. IgA, similarly to IgG, contains three constant domains (CA1-CA3), with a hinge region between the CA1 and CA2 domains. The main difference between lgA1 and lgA2 resides in the hinge region that lies between the two Fab arms and the Fc region. lgA1 has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2. Both forms of IgA have the capacity to form dimers, in which two monomer units, are arranged in an end- to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. J-chains are also part of IgM pentamers and are discussed in more detail below.

[0150] Both IgA and IgM (discussed further below in relation to pentamers and hexamers) possess an 18-amino acid extension in the C terminus called the "tailpiece" (tp). The IgA and IgM tp is highly conserved among various animalspecies. The conserved penultimate cysteine residue in the IgA and IgM tp has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tp contain an N- linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition of the N-linked carbohydrates in the tp differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases. Particularly, the IgA (atp) and IgM (ptp) tp differ at seven amino acid positions.

[0151] The human lgA1 constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNWIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLA GKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVT FTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSE ELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGH EALPLAFTQKTIDRLAGKPTHVNVSWMAEVDGTCY (SEQ ID NO: 119). Referring to this SEQ ID NO: 119, the human CA1 domain extends from amino acid 6 to amino acid 98; the human lgA1 hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tp extends from amino acid 331 to amino acid 352.

[0152] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNVWACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPD GKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAV QGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCL ARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTID RLAGKPTHVNVSWMAEVDGTCY (SEQ ID NO: 120). Referring to this SEQ ID NO: 120, the human CA1 domain extends from amino acid 6 to amino acid 98, the human lgA2 hinge region extends from amino acid 102 to amino acid 111, the human CA2 domain extends from amino acid 113 to amino acid 206, the human CA3 domain extends from amino acid 215 to amino acid 317, and the tp extends from amino acid 318 to amino acid 340.

[0153] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 121, the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule. An exemplary precursor secretory component includes the sequence MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGR ANLTNFPENGTFWNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQK RKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSWINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEP ELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVWNTLGKRAPAFEGRILLNPQDKDGSFSWITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTWKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVD SEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVP CHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAV YVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEEQGGSS RALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGANDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEAQDGPQEA (SEQ ID NO: 122). An exemplary mature secretory component includesKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITUSSEGYVSSKYAGRANLTNFPENGTFWNIA QLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDS SGYVNPNYTGRIRLDIQGTGQLLFSWINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCAL GPEVANVAKFLCRQSSGENCDVWNTLGKRAPAFEGRILLNPQDKDGSFSWITGLRKEDAGRYLCGAHSDGQLQEGSP IQAWQLFVNEESTIPRSPTWKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLE EPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCK WNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVS LAKADAAPDEKVLDSGFREIENKAIQDPR (SEQ ID NO: 123). While not wishing to be bound by theory, and as indicated above, the assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the CA3 and tp domains. See, e.g., Braathen, R., el al., J. Biol. Chem. 277:42755-42762 (2002). Accordingly, a multimerizing dimeric IgA-derived binding molecule provided in this disclosure typically includes IgA constant regions that include at least the CA3 and tp domains.

[0154] An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CA1 domain or a fragment thereof, and / or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region. In certain embodiments, a binding molecule as provided herein can include a complete IgA heavy chain constant region (e.g., SEQ ID NO: 119 or SEQ ID NO: 120), or a variant, derivative, or analog thereof.

[0155] In particular embodiments, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 119 or amino acids 113 to 340 of SEQ ID NO: 120. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA or IgG hinge region situated N-terminal to the IgA CA2 domains. For example, the IgA heavy chain constant regions can include amino acids 102 to 353 of SEQ ID NO: 119 or amino acids 102 to 340 of SEQ ID NO: 120. In particular embodiments, the IgA heavy chain constant regions can each further include an IgACAI domain situated N-terminal to the IgA hinge region.

[0156] Each of the strategies discussed above can be used to create IgA antibody-based dimers.

[0157] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0158] Particular embodiments include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers. Embodiments with an IgM constant region typically include at least the Cpi4-tp domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. In particular embodiments, one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and / or pentamers. Thus, an IgM antibody can be, e.g., a hybrid IgM / IgG antibody or can be a “multimerizing fragment” of an IgM- derived binding molecule.

[0159] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the Cpi4 and tp domains. See, e.g., Braathen, R., eta / ., J Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the Cpi4 and / or tp domains (also referred to herein collectively as Cpi4-tp). A “multi merizi ng fragment” of an IgM heavy chain constant region thus includes at least the Cpi4-tp domains. An IgM heavy chain constant region can additionally include a Cpi3 domain or a fragment thereof, a Cpi2 domain or a fragment thereof, a Cpi1 domain or a fragment thereof, and / or other IgM heavy chain domains.

[0160] Five IgM monomers form a complex with a J-chain to form a native IgM molecule. The J-chain is considered to facilitate polymerization of chains before IgM is secreted from antibody-producing cells. Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487). The J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35): 14960-14965, 2009) published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroom-shaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: Asn-171, Asn-332, Asn-395, Asn-402 and Asn-563. In an IgM antibody where each binding unit is bivalent, the binding molecule itself can have 10 or 12 valencies.

[0161] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. "Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region) or by using the Kabat numbering scheme.

[0162] A "full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cpi 1 ), an antibody heavy chain constant domain 2 (CM2 or Cpi2), an antibody heavy chain constant domain 3 (CM3 or Cpi3), and an antibody heavy chain constant domain 4 (CM4 or Cpi4) that can include a tp, as indicated above.

[0163] In particular embodiments, each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM C 4 domain and an IgM tp domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM Cpi3 domain situated N-terminal to the IgM Cpi4 and IgM tp domains.

[0164] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM Cpi2 domain situated N-terminal to the IgM Cpi3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human Cpi2, Cpi3, and Cpi4-tp domains as follows: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIK ESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEA VKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTE RTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 124).

[0165] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include, instead of, or in addition to an IgM C 2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM C 3 domain. An exemplary variant human IgG 1 hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 125). An exemplary IgM constant region of this type includes the variant human lgG1 hinge region fused to a multimerizing fragment of the human IgM constant region including the Cp3, Cp4, and tp domains, and includes the amino acid sequence:VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNAT FSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYN VSLVMSDTAGTCY (SEQ ID NO: 126).

[0166] Human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif" includes the amino acid sequence N-X1-S / T, wherein N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 127 or SEQ ID NO: 128 starting at positions 46 (“N1”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Each of these sites in the human IgM heavy chain constant region, except for N4, can be mutated to prevent glycosylation at that site, while still allowing IgM expression and assembly into a hexamer or pentamer.

[0167] The human IgM heavy chain constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 127; identical to, e.g., Gen Bank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1). Referring to this SEQ ID NO: 127, the human C 1 region ranges from amino acid 5 to amino acid 102; the human Cpi2 region ranges from amino acid 114 to amino acid 205, the human C 3 region ranges from amino acid 224 to amino acid 319, the C 4 region ranges from amino acid 329 to amino acid 430, and the tp ranges from amino acid 431 to amino acid 453.

[0168] In particular embodiments, an IgM heavy chain constant region includes the sequence:GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 128; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 127 by one amino acid at position 191.

[0169] Other forms of the human IgM constant region with minor sequence variations exist, including GenBank Accession Nos. P01871.4, CAB37838.1, and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 127 described herein can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species, e.g., those shown in FIG. 1 of PCT / US2019 / 020374.

[0170] In certain aspects, a variant human IgM constant region includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311, P313, R344, E345, S401, E402, and / or E403 of SEQ ID NO: 127. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 127 corresponds to S524 of Kabat; E402 of SEQ ID NO: 127 corresponds to E525 of Kabat; E403 of SEQ ID NO: 127 corresponds to E526 of Kabat; R344 of SEQ ID NO: 127 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 127 corresponds to E468 of Kabat.

[0171] In particular embodiments, “corresponds to” means the designated position of SEQ ID NO: 127 and the amino acid in the sequence of the IgM constant region of any species which is homologous to the specified position. See FIG. 1 of PCT / US2019 / 020374.

[0172] In particular embodiments, P311 of SEQ ID NO: 127 can be substituted, e.g., with alanine (P311A), serine (P311S), or glycine (P311G) and / or P313 of SEQ ID NO: 127 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 127 can be substituted with alanine (P311A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 129).

[0173] In certain aspects, S401 of SEQ ID NO: 127 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 127 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVA EEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 130).

[0174] In certain aspects, E402 of SEQ ID NO: 127 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 127 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS AEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 131).

[0175] In certain aspects, E403 of SEQ ID NO: 127 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 127 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EAEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 132).

[0176] In certain aspects, R344 of SEQ ID NO: 127 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 127 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 133).

[0177] In certain aspects, E345 of SEQ ID NO: 127 can be substituted with any amino acid. In certain aspects, E345 of SEQ ID NO: 127 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQ GTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRP KGVALHRPDVYLLPPAREQLNLRASATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 134).

[0178] As indicated, five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody. The precursor form of the human J-chain includes: MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFV YHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQID NO: 135). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 135 and the mature human J- chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 135.

[0179] The mature human J-chain includes the amino acid sequenceQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIV TATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 121).

[0180] The term "J-chain” as used herein refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and / or variant thereof, including a mature human J-chain amino acid sequence provided herein as SEQ ID NO: 121. A functional fragment, derivative, and / or variant of a J-chain has at least 90% sequence identity to the reference J-chain and retains the multimerizing function of the reference J-chain.

[0181] In certain aspects, the J-chain of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid Y102, T103, N49 or S51 of SEQ ID NO: 121.

[0182] By “an amino acid corresponding to" a position of SEQ ID NO: 121 is meant the amino acid in the sequence of the J-chain of any species which is homologous to the referenced residue in the human J-chain. For example, the position corresponding to Y102 in SEQ ID NO: 121 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 121 is conserved in the J-chain amino acid sequences of at least 37 other species. The positions corresponding to N49 and S51 in SEQ ID NO: 121 are conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S. Patent No. 9,951,134 and FIG. 2 of PCT / US2019 / 020374.

[0183] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 121 can be substituted with any amino acid. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 121 can be substituted with alanine (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIV TATQSNICDEDSATETCATYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 136),With serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIV TATQSNICDEDSATETCSTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 137),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIV TATQSNICDEDSATETCRTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 138).

[0184] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 121 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 121 can be substituted with alanine as follows (alanine substitution indicated by bold underline): QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIV TATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 139).

[0185] In certain aspects, the variant J-chain or functional fragment thereof of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO:121, provided that S51 is not substituted with threonine (T), or wherein the J-chain includes amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 121.

[0186] The amino acids corresponding to N49 and S51 of SEQ ID NO: 121 along with the amino acid corresponding to 150 of SEQ ID NO: 121 include an N-linked glycosylation motif in the J-chain. Accordingly, mutations at N49 and / or S51 (with the exception of a single threonine substitution at S51) can prevent glycosylation at this motif. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 121 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 121 can be substituted with alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect the position corresponding to N49 of SEQ ID NO: 121 can be substituted with alanine (A). In a particular aspect the J-chain is a variant human J-chain and includes the amino acid sequence:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVT ATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 140).

[0187] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 121 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 121 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 121 can be substituted with alanine (A). In a particular aspect the variant J-chain or functional fragment thereof is a variant human J-chain and includes the amino acid sequence:EDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTA TQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 141).

[0188] Particular embodiments include a heterologous polypeptide (e.g., a single-domain antibody binding domain) fused to the J-chain or functional fragment thereof via a peptide linker, e.g., a peptide linker including at least 5 amino acids, but no more than 25 amino acids. In certain aspects, the peptide linker includes (GGGGS)n (SEQ ID NO: 105) wherein n is 1-5.

[0189] A single-domain antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, Ig M, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible. In certain aspects, the antigen-binding domain can be introduced into the mature human J-chain of SEQ ID NO: 121 between cysteine residues 92 and 101 of SEQ ID NO: 121. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 121 at or near a glycosylation site. In a further aspect, the antigen-binding domain can be introduced into the human J- chain of SEQ ID NO: 121 within 10 amino acid residues from the C- terminus, or within 10 amino acids from the N-terminus.

[0190] In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 121 by chemical or chemo-enzymatic derivatization. In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 121 by a chemical linker. In some embodiments, the chemical linker is a cleavable or non-cleavable linker. In particular embodiments, the cleavable linker is a chemically labile linker or an enzyme-labile linker. In some embodiments, the linker is selected from the group including N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), afunctional derivatives of imidoesters, active esters, aldehydes, bis-azido compounds, bis-diazonium derivatives, diisocyanates, and bis-active fluorine compounds. In particular embodiments, the modified J-chain is modified by insertion of an enzyme recognition site, and by post- translational ly attaching a binding moiety at the enzyme recognition site through a peptide or non-peptide linker.

[0191] In certain aspects the modified J-chain can include the formula X[Ln] J or J[Ln]X, where J includes a mature native J-chain or functional fragment thereof, X includes a heterologous binding domain, and [Ln] is a linker sequence including n amino acids, where n is a positive integer from 1 to 100, 1 to 50, or 1 to 25. In certain aspects N is 5, 10, 15, or 20.

[0192] J-chains from the following species can also be used in certain embodiments: Pan troglodytes, Pongo abelii, Callithrix jacchus, Macaca mulatta, Papio Anubis, Saimiri boiiviensis, Tupaia chinensis, Tursiops truncates, Orcinus orca, Loxodonta Africana, Leptonychotes weddellii, Ceratotherium simum, Felis catus, Canis familiaris, Ailuropoda melanoleuca, Mustela furo, Equus caballus, Cavia porcellus, Camelus ferus, Capra hircus, Chinchilla lanigera, Mesocricetus auratus, Ovis aries, Myotis lucifugus, Pantholops hodgsonii, Bos taurus, Mus musculus, Rattus norvegicus, Echinops telfairi, Oryctolagus cuniculus, Monodelphis domestica, Alligator mississippiensis, Chrysemys picta, Sarcophilus harrisii, Ornithorhynchus anatinus, Melopsittacus undulatus, Anas platyrhynchos, Gallus gallus, Meleagris gal / opavo, Falco peregrines, Zonotrichia albicollis, and Pteropus alecto.

[0193] In particular embodiments, the antibodies can multimerize by optionally including a multimerization domain. A "multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0194] In particular embodiments, the multimerization domain is a dimerization domain that allows binding of two complementary monomers to form a dimer. In particular embodiments, a dimerization and docking domain (ODD) can be derived from the cAMP-dependent protein kinase (PKA) regulatory subunits and can be paired with an anchoring domain (AD). The AD can be derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. Additional DDDs and ADs include: the 4-helix bundle type DDD (Newlon, et al. EMBO J. 2001; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) domains obtained from p53, DCoH (pterin 4 a carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 a (TCF1)) and HNF-1 (hepatocyte nuclear factor 1) (Rose, etal. Nature Struct Biol. 2000; 7: 744-748). Other AD sequences of potential use may be found in US 2003 / 0232420A1.

[0195] In particular embodiments, complementary binding domains can dimerize. In particular embodiments, the binding domain is a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, an antibody or fragment thereof can include a part of a FCERI O chain and another antibody or fragment thereof can include a part of an FCERI |3 chain such that said FCERI chains spontaneously dimerize together to form a dimeric antibody (e.g., bispecific antibody). In particular embodiments, an antibody or fragment thereof can include a part of a FceRI a chain and another antibody or fragment thereof part of a FCERI y chain such that said FCERI chains spontaneously trimerize together to form a trimeric antibody, and in another embodiment the multi-domain binding molecule can include a part of FCERI a chain, a part ofFCERI p chain and a part of FCERI y chain such that said FCERI chains spontaneously tetramerize together to form a tetrameric multi-domain binding molecule.

[0196] Leucine zippers are described in US 5932448; SH2 and SH3 are described in Vidal eta!., Biochemistry, 43:7336- 44, 2004); PTB is described in Zhou et al., Nature, 378:584- 592, 1995); WW is described in Sudol Prog Biochys MoL Bio, 65:113-132, 1996; PDZ is described in Kim et al., Nature, 378: 85-88, 1995 and Komau et a!., Science, 269:1737-1740, 1995; and WD40 is described in Hu et al., J Biol Chem., 273:33489- 33494, 1998.

[0197] Additional multimerization domains and systems are described in, for example, Hodneland, etal. Proc Natl Acd Sci USA. 2002; 99: 5048-5052; Arakawa eta / ., J Biol. Chem., 269:27833-27839, 1994; Radziejewski etal., Biochem, 32: 1350, 1993; WO2012001647A2; US 5821333; GenBank Accession no. AAF73912.1 (Nishi eta!., Mol Cell Biol, 25: 2607-2621, 2005), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006), the PTB domain of human DOK-7 from GenBank Accession no. NP_005535.1 (Wagner etal., Cold Spring Harb Perspect Biol. 5: a008987, 2013), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande etal., Mol Cell Biol. Aug; 21: 5591-5604, 2001), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079— 4088, 1998) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014). C4b, dextrameric, and ferritin-based multimerization can be used.

[0198] In particular embodiments, complementary binding domains can be induced using a third molecule or chemical inducer. This method of dimerization requires that one antibody or fragment thereof include a chemical inducer of dimerization binding domain 1 (CBD1) and the second antibody or fragment thereof include the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). CBD1 may include a rapamycin binding domain of FK-binding protein 12 (FKBP12) and CBD2 may include a FKBP12-Rapamycin Binding (FRB) domain of mTOR.

[0199] (III) Recombinant Production. In particular embodiments, the ai-mAbs disclosed herein are produced from a gene using a protein expression system. Protein expression systems can utilize DNA constructs (e.g., chimeric genes, expression cassettes, expression vectors, recombination vectors) including a nucleic acid sequence encoding the protein or proteins of interest operatively linked to appropriate regulatory sequences. In particular embodiments, such DNA constructs are not naturally-occurring DNA molecules and are useful for introducing DNA into host-cells to express selected proteins of interest. In particular embodiments, a DNA construct that encodes an antibody disclosed herein can be inserted into cells (e.g., bacterial, mammalian, insect, etc.), which can produce the antibody encoded by the DNA construct.

[0200] Operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded protein is expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1982; and Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989.

[0201] Expression control sequences are DNA sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art. Expressioncontrol sequences generally include a promoter. The promoter may be inducible or constitutive. It may be naturally- occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-2361, 1987. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760-764, 1979.

[0202] The promoter may include, or be modified to include, one or more enhancer elements. In particular embodiments, the promoter will include a plurality of enhancer elements. Promoters including enhancer elements can provide for higher levels of transcription as compared to promoters that do not include them.

[0203] For efficient expression, the coding sequences can be operatively linked to a 3' untranslated sequence. In particular embodiments, the 3' untranslated sequence can include a transcription termination sequence and a polyadenylation sequence. The 3' untranslated region can be obtained, for example, from the flanking regions of genes.

[0204] In particular embodiments, a 5' untranslated leader sequence can also be employed. The 5' untranslated leader sequence is the portion of an mRNA that extends from the 5' CAP site to the translation initiation codon.

[0205] In particular embodiments, a “hisavi” tag can be added to the N-terminus or C-terminus of a gene by the addition of nucleotides coding for the Avitag amino acid sequence, “GLNDIFEAQKIEWHE” (SEQ ID NO: 48), as well as the 6xhistidine tag “HHHHHH” (SEQ ID NO: 49). The Avitag avidity tag can be biotinylated by a biotin ligase to allow for biotinavidin or biotin-streptavidin based interactions for protein purification, as well as for immunobiology (such as immunoblotting or immunofluorescence) using anti-biotin antibodies. The 6xhistidine tag allows for protein purification using Ni-2+affinity chromatography. Other tags include: Flag tag (DYKDDDDK; SEQ ID NO: 50), Xpress tag (DLYDDDDK; SEQ ID NO: 51), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 52), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 53), Myc tag (EQKLISEEDL; SEQ ID NO: 54), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 55), STREP® tag II (WSHPQFEK SEQ ID NO: 56 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 57), Softag 3 (TQDPSRVG; SEQ ID NO: 58), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 59).

[0206] In particular embodiments, ai-mAbs disclosed herein can be produced using, for example, human suspension cells and / or the Daedalus expression system as described in Pechman et al., Am J Physiol 294: R1234-R1239, 2008. The Daedalus system utilizes inclusion of minimized ubiquitous chromatin opening elements in transduction vectors to reduce or prevent genomic silencing and to help maintain the stability of decigram levels of expression. This system can bypass tedious and time-consuming steps of other protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg / l of correctly folded and post-translationally modified, endotoxin-free protein of up to 70 kDa in size, can be achieved in conventional, small-scale (100 ml.) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. Bandaranayake etal., Nucleic Acids Res., 2011 (Nov); 39(21). In some instances, purification by chromatography may not be needed due to the purity of manufacture according the methods described herein.

[0207] In particular embodiments, the DNA constructs can be introduced by transfection, a technique that involves introduction of foreign DNA into the nucleus of eukaryotic cells. In particular embodiments, the proteins can be synthesized by transient transfection (DNA does not integrate with the genome of the eukaryotic cells, but the genes are expressed for 24-96 hours). Various methods can be used to introduce the foreign DNA into the host-cells, and transfection can be achieved by chemical-based means including by the calcium phosphate, by dendrimers, by liposomes, and by the use of cationic polymers. Non-chemical methods of transfection include electroporation, sono-poration , optical transfection, protoplast fusion, impalefection, and hydrodynamic delivery. In particular embodiments, transfection can be achieved by particle-based methods including gene gun where the DNA construct is coupled to a nanoparticle of an inert solid which is then "shot" directly into the target-cell's nucleus. Other particle-based transfection methods include magnet assisted transfection and impalefection.

[0208] Nucleic acid sequences encoding proteins disclosed herein can be derived by those of ordinary skill in the art. Nucleic acid sequences can also include one or more of various sequence polymorphisms, mutations, and / or sequence variants (e.g., splice variants or codon optimized variants). In particular embodiments, the sequence polymorphisms, mutations, and / or sequence variants do not affect the function of the encoded protein.

[0209] Sequence information provided by public databases can be used to identify additional gene and protein sequences that can be used with the systems and methods disclosed.

[0210] (IV) Compositions for Administration. Active ingredients (ai-mAbs disclosed herein) can be formulated alone or in combination into compositions for administration to subjects. In particular embodiments, the active ingredients include immunogenic compositions. An immunogenic composition refers to an agent that stimulates an innate and / or an adaptive immune response in a subject.

[0211] Salts and / or pro-drugs of the active ingredients can also be used.

[0212] A pharmaceutically acceptable salt includes any salt that retains the activity of the RSV therapeutic and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0213] Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0214] Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

[0215] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of an active ingredients or by hydrolysis of a biologically labile group.

[0216] In particular embodiments, compositions disclosed herein include an active ingredients of at least 0.1 % w / v or w / w of the composition; at least 1 % w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / wof composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0217] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0218] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0219] Exemplary buffering agents 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.

[0220] An exemplary chelating agent is EDTA.

[0221] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0222] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0223] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredients or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can 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, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium th ioglycol ate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or 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. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0224] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous injection.

[0225] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the formulation can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0226] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid formulations such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0227] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredients and a suitable powder base such as lactose or starch.

[0228] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.

[0229] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more active ingredients following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0230] Depot formulations can include a variety of bioerodible polymers including poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0231] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0232] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas,Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0233] Excipients that partition into the external phase boundary of microparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0234] Additional processing of the disclosed sustained release depot formulations can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0235] In certain examples, compositions include a vaccine adjuvant, an anti-infective agent, and / or a secondary vaccine or antibody. Examples of vaccine adjuvants, anti-infective agents, and secondary vaccines or antibodies are described elsewhere herein.

[0236] Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0237] (V) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0238] An "effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model or clinical study relevant to the assessment of an infection’s development, progression, and / or resolution, as well as the effects of the infection. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0239] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of an infection or displays only early signs or symptoms of an infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the infection. Thus, a prophylactic treatment functions as a preventative treatment against an infection and / or the potential effects of an infection.

[0240] Particular uses of the compositions include use as a prophylactic vaccine. Vaccines increase the immunity of a subject against a particular infection. Therefore, " vaccine" can refer to a treatment that increases the immunity of a subject against RSV and / or HMPV. In certain examples, a vaccine may be administered prophylactical ly, for example to a subjectthat is immunologically naive (e.g., no prior exposure or experience with RSV or HMPV). In particular embodiments, a vaccine may be administered therapeutically to a subject who has been exposed to RSV and / or HMPV. Thus, a vaccine can be used to ameliorate a symptom and / or complication associated with RSV and / or HMPV, examples of each of which are described elsewhere herein. In particular embodiments, a vaccine includes the ai-mAb disclosed herein and a pharmaceutically acceptable carrier.

[0241] In particular embodiments, a vaccine is a therapeutically effective composition including binding domains derived from ai-mAb that bind VH3-21 / L1 -40 B cell receptors (BCRs) that induce an immune response in a subject against RSV and / or HMPV. The skilled artisan will appreciate that the immune system generally is capable of producing an innate immune response and an adaptive immune response. An innate immune response generally can be characterized as not being substantially antigen specific and / or not generating immune memory. An adaptive immune response can be characterized as being substantially antigen specific, maturing over time (e.g., increasing affinity and / or avidity for antigen), and in general can produce immunologic memory. Even though these and other functional distinctions between innate and adaptive immunity can be discerned, the skilled artisan will appreciate that the innate and adaptive immune systems can be integrated and therefore can act in concert.

[0242] In particular embodiments, administration of a vaccine disclosed herein can further include administration of one or more adjuvants. The term “adjuvant" refers to material that enhances the immune response to a vaccine antigen and is used herein in the customary use of the term. The precise mode of action is not understood for all adjuvants, but such lack of understanding does not prevent their clinical use for a wide variety of vaccines.

[0243] Exemplary vaccine adjuvants, include any kind of Toll-like receptor ligand or combinations thereof (e.g. CpG, Cpg- 28 (a TLR9 agonist), polyriboinosinic polyribocytidylic acid (Poly(l:C)), o-galactoceramide, MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081), TMX-101 (imiquimod), MGN1703 (a TLR9 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), Entolimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and Imiquimod), and / or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0244] In particular embodiments a squalene-based adjuvant can be used. Squalene is part of the group of molecules known as triterpenes, which are all hydrocarbons with 30 carbon molecules. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seeds, and olives, as well as from animal sources, such as shark liver oil. In particular embodiments, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant that is similar to MF59® but is designed for preclinical research use is Addavax™ (I nvivoGen, San Diego, CA). MF59 has been FDA approved for use in an influenza vaccine, and studies indicate that it is safe for use during pregnancy (Tsai etal. Vaccine. 2010. 17:28(7): 1877-80; Heikkinen etal. Am J Obstet Gynecol. 2012. 207(3):177). In particular embodiments, squalene based adjuvants can include 0.1 %-20% (v / v) squalene oil. In particular embodiments, squalene based adjuvants can include 5%(v / v) squalene oil.

[0245] In particular embodiments the adjuvant alum can be used. Alum refers to a family of salts that contain two sulfate groups, a monovalent cation, and a trivalent metal, such as aluminum or chromium. Alum is an FDA approved adjuvant. In particular embodiments, vaccines can include alum in the amounts of 1-1000pig / dose or 0.1mg-10mg / dose.

[0246] In particular embodiments, one or more STING agonists are used as a vaccine adjuvant. "STING" is an abbreviation of "stimulator of interferon genes", which is also known as "endoplasmic reticulum interferon stimulator (ERIS)", "mediator of IRF3 activation (MITA)", "MPYS" or "transmembrane protein 173 (TM173)".

[0247] In particular embodiments, STING agonists include cyclic molecules with one or two phosphodiester linkages, and / or one or two phosphorothioate diester linkages, between two nucleotides. This includes (3',5')-(3',5') nucleotide linkages (abbreviated as (3', 3')); (3',5')-(2',5') nucleotide linkages (abbreviated as (3', 2')); (2',5')-(3',5') nucleotide linkages (abbreviated as (2', 3')); and (2',5')-(2',5') nucleotide linkages (abbreviated as (2', 2')). "Nucleotide" refers to any nucleoside linked to a phosphate group at the 5', 3' or 2' position of the sugar moiety.

[0248] In particular embodiments, STING agonists include c-AIMP; (3’,2’)c-AIMP; (2’,2’)c-AIMP; (2',3’)c-AIMP; c-AIMP(S); c-(dAMP-dlMP); c-(dAMP-2’FdlMP); c-(2’FdAMP-2’FdlMP); (2’,3’)c-(AMP-2’FdlMP); c-[2’FdAMP(S)-2’FdlMP(S)]; c- [2’FdAMP(S)-2’Fdl MP(S)](POM)2; and DMXAA. Additional examples of STING agonists are described in WO2016 / 145102.

[0249] Other immune stimulants can also be used as vaccine adjuvants. Additional exemplary small molecule immune stimulants include TGF-p inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNA capable of down-regulating immune-suppressive signals or oncogenic pathways (such as kras) can be used whereas any plasmid DNA (such as minicircle DNA) encoding immune-stimulatory proteins can also be used.

[0250] In particular embodiments, the immune stimulant may be a cytokine and or a combination of cytokines, such as IL- 2, IL-12 or IL-15 in combination with IFN-a, IFN-p or IFN-y, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The above-identified cytokines stimulate TH1 responses, but cytokines that stimulate TH2 responses may also be used, such as IL-4, IL-10, IL-11, or any effective combination thereof. Also, combinations of cytokines that stimulate TH1 responses along with cytokines that stimulate TH2 responses may be used.

[0251] "Immune response" refers to a response of the immune system to produce RSV neutralizing antibodies and / or HMPV neutralizing antibodies in response to a ai-mAb as described herein binding a VH3-21 / VL 1-40 B cell receptor (BCR). In particular embodiments, the immune response causes production of VH3-21 / VL1 -40 to neutralize RSV and / or HMPV. In particular embodiments, an immune response to an ai-mAb can be an innate and / or adaptive response. In particular embodiments, an adaptive immune response can be a "primary immune response" which refers to an immune response occurring on the first exposure of a "naive" subject to RSV and / or HMPV or vaccine. For example, in the case of a primary antibody response, after a lag or latent period of from 3 to 14 days depending on, for example, the composition, dose, and subject, antibodies to RSV and / or HMPV can be produced. Generally, IgM production lasts for several days followed by IgG production and the IgM response can decrease. Antibody production can terminate after several weeks but memory cells can be produced. In particular embodiments, an adaptive immune response can be a "secondary immune response", "anamnestic response," or "booster response" which refer to the immune response occurring on a second and subsequent exposure of a subject to a virus (e.g., RSV and / or HMPV) or vaccine. Generally, in a secondary immune response, memory cells respond to the virus or vaccine and therefore the secondary immune response can differ from a primary immune response qualitatively and / or quantitatively. For example, in comparison to a primary antibody response, the lag period of a secondary antibody response can be shorter, the peak antibody titer can be higher, higher affinity antibody can be produced, and / or antibody can persist for a greater period of time.

[0252] In particular embodiments, an immune response against RSV and / or HMPV will include antibody production against: the F protein. In particular embodiments, an immune response will include antibody production of VH3-21 / VL1-40 to neutralize RSV.

[0253] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of an infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the infection or effects of the infection. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the infection and / or reduce, control or eliminate side effects of the infection.

[0254] In particular embodiments a therapeutic treatment can reduce, control, or eliminate a primary infection with RSV. In particular embodiments a therapeutic treatment can reduce or eliminate the symptoms of RSV. In particular embodiments, a therapeutically effective amount reduces or prevents transmission of RSV.

[0255] In particular embodiments a therapeutic treatment can reduce, control, or eliminate a primary infection with HMPV. In particular embodiments a therapeutic treatment can reduce or eliminate the symptoms of HMPV. In particular embodiments, a therapeutically effective amount reduces or prevents transmission of HMPV.

[0256] In particular embodiments a therapeutic treatment can reduce, control, or eliminate a primary infection with RSV and HMPV. In particular embodiments a therapeutic treatment can reduce or eliminate the symptoms of RSV and HMPV. In particular embodiments, a therapeutically effective amount reduces or prevents transmission of RSV and HMPV.

[0257] In particular embodiments, a therapeutically effective amount alleviates or reduces the severity or occurrence of symptoms and / or complications associated with RSV and / or HMPV infection. Exemplary symptoms include fever, bluish tint to skin, wheezing, cough, runny nose, congestion, sore throat, apnea, decreased appetite, and difficulty breathing. Exemplary complications include bronchiolitis, pneumonia, acute bronchitis, worsening congestive heart failure or chronic obstructive pulmonary disease (COPD), worsening asthma, respiratory failure, hypoxia, dehydration, and ear infection.

[0258] In particular embodiments, a therapeutically effective amount reduces the duration of hospitalization for a subject infected with RSV and / or HMPV as compared to a subject that has not received a vaccine disclosed herein.

[0259] In particular embodiments, a prophylactic and / or therapeutic treatment is administered to a pediatric patient. A pediatric patient can refer to patient who is 18 years of age or younger. In particular embodiments, a pediatric patient includes a subject under 5 years of age. In particular embodiments, a pediatric patient includes a subject under 1 year of age.

[0260] In particular embodiments, a therapeutically effective amount reduces the time to sustained non-detectable RSV and / or HMPV in the nose and / or throat in a patient infected with the virus as compared to a subject that has not received a vaccine disclosed herein.

[0261] In particular embodiments, a therapeutically effective amount reduces respiratory failure or death as compared to a subject that has not received a vaccine disclosed herein.

[0262] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0263] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can bedetermined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of infection, stage of infection, effects of infection (e.g. , IM, lymphoproliferative disorders), previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0264] Useful doses can range from 0.1 to 5 pg / kg or from 0.5 to 1 g / kg. In other examples, a dose can include 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can 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.

[0265] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, 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 yearly).

[0266] In some embodiments, the vaccine described herein can be administered in combination or alternation with a secondary vaccine (e.g., RSV and / or HMPV vaccine). In particular embodiments, the secondary vaccine includes a prophylactic mAb transfer or a maternal vaccination. In some embodiments, the secondary vaccine can be selected from an mRNA-based vaccine, an adenovirus vaccine, a non-replicating vaccine, a DNA vaccine, a live attenuated vaccine, a plant-based adjuvant vaccine, a multi-epitope peptide-based vaccine, an inactivated virus, and a peptide vaccine, or combinations thereof. Additional exemplary secondary vaccines suitable for use with the vaccines and methods described herein include AREXVY™ (GSK, United Kingdom), ABRYSVO™ (Pfizer, New York, NY), SYNAGIS™ (Sobi, Sweden), Beyfortus™ (Sanofi, France), or combinations thereof.

[0267] In some embodiments, vaccines disclosed herein can be used as a booster vaccine, to increase or modify or alter immune responses induced by a prior RSV and / or HMPV vaccine; such as an RNA vaccine, a DNA vaccine, a viral vector vaccine, or a protein-based vaccine. In some embodiments, the vaccine disclosed herein can be used as a booster vaccine following infection and recovery from RSV and / or HMPV.

[0268] The vaccine described herein can be administered on top of the current standard of care for RSV and / or HMPV patients, or in combination or alternation with any other compound or therapy that the healthcare provider deems beneficial for the patient. The combination and / or alternation therapy can be therapeutic, adjunctive, or palliative.

[0269] In some embodiments, the vaccine disclosed herein is administered with an anti-infective agent, for example, ribavirin, 4'-fluorouridine, small molecule TMC353121, or AVG-388. Any of these drugs or vaccines can be used in combination or alternation with the vaccine provided herein to treat an RSV and / or HMPV viral infection.

[0270] The pharmaceutical compositions described herein can be administered by, without limitation, injection, inhalation, infusion, perfusion, lavage or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual administration and more particularly by intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual injection.

[0271] (VI) Kits. Also disclosed herein are kits including one or more containers including one or more of the ai-mAb described herein, vaccines described herein, modified cells (e.g. cells modified to express an ai-mAb disclosed herein), and / or compositions and / or adjuvants, anti-infective agents, or secondary vaccines described herein. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[0272] The Exemplary Embodiments and Examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0273] (VI I) Exemplary Embodiments.1. A multi-specific binding molecule including a first arm and a second arm, wherein the first arm includes a heavy chain including the sequence of SEQ ID NO: 6 and a light chain including the sequence of SEQ ID NO: 7; and wherein the second arm includes an scFv including the sequence of SEQ ID NO: 44 fused to a constant region including the sequence of SEQ ID NO: 43.2. A multi-specific binding molecule including a first binding domain that binds a VH3-21 heavy chain and a second binding domain that binds a VL1-40 light chain, wherein(i) the first binding domain includes a complementarity determining region (CDR) heavy (H)1, CDRH2, and CDRH3 and a variable light chain including a CDR light (L) 1 , CDRL2, and CDRL3 wherein the CDRH1 includes the sequence of SEQ ID NO: 13, the CDRH2 includes the sequence of SEQ ID NO: 14, and the CDRH3 includes the sequence of SEQ ID NO: 15, and the CDRL1 includes the sequence of SEQ ID NO: 16, the CDRL2 includes the sequence of SEQ ID NO: 17, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 61, the CDRH2 includes the sequence of SEQ ID NO: 62, and the CDRH3 includes the sequence of SEQ ID NO: 15, and the CDRL1 includes the sequence of SEQ ID NO: 16, the CDRL2 includes the sequence of SEQ ID NO: 17, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 63, the CDRH2 includes the sequence of SEQ ID NO: 64, and the CDRH3 includes the sequence of SEQ ID NO: 65, and the CDRL1 includes the sequence of SEQ ID NO: 66, the CDRL2 includes the sequence YAS, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to I MGT; the CDRH1 includes the sequence of SEQ ID NO: 67, the CDRH2 includes the sequence of SEQ ID NO: 68, and the CDRH3 includes the sequence of SEQ ID NO: 65, and the CDRL1 includes the sequence of SEQ ID NO: 16, the CDRL2 includes the sequence of SEQ ID NO: 69, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to North; or the CDRH1 includes the sequence of SEQ ID NO: 70, the CDRH2 includes the sequence of SEQ ID NO: 71, and the CDRH3 includes the sequence of SEQ ID NO: 72, and the CDRL1 includes the sequence of SEQ ID NO: 73, theCDRL2 includes the sequence of SEQ ID NO: 74, and the CDRL3 includes the sequence of SEQ ID NO: 75 according to Contact; and(ii) the second binding domain includes a CDRH1, CDRH2, and CDRH3 and a CDRLI, CDRL2, and CDRL3 wherein: the CDRH1 includes the sequence of SEQ ID NO: 19, the CDRH2 includes the sequence of SEQ ID NO: 20, and the CDRH3 includes the sequence of SEQ ID NO: 21, and the CDRL1 includes the sequence of SEQ ID NO: 22, the CDRL2 includes the sequence of SEQ ID NO: 23, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 76, the CDRH2 includes the sequence of SEQ ID NO: 77, and the CDRH3 includes the sequence of SEQ ID NO: 21, and the CDRL1 includes the sequence of SEQ ID NO: 22, the CDRL2 includes the sequence of SEQ ID NO: 23, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 78, the CDRH2 includes the sequence of SEQ ID NO: 79, and the CDRH3 includes the sequence of SEQ ID NO: 80, and the CDRL1 includes the sequence of SEQ ID NO: 81, the CDRL2 includes the sequence SAS, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to I MGT; the CDRH1 includes the sequence of SEQ ID NO: 82, the CDRH2 includes the sequence of SEQ ID NO: 83, and the CDRH3 includes the sequence of SEQ ID NO: 80, and the CDRL1 includes the sequence of SEQ ID NO: 22, the CDRL2 includes the sequence of SEQ ID NO: 84, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to North; or the CDRH1 includes the sequence of SEQ ID NO: 85, the CDRH2 includes the sequence of SEQ ID NO: 86, and the CDRH3 includes the sequence of SEQ ID NO: 87, and the CDRL1 includes the sequence of SEQ ID NO: 88, the CDRL2 includes the sequence of SEQ ID NO: 89, and the CDRL3 includes the sequence of SEQ ID NO: 90 according to Contact.3. The multi-specific binding molecule of embodiment 2, wherein the first binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity to SEQ ID NO: 1; and a variable light chain including the sequence of SEQ ID NO: 2 or a sequence having at least 95% sequence identity to SEQ ID NO: 2.4. The multi-specific binding molecule of embodiments 2 or 3, wherein the first binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 93 or a sequence having at least 95% sequence identity to SEQ ID NO: 93; and a variable light chain encoded by the sequence of SEQ ID NO: 94 or a sequence having at least 95% sequence identity to SEQ ID NO: 94.5. The multi-specific binding molecule of any of embodiments 2-4, wherein the second binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity to SEQ ID NO: 3; and a variable light chain including the sequence of SEQ ID NO: 4 or a sequence having at least 95% sequence identity to SEQ ID NO: 4.6. The multi-specific binding molecule of any of embodiments 2-5, wherein the second binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 95 or a sequence having at least 95% sequence identity toSEQ ID NO: 95; and variable light chain encoded by the sequence of SEQ ID NO: 96 or a sequence having at least 95% sequence identity to SEQ ID NO: 96.7. The multi-specific binding molecule of any of embodiments 2-6, wherein the multi -specific binding molecule includes a first arm and a second arm.8. The multi-specific binding molecule of embodiment 7, wherein the first arm includes a heavy chain and a light chain; and the second arm includes the second binding domain and an IgG Fc constant region.9. The multi-specific binding molecule of embodiment 8, wherein the heavy chain includes the variable heavy chain of the first binding domain and a heavy chain constant region.10. The multi-specific binding molecule of embodiment 9, wherein the heavy chain constant region includes the sequence of SEQ ID NO: 42 or a sequence having at least 95% sequence identity to SEQ ID NO: 42.11. The multi-specific binding molecule of any of embodiments 8-10, wherein the heavy chain includes the sequence of SEQ ID NO: 6 or a sequence having at least 95% sequence identity to SEQ ID NO: 6.12. The multi-specific binding molecule of any of embodiments 8-11 , wherein the light chain includes the variable light chain of the first binding domain and a light chain constant region.13. The multi-specific binding molecule of embodiment 12, wherein the light chain constant region includes the sequence of SEQ ID NO: 41 or a sequence having at least 95% sequence identity to SEQ ID NO: 41.14. The multi-specific binding molecule of any of embodiments 8-13, wherein the light chain include the sequence of SEQ ID NO: 7 or a sequence having at least 95% sequence identity to SEQ ID NO: 7.15. The multi-specific binding molecule of any of embodiments 8-14, wherein the second binding domain is an scFv.16. The multi-specific binding molecule of embodiment 15, wherein the scFv includes the sequence of SEQ ID NO:44 or SEQ ID NO: 45 or a sequence having at least 95% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45.17. The multi-specific binding molecule of any of embodiments 8-16, wherein the second arm includes the sequence of SEQ ID NO: 5 or SEQ ID NO: 8 or a sequence having at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 8.18. The multi-specific binding molecule of any of embodiments 8-17, wherein the second arm is encoded by the sequence of SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 97.19. The multi-specific binding molecule of any of embodiments 8-18, wherein the first arm includes a heavy chain including the sequence of SEQ ID NO: 6 and a light chain including the sequence of SEQ ID NO: 7; and the second arm includes the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.20. The multi-specific binding molecule of any of embodiments 8-19, wherein the first arm includes a heavy chain encoded by the sequence of SEQ ID NO: 98 and a light chain encoded by the sequence of SEQ ID NO: 99; and the second arm is encoded by the sequence of SEQ ID NO : 97.21. The multi-specific binding molecule of embodiment 7, wherein the first arm includes a heavy chain and a light chain; and the second arm includes the first binding domain and an IgG Fc constant region.22. The multi-specific binding molecule of embodiment 21 , wherein the heavy chain includes the variable heavy chain of the second binding domain and a heavy chain constant region.23. The multi-specific binding molecule of embodiment 22, wherein the heavy chain constant region includes the sequence of SEQ ID NO: 42 or a sequence having at least 95% sequence identity to SEQ ID NO: 42.24. The multi -specific binding molecule of any of embodiments 21-23, wherein the heavy chain includes the sequence of SEQ ID NO: 11 or a sequence having at least 95% sequence identity to SEQ ID NO: 11.25. The multi-specific binding molecule of any of embodiments 21-24, wherein the light chain includes the variable light chain of the second binding domain and a light chain constant region.26. The multi-specific binding molecule of embodiment 25, wherein the light chain constant region includes the sequence of SEQ ID NO: 41 or a sequence having at least 95% sequence identity to SEQ ID NO: 41.27. The multi-specific binding molecule of any of embodiments 21-26, wherein the light chain include the sequence of SEQ ID NO: 12 or a sequence having at least 95% sequence identity to SEQ ID NO: 12.28. The multi-specific binding molecule of any of embodiments 21-27, wherein the first binding domain is an scFv.29. The multi-specific binding molecule of embodiment 28, wherein the scFv includes the sequence of SEQ ID NO: 46 or SEQ ID NO: 47 or a sequence having at least 95% sequence identity to SEQ ID NO: 46 or SEQ ID NO: 47.30. The multi-specific binding molecule of any of embodiments 21-29, wherein the second arm includes the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.31. The multi-specific binding molecule of any of embodiments 21-30, wherein the first arm includes a heavy chain including the sequence of SEQ ID NO: 11 and a light chain including the sequence of SEQ ID NO: 12; and the second arm includes the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.32. The multi-specific binding molecule of any of embodiments 7-31 , wherein the first arm includes a first Fc region or fragment thereof and the second arm includes a second Fc region or fragment thereof.33. The multi-specific binding molecule of embodiment 32, wherein the first Fc region or fragment thereof includes mutations that lower the isoelectric point.34. The multi-specific binding molecule of embodiments 32 or 33, wherein the second Fc region or fragment thereof includes mutations that lower the isoelectric point.35. The multi-specific binding molecule of any of embodiments 32-34, wherein the first Fc region or fragment thereof includes Fc silencing mutations.36. The multi-specific binding molecule of embodiment 35, wherein the Fc silencing mutations include E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.37. The multi-specific binding molecule of any of embodiments 32-36, wherein the second Fc region or fragment thereof includes Fc silencing mutations.38. The multi-specific binding molecule of embodiment 37, wherein the Fc silencing mutations include E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.39. A binding domain that binds a VH3-21 heavy chain, wherein the binding domain includes a complementarity determining region (CDR) heavy (H)1, CDRH2, and CDRH3 and a variable light chain including a CDR light (L)1, CDRL2, and CDRL3;wherein the CDRH1 includes the sequence of SEQ ID NO: 13, the CDRH2 includes the sequence of SEQ ID NO: 14, and the CDRH3 includes the sequence of SEQ ID NO: 15, and the CDRL1 includes the sequence of SEQ ID NO: 16, the CDRL2 includes the sequence of SEQ ID NO: 17, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to Kabat; wherein the CDRH1 includes the sequence of SEQ ID NO: 61, the CDRH2 includes the sequence of SEQ ID NO: 62, and the CDRH3 includes the sequence of SEQ ID NO: 15, and the CDRL1 includes the sequence of SEQ ID NO: 16, the CDRL2 includes the sequence of SEQ ID NO: 17, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to Chothia; wherein the CDRH1 includes the sequence of SEQ ID NO: 63, the CDRH2 includes the sequence of SEQ ID NO: 64, and the CDRH3 includes the sequence of SEQ ID NO: 65, and the CDRL1 includes the sequence of SEQ ID NO: 66, the CDRL2 includes the sequence YAS, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to IMGT; wherein the CDRH1 includes the sequence of SEQ ID NO: 67, the CDRH2 includes the sequence of SEQ ID NO: 68, and the CDRH3 includes the sequence of SEQ ID NO: 65, and the CDRL1 includes the sequence of SEQ ID NO: 16, the CDRL2 includes the sequence of SEQ ID NO: 69, and the CDRL3 includes the sequence of SEQ ID NO: 18 according to North; or wherein the CDRH1 includes the sequence of SEQ ID NO: 70, the CDRH2 includes the sequence of SEQ ID NO: 71, and the CDRH3 includes the sequence of SEQ ID NO: 72, and the CDRL1 includes the sequence of SEQ ID NO: 73, the CDRL2 includes the sequence of SEQ ID NO: 74, and the CDRL3 includes the sequence of SEQ ID NO: 75 according to Contact.40. The binding domain of embodiment 39, wherein the binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 1 or having at least 95% sequence identity to the sequence of SEQ ID NO: 1 and a variable light chain including the sequence of SEQ ID NO: 2 or having at least 95% sequence identity to the sequence of SEQ ID NO: 2.41. The binding domain of embodiments 39 or 40, wherein the binding domain includes a variable heavy chain encoded by the sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.42. The binding domain of any of embodiments 39-41 , wherein the binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence of SEQ ID NO: 94.43. The binding domain of any of embodiments 39-42, wherein the binding domain is an scFv.44. The binding domain of embodiment 43, wherein scFv includes a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 46 or SEQ ID NO:47.45. The binding domain of embodiments 43 or 44, wherein scFv includes the sequence of SEQ ID NO: 46 or SEQ ID NO:47.46. The binding domain of any of embodiments 43-45, wherein the scFv is attached to a fragment of a constant heavy chain.47. The binding domain of embodiment 46, wherein the constant heavy chain includes an lgG1 heavy chain, an lgG2 heavy chain, an lgG3 heavy chain, or an lgG4 heavy chain.48. The binding domain of embodiments 46 or 47, wherein the constant heavy chain includes an lgG1 heavy chain.49. A binding domain that binds a VL1-40 light chain, wherein the binding domain includes a complementarity determining region (CDR) heavy (H)1 , CDRH2, and CDRH3 and a variable light chain including a CDR light (L)1 , CDRL2, and CDRL3; wherein the CDRH1 includes the sequence of SEQ ID NO: 19, the CDRH2 includes the sequence of SEQ ID NO: 20, and the CDRH3 includes the sequence of SEQ ID NO: 21, and the CDRL1 includes the sequence of SEQ ID NO: 22, the CDRL2 includes the sequence of SEQ ID NO: 23, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 76, the CDRH2 includes the sequence of SEQ ID NO: 77, and the CDRH3 includes the sequence of SEQ ID NO: 21, and the CDRL1 includes the sequence of SEQ ID NO: 22, the CDRL2 includes the sequence of SEQ ID NO: 23, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 78, the CDRH2 includes the sequence of SEQ ID NO: 79, and the CDRH3 includes the sequence of SEQ ID NO: 80, and the CDRL1 includes the sequence of SEQ ID NO: 81, the CDRL2 includes the sequence SAS, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to I MGT; the CDRH1 includes the sequence of SEQ ID NO: 82, the CDRH2 includes the sequence of SEQ ID NO: 83, and the CDRH3 includes the sequence of SEQ ID NO: 80, and the CDRL1 includes the sequence of SEQ ID NO: 22, the CDRL2 includes the sequence of SEQ ID NO: 84, and the CDRL3 includes the sequence of SEQ ID NO: 24 according to North; or the CDRH1 includes the sequence of SEQ ID NO: 85, the CDRH2 includes the sequence of SEQ ID NO: 86, and the CDRH3 includes the sequence of SEQ ID NO: 87, and the CDRL1 includes the sequence of SEQ ID NO: 88, the CDRL2 includes the sequence of SEQ ID NO: 89, and the CDRL3 includes the sequence of SEQ ID NO: 90 according to Contact.50. The binding domain of embodiment 49, wherein the binding domain includes a variable heavy chain having at least 95% sequence identity to SEQ ID NO: 3 and a variable light chain having at least 95% sequence identity to SEQ ID NO: 4.51. The binding domain of embodiments 49 or 50, wherein the binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 3 and a variable light chain including the sequence of SEQ ID NO: 4.52. The binding domain of any of embodiments 49-51, wherein the binding domain includes a variable heavy chain encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 95 and a variable light chain encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 96.53. The binding domain of any of embodiments 49-52, wherein the binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and a variable light chain encoded by the sequence of SEQ ID NO: 96.54. The binding domain of any of embodiments 49-53, wherein the binding domain is an scFv.55. The binding domain of embodiment 54, wherein the scFv includes a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.56. The binding domain of embodiments 54 or 55, wherein the scFv includes the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.57. The binding domain of any of embodiments 54-56, wherein the scFv is attached to a fragment of a constant heavy chain.58. The binding domain of embodiment 57, wherein the constant heavy chain includes an lgG1 heavy chain, an lgG2 heavy chain, an lgG3 heavy chain, or an lgG4 heavy chain.59. The binding domain of embodiments 57 or 58, wherein the constant heavy chain includes an lgG1 heavy chain.60. A binding molecule including a binding domain that binds a VH3-21 1 VL1-40 B cell receptors (BCRs).61. The binding molecule of embodiment 60, wherein the binding molecule is an anti-idiotypic antibody.62. The binding molecule of embodiments 60 or 61 , wherein the binding molecule is a multi-specific binding molecule.63. The binding molecule of embodiment 62, wherein the multi-specific binding molecule is a bispecific antibody.64. The binding molecule of embodiments 62 or 63, wherein the multi-specific binding molecule includes a binding domain that binds a VH3-21 and a binding domain that binds VL1-40.65. The binding molecule of embodiment 64, wherein the binding domain that binds VH3-21 includes the binding domain of any of embodiments 39-48.66. The binding molecule of embodiments 64 or 65, wherein the binding domain that binds VL1-40 includes the binding domain of any of embodiments 49-59.67. The binding molecule of any of embodiments 60-66, further including a first arm and a second arm.68. The binding molecule of embodiment 67, wherein the first arm includes a variable light chain, a constant light chain, a variable heavy chain, and a constant heavy chain; and wherein the second arm includes an scFv fused to a portion of an Fc region.69. The binding molecule of embodiment 68, wherein the variable light chain and variable heavy chain of the first arm include the binding domain that binds VH3-21; and the scFv of the second arm includes the binding domain that binds VL1-40.70. The binding molecule of embodiments 68 or 69, wherein the variable light chain and variable heavy chain of the first arm include the binding domain that binds VL1-40; and the scFv of the second arm includes the binding domain that binds VH3-21.71. The binding molecule of any of embodiments 68-70, wherein the constant light chain of the first arm includes a kappa light chain or lambda light chain.72. The binding molecule of any of embodiments 68-71, wherein the constant light chain of the first arm includes a kappa light chain.73. The binding molecule of any of embodiments 68-72, wherein the constant light chain includes the sequence of SEQ ID NO: 41 or a sequence having at least 95% sequence identity to SEQ ID NO: 41.74. The binding molecule of any of embodiments 68-73, wherein the constant heavy chain of the first arm includes an IgG heavy chain.75. The binding molecule of embodiment 74, wherein the IgG heavy chain includes mutations that lower the isoelectric point.76. The binding molecule of embodiment 75, wherein the mutations that lower the isoelectric point include N208D, Q295E, N384D, Q418E, and N421D in reference to SEQ ID NO: 27 with position numbering of SEQ ID NO: 27 starting at 118.77. The binding molecule of any of embodiments 74-76, wherein the IgG heavy chain includes heterodimer mutations.78. The binding molecule of embodiment 77, wherein the heterodimer mutations include L368D and K370S in reference to SEQ ID NO: 27 with position numbering of SEQ ID NO: 27 starting at 118.79. The binding molecule of any of embodiments 74-78, wherein the IgG heavy chain includes Fc silencing mutations.80. The binding molecule of embodiment 79, wherein the Fc silencing mutations include E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.81. The binding molecule of any of embodiments 68-80, wherein the constant heavy chain of the first arm includes the sequence of SEQ ID NO: 42 or a sequence having at least 95% sequence identity to SEQ ID NO: 42.82. The binding molecule of any of embodiments 68-81, wherein the portion of the Fc region of the second arm includes a fragment of an lgG1 antibody, fragment of an I gG2 antibody, fragment of an lgG3 antibody, or fragment of an lgG4 antibody.83. The binding molecule of any of embodiments 68-82, wherein the portion of the Fc region of the second arm includes a fragment of an lgG1 antibody.84. The binding molecule of embodiment 83, wherein the fragment of the lgG1 antibody includes the sequence of SEQ ID NO: 43 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 43.85. The binding molecule of any of embodiments 68-84, wherein the portion of the Fc region of the second arm includes mutations C220S, E357Q, and S364K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.86. The binding molecule of any of embodiments 68-85, wherein the portion of the Fc region of the second arm includes Fc silencing mutations.87. The binding molecule of embodiment 86, wherein the Fc silencing mutations include E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.88. The binding molecule of any of embodiments 60-87, including (i) a first arm including variable light chain that binds VH3-21 , a constant light chain, variable heavy chain that binds VH3-21 , and a constant heavy chain; and (ii) a second arm including an scFv that binds VL1-40 fused to a portion of an Fc region.89. The binding molecule of embodiment 88, wherein the first arm includes a variable and constant heavy chain including the sequence of SEQ ID NO: 6 and a variable and constant light chain including the sequence of SEQ ID NO: 7; and the second arm including an scFv-Fc including the sequence of SEQ ID NO: 5.90. The binding molecule of embodiments 88 or 89, wherein the first arm includes a variable and constant heavy chain including the sequence of SEQ ID NO: 6 and a variable and constant light chain including the sequence of SEQ ID NO: 7; and the second arm including an scFv-Fc including the sequence of SEQ ID NO: 8.91. The binding molecule of any of embodiments 88-90, wherein the first arm includes a variable and constant heavy chain encoded by the sequence of SEQ ID NO: 98, a variable and constant light chain encoded by the sequence of SEQ ID NO: 99, and the second arm includes an scFv-Fc encoded by the sequence of SEQ ID NO: 97.92. The binding molecule of any of embodiments 60-91, including (I) a first arm including variable light chain that binds VL1 -40, a constant light chain, a variable heavy chain that binds VL1 -40, a constant heavy chain; and (ii) a second arm including an scFv that binds VH3-21 fused to a portion of an Fc region.93. The binding molecule of embodiment 92, wherein the first arm includes a variable and constant heavy chain including the sequence of SEQ ID NO: 11 and a variable and constant light chain including the sequence of SEQ ID NO: 12; and the second arm includes an scFv-Fc including the sequence of SEQ ID NO: 9.94. The binding molecule of embodiments 92 or 93, including a variable and constant heavy chain including the sequence of SEQ ID NO: 11 and a variable and constant light chain including the sequence of SEQ ID NO: 12; and the second arm includes an scFv-Fc including the sequence of SEQ ID NO: 10.95. A composition including the multi-specific binding molecule of any of embodiments 2-38 or the binding molecule of any of embodiments 60-94 and a pharmaceutically acceptable carrier.96. The composition of embodiment 95, further including one or more adjuvants.97. The composition of embodiment 96, wherein the one or more adjuvants are selected from alum, a squalene- based adjuvant, a STING agonist, or a liposome-based adjuvant.98. The composition of any of embodiments 95-97, wherein the composition is a vaccine.99. A nucleic acid sequence encoding the multi-specific binding molecule of any of embodiments 2-38, the binding molecule of any of embodiments 60-94, or the binding domain of any of embodiments 39-59.100. The nucleic acid sequence of embodiment 99, including a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.101. The nucleic acid sequence of embodiments 99 or 100, including a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence of SEQ ID NO: 94.102. The nucleic acid sequence of any of embodiments 99-101, including a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 95 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 96.103. The nucleic acid sequence of any of embodiments 99-102, including a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and a variable light chain encoded by the sequence of SEQ ID NO: 96.104. The nucleic acid sequence of any of embodiments 99-103, including an scFv fused to an Fc region or fragment thereof encoded a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 97.105. The nucleic acid sequence of any of embodiments 99-104, including an scFv fused to an Fc region or fragment thereof encoded the sequence of SEQ ID NO: 97.106. The nucleic acid sequence of any of embodiments 99-105, including a heavy chain encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 98.107. The nucleic acid sequence of any of embodiments 99-106, including a heavy chain encoded by the sequence of SEQ ID NO: 98.108. The nucleic acid sequence of any of embodiments 99-107, including a light chain encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 99.109. The nucleic acid sequence of any of embodiments 99-108, including a light chain encoded by the sequence of SEQ ID NO: 99.110. A vector including the nucleic acid sequence of any of embodiments 99-109.111. A cell genetically modified to express the multi-specific binding molecule of any of embodiments 2-38 or the binding molecule of any of embodiments 60-94.112. The cell of embodiment 111, wherein the cell is a 293E cell.113. A method of stimulating a respiratory syncytial virus (RSV) or human metapneumovirus (HMPV) immune response in a subject including administering to the subject a therapeutically effective amount of a composition including a binding molecule that binds VH3-21 / L1-40 B cell receptors (BCRs), thereby stimulating an RSV or HMPV immune response in the subject.114. The method of embodiment 113, wherein the binding molecule includes an anti-idiotypic antibody.115. The method of embodiment 114, wherein the binding molecule includes the multi-specific binding molecule of any of embodiments 2-38 or the binding molecule of any of embodiments 60-94.116. The method of any of embodiments 113-115, wherein the binding molecule includes a multi-specific antibody including a first arm including a first binding domain that binds a VH3-21 heavy chain and a second arm including a second binding domain that binds a VL1-40 light chain.117. The method of embodiment 116, wherein the first binding domain includes a variable heavy chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1 and variable light chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2.118. The method of embodiments 116 or 117, wherein the first binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 1 and variable light chain including the sequence of SEQ ID NO: 2.119. The method of any of embodiments 116-118, wherein the first binding domain includes a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.120. The method of any of embodiments 116-119, wherein the first binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence of SEQ ID NO: 94.121. The method of any of embodiments 116-120, wherein the first arm includes a full heavy chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6 and a full light chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 7.122. The method of any of embodiments 116-121, wherein the first arm includes a full heavy chain including the sequence of SEQ ID NO: 6 and a full light chain including the sequence of SEQ ID NO: 7.123. The method of any of embodiments 116-122, wherein the first arm includes a full heavy chain encoded by a sequence having at least 95% sequence identity to sequence of SEQ ID NO: 98 and a full light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 99.124. The method of any of embodiments 116-123, wherein the first arm includes a full heavy chain encoded by the sequence of SEQ ID NO: 98 and a full light chain encoded by the sequence of SEQ ID NO: 99.125. The method of any of embodiments 116-124, wherein the second binding domain includes an scFv including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.126. The method of any of embodiments 116-125, wherein the second binding domain includes an scFv including the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.127. The method of any of embodiments 116-126, wherein the second binding domain includes an scFv including a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 95 and variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 96.128. The method of any of embodiments 116-127, wherein the second binding domain includes an scFv including a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and variable light chain encoded by the sequence of SEQ ID NO: 96.129. The method of any of embodiments 116-128, wherein the second arm includes an scFv and fragment of an Fc region.130. The method of any of embodiments 116-129, wherein the second arm includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.131. The method of any of embodiments 116-130, wherein the second arm includes the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.132. The method of any of embodiments 116-131, wherein the second arm is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 97.133. The method of any of embodiments 116-132, wherein the second arm is encoded by the sequence of SEQ ID NO: 97.134. The method of any of embodiments 113-133, wherein the subject is a pediatric patient.135. The method of embodiment 134, wherein the pediatric patient is 5 years of age or under.136. The method of embodiments 134 or 135, wherein the pediatric patient is 1 year of age or under.137. The method of any of embodiments 113-136, wherein the subject has RSV.138. The method of any of embodiments 113-137, wherein the subject has HMPV.139. The method of any of embodiments 113-138, wherein the subject does not have RSV, and wherein the therapeutically effective amount reduces the likelihood of the subject becoming infected with RSV.140. The method of any of embodiments 113-139, wherein the subject does not have HMPV, and wherein the therapeutically effective amount reduces the likelihood of the subject becoming infected with HMPV.141. The method of any of embodiments 113-140, wherein the subject does not have RSV or HMPV, and wherein the therapeutically effective amount reduces the likelihood of the subject becoming infected with RSV or HMPV.142. The method of any of embodiments 113-141, wherein the therapeutically effective amount provides a prophylactic or therapeutic treatment against RSV or HMPV.143. The method of any of embodiments 113-142, wherein the therapeutically effective amount provides a prophylactic or therapeutic treatment against RSV and HMPV.144. The method of any of embodiments 113-143, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.145. The method of any of embodiments 113-144, wherein the administering precedes or follows administration of a different RSV vaccination protocol.146. The method of any of embodiments 113-145, wherein the administering precedes or follows administration of a different HMPV vaccination protocol.147. A method of enhancing production of RSV-neutralizing antibodies or HMPV-neutralizing antibodies including contacting the multi-specific binding molecule of any of embodiments 2-38 or the binding molecule of any of embodiments 60-94 with a population of cells including a VH3-21 / L1 -40-expressing B cell.148. The method of embodiment 147, wherein the method enhances production of RSV-neutralizing antibodies and HMPV-neutralizing antibodies.149. A method of binding VH3-21 / VL1-40 neutralizing B cell receptors (BCRs) including administering a binding molecule that binds VH3-21A / L1-40 to a population of cells including a VH3-21 / VL1 -40-expressing B cell.150. The method of embodiment 149, wherein the binding molecule includes an anti-idiotypic antibody.151. The method of embodiments 149 or 150, wherein the binding molecule includes the multi-specific binding molecule of any of embodiments 2-38 or the binding molecule of any of embodiments 60-94.152. The method of any of embodiments 149-151, wherein the binding molecule includes a multi-specific antibody including a first arm including a first binding domain that binds a VH3-21 heavy chain and a second arm including a second binding domain that binds a VL1-40 light chain.153. The method of embodiment 152, wherein the first binding domain includes a variable heavy chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1 and variable light chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2.154. The method of embodiments 152 or 153, wherein the first binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 1 and variable light chain including the sequence of SEQ ID NO: 2.155. The method of any of embodiments 152-154, wherein the first binding domain includes a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.156. The method of any of embodiments 152-155, wherein the first binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and variable light chain encoded by the sequence of SEQ ID NO: 94.157. The method of any of embodiments 152-156, wherein the first arm includes a full heavy chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6 and a full light chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 7.158. The method of any of embodiments 152-157, wherein the first arm includes a full heavy chain including the sequence of SEQ ID NO: 6 and a full light chain including the sequence of SEQ ID NO: 7.159. The method of any of embodiments 152-158, wherein the first arm includes a full heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 98 and a full light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 99.160. The method of any of embodiments 152-159, wherein the first arm includes a full heavy chain encoded by the sequence of SEQ ID NO: 98 and a full light chain encoded by the sequence of SEQ ID NO: 99.161. The method of any of embodiments 152-160, wherein the second binding domain includes an scFv including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.162. The method of any of embodiments 152-161, wherein the second binding domain includes an scFv including the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.163. The method of any of embodiments 152-162, wherein the second binding domain includes an scFv including a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 95 and variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 96.164. The method of any of embodiments 152-163, wherein the second binding domain includes an scFv including a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and variable light chain encoded by the sequence of SEQ ID NO: 96.165. The method of any of embodiments 152-164, wherein the second arm includes an scFv and fragment of an Fc region.166. The method of any of embodiments 152-165, wherein the second arm includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.167. The method of any of embodiments 152-166, wherein the second arm includes the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.168. The method of any of embodiments 152-167, wherein the second arm is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 97.169. The method of any of embodiments 152-168, wherein the second arm is encoded by the sequence of SEQ ID NO: 97.

[0274] (VIII) Examples.

[0275] Example 1 (Experimental). An RSV vaccine derived from anti-idiotypic antibodies targets neutralizing B-cells in bulk PBMCs.

[0276] Respiratory Syncytial Virus (RSV) is a common pathogen that causes lower respiratory tract infections (LRTI) leading to significant morbidity and mortality at the extremes of age. Although an RSV vaccine was recently approved for use in adults over 60, a vaccine that can prevent infection in the infant population remains lacking. Current strategies to protect infants from RSV rely on passive immunity, either by the transfer of neutralizing monoclonal antibodies (mAbs) or maternal vaccination and in utero antibody transfer. A vaccine that could elicit rapidly protective neutralizing antibodies and establish immunological memory in the infant population would fulfill a critical unmet need. Antibodies that arise from the chromosomally encoded VH3-21 / L1 -40 gene pairing are unique in that they are structurally pre-configured to bind to and neutralize RSV, without the need to undergo affinity maturation. These protective germline antibodies have been identified in both cord blood samples as well as naive adult PBMCs, establishing them as a reproducible antibody class that could be targeted through vaccination. Here, the generation of a vaccine immunogen derived from anti-idiotypic monoclonal antibodies (ai-mAbs) that display a high degree of binding-specificity for unmutated VH3-2WL1-40 B cell receptors (BCRs) is described. When used as bait in B cell sorting experiments, the immunogen selected for these BCRs over 200-fold above the estimated frequency of this gene pairing. Furthermore, antibodies derived from sorted BCRs neutralized RSV, despite being sorted by an immunogen that is antigenically distinct from the pre-fusion F protein (preF) which is the basis for current vaccines. It is also shown that the immunogen specifically binds and activates B cell lines engineered to express target BCRs, but not off-target, non-neutralizing BCRs that are readily activated by preF. When used as a vaccine antigen, this ai-mAb selectively engages and stimulate B cells predisposed to neutralize RSV. This vaccine strategy circumvents the need for the lengthy process of somatic hypermutation, often used to achieve potent neutralization, and can rapidly elicit neutralizing serum antibodies following a single immunization in infants.

[0277] Introduction. Respiratory syncytial virus (RSV) is a common seasonal pathogen and infection generally causes mild respiratory symptoms in adults but can cause serious lower respiratory infection in infants and older adults. RSV infection is responsible for 60,000 recorded in-hospital deaths annually in children under 5 (Shi et a / ., Lancet 390, 946- 958, 2017 ("Shi 1”); Lozano eta / ., Lancet 380, 2095-2128, 2012) and accounts for a substantial hospitalization burden in infants as well as aged adults (Shi 1; Widmer eta / ., Influenza Other Respir Viruses 8, 347-352, 2014; Hall eta / ., The New England journal of medicine 360, 588-598, 2009). In 2023, two RSV vaccines, AREXVY® (GlaxoSmithKline Biological SA, United Kingdom) and ABRYSVO® (Pfizer, New York, NY), were approved for use in adults over 60 years old. However, the current treatments available for infant protection are prophylactics based on the transfer of neutralizing antibodies. Historically, the mAb palivizumab (SYNAGIS™ (Sobi, Sweden)), has been available for infants at high risk of infection, such as those born prematurely (Pediatrics; Homaira). More recently, the extended half-life mAb nirsevimab (BEYFORTUS™ (Sanofi, France)), has been approved for all newborns and babies under 1 year of age entering their first RSV season (Hammitt eta / ., N Engl J Med 386, 837-846, 2022 (“Hammitt”); Muller et a / ., N Engl J Med 388, 1533-1534, 2023). These mAbs target the virally encoded fusion protein, F. As an alternative prophylactic strategy, ABRYSVO™ (Pfizer) was recently approved for use in pregnant individuals in the final month of gestation to protect infants from birththrough the first 6 months of age (ed FDA Office of Media Affairs, FDA, 2023). This strategy relies on the transplacental transfer of protective antibodies from the mother to the fetus (Kampmann et al., N Engl J Med 388, 1451-1464, 2023). These prophylactic strategies afford transient protection to the infant for the duration serum half-life of the antibodies, but this protection wanes as these antibodies are cleared from the infant's system.

[0278] Prevention of severe RSV infection in infants with palivizumab and nirsevimab demonstrate that neutralizing antibodies against F are a clear correlate of protection against RSV-mediated disease. The viral F protein exists in a metastable pre-fusion (preF) state and a highly stable post-fusion state (postF). Most neutralizing antibodies in the sera of infected individuals map to the energetically unfavorable pre F configuration highlighting the importance of preF for vaccine design (Gilman ef al., Science immunology 1, eaaj1879, 2016 ("Gilman”); Ngwuta ef al., Sci Transl Med 7, 309ra162, 2015). Epitope mapping of mAbs has identified distinct antigenic sites on RSV F termed 0-V. Some sites such as 0, III, and V, are present on preF, while the others are present on both conformations (Ruckwardt ef al., Immunity 51, 429-442, 2019; Battles & McLellan, Nature reviews. Microbiology 17, 233-245, 2019). Multiple neutralizing mAbs have been identified that bind exclusively or preferentially to preF, including nirsevimab (Hammitt; Gilman; Corti ef al., Nature 501, 439-443, 2013 (“Corti”); Goodwin ef a / ., Immunity 48, 339-349. e335, 2018 (“Goodwin 1”); Kwakkenbos ef al., Nature medicine 16, 123-128, 2010; McLellan ef al., Science 340, 1113-1117, 2013; Wen ef al., Nature microbiology 2, 16272- 16272, 2017 (“Wen”))) Indeed, this viral protein is the basis for the approved subunit vaccines, AREXVY and ABRYSVO, which have been made possible by the recent advantages in antigen engineering leading to the stabilization of RSV F in its pre-fusion conformation (McLellan et al., Science 342, 592-598, 2013 (“McLellan 1”)). The earliest RSV vaccine was based on formalin inactivated whole virus in which, unintentionally, RSV F preferentially adopted the post-fusion conformation (Killikelly ef al., Scientific reports 6, 34108-34108, 2016 (“Killikelly”)). Unfortunately, this vaccine led to enhanced respiratory disease (ERD) in infants which caused hospitalization and in some cases lead to death (Kim ef al., American journal of epidemiology 89, 422-434, 1969 (“Kim")). The ERD identified has been attributed in part due to the elicitation F-binding but non-neutralizing antibodies targeting the postF protein (Killikelly; Kim; Kapikian ef al., American journal of epidemiology 89, 405-421, 1969 (“Kapikian”)). Due to this failure, infant RSV vaccine research has proceeded cautiously, and has sought to avoid subunit-based strategies in RSV-na'ive populations.

[0279] In general, the infant immune system is inefficient at generating somatically mutated antibodies (Prelog, Gerontology 59, 230-239, 2013; Siegrist & Aspinall, Immunology 9, 185-194, 2009) which are often used for high affinity binding and / or neutralizing potency. Maternal antibody can bind to vaccine antigens and suppress the infant response to vaccination ((Siegrist; Vono ef al., Cell reports 28, 1773-1784.e1775, 2019 (“Vono”)). Indeed, infants tend to elicit higher neutralizing titers following RSV infection when maternal antibody is low (Shinoff etal., The Journal of infectious diseases 198, 1007-1015, 2008; Murphy)) These inherent challenges complicate the development of an effective infant vaccine that can generate humoral protection and establish immunological memory.

[0280] Characterization of infant humoral responses to natural RSV infection has identified interesting immunization targets. The isolation of RSV F reactive mAbs from infected infants (Goodwin 1) revealed that multiple potently neutralizing mAbs were derived from the same antibody variable heavy (VH) and variable light (VL) chain genes: VH3-21 and VL1-40 (Goodwin 1). These VH3-21 / VL 1-40 mAbs mapped to an epitopeon antigenic site III present on preF (Corti; Goodwin 1) and were able to potently neutralize RSV without undergoing affinity maturation. Unmutated, site Ill-directed neutralizing VH3-21 / VL1 -40 mAbs were further isolated from cord blood and naive B cells from adults (Goodwin 1). Similarly potent VH3-21 / VL1-40 mAbs with <1% somatic mutation were isolated from six of eight clinical trial participants immunized with a stabilized preF vaccine (Mukhamedova et al., Immunity 54, 769- 78O.e766, 2021). The crystal structure of an unmutated neutralizing VH3-21 / VL1 -40 mAb in complex with preF revealed that the heavy chain contacts are primarily made with the CDRH1 and CDRH2, which are chromosomally encoded by VH3-21 (Goodwin 1). Similarly, the light chain contacts are made by the CDRL1 and CDRL2 regions encoded by the VL1- 40 gene. Collectively this data identified germline VH3-21 A / L1-40-derived antibodies as a public clonotype that is preconfigured to bind to and neutralize RSV, and established them as an attractive vaccine target.

[0281] Vaccine antigens derived from ai-mAbs raised against unmutated precursor versions of neutralizing antibodies against HIV-1 and influenza can activate B cells in vitro and generate robust on-target B cell and serum responses using knock-in mice in vivo (Avnir et al., Cell reports 21, 3243-3255, 2017; Avoir et al., PLoS pathogens 10, e1004103, 2014; Dosenovic et al., The Journal of experimental medicine 216, 2316-2330, 2019 (“Dosenovic"); Bancroft et al., The Journal of experimental medicine 216, 2331-2347, 2019 (“Bancroft”); Seydoux et al., Cell reports 35, 109084, 2021 “Seydoux 1”)). Given that germline-encoded VH3-21 / VL1 -40 BCRs are hard wired to neutralize RSV without a need for somatic mutation, it was hypothesized that they may be amenable to elicitation by an anti-idiotype vaccine. To this end, ai-mAbs were raised to VH3-21A / L1-40 BCRs. By integrating binding, single B cell sorting with fluorescently labeled ai-mAbs, and structural analysis, one ai-mAb was identified that bound with high affinity and specificity to VH3-21 heavy chains and another that similarly bound VL1-40 light chains. The binding properties of both ai-mAbs were combined in a bispecific platform that specifically cross-linked B cells engineered to express VH3-21 / VL1 -40 BCRs on their surface. 40% of B cells that were sorted from multiple donors using the fluorescently-labeled ai-mAb as bait expressed VH3-21 / VL1-40 BCRs. Of these VH3- 21 / VL1 -40 BCRs that were expressed as full length IgGs, 30% were cross reactive with RSV preF but not postF, and 5% were able to neutralize RSV to some degree. These results provide proof of concept that an anti-idiotype vaccine can rapidly elicit RSV neutralizing antibodies by specifically targeting VH3-21 / VL1 -40 BCRs.

[0282] Results. Isolation of ai-mAbs Against mAbs Encoded by VH3-21 and VL1-40 Genes. Hybridomas were generated from splenocytes of mice immunized with a cocktail of three RSV F site Ill-directed mAbs derived from unmutated VH3-21 and VL1-40 genes: ADI-19425, ADI-25532 and ADI-14337. 4 hybridomas, 2C2, 2C1, 2F1, and 1D3 that showed binding to the mAbs used to immunize, and weaker or no binding to negative control mAbs, were further subcloned to ensure the establishment of monoclonal cell lines. ai-mAbs purified from culture supernatants were evaluated for their ability to bind to a panel of recombinant mAbs by ELISA (FIG. 1). The panel included ADI-19425, ADI-25532 and ADI-14337, as well as chimeric antibodies with a VH3-21 -encoded heavy chain paired with a non-VL1-40 light chain or a VL1-40-encoded light chain but a non VH3-21 -derived heavy chain, and 37 other anti-EBV and anti-HIV-1 mAbs with diverse VH and VL gene usage (Dosenovic; Bancroft; Seydoux 1; Snijder et al., Immunity 48, 799-811 e799, 2018 ("Snijder”)). All four ai-mAbs bound to ADI-19425, ADI-25532 and ADI-14337. 2C2, 2C1, and 2F1 bound to chimeric mAbs with a VL1-40 light chain (LC), while 1D3 bound to chimeric mAbs with a VH3-21 heavy chain (HC) (FIG.1). None of the ai-mAbs bound to any of the control antibodies (FIG.1).

[0283] Fluorescently Labeled ai-mAbs as Bait for Bulk Sorting of Naive B cells and BCR Gene Analysis. Whether the hybridomas could recognize B cells expressing bona-fide VH3-21 / VL1-40 B cell receptors (BCRs) on naive B cells was determined. 2C1 , 2C2, 2F1 , and 1 D3 ai-mAbs were biotinylated and individually complexed with a streptavidin fluorophore(APC) conjugated to a unique oligonucleotide barcode. To counter screen against B cells that bind outside of the antiidiotype paratope, a biotinylated isotype control iv2, which was raised against iGL-VRC01 (Seydoux 1)j was included and conjugated to SA-APC with a unique oligonucleotide barcode. 2-3% of naive B cells stained with each labeled ai-mAb while <0.5% of B cells stained with a control streptavidin APC barcode reagent (FIG. 2A and FIG. 3). APC+naive B cells were sorted and single cell VDJ antigen-barcode libraries were generated using paired heavy / light chain next generation sequencing. The BCRs sorted by each ai-mAb were identified by bioinformatic demultiplexing of the libraries (FIGs. 4A, 4B) (Fong et al., BMC Bioinformatics 18, 454, 2017 (“Fong”)). The percentage of ai-mAb sorted B cells that expressed VH3-21 / VL1 -40 pairs ranged from 0.9 to 5% (FIG. 2B), which is marginally higher than would be expected by chance (0.1- 0.35%) based on high-throughput unbiased sequencing of paired antigen inexperienced BCRs (DeKosky).

[0284] 95% of BCRS sorted with 1D3 as bait expressed BCRs encoded by VH3-21 heavy chains (FIG. 2C), and 85-95% of BCRs sorted with 2C1, 2C2 and 2F1 expressed VL1-40 encoded light chains (FIG. 2D). Sequencing of the heavy and light chain transcripts from the hybridomas revealed that 2C1, 2C2, and 2F1 were all clonal variants, explaining the similarity of their binding and sorting properties. Collectively these results indicate that the epitope of 1D3 is primarily encoded by the VH3-21 gene, and those bound by 2C1, 2C2 and 2F1 are primarily encoded by the VL1-40 gene.

[0285] Crystal Structures of ADI-19425 Complexed with 1D3 and 2C1. To understand the molecular basis behind the observed interactions between the ai-mAbs and B cells derived from VH3-21 and VL1-40, their co-crystal structures with ADI-19425 were determined. 1D3 and 2C1 were produced as recombinant IgGs and their high affinity binding to ADI- 19425 Fab was measured by biolayer interferometry (BLI) (KD = 0.4nM for 1 D3 and 1.5 nM for 2C1, FIGs. 5A, 5B). The ai- mAbs were then digested into Fabs and complexed with ADI-19425 Fab to enable structural analysis. The crystal structures of the ADI-19425 Fab in complex with 1D3 Fab at 2.67 A and ADI-19425 Fab in complex with 2C1 Fab at 2.41 A (FIGs. 6, 7A, 7C, 8A-8C) were solved. ADI-19425 binds 1D3 with a total buried surface area (BSA) of 1019 A2, primarily through its heavy chain with 579 A2(57% of total BSA) from the VH3-21 encoded region and 190 A2(19% of total BSA) from the CDRH3. The remainder, 208 A2(20% of total BSA), is contributed by ADI-19425 VL1-40 encoded germline gene and 42 A2from the lambda J gene. ADI-19425 binds 2C1 with a total BSA of 994 A2, 471 A2(47% of the total BSA) of which is contributed from its VL1-40 encoded germline gene, while 248 A2(25% of the total BSA) comes from its VH3-21. The CDHR3 contributes 275 A2(28%) of the total BSA.

[0286] Superposition of the crystal structures showed that both ai-Fabs bound the ADI-19425 head on with some overlap (FIGs. 8A-8C). Five ADI-19425 heavy chain residues (Tyr97 - Gly 100A, kabat numbering) and one light chain residue (Tyr91) interacted with both ai-Fabs. These structural analyses support the observation that 1D3 and 2C1 can engage BCRs derived from the VH3-21 and VL1-40 genes, respectively.

[0287] Generation of a Bispecific ai-mAb Specific for VH3-21 / VL1-40 BCRs. Based on the ai-mAb binding (FIG. 1) and B cell sorting results (FIGs. 2A-2D), none of the ai-mAbs show the desired specificity for paired VH3-21 / L1 -40 BCRs. It is feasible that if any of these ai-mAbs were to be used as an immunogen they would bind and cross-link BCRs encoded byeither VH3-21 or VL1-40, but not efficiently enrich for BCRs derived from both, leading to off-target activation. To avoid this and enhance the specificity of the ai-mAb immunogen to target VH3-21 / VL1 -40 BCR pairs, a bispecific platform to combine the binding modalities of 2C1 and 1D3 ai-mAbs was developed. It is demonstrated that a bispecific ai-mAb that binds specifically to unmutated VRC01-class heavy chains with one arm and VRC01-class light chains with the other, selectively activated target B cells in vitro in and in vivo (Seydoux 1). A similar approach to generate a 1D3 / 2C1 bispecific molecule to target and activate germline VH3-21 / VL1-40 BCRs was used. The VH and VL sequences of 1D3 and 2C1 were cloned into an engineered heterodimeric Fc platform that permits the efficient purification of bispecific mAbs (Moore et al., Methods 154, 38-50, 2019 (“Moore”)). To generate the bispecific, 2C1 was produced as an scFv and fused to an Fc, while the 1D3 VL was cloned in frame with a kappa light chain constant region and the 1D3 VH was cloned into an IgG heavy chain expression vector that contained mutations in the Fc region that lower the isoelectric point (FIG. 9A, white stars). The bispecific construct also includes mutations that disrupt binding to Fc receptors (FIG. 9A, black stars). The expression plasmids were co-transfected into 293E cells and purified by protein A-affinity followed by anion exchange chromatography, the latter of which yielded two peaks at 13.1 mS and 24.8 mS (FIG. 9B). The binding of each peak was evaluated, as well as the parental recombinant 2C1 and 1D3 ai-mAbs to the VH3-21 / L1 -40 encoded anti-RSV Fab ADI- 19425 as well as chimeric Fabs with an ADI-19425 heavy chain paired with a non-VL1-40 light chain, or a Fab with an ADI- 19425 light chain paired with a non-VH3-21 heavy chain (FIGs. 9C-9F). 2C1 bound ADI-19425 and the ADI-19425 LC chimera (FIG. 9C) while 1D3 bound ADI-19425 and the ADI-19425HC chimera (FIG. 9D). The ai-mAb that eluted in the first peak of the anion exchange column bound ADI-19425 and both chimeras, while the ai-mAb from the second peak bound the ADI-19425HC chimera. The desired bispecific ai-mAb eluted in the first peak while the second peak contained an ai-mAb in which both Fab arms are 1 D3. This was further confirmed by running both peaks on an SDS-PAGE gel under non-reducing conditions (FIG. 10). While the second peak demonstrated a molecular weight equivalent to an IgG standard (150 kDa), the first peak had a lower weight (<150 kDa) indicative of heterodimeric pairing of a standard IgG HC-LC pair with a smaller scFv-Fc fusion protein, 126kDa (FIG. 10). The desired 2C1-1D3 bispecific ai-mAb elutes at 13 mS which was used in subsequent experiments.

[0288] Fluorescently Labeled Bispecific ai-mAb as Bait for Single Cell Sorting and BCR Gene Analysis. To determine whether the bispecific ai-mAb could more efficiently engage naive human B cells expressing VH3-21A / L1 -40-encoded BCRs, fluorescently labeled bispecific was used as bait in single B cell sorting experiments (FIGs. 11 A, 12, and 17). Naive B cells that were labeled with the bispecific ai-mAb (FIG. 11A right) were single cell sorted, their VH and VL transcripts recovered by RT-PCR (Tiller et al., J Immunol Methods 329, 112-124, 2008 (“Tiller”)), and sequenced to determine their gene usage using the IMGTA / -QUEST online tool (Brochet ef al., Nucleic Acids Res 36, W503-508, 2008 (“Brochet”); Giudicelli et al., Cold Spring Harbor protocols 2011, 695-715, 2011). Sequencing results from 3 independent sorting experiments each using PBMC samples from a unique donor were combined for analysis, representing 270 sorted B cells yielding 211 productive HCs, 216 productive LCs and 159 productive paired BCR sequences (FIG. 11 B). The ai-mAb selected for BCRs that individually expressed the VH3-21 HC at a rate of 87% (± 3.5%) and the VL1-40 LC at a rate of 43.2% (± 9.6%) (FIG. 11 B). These represent 26.5- and 7.5-fold enrichment, respectively, over estimated gene frequencies determined by unbiased high-throughput sequencing of naive paired BCRs from 3 unique donors (DeKosky) (FIG. 11 B).

[0289] Moreover, the bispecific was able to efficiently engage B cells expressing the desired VH3-21 / VL1-40 BCR gene pairing. Among ai-mAb sorted B cells for which paired sequences of the heavy and light chain were recovered, 40.6% (± 6%) expressed VH3-21 / VL1-40 pairs, representing a 209-fold enhancement over estimated gene frequencies (FIG. 11 B). This dramatic enrichment demonstrates that the bispecific antibody has a high-degree of specificity for VH3-21 / L1-40 BCRs and selectively targets B cells expressing this desired gene pair.

[0290] VH3-21A / L1 -40 BCRs Sorted with the Bispecific ai-mAb Cross React with Pre-fusion RSV F. In all, 65 unique VH3- 21 / VL1 -40 BCRs were recovered over 3 independent sorting experiments. To determine the functional properties of these sorted B cells, mAbs were produced using a high throughput expression platform (FIGs. 13A, 13B) (Liao et al., J Virol Methods 158, 171-179, 2009 ("Liao”)). Culture supernatants were screened by BLI for binding to 2C1 Fab to confirm mAb expression, and against RSV preF (DS-Cav1)(McLellan 1) and postF (McLellan et al., J Virol 85, 7788-7796, 2011 ("McLellan 2”)) to determine functionality (FIGs. 13A, 13B). Antibodies that demonstrated binding to preF, as well as those that were not successfully expressed in this HTP method, were subsequently cloned into lgG1 expression plasmids, expressed in 293 cells, and purified by protein A affinity chromatography. 60 of the 65 sorted BCR sequences were successfully purified as recombinant mAbs. Overall, 18 / 60 or 30% of the sorted germline VH3-21 / VL1 -40 mAbs showed binding to RSV preF while none exhibited reactivity to postF (FIGs. 11 C, 13B).

[0291] The majority of RSV-binding mAbs that use the VH3-21 / VL1 -40 gene pairing target antigenic site III on RSV preF (Gilman; Corti; Goodwin 1; Caban etal., Nat Commun 14, 798, 2023 ("Caban”)). To evaluate whether the VH3-21 / L1-40 mAbs sorted with the bispecific ai-mAb target site III on RSV preF, competitive binding assays were performed by BLI. The majority of VH3-21 / VL1-40 sorted mAbs competed with ADI-14337 for binding to preF, consistent with site III binding (FIG. 11D). Two antibodies showed incomplete inhibition by ADI-14337, indicating that they may target an epitope that partially overlaps with ADI-14337 on preF (FIG. 11 D). Collectively, these data demonstrate that the bispecific ai-mAb selectively engages VH3-21 / VL1-40 BCRs that target a site III epitope present on preF.

[0292] Sorted VH3-21 / VL1 -40 Germline mAbs Show Neutralization Against Both RSV Subtypes. To determine whether the sorted VH3-21 / VL1-40 mAbs were neutralizing, they were tested against RSV subtypes A (A2) and B (B1) using plaque reduction assays (Caban). All mAbs were initially tested at a set concentration of 500 pg / mL (FIGs. 14A, 14B). Three mAbs (MLR_15, MLR_24, MLR_55) showed neutralization and were titrated to determine their half maximal inhibitory concentration (IC50, FIGs. 15A, 15B). Two of these antibodies exhibited weak or undeterminable potency against RSV-A (MLR_55: > 500 g / mL, MLR_15: 173.8 pg / mL), while the third showed significantly higher activity (MLR_24: 24.2 pg / mL) (FIGs. 15A, 15B). The mAbs showed a similar hierarchy but were more potent against RSV-B (MLR_55: 229.6 pg / mL; MLR_15: 24.5 pg / mL; MLR_24: 0.23 pg / mL) (FIGs. 15A, 15B). Interestingly, MLR_24 demonstrated 100 fold more potency against RSV-B than RSV-A. MLR_24 showed comparable potency to the contemporary prophylactic, palivizumab, and the VH3-21 / VL1 -40 mAb AD114337 against RSV-B (Pediatrics; Goodwin 1) (FIGs. 15A, 15B). Thus, among the population of naive VH3-21 / VL1-40 antibodies sorted with the bispecific ai-mAb, 3 out of 60 (5%) had neutralizing activity against RSV. This is the first instance of targeting an RSV-neutralizing B cell using an anti-idiotype immunogen and demonstrates the feasibility of using this vaccine platform to target defined, protective, B cell lineages.

[0293] Binding Kinetics of VH3-2WL 1-40 mAbs Dictate Neutralization Potential. It was next assessed whether differences in avidity among VH3-21 / VL1 -40 mAbs could discriminate between those that neutralize and those that do not. To this end, their relative steady-state binding to RSV-A preF was measured by BLI. mAbs MLR_15 and MLR_24 demonstrated the highest binding responses over a range of mAb dilutions (FIG. 14C), while MLR_55 was relatively lower in the response hierarchy (FIG. 14C). An examination of the dissociation rates (kd) from this data set revealed that MLR_55 had the slowest dissociation rate of all expressed mAbs (FIG. 14D). From this analysis, the neutralizing mAbs separate themselves from the non-neutralizers based on overall binding avidity and / or slow dissociation rates, demonstrating that avidity is a key driver of neutralization potency.

[0294] Tofurtherrefinethe avidity analysis of the neutralizing mAbs (MLR_15, MLR_24, MLR_55), the steady state binding to RSV-B (Joyce et al., Pathog Immun 4, 294-323, 2019 (“Joyce”)) preF and RSV-A were compared (FIG. 15C, dotted vs. solid line). This analysis revealed that the neutralizing VH3-2WL1-40 mAbs bound stronger to RSV-B preF compared to RSV-A (FIG. 15C). At the higher end of the dilution series, M LR_15 showed higher binding to RSV-A preF, but this reversed at the lower end of the dilution series (FIG. 15C left). The apparent affinity (KD) for each neutralizing mAb against RSV-A / RSV-B preF (FIG. 15D) was also determined. All mAbs demonstrated higher apparent affinity for RSV-B vs. RSV-A, with MLR_24 showing a 2.8-fold increase in apparent affinity. The stronger binding to RSV-B preF by these mAbs supports their higher potency to neutralize this subtype vs. RSV-A.

[0295] Bispecific ai-mAb Discriminatively Activates B cells with Desired BCR Gene Pairing. B cells sorted with an antigen are representative of those that will respond to a matched immunization in humans (Jardine et al., Science 351, 1458- 1463, 2016 (“Jardine”); Havenar-Daughton et al., Sci Transl Med 10, 2018 (“Havenar-Daughton”); Leggat etal., Science 378, eadd6502, 2022 (“Leggat”)). Because the bispecific ai-mAb engages natively paired BCRs with the VH3-21 / VL1 -40 gene pairing (FIGs. 11A-11D), it was hypothesized that this could be used as an immunogen to activate these target B cells. However, the binding and sorting data in FIGs. 9A-9F and 11A-11D also indicate that the bispecific ai-mAb can bind off-target BCRs with either target HC or LC, but without the desired pairing. Since B cell activation calls for cross-linking of adjacent cell-surface BCRs, it was hypothesized that the bispecific ai-mAb will preferentially activate paired, target B cells. To test this directly, B cell lines were generated stably expressing the ADI-19425 BCR, the ADI-19425 HC paired with an irrelevant light chain, or an irrelevant heavy chain paired with the ADI-19425 light chain. The B cell lines were stained with fluorescently labeled 1D3 and 2C1 ai-mAbs, as well as the 1D3 / 2C1 bispecific (FIGs. 16A-16C). 1D3 bound to B cells expressing the ADI-19425 BCR (black) and the B cells expressing the BCR derived from the ADI-19425 HC paired with an irrelevant light chain (blue), while 2C1 bound to the ADI-19425 BCR and the irrelevant HC / ADI-19425 LC chimera (green) (FIGs. 16A-16C). The bispecific ai-mAb showed bright staining of the ADI-19425 cells (FIG. 16C, black A), but modest staining of the ADI-19425 HC I irrelevant LC chimera cell line (FIG. 16C, upside down triangle). The bispecific ai-mAb did not bind the irrelevant HC I ADI-19425 LC chimeric cells, likely due to the fact that the chimeric BCR lacks contact residues encoded by the VH3-21 heavy chain (FIGs. 7C and 7D), which results in an increased dissociation rate and weaker binding (FIGs. 9C and 9E).

[0296] The ability of the mono- and bispecific ai-mAbs to activate the BCR-expressing cell lines from FIGs. 16A-16C was measured using a flow cytometry-based calcium flux assay (FIGs. 16D-16F) (Hoot et al., PLoS pathogens 9, e1003106,2013 ("Hoot"); McGuire et al., Science 346, 1380-1383, 2014 ("McGuire")). 1D3 and 2C1 activated cells expressing the ADI-19425 BCR, as well as the VH3-21 and VL1-40 chimeric BCRs, respectively (FIGs. 16D, 16E). In contrast, the bispecific ai-mAb activated the ADI-19425 cell line (FIG. 16F). None of the ai-mAbs activated DG75 cells expressing a non-VH3-21 / non-VL1-40 control BCR (FIGs. 16D-16F) (Seydoux 1).

[0297] To further evaluate the specificity of the bispecific ai-mAb to activate B cells harboring the VH3-21 / VL1 -40 gene pair, three additional B cell lines were engineered expressing unmutated, non-neutralizing, non-site III, preF-specific BCRs that are from neither VH3-21 or VL1-40 (ADI-19470, ADI-24811, and ADI-25542) (Goodwin 1). All cell lines including ADI- 19425 were activated by the addition of RSV-A preF, however ADI-19425 was activated by the addition of the bispecific ai-mAb (FIGs. 16G, 16H). Collectively these data indicate that the bispecific ai-mAb preferentially activates B cells harboring paired VH3-21 / VL1-40 BCRs, and not off target B cells that would be activated by traditional subunit vaccines such as ABRYSVO™ (Pfizer) or AREXVY™ (GSK) based on RSV proteins.

[0298] These sorting data and the binding data in FIG. 9E indicate that the bi-specific antibody can also bind BCRs with either VH3-21 or VL1 -40 without the desired pairing. However, since B cell activation calls for cross-linking of adjacent cellsurface BCRs the bi-specific ai-mAb should preferentially activate target B cells (Seydoux 1). This concept is depicted in FIGs. 18A-18C.

[0299] Discussion. The work presented here demonstrates the efficacy of using an ai-mAb based approach to target a conserved B cell class capable of neutralizing RSV without the need for affinity maturation. This approach has several advantages over the traditional vaccine strategies to protect infants from RSV infection. First, this immunogen is antigenically distinct from RSV, and thus may prevent epitope masking and antigen clearance that has been seen in the presence of maternal antibodies. In line with this, the vaccine approach complements and could be implemented in the context of maternal vaccination or prophylactic mAb transfer, priming the infants’ immune system to prevent infection at the point that transferred antibodies no longer provide protection. Secondly, the immunogen targets a unique class of B cells that are primed to produce neutralizing antibodies without the need for affinity maturation. This is a dramatic benefit over existing methodologies because it circumvents the inherent challenges of infant vaccination, such as their diminished ability to generate somatically mutated antibodies. Further, the vaccination can confer protection after a single dose.

[0300] This example represents the first instance of using an ai-mAb to select for RSV neutralizing B cells from a naive cell population. An important observation in the development of the anti-idiotypes, is that no single ai-mAb is able to simultaneously select for VH3-21 / V1 -40 paired BCRs. While the anti-idiotype 2C1 was able to sort out B cells with VL1-40 LCs and 1D3 B cells with VH3-21 HCs, neither was able to efficiently select for VH3-21 / L1 -40 paired BCRs. Here, the use of an Fc platform that allows for discrimination and purification of bispecific antibodies is described (Moore). Compared to previous work using the spy-catcher I spy-tag bispecific format, the presence of the Fc on the current immunogen allows for an extended half-life and recirculation through the FcRn. Importantly, in this format, Fey interactions have been silenced with mutations introduced in the constant Fc region. This silencing can be important as the presence of an Fc on an ai- mAb can lead to the death of target B cells, presumably through Fc-mediated killing (Dosenovic).

[0301] The historic failure of the first formalin inactivated RSV vaccine trial set back the infant vaccination field drastically. However, this trial also identified outcomes that should be avoided in next generation RSV vaccines. Within this, theelicitation of non-neutralizing antibodies targeting RSV postF is postulated to be responsible in some part for the ERD seen in participants from this initial trial (Killikelly; Kim; Kapikian) following subsequent natural infection. While recent antigen engineering advances have been able to structurally lock the fusion protein in its pre-fusion conformation, it is widely determined that subunit-based vaccines are less desirable for RSV-na'ive infants. Alternatively, it has been proposed that live-attenuated and vectored vaccines exhibit an acceptable safety profile for use in infants. This is due to multiple factors, such as the ability for intranasal deliveries, eliminating injections and activating immune responses at the point of infection. Indeed, live-attenuated and vectored vaccines to protect infants from RSV-related LRTI are advancing through clinical trials (Stuart et al., J Infect Dis 227, 71-82, 2022; Karron et al., J Infect Dis, 2023). Despite these promising results, questions remain surrounding antigenic match between delivered RSV strain immunogens and those that are contemporarily circulating as well as doses needed to confer protection.

[0302] Data Availability. Coordinates and structure factors for both ADI-19425 Fab - 1D3 Fab and ADI-19425 Fab - 2C1 Fab structures have been deposited to the Protein Data Bank (PDB) under accession codes 8VS8 (ADI-19425 Fab - 1D3 Fab) and 8VS7 (ADI-19425 Fab- 2C1 Fab). In addition, the following publicly available datasets are mentioned: PDB ID: 1D5I, PDB ID: 5I76, PDB ID: 6P67 and PDB ID: 6APC.

[0303] Material and Methods. Recombinant Antibodies. The heavy and light chain variable regions of ADI-19425 (VH Genbank: MG524063, VL Genbank: MG524528), ADI-25532 (VH Genbank: MG524182, VL Genbank: MG524647) and ADI-14437 (VH Genbank: MG524251, VL Genbank: MG524716) were codon optimized and cloned into pTT3-based lgG1 expression vectors with human constant regions (Snijder) using 5x In-Fusion HD Enzyme Premix (Takara Bio Cat#639650) according to the manufacturer’s instructions.

[0304] VH3-21 / VL1-40 sorted BCRs were expressed as recombinant antibodies in a high-throughput, amplicon-based expression system as described below. Those antibodies which showed measurable binding to RSV-A preF by BLI were further cloned into expression systems as described above, as well as those that did not express in this high-throughput system.

[0305] Generation of ai-mAb. Mice were injected 3 or 5 times with a cocktail of ADI-19425, ADI-25532 and ADI-14337. 3 days after the final injection spleens were harvested and used to generate hybridomas. Hybridoma supernatants were initially screened for binding to a panel of mAbs including ADI-19425, ADI-25532, ADI-14337, the anti-EBV mAb AMMO1 , a chimeric mAb with a VL1-40 light chain and a non-VH3-21 heavy chain (AMM01 -HC / ADI-19425LC) and a chimeric mAb with a VH3- 21 heavy chain and a non-VL1-40 light chain (ADI-19425LC / CL40 light chain). Hybridomas from wells containing supernatant that showed a strong binding signal to ADI-19425, ADI-25532, ADI-14337 and weaker or no binding to the control mAbs were plated onto semi-solid media and single colonies were used to establish monoclonal cell lines using a Clonepix instrument. To purify mAbs, hybridomas were cultured in serum free media cells and cellular debris was removed by centrifugation at 4,000 x g followed by filtration through a 0.22 m filter. The clarified supernatant was diluted with an equal volume of Pierce™ Protein G IgG Binding Buffer (ThermoFisher Scientific Cat# 21011) and then passed over a Protein G Agarose (ThermoFisher Scientific Cat # 15920010) column, washed with 5 column volumes of Protein G IgG Binding Buffer and then eluted in 1 ml fractions of Pierce IgG Elution Buffer, pH 2.0 (ThermoFisher cat. # 21004) into 0.1 ml of Tris HCI, pH 8.0. Purified mAbs were then concentrated and buffer exchanged into PBS using an Amicon filter unit (UFC903024).Aliquots of purified mAbs were biotinylated using the EZ-Link™ NHS-PEG4-Biotin kit (ThermoFisher Scientific Cat# A39259) at a theoretical 1:1 ratio of mAb to biotin. Purified mAbs were flash frozen and stored at -20°C until use.

[0306] To produce recombinant ai-mAb, RNA was extracted from 1 x 106cells using the Monarch® Total RNA Miniprep Kit (New England Biolabs Cat # T2010S, Ipswich, MA) and cDNA encoding the heavy and light chain variable regions of the murine hybridomas were reverse transcribed and amplified using the procedures outlined in Meyer et al., PloS one 14, e0218717, 2019. Amplicons were sanger sequenced directly or TOPO cloned (ThermoFisher Scientific Cat# 450245) and then sequenced. The sequences were codon optimized and cloned into pTT3-based IgG expression vectors with human constant regions (Snijder) using 5x In-Fusion HD Enzyme Premix (Takara Bio Cat#639650), expressed in 293E cells and purified using Protein A chromatography.

[0307] Recombinant Protein Expression. Plasmids encoding antibody heavy and light chains were transfected into 293E cells at a density of 106cells / ml in Freestyle 293 media (ThermoFisher Cat# 12338018) using the 293Free transfection reagent (EMD Millipore Cat # 72181) according to the manufacturer’s instructions. Expression was carried out in Freestyle 293 media for 6 days after which cells and cellular debris were removed by centrifugation at 4,000 x g followed by filtration through a 0.22 m filter. Clarified cell supernatant containing recombinant mAbs were passed over Protein A resin (Goldbio Cat# P-400-50), pre-equilibrated with phosphate buffered saline (PBS; 1 mM KH2PO4, 155 mM NaCI, 3mM Na2HPO4) washed with 10 column volumes of PBS and then eluted in 1 ml_ aliquots of Pierce IgG Elution Buffer, pH 2.0 (ThermoFisher Cat# 21004) into 0.1 mL of Tris HCI, pH 8.0. Purified antibodies were concentrated, and buffer exchanged into PBS using an Amicon filter unit (UFC903024). Recombinant mAbs were flash frozen and stored at -20°C until use.

[0308] Plasmids encoding RSV-A preF (DS-Cav1) and postF and the plasmid encoding RSV strain B1 preF were gifts (McLellan 1; McLellan 2; Joyce). All RSV viral protein-encoding plasmids were transfected into 293E cells as described above. Imidazole and sodium chloride were added to the cellular supernatant prior to resin capture at final concentrations of 0.01 M and 0.5M respectively. Clarified cell supernatants were then passed over Ni-NTA resin (ThermoFisher Cat# 88221), pre-equilibrated with NTA-A buffer (500mM NaCI, 10mM Tris, 0.02% azide, 10mM Imidazole, pH 7.1), washed with 10 column volumes of NTA-A buffer and then eluted in 1mL aliquots of NTA-B buffer (500mM NaCI, 10mM Tris, 0.02% azide, 500mM Imidazole, pH 8). Purified viral proteins were then concentrated and purified by size exclusion chromatography using a HILoad 16 / 600 Superdex S200 (GE) column fitted to a BioRad NGC system. Final fractions containing the RSV proteins were concentrated, analyzed by SDS-Page and BLI, and flash frozen for future use.

[0309] ADI-19425, 1D3 and 2C1 IgGs were digested into Fabs using Endoproteinase Lys-C (New England BioLabs Cat# P8109S) by adding 1 pig of LysC per 10mg of IgG and the mixture incubated overnight at 37°C. Following digestion, mixtures were incubated with Protein A resin for 1-2 hours to remove any remaining Fc or undigested IgG. Resulting Fabs were further purified using an Enrich SEC 650 (BioRad) column and concentrated.

[0310] Generation of a VH3-21 / VL1-40 Targeting Bispecific ai-mAb. A recombinant VH3-21 and VL 1-40 targeting bispecific ai-mAb with a “silent Fc" was developed using the platform described in Moore. Codon optimized cDNA encoding a signal peptide, the 1D3 variable heavy chain region and the human lgG1 constant domain harboring N208D, Q295E, N384D, Q418E, N421D, L368D, K370S, E233P, L234V, L235A, G236del, and S267K mutations (Chain A) was synthesized by Twist Biosciences and cloned into pTT3. A leader peptide followed by a 2C1 scFv (VH-GKPGS4 (SEQ ID NO: 60)-VL)fused to Human lgG1 residues 216 through 447 containing a C220S, E357Q, and S364K mutations (Chain B) was synthesized by Twist Biosciences and cloned into pTT3. pTT3 plasmids encoding Chain A, Chain B and the 1 D3 light chain were co-transfected into 293E cells and purified using Protein A chromatography as described in “recombinant protein expression”. Purified antibodies were then concentrated, buffer exchanged into 50 mM Tris pH 8.5, and purified on a Hi- Trap Q HP (Cytiva) anion exchange column pre-equilibrated with 50 mM Tris, pH 8.5 and eluted over a gradient to 50% elution buffer (50 mM Tris, 1 M NaCI, pH 8.5) over 10 column volumes. Fractions containing the bispecific antibody were concentrated and then further purified by size exclusion chromatography on a HILoad 16 / 600 Superdex 200 (GE) column. Final fractions containing the bispecific antibody were analyzed by SDS-PAGE, tested for binding specificity, concentrated, buffer exchanged into PBS, snap frozen and stored at -80°C until use.

[0311] B Cell Sorting. Bulk Human B cell sorting. Cryopreserved PBMCs were thawed and resuspended in 200pil of EasySep buffer (1X PBS, 2% heat inactivated fetal bovine serum, 1 M EDTA). B cells were isolated using the Human B Cell Enrichment Kit (Stem Cell Cat # 19054) according to the manufacturer’s instructions. Enriched B cells were resuspended in 200 L EasySep buffer and incubated with 10 L rat serum, 10 L Mouse Serum, 10 piL mouse-anti-human CD32(BD Cat# 551900), W L mouse-anti-human CD23(BD Cat# 555707) and W L mouse-anti-human CD16 (BD Cat# 550383). Cells were then washed with EasySep buffer and resuspended in 200piL EasySep buffer containing CD19-BV711 (Biolegend Cat# 302246) at a 1:200 dilution, CD27-PE-Cy7 (eBiosciences Cat# 25-0271-82) at a 1:600 dilution, CD14- FITC (BD Pharmingen Cat# 557153) at a 1:60 dilution, CD3-FITC (BD Pharmingen, 556611) at a 1:60 dilution, CD20- AF700 (Biolegend Cat# 302322) at a 1 :300 dilution, I gD-PerCP-Cy5.5 (eBiosciences Cat# 46-9868-42) at a 1 : 120 dilution, lgM-BV605 (Biolegend Cat# 314524) at a 1:120 dilution, Fixable Viability Dye, V500 (eBiosciences Cat# 65-0866-14) at a 1:300 dilution, and I) biotinylated murine ai-mAb conjugated to Total SeqC-0960 APC streptavidin (Biolegend Cat# 405157), Total SeqC-0959 APC streptavidin (Biolegend Cat # 405159), Total SeqC-0958 APC streptavidin (Biolegend Cat# 405293), Total SeqC-0957 APC streptavidin (Biolegend Cat# 405285), or Total SeqC-0956 APC streptavidin (Biolegend Cat# 405293), ii) recombinant ai-mAb labeled with APC and PE (separately labeled pools) using Zenon™ Human IgG Labeling Kits (ThermoFisher Scientific Cat# Z25451 and Z25455) murine ai-mAb labeled with Zenon-PE. Cells were then washed with 5mL of EasySep buffer and suspended in 0.5 mL of EasySep buffer and subjected to analysis on a FACSymphony S6 (BD Biosciences). Naive B cells were defined as live (V500-), CD14-, CD3-, CD19+, CD20+, lgM+, lgD+, CD27-. Naive ai-mAb+(APC+, PE+) B cells were bulk sorted and BCR libraries were prepared using the Chromium Single Cell Human BCR Amplification Kit (10X genomics Cat# 1000253).

[0312] Libraries from naive B cells that were positive for murine ai-mAb conjugated to TotalseqC streptavidin APC were generated using the Chromium Single Cell Human BCR Amplification Kit (10X genomics Cat# 1000253) and the 5' Feature Barcode Kit (1 OX genomics Cat # 1000256). All single cell libraries were separately indexed using the Dual Index Kit TT Set A (10X genomics Cat# 1000215) and sequenced on an Illumina NovaSeq or NextSeq instrument. Analysis of flow data was processed using FlowJo 10.8.1 software (Tree Star).

[0313] Analysis of Barcoded NGS Sequences. Sequence files were obtained and FASTQ data was analyzed using Cell Ranger (10x Genomics Cell Ranger 3.0.0) on an independent computing cluster. Subsequently, enclone (Jaffe et al., bioRxiv, 2022.2004.2021.489084, 2022) was used for clustering and visualization of B cell clonotypes in association withspecific barcodes. Both enclone and Cell Ranger are programs developed and published by 10x Genomics used for downstream analysis of 10x compatible sequencing datasets (Pleasanton, CA). Briefly, Cell Ranger was run using sequencing data mentioned above in order to demultiplex using unique molecular identifier (UMI), index, and unique 10x compatible barcodes (totalSeqC Barcodes) present in the sequencing data. The output of Cell Ranger is demultiplexed data compatible with downstream use in enclone software. Enclone was then used to enumerate and visualize the grouping of VDJ sequences in association with respective 10x feature barcode. By extension, BCRs were assigned to the ai-mAB they were tagged with during sorting. Datasets produced in enclone were exported as CSV files and R studio was used to visualize data further.

[0314] Analysis focused on B cells with a single heavy-light chain pair. The feature barcode counts for each antigen were analyzed using the chngpt package in R (Fong) for a segmented generalized linear model to determine a threshold for positivity (FIGs. 4A, 4B). B cells above the threshold were considered positive for a given ai-mAb. The iv2 mAb labeled with a barcoded APC-barcode conjugate was included as a control for non-specific binding to murine IgG constant regions, or the SA-APC reagents. B cells that were above the threshold for the iv2 barcode were excluded from subsequent analysis.

[0315] Single B Cell Sorting and Sequencing. In single B cell sorting experiments, B cells were thawed and enriched using magnetic beads from cryopreserved PBMCs as described above. Enriched B cells were resuspended in 200piL EasySep buffer and incubated with 10 L rat serum, 10 L mouse serum, 10 L mouse-anti-human CD32 (BD Cat# 551900), 10 L mouse-anti-human CD23 (BD Cat# 555707) and 10piL mouse-anti-human CD16 (BD Cat# 550383). Cells were then washed with EasySep buffer and resuspended in 200 L EasySep buffer containing CD19-BV711 (Biolegend Cat# 302246) at a 1:200 dilution, CD27-PE-Cy7 (eBiosciences Cat# 25-0271-82) at a 1:200 dilution, CD14- PE (BD Pharmingen Cat# 555398) at a 1:100 dilution, CD3-PE (BD Pharmingen Cat# 556612) at a 1:100 dilution, CD20-AF700 (Biolegend Cat# 302322) at a 1:250 dilution, lgD-PerCP-Cy5.5 (eBiosciences Cat# 46-9868-42) at a 1:100 dilution, lgM-BV605 (Biolegend Cat# 314524) at a 1:100 dilution, and Fixable Viability Dye-V500 (eBiosciences Cat# 65-0866-14) at a 1:200 dilution. The bispecific antibody was conjugated to DyLight 488 NHS Ester (ThermoFisher Scientific Cat # 46403) according to the manufacturer's instructions, and used at a 1:5,000 dilution. After a 30-minute incubation at 4°C, cells were washed with 5mLs of EasySep buffer, resuspended in 400pL PBS + 2% BSA, and subjected to analysis on a FACSARIA II (BD Biosciences). Naive B cells that stained positive for the bispecific antibody (AF488j were single-cell sorted into individual wells of 96 well plates and then were snap frozen on dry ice. Analysis of flow data was processed using FlowJo 10.8.1 software (Tree Star).

[0316] To sequence the VH / VL pairs from single sorted B cells, cDNAwas generated using Superscript IV (ThermoFisher Scientific Cat# 18091050) and the VH and VL sequences were recovered using gene specific primers and cycling conditions previously described (Tiller). VH and VL amplicons were sanger sequenced (Genewiz) and assigned antibody gene usage using IMGT V-quest (Brochet).

[0317] High Throughput mAb Production. To assess the functionality of BCRs identified in single cell sorting experiments, a high-throughput antibody production and screening method was adapted from Liao. The CMV promoter and leader sequence from the pTT3 lgG1 expression vector was amplified by PCR with the Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific Cat#12368050) and gel purified with the Monarch Gel Extraction Kit (New England BioLabsCat#T1020S). Further, the heavy chain and light chain constant regions up to the Poly A tail were amplified and purified from the pTT3 lgG1 and IgL expression vectors in the same way. Paired heavy-chain and light chain variable regions derived from the VH3-21 and VL1-40 genes were codon optimized and synthesized by IDT. Homology arms complimentary to the leader sequence or lgG1 1 IgL constant regions were added to the 5' and 3' ends, respectively. In cases where nucleotide sequence reads from the sorting experiment had uncalled bases, these were reverted to the germline sequence. The components (the CMV promoter, mAb variable region, mAb constant region and Poly A tail) were assembled with the 5x In-Fusion HD Enzyme Premix (Takara Bio Cat# 639650) according to the manufacturer's instructions, further amplified by PCR with flanking primers, and purified with the Monarch PCR & DNA Clean Up Kit (New England BioLabs Cat# T1030S). Heavy and light chain antibody amplicons were then co-transfected into 293E cells (2mL at 1X106cells / ml) in 24 deep well plates, and the supernatants were harvested after 5 days in culture with shaking at 250rpm.

[0318] Biolayer Interferometry. BLI assays were performed on the Octet Red Instrument (Forte Bio) at 30°C with shaking at 1,000 RPM.

[0319] Bispecific ai-Ab Binding Screens. The FPLC-purified bispecific antibody was captured using Anti-Human IgG capture (AHC, Sartorius Cat#18-5060) biosensors by immersing sensors into 250 L KB buffer (1X PBS, 0.01% BSA, 0.02% Tween 20, and 0.005% NaNs) with the bispecific at a concentration of 80nM for 200s. 1D3 IgG and 2C1 IgG were captured in the same fashion in parallel. After loading, the baseline signals were recorded for 60s in KB. The sensors were then immersed into wells containing ADI-19425 Fab, ADI-19425HC I AMMO1 LC chimeric Fab, iv8 HC I ADI-19425 LC chimeric Fab, or a negative control Fab at concentrations of 200nM for 300s (association phase). Following this first immersion, sensors were next immersed in KB for an additional 300s (dissociation phase). The background signal obtained from sensors immersed in the negative control Fab were subtracted from each individual ai-mAb or the bispecific antibody.

[0320] High-Throughput mAb Screens. Total antibody recovered from the high-throughput production pipeline was assayed for binding to RSV-ApreF (DS-Cavl) and postF by BLI (McLellan 1; McLellan 2). Briefly, antibodies were captured by immersing AHC biosensors into 250 L of supernatant harvested from mAb-transfected 293E cultures on day 5 for 200s. After loading, baseline signals were recorded for 60s in KB. The sensors were then immersed in wells containing RSV-A preF or postF at concentrations of 140nM in 250ul KB for 300s. Following this, sensors were immersed in KB for an additional 300s. The binding screens included ADI-19425 and palivizumab as positive controls and VRC01 (Pediatrics; Goodwin 1; Wu et al., Science 329, 856-861, 2010 (“Wu”)) as a negative control.

[0321] Kinetic Analysis of ai-mAbs and Neutralizing VH3-21 / VL1-40 mAbs. AHC sensors were immersed in KB containing 67nM of 1D3 IgG or 2C1 IgG for 300s. After loading, the baseline signal was recorded for 60s in KB. Next, sensors were immersed in wells containing ADI-19425 Fab at serial dilutions ranging from 250nM to OnM for 300s (association), followed by dissociation in KB for an additional 300s. As a negative control, wells containing no mAb were used, and the background signal from these wells were subtracted from the mAb containing wells. Curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software. Mean kaand kd values were determined by averaging all binding curves that matched the theoretical fit with an R2value of > 0.99. The average KD for each ai-mAb was determined by dividing the average kd by the average ka.

[0322] Steady State Kinetic Analysis of Sorted VH3-21 / VL1-40 mAbs. Purified VH3-21 / VL1-40 mAbs recovered from sorting experiments that showed measurable binding to RSV-A preF were further analyzed for steady state binding kinetics by BLI. VH3-21 / VL1-40 mAbs were immobilized on AHC sensors at a concentration of 67nM in KB for 300s. Following loading, baseline signals were recorded for 60s in KB. As the association step, sensors were immersed in wells containing serial dilutions of RSV-A preF or postF from 250 nM to OnM in KB for 300s, followed by immersion into wells containing KB to measure dissociation for 300s. Double subtractions of parallel wells containing an irrelevant EBV mAb (E1D1) (Sathiyamoorthy eta / ., Nat Commun 7, 13557, 2016) as well as non-mAb loaded sensors associated in RSV-A preF were applied to each sample. Curve fitting was performed using a 1:1 binding model in the ForteBio data analysis software. Steady state Response values (average Rmaxvalues for final 5s of association step) were plotted for each mAb concentration in the dilution series. All experiments were performed twice, and the mean Response was plotted. Additionally, the average dissociation rates (kd) measured over the whole concentration series under these conditions were plotted for each mAb.

[0323] For RSV-neutralizing VH3-21 / L1-40 mAbs (MLR_15, MLR_24, MLR_55), steady state binding kinetics were calculated for RSV-B preF. The assay described above was repeated using RSV-B preF, after which curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software. Steady state Response values (average values for final 5s of association step) were plotted for each mAb concentration in the dilution series. In addition, average apparent affinity (bivalent KD) was calculated for these mAbs as described above for ai-Fabs to ADI-19425.

[0324] Competition Assays. VH3-21 / VL1-40 mAbs were biotinylated using the EZ-Link™ NHS-PEG4-Biotin kit (ThermoFisher Scientific Cat# A39259) at a theoretical 1:1 ratio of mAb to biotin. Biotinylated mAbs were immobilized on SA sensors at 10OnM in KB buffer for 300s, after which a baseline signal was read for 60s in KB. Sensors were then immersed for 300s in wells containing 25nM of RSV-A preF that had been pre-incubated with 250nM of the previously characterized RSV mAb ADI-14337(site III) or VRC01 (control mAb) in KB for 1 hour at 37°C (Goodwin 1; Wu). Following this, the dissociation rate was measured in KB for 300s. Assays were repeated twice, and mean values shown. The following equation was used to calculate the % of Max Binding in the presence of competing mAbs: % of Max Binding = (RmaxpreF + ADI-143371 RmaxpreF +VRC01)*100

[0325] Generation of B Cell Lines. BCR expression constructs were designed as previously described (Hoot; McGuire et al., The Journal of experimental medicine 210, 655-663, 2013). cDNA including a signal peptide-rearranged VDJ-IgG constant region (membrane anchored splice variant)-furin cleavage site-T2A self-cleaving peptide-signal peptide- rearranged VK or VL and kappa or lambda constant regions were codon-optimized and synthesized, and cloned into pTwist Len SFFV Puro WPRE to create pTwist Lenti SFFV Puro WPRE-ADI-19425-BCR, pTwist Lenti SFFV Puro WPRE-ADI- 19425HC / AMMO1LC-BCR, pTwist Lenti SFFV Puro WPRE-iv8HC / ADI-19425LC-BCR, pTwist Lenti SFFV Puro WPRE- ADI-19470-BCR, pTwist Lenti SFFV Puro WPRE-ADI-24811-BCR and pTwist Lenti SFFV Puro WPRE-ADI-25542-BCR. pTwist Lenti-based BCR expression constructs were co-transfected with psPAX2 (Addgene plasmid # 12260, n2t.net / addgene:12260, RRID:Addgene_12260) and pMD2.G (Addgene plasmid # 12259, n2t.net / addgene: 12259, RRID:Addgene_12259) at a 4:2:1 ratio into suspension adapted 293Tcells in Freestyle 293 media (ThermoFisher Cat# 12338018) using 293-Free Transfection reagent (Millipore Sigma Cat# 72181 ) according to the manufacturer’s instructions.4 days later the culture was centrifuged at 300X g to remove cells and debris and the supernatant was passed through a 0.22pm filter. 1 mL of clarified supernatant was added to 10 mL DG-75 cells (ATCC Cat # CRL-2625, RRID:CVCL_0244) at 1X106cells / ml in cRPMI plus 2 pg / mL polybrene. 24-48 hours post-transduction and indefinitely thereafter, puromycin was added to the cell cultures at a concentration of 1 pg / mL. Generation of the DG75 cell line transduced with the gl 12A21 BCR is described in Seydoux 1.

[0326] Ai-mAb Staining of BCR-Transduced Cell Lines. Surface expression of BCR-transduced cell lines were confirmed by staining with fluorescently labeled ai-mAbs and subsequent analysis via flow cytometry. Briefly, parental ai-mAbs 1D3 and 2C1 were labeled with PE and APC using the Zenon™ (Punzenberger, Austria) Human IgG Labeling Kits (ThermoFisher Scientific Cat # Z25451 and Z25455) respectively. These labeled antibodies in addition to the DyLight-488 labeled bispecific were used to surface stain BCR-transduced cell lines. BCR expression was assessed on a FACSymphony A5 (BD Biosciences) flow cytometer and analyzed using FlowJo 10.8.1 software (Tree Star).

[0327] B Cell Activation in BCR-Transduced Cell Lines. Calcium mobilization in B cells stably expressing BCRs upon bispecific antibody stimulation was monitored as previously described (Hoot; McGuire). Briefly, cells were loaded with Fluo- 4 Direct calcium indicator (Invitrogen, Carlsbad CA), mixed 1:1 with complete RPMI at 37°C for 1 hour. Cells were pelleted and stained with PE-conjugated anti-human Fey Fab (Jackson ImmunoResearch Cat# 109-117-008) (1:100 dilution in 100 pL complete RPMI plus Fluo-4 Direct) for 30 min. The cells were washed with 5 mL of complete RPMI and resuspended at 2X106cells / mL in complete RPMI and subjected to Ca2+flux analysis at a medium flow rate on an FACSymphony A5 (BD Biosciences) flow cytometer. In all cases BCR transduced cells were mixed with untransduced cells as an internal control at a 5:2 ratio.

[0328] Levels of background fluorescence (M i n FL) were determined by averaging the background Fluo-4 absorbance in cells for 30s. After that, activation of B cells expressing exogenous BCRs by the various immunogens was determined by monitoring changes in Fluo-4 fluorescence associated with cells expressing the exogenous BCRs (PE+cells) for 210s. The bispecific ai-mAb, parental ai-mAbs (1D3, 2C1), and control mAb (VRC01), were added at a final concentration of 100 nM. RSV-A preF was added to final concentration of 1 piM. An a-IgG Fey F(ab')2 (Jackson ImmunoResearch Cat: 109-006-008) was added at a final concentration of 10OnM as a positive control, lonomycin was added to a final concentration of 150 nM for 60s following the immunogen addition and maximum Fluo-4 fluorescence (Maxpi) was established by averaging the Fluo- 4 fluorescence signal recorded during the last 10s. The percent of maximum Fluo-4 fluorescence at each time point t was determined using the formula:(Fluorescence at f-Minpi) I (MaxpL-Minpi) 100.This analysis was performed on both the BCR positive (anti-Human Fcy-PE j and BCR negative cells (anti-Human Fcy-PE ) simultaneously. The background Fluo-4 fluorescence signal from the BCR negative cells was subtracted from that of the BCR positive population at each time point. All flow analysis was done with FlowJo 10.8.1 software (Tree Star).

[0329] Fab-Fab Complex Formation. ADI-19425 and 1D3 Fabs were mixed at a 1.5:1 (ADI-19425: 1D3) molar ratio and kept at 4°C rotating overnight. The resulting complex was purified using a HiLoad 16 / 600 Superdex200 (GE) SEC column, resulting in a major peak including the Fab-Fab complex and a minor peak including the excess ADI-19425 Fab. Fractions from the major peak were pooled and concentrated to 10mg / mL. ADI-19425 and 2C1 Fabs were mixed at a 1.2:1 (ADI-19425:2C1 ) molar ratio and complexed and purified the same way as the ADI-19425 - 1D3 complex. The ADI-19425 - 2C1 complex was concentrated to 9.8mg / mL.

[0330] Crystallization and Data Collection. For both complexes, crystallization conditions were screened using the vapor diffusion method and monitored with a Formulatrix NT8 drop setting and Rock Imager.

[0331] ADI-19425 Fab - 1D3 Fab complex screening was done with Clear Strategy I and II, MCSG1-3, ProPlex (Molecular Dimensions), WPS1 and 2 (Rigaku), Xtal HT, and an MCSG2 Additive screen (Hampton Research). Final crystals were grown in a solution of 0.1 M MES pH 6.5, 12% PEG 20K, 4% formamide and cryoprotected in solutions containing MCSG2 B1 components with 4% formamide and 40% ethylene glycol. Crystals diffracted to 2.67 A. Data was collected at ALS beamline 5.0.2 and processed using XDS (Kabsch, Acta Crystallogr D Biol Crystallogr 66, 125-132, 2010). ADI-19425 Fab- 2C1 Fab complex screening was done with MCSG1-3, Morpheus, ProPlex (Molecular Dimensions), WPS1 (Rigaku) and Xtal HT (Hampton Research). Final crystals were grown in a solution of 0.1 M Imidazole pH 6.5, 30% MPD, 12% PEG 3350, and 0.2M (NH^SO and cryoprotected in solutions containing Xtal HT D3 components with 30% ethylene glycol. Crystals diffracted to 2.41 A. Data was collected at Advanced Photon Source beamline 19-ID and processed using HKL3000 (Minor et al., Acta Crystallogr D Biol Crystallogr 62, 859-866, 2006). The data collection statistics are summarized in FIG. 6.

[0332] Structure Solution, Refinement and Analysis. The structures of both ADI-19425 Fab - 1D3 Fab and ADI-19425 Fab- 2C1 were solved by molecular replacement with Phaser in Phenix (Adams et al., Acta crystallographica. Section D, Biological crystallography 66, 213-221, 2010 ("Adams")). Chain H (heavy chain) of PDB ID: 1D5I (Mundorff et al., Biochemistry 39, 627-632, 2000) and Chain A (light chain) of PDB ID: 5I76 (Kim ef al., Appl Microbiol Biotechnol 100, 10521-10529, 2016) were used as the search model for 1D3 Fab. The anti-IL-7Ralpha 2B8 Fab of PDB ID: 6P67 (Hixon et al., Leukemia 34, 35-49, 2020) was used as the search model for 2C1 Fab. An existing ADI-19425 structure (PDB ID: 6APC (Goodwin 1)) was used as the search model for ADI-19425 Fab. All search models were divided into VL:VH and CL:CH1 components. Model building was done using Coot (Emsley & Cowtan, Acta crystallographica. Section D, Biological crystallography 60, 2126-2132, 2004; Emsley et al., Acta crystallographica. Section D, Biological crystallography 66, 486- 501, 2010) and refinement was performed in Phenix (Adams). The ADI-19425 Fab - 1D3 Fab structure had eight Fab-Fab complexes in the unit cell, but electron density was missing for four ADI-19425 CL:CH1 domains, which were not placed in the structure. The ADI-19425 Fab - 2C1 Fab structure had one Fab-Fab complex in the unit cell. The data collection and refinement statistics are summarized in FIG. 6. Structural figures were made in PyMOL (DeLano, www.pymol.org / , 2002) and Inkscape (Harrington & Engelen, www.inkscape. org, 2004). BSA analysis was done using PISA (Krissinel & Henrick, J Mol Biol 372, 774-797, 2007). For the ADI-19425 Fab - 1D3 Fab complex structure, chains E, F, G and H were used for BSA analysis.

[0333] Virus Production. Recombinant viruses RSV subtype strain A2-GFP (Cat# RSV-GFP1) and subtype strain B1-GFP (Cat# RSVB-GFP3) were uptrained from ViraTree. Viruses were cultured and viral titers determined as previously described (Caban). Briefly, RSV viruses were cultured on HEp-2 cells, and harvested by physical disruption. Viral supernatant was purified from cell pellets by centrifugation at 4,000X , followed by decanting. Viral supernatant was aliquoted in 1 mL vials, flash frozen, and stored at -80°C. Viral titers were determined through infection of Vero cells in 24- well plates over serial dilutions of the virus, overlaying with DMEM including 0.8% methylcellulose (Sigma-AldrichCAT#M0387). Fluorescent scans were completed using a Typhoon imager (GE Life Sciences) at 5 days post-infection, and plaques were quantified using Imaged (LOCI, University of Wisconsin).

[0334] Plaque Reduction Neutralization Assays. As a primary screen of neutralization activity of VH3-21 / VL1-40 mAbs, plaque reduction assays were run on Vero cells at a set mAb concentration of 500|jg / mL. Vero cells were seeded at 100,000 cells / mL in 24 well plates and cultured for 48-72 hours. VH3-21 / VL1 -40 mAbs were diluted to 500|jg / mL in DMEM and then mixed with an equal volume of RSV-A-GFP or RSV-B-GFP diluted to 2,000 pfu / mL and incubated for 1 hour at 37°C. Vero cells were then incubated with 10O L of the antibody / virus mixture for 1 hour at 37°C, followed by addition of 500|JL DMEM containing 0.8% methylcellulose. Fluorescent images were taken at 5 days post-infection using a Typhoon imager, and plaques were counted using Imaged. Plaque counts were transformed into % infectivity by dividing the well counts by those from no antibody control wells and multiplying by 100%. Antibodies that had visible neutralization capacity, reduced % infectivity vs. no antibody containing wells, were followed up for IC50 titer calculation. All antibodies were run in triplicate over a single experiment for these initial screens.

[0335] Neutralizing I C50 titers of mAbs were further determined by plaque reduction assays. Vero cells were prepared as above, and mAbs were serially diluted in 80 L of DMEM mixed with 80|uL of RSV-A-GFP or RSV-B-GFP diluted to 2,000 pfu / mL and incubated for 1 hour at 37°C. Vero cells were then incubated with 10OpiL of the antibody / virus mixture for 1 hour at 37°C, followed by addition of 500|JL DMEM containing 0.8% methylcellulose. Fluorescent images were taken at 5 days post-infection using a Typhoon imager, and plaques were counted using Imaged. Plaque counts were transformed into % infectivity by dividing the well counts by those from the highest antibody dilution for each respective antibody multiplied by 100%. % infectivity was plotted as a function of antibody dilution. The neutralization curves were fit using the log(inhibitor) versus response - variable slope (four parameters) analysis in Prism 9.4.0. The half maximal inhibitory antibody dilution I C50 was interpolated from the curve in Prism 9.5.0. Within each assay antibodies were run in duplicate, and assays were repeated 3-4 times.

[0336] Statistical Analysis. Data were analyzed, and graphs created using GraphPad Prism 9.5.0 software. Statistical tests used are included in figure legends where applicable.

[0337] Example 2 (Experimental). Generation of a knock-in mouse model to evaluate VH3-21 / L1 -40 B cell responses in vivo. Knock-in mice where pre-arranged antibody heavy and light chain variable regions are edited into the murine IgG or IgK loci were established (FIG. 26C), such that nearly all B cells in the mice express a desired heavy / light chain pair. The knock-in cells were activated to secrete antibodies by immunization of the bispecific ai-mAb (FIG. 25D).

[0338] Since mice do not express human VH3-21, or VL1-40 genes, CRISPR-Cas9 was used to cleave the IgG and IgK locus in fertilized murine eggs and knock in a single stranded DNA template encoding the rearranged ADI-14337 VDJ (FIG. 19A) and VJA regions (FIG. 19B). Although the ADI-14337 light chain is lambda, the kappa chain locus was edited since mice predominately express kappa light chains, and other lambda VL sequences have been successfully knocked in at the kappa locus in different mouse lines (Wang; Escolano; Saunders et al., 2019;366(6470).).The eggs were then implanted in pseudo-pregnant females. Pups heterozygous for the knocked in VDJ and VJA at the IgG and IgK loci were identified using diagnostic PCR. Peripheral B cells from a wildtype and knock-in mouse were co-stained with the VH3-21- specific 1D3, and VL1 -40-specific 2C1 mAbs. No B cells from a wildtype mouse stained positive with 1D3 and 2C1 (FIG.19C) while 5% of the B cells from the knock-in mouse stained double positive (FIG. 19D), indicating that the ADI-14337 BCR was successfully knocked in and expressed.

[0339] Similarly, CRISPR-Cas9 were used to knock the rearranged ADI-19425 VD J and VJA into the murine IgG and kappa loci. Peripheral B cells from a wildtype and mouse heterozygous for the knock-in BCR were stained with fluorescently labeled PreF (DS-Cav1). No B cells from the wildtype mouse stained positive, while 65% of the B cells from the knock-in mouse bound PreF (FIG. 26C, bottom), indicating that the ADI-19425 BCR was successfully knocked in and expressed. Edited B cells harvested from these knock-in mice were transferred into MD4 (anti-HEL) mice (Mason, eta / ., Int Immunol. 1992;4(2): 163-75) and immunized with the ai-mAb or an isotype control. Serum collected from mice immunized with the ai-mAb but not the isotype control bound DS Cav-1 five days after immunization demonstrating that the B cells respond to ai-mAb immunization.

[0340] Example 3 (Prophetic). This example seeks to determine how efficiently the bi-specific ai-mAb specifically engages naive B cells expressing VH3-21 / VL1 -40 pairs. This example will go one step further and determine how efficiently the bispecific ai-mAb activates target B cells in a pool of B cells with diverse BCRs. Sanjuan Nandin etal. developed a platform to monitor the antigen-specific activation of primary B cells ex vivo. They demonstrated that direct coupling an antigen to CpG, a TLR 9 agonist, and delivering it to purified B cells induced proliferation and plasma cell differentiation of antigenspecific B cells that secreted detectable levels of antigen-specific IgG and enabled the isolation of antigen specific plasma cells against tetanus toxoid, influenza HA and HIV-1 gp120 (The Journal of experimental medicine. 2017;214(8):2471 -90 ("Sanjuan Nandin”)). Here CpG will be coupled to the bi-specific ai-mAb and evaluate the ability of this antigen to stimulate an RSV-reactive B cell response ex vivo will be evaluated.

[0341] A key to this method of ex vivo B cell activation is the conjugation of CpG directly to the antigen to avoid nonspecific TLR simulation of off-target B cells. To achieve this, expression constructs were engineered where a monomeric streptavidin (Lim et a / ., Biotechnology and bioengineering. 2013;110(1):57-67) is fused to the C-terminus of each heavy chain of the bi-specific ai-mAb (FIG. 20A). This construct was expressed and purified and verified that it runs a higher molecular weight as compared to the original bi-specific design (FIG. 20B), while retaining the expected binding properties was verified (FIG. 20C, compare to FIG. 9E). The bi-specific-monovalent-streptavidin fusion molecule will be incubated with biotinylated CpG which is commercially available from I nvivoGen . Following conjugation, excess CpG will be removed by size exclusion chromatography.

[0342] Naive B cells will be purified from PBMC from healthy donors and conjugated to Cell T race Violet (CTV) and cultured in media in the presence of IL-15, IL-6 and 10Ong / ml of the CpG-conjugated bi-specific mAb. These culture conditions were established to result in proliferation of antigen-specific B cells ex vivo (Sanjuan Nandin). As a control, B cells will be stimulated by adding unconjugated CpG alone which will activate the cells non-specifically. 3 days later cell supernatant will be harvested and IgG levels (to confirm mAb secretion) and binding to pre- and PostF will be measured using a custom Luminex binding assay. Here the bulk of stimulated cells will have high levels of IgG but very few (if any) of the secreted antibodies will bind RSV F and a very weak or absent binding signal will be observed. In contrast, the bi-specific will preferentially stimulate B cells expressing VH3-21 / VL1 -40 BCRs, resulting in an overall lower level of IgG secretion in the cultures but the majority will bind to PreF resulting in a detectable signal for binding to PreF but not PostF.

[0343] It is possible that the ai-mAb is capable of binding and activating target B cells, but they do not secrete levels of antibodies above the threshold of detection in the Luminex assay. Therefore the cultured B cells will also be analyzed by ELISPOT. B cells that respond to stimulation will divide, diluting the CTV signal. Therefore, CTVLowcells will be sorted and subjected to ELISPOT assays to enumerate the frequency of B cells that are secreting antibodies specific to PreF. These results will mirror those in the Luminex assay; a majority of the bulk stimulated cells will undergo division resulting in a high frequency of CTV|OWcells, but the overwhelming majority will not bind to PreF and very few (if any) will score positive in the ELISPOT assays. In contrast, the bi-specific will preferentially stimulate B cells expressing VH3-21 / VL1-40 BCRs resulting in a much lower frequency of CTV|OWcells relative to the bulk stimulated control but a substantial portion of these cells will secrete PreF binding antibodies in the ELISPOT assays.

[0344] To unequivocally confirm that the B cells that respond to stimulation with the bi-specific do indeed express VH3- 21 / VL1-40 BCRs, the experiment will be repeated and CTVlowcells will be sorted into 96 well plates and their VH and VL transcripts will be recovered by RT PCR (Seydoux 2; Tiller; Jennewein). Here the frequency of VH3-21 heavy chains, VL1 - 40 light chains and VH3-21 / VL1 -40 pairs among the sorted CTVlowcells will be quantified. Paired VH3-21 / L1 -40 sequences will be produced as recombinant mAbs and tested for RSV-F cross reactivity, epitope specificity and neutralizing potential.

[0345] Example 4 (Prophetic). Determining that ai-mAb Vaccines Induce Protective Antibodies using a Small Animal RSV Challenge Model. ai-mAb vaccines can induce protective antibodies in a small animal RSV challenge model. Rationale. The bi-specific ai-mAb described above will selectively engage B cell receptors (BCRs) derived from VH3-21 / VL1-40 pairs on naive B cells, and these B cells will become activated and secrete antibodies capable of neutralizing RSV. In this prophetic example, this will be confirmed by demonstrating the ability of the bi-specific mAb to induce a protective antibody response when target B cells are present at physiological frequencies in vivo using a murine adoptive transfer system.

[0346] Pre-existing antibody responses can drastically affect primary antibody responses to vaccination (Tas; Heyman, Annual review of immunology. 2000;18:709-37; Hjelm et al., Scandinavian journal of immunology. 2006;64(3): 177-84 (“Hjelm”)). In some contexts, the presence of maternal antibodies can inhibit infant responses to vaccination (Siegrist; Vono; Crowe, Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2001 ;33(10):1720-7). Epitope masking, antigen clearance and Fc-FcR interactions with immune complexes have all been proposed as potential mechanisms whereby pre-existing antibodies can exert inhibitory effects on vaccine responses (Hjelm). Importantly, pre-existing antibodies may be particularly problematic in the context of maternal immunization. The strategy to elicit high titers of antibody in pregnant women, who will in turn confer immunity to infants though transplacental antibody transfer, may be a double-edged sword whereby maternal antibodies provide short term protection against RSV in newborns but may also thwart the development of B cell responses in infants, creating a barrier to direct immunization. In line with this notion, passive delivery of F-specific antibodies; mimicking maternal transfer, inhibits the de novo response to RSV F immunization in rodents, resulting in lower neutralizing titers and reduced protection from viral challenge (Murphy et al., Vaccine. 1991;9(3):185-9). In this prophetic example, a similar model will be used where polyclonal F-specific antibodies are transferred to mice harboring physiological frequencies of ADI14337 B cells. The ability of the bi-specific ai- mAb and a PreF protein subunit vaccine to specifically target VH3-21 / VL 1-40 B cells and elicit neutralizing antibodies that protect against RSV challenge will then be confirmed.

[0347] Edited B cells harvested from knock in mice can be transferred into wildtype mice at physiological frequencies and serve as a model for studying antigen-specific B cell responses in the context of a diverse polyclonal B cell repertoire (Abbott eta / ., Immunity. 2018;48(1):133-46 e6 ("Abbott”); Lin eta / ., The EMBO journal. 2018;37(18); Tas eta / ., Immunity. 2022. Epub 20220804 ("Tas”); Wang et a / „ The EMBO journal. 2021 ;40(2):e 105926 ("Wang”); Escolano et a / „ Cell. 2016;166(6):1445-58 e12 (“Escolano”); McGuire et a / ., Nature communications. 2016, 7:10618). Three such adoptive transfer models were previously employed to demonstrate robust in vivo activation of target B cells with vaccines derived from ai-mAbs raised against unmutated neutralizing HIV-1 antibody precursors (Bancroft; Dosenovic; Seydoux 1).

[0348] Experimental Approach. Generation and characterization of knock-in mice. To generate mice appropriate for these studies AD114337 knock-in mice will be crossbred to homozygosity. Gene specific primers will be used to identify homozygous pups. B cells from the knock-in mice will undergo an in-depth phenotypic analysis to confirm that they exhibit normal B cell development. The frequency of mature (CD93-) and immature (CD93j B cells (CD19+B220+CD3" F4 / 80" Gr-1") in the spleen and bone marrow will be analyzed and compared to wild type mice. To verify expression of the BCR, B cells from knock-in mice will be stained with 1D3, 2C1 (e.g., FIG. 19D) and the bi-specific to verify expression of both chains and also with PreF (DS-Cav1) and PostF to verify they stain positive for Pre- but not PostF. Single B cells will be sorted and the sequences of the expressed BCRs will be verified by RT-PCR. To verify that the BCRs are functional, splenocytes will be harvested and subjected to the calcium flux assay shown in FIG. 17 with the bi-specific mAb, and pre- and postF. The cells will be activated by the bi-specific mAb and PreF but not PostF.

[0349] An adoptive transfer system will be employed that has previously been used to demonstrate robust in vivo activation of target B cells with vaccines derived from ai-mAbs raised against HIV-1 bnAb precursors (Bancroft; Dosenovic; Seydoux 1). The AD114337 knock-in mice engineered (FIGs. 19A and 19B) to express the CD45.2 allele.

[0350] B cells from the AD114337 BCR knock-in mice will be transferred into wildtype mice that express the CD45.1 allele and differential CD45.1 / CD45.2 staining will be used to identify adoptively transferred B cells, determine their frequency, and track theirfate. As noted above, 0.1-0.35% naive B cells in healthy donors express a VH3-21 / VL1 -40 pair. Therefore, a pilot experiment will be performed to transfer increasing amounts of B cells to groups of 5 mice via retro-orbital transfer and then the frequency of CD45.2 B cells will be measured 24 hours later. This timing will allow for the determination of the number of transferred cells needed to achieve the desired frequency.

[0351] Specific targeting of ADI14337 B cells through immunization with ai-mAbs. After establishing the conditions to transfer the appropriate amount of engineered B cells, B AD114337 B cellswill next be labeled with CellTrace Violet (CTV) and transferred in to two groups of 5 mice. 24 hours later one group of mice will be immunized with the 1D3 / 2C1 bi-specific mAb and another with an isotype control. Initially immunization will include 10pg of mAb formulated with Sigma Adjuvant System administered bilaterally in the thigh muscles. 5 Days later the spleen as well as the inguinal axillary, and lumbar lymph nodes will be harvested and the frequency of CD45.2 AD114337 B cells and the intensity of CTV staining will be measured. An increase in the frequency of CD45.2 cells and a corresponding dilution CTV in CD45.2 B cells from animals immunized with the bi-specific, but not the isotype control will demonstrate that the bi-specific mAb engaged and activated the target B cells in vivo.

[0352] Next, a series of experiments to optimize the immunization regimen will be conducted. The ai-mAb will be delivered to groups of 5 mice in 0.1, 1, and 10 pg doses. In addition, different commercially available adjuvants: Sigma Adjuvant System (a squalene emulsion formulated with monophosphoryl lipid A), Addavax (a squalene emulsion), and QuilA (a saponin-based adjuvant) (9 immunized groups, plus 3 adjuvant only groups for 12 total) will be evaluated. Mice will be immunized once with each dose / adjuvant combination and then bled weekly for up to 105 weeks. ELISA will be used to measure serum antibody binding to the bi-specific mAb. Since the bi-specific mAb is built on a human IgG backbone (Moore) some off-target responses against the constant regions that arise from the endogenous CD45.1 B cells are expected. Therefore, to assess how much of the binding is specific to the ai-mAb paratopes, binding to an isotype matched control will also be measured. To complement these analyses, serum antibody binding to PreF and PostF will be measured. The bi-specific ai-mAb is expected to elicit high titers of AD114337 antibodies in the serum resulting in strong binding titers to PreF and none to PostF. The functional activity of the immune sera will be demonstrated by its ability to neutralize infection of GFP-labeled RSV (strains A2 and B1) Vero cells.

[0353] Upon identifying the dose and adjuvant combination that yields the highest neutralizing titers, the immunization regimen will be repeated alongside a control group of mice immunized with DS-Cav1 and the serological assays described above will be conducted. Here it is expected that animals immunized with DS-Cav1 will develop antibodies that will bind both PreF and PostF due to the presence of epitopes shared on the two conformations and the presence of a diverse wildtype BCR repertoire. DS-Cav1 will also activate target B cells resulting in a serum response that binds weakly to the bi-specific ai-mAb. The ai-mAb and DS-Cav1 will both elicit neutralizing antibodies, but the ai-mAb will be superior to DS- Cav1 as the latter will elicit antibodies to all antigenic regions of RSV F, not all of which will be neutralizing, while the bi- specific ai-mAb will activate PreF-specific B cells that produce potently neutralizing antibodies. The predicted outcome of the serological analyses are summarized in FIG. 21.

[0354] To map the epitope specificities of the serum antibodies, competitive binding assays will be carried out by ELISA. Serum from immunized animals will be titrated over PostF immobilized on ELISA plates. Human mAb that bind to known epitopes on PostF, such as D25 (site 0), 131 -2a (site I), palivizumab (site II), AD114337 (Site III), 101 F (IV), and RB1 (site V), and their binding will be detected with an anti-human IgG secondary. Animals that are immunized with the bi-specific ai-mAb will elicit high titers of antibodies that inhibit binding of VH3-21 / VL1 -40 mAbs and other site III directed antibodies, but because the ai-mAb will elicit AD114337 Abs, these sera will not compete with the binding of mAbs targeting other epitopes on PreF. Relative to the mice immunized with the ai-mAb, mice immunized with PreF will elicit a more diverse polyclonal B cell response with lower titers of serum antibodies that inhibit binding of VH3-21 / VL1-40 mAbs and other site III directed antibodies. These sera will compete with other mAbs, both neutralizing and non- that bind to epitope regions outside of site III.

[0355] To gain a better understanding of the immune responses at the B cell level, an in-depth analysis of the B cell responses to immunization will be conducted with the bi-specific ai-mAb and PreF. Groups of 5 mice will again be immunized with both the bi-specific ai-mAb and PreF using the optimal dose and adjuvant (determined above) but mice will be euthanized at 10, 21 and 105 days post immunization and peripheral blood, spleen, lymph nodes and bone marrow will be collected. B cells from immunized mice will be co-stained with fluorescently labeled PreF (e.g., PE), and with PostFlabeled with a distinct fluorophore (e.g,, APC). All the ai-&F-specific B cells from mice immunized with the bi-specific will be CD45.2+ and they will be PreF+ / PostF-. In contrast, B cells from mice immunized with DS-Cav1 will have a mix of VH3- 21 / VL1 -40 (CD45.2+ / PreF+ / PostF-), but also endogenous B cells capable of recognizing both pre- and PostF conformations (CD45.1+ / PreF+ / PostF+) as well as some that recognize PreF (CD45.1+ / PreF+ / PostF). The phenotype of the antigen specific B cells will also be analyzed. Plasmablasts (CD138+B220|OWantigenh'9h), early memory B cells (B220+CTV|OWGL7"CD38+) and germinal center (B220+CD95+CD38_) B cells will be enumerated. Long-lived plasma cells will be identified as CD20+CD197B220’ CD138+antigen11'^ using intracellular staining.

[0356] Based on the previous results using a similar adoptive transfer system and ai-mAb-derived immunogens (Dosenovic), immunization with the bi-specific ai-mAb will elicit a higher frequency of plasmablasts and early memory B cells derived from the knock-in cells as compared to immunization with DS-Cav1 at timepoints early after infection. The kinetics of knock-in-derived plasmablast cell expansion will correlate with a peak in serum neutralizing antibody titers. At day 105, animals immunized with the bi-specific ai-mAb will have higher frequencies of knock-in derived long-lived plasma cells in the bone marrow as compared to animals immunized with DS-Cav1 and that these will correlate with serum neutralizing antibodies at later timepoints.

[0357] Challenge studies. Upon identifying the regimen that elicits the highest serum antibody titers, the capacity of the bi- specific ai-mAb to elicit protective titers of neutralizing antibodies will be confirmed by conducting challenge studies. Adoptive transfer experiments will be designed to deliver target B cells at physiological frequencies in groups of 5 mice as outlined above. 24 hours later, one group will be immunized with ai-mAb + the adjuvant at a dose that yielded the highest in vitro neutralizing titer determined above (FIG. 22, Group 2). A control group will receive an immunization with saline (FIG. 22, Group 1). A third group will be immunized with PreF (DS-Cav1, FIG. 22, Group 3).

[0358] At a timepoint corresponding to the peak neutralizing serum response following immunization with the bi-specific ai-mAb (determined above), mice will be challenged intranasally with 106plaque-forming units (PFU) of RSV containing a fluorescent reporter. 5 days later, viral titers will be measured in supernatants from lung homogenates (Moffett et al., Science immunology. 2019;4(35)). Here, high viral titers will be measured in the lungs from animals in Group 1 which will serve as an infection control. In contrast, immunization with the bi-specific ai-mAb will elicit high titers of AD114337 serum antibodies that will protect the mice from challenge, and no or low viral titers in the lungs of Group 2 will be observed. Immunization with PreF will elicit neutralizing antibodies that afford some protection from challenge in Group 3 but they will target both the AD114337 epitope and other neutralizing epitopes on F. However, specific targeting of the AD114337 B cells with a single immunization with the ai-mAb in Group 2 will result in better protection compared Group 3, evidenced by lower viral titers in the lungs from animals in the former group for two related reasons. The first is that non-VH3-21 / VL1 -40 antibodies typically use somatic mutation to achieve tight binding and potent neutralization and the short timeline of the proposed immunization regimen would not allow for adequate time for somatic hypermutation. Although PreF will almost certainly activate ADI14337 B cells, they will also activate other F-specific B cells which will decrease the proportion of AD14337 cells and increase inter-clonal B cell competition in Group 3. To interrogate this further, 2 additional groups will be formed where B cells expressing a non-RSV BCR (inferred germline from of VRC01 (Seydoux 1; Abbott)), are transferred instead of AD114337 (FIG. 22, Groups 4 and 5). Group 4 will allow for the determination of how much (if any)of the early serum response arising from non-VH3-21 / VL1 -40 B cells contributes to protection after vaccination with pre-F. If VH3-21 / VL1-40 antibodies contribute substantially following immunization with PreF, then the protection in Group 3 will be superior to Group 4. Alternatively, if VH3-21 / VL1-40 antibodies constitute a minor fraction of the polyclonal response, then the outcomes of Groups 3 and 4 will be indistinguishable. Group 5 is not expected to elicit any antibodies that crossreact with RSV F since the ADI14337 B cells are absent and the bi-specific ai-mAb is antigenically distinct from F. Therefore, the fate of the mice upon challenge will be indistinguishable from Group 1. The challenge studies in groups 1- 5 will be carried out with both RSV subtypes A and B. Both are clinically relevant. Subtype A and B circulation shows alternating periodicity in different geographic regions (Zlateva et al., Journal of clinical microbiology. 2007; 45(9): 3022-30; and Waris, The Journal of infectious diseases. 1991 ; 163(3):464-9). DS-Cav1 is derived from subtype A, and antibodies elicited by DS-Cav1 are less effective at neutralizing subtype B (McLellan 1), so higher viral titers in animals immunized with DS-Cav1 and challenged with subtype B (e.g., groups 3 and 4) are predicted. In contrast unmutated VH3-21 / VL1 -40 mAbs show comparable potency against both subtypes (Goodwin 2), comparable efficacy against both subtypes in Group 2 will be observed.

[0359] Example 5 (Prophetic). Determining the Effect of Pre-Existing Antibodies on the Activation of VH3-21 / VL1-40 B Cells. To mimic the elicitation of both pre and post F-specific antibodies by natural infection or vaccination in the mother, groups of CD45.1 mice will receive transfer of physiological frequencies of AD114337 B cells followed by three immunizations with a cocktail of pre- and PostF. 2 weeks after the final immunization the mice will be euthanized. Their sera will be collected and transferred into naive CD45.1 mice alongside CTV-labeled AD114337 B cells. Groups of mice will be immunized with adjuvant alone, (FIG. 23, Group 1) the bi-specific ai-mAb (FIG. 23, Group 2), or PreF (FIG. 23, Group 3). A control group of animals will receive passive transfer of polyclonal IgG from unimmunized RSV-F-na'ive animals (FIG. 23, Group 4). 14 days later, the serum and B cell responses to immunization will be evaluated as described elsewhere. Here, transferred antibodies will bind to PreF and inhibit antigen-specific B cell responses in Group 3. Specifically, a reduction in the frequency of both endogenous (CD45.1) and transferred AD114337 (CD45.2) B cells that stain positive in animals that received polyclonal F-specific mAb prior to immunization with PreF (FIG. 23, Group 3) as compared to the group that received control antibodies (FIG. 23, Group 4) will be observed.

[0360] Because of the antigenic disparity between the bi-specific ai-mAb and PreF, the majority of F-specific transferred antibodies will not bind to or inhibit the activation of AD114337 B cells in Group 2. The exception will be the AD114337 antibodies present in the polyclonal “maternal” sera, but they will constitute a small fraction of the polyclonal “maternal” antibodies and not be of sufficient titer to substantially affect activation of AD114337 B cells. Thus, immunization with the bi-specific ai-mAb will lead to robust activation of AD 114337 B cells in Group 2 and a high (compared to Group 3) frequency of PreF specific B cells that are AD114337 will be observed. Animals immunized with the bi-specific ai-mAb (FIG. 23, Group 3) will have the highest neutralizing titers following immunization.

[0361] The transfer / immunization experiments will also be repeated as depicted above and in FIG. 23, but rather than euthanizing the animals to analyze B cells at 14 days, they will be challenged with RSV at a timepoint equivalent to that determined in FIG. 22. For these experiments, an additional infection control group (FIG. 23, Group 5) will be included that receives transfer of control IgG along with AD114337 B cells prior to immunization with adjuvant alone. Following challenge,mice in Group 5, will have the highest viral titers, since they will have no humoral RSV immunity. Group 1 will have the second highest titers as residual "maternal" antibodies will confer some protection. Next in the ranking, Group 3 will have higher viral titers than Group 4 because the "maternal” antibodies will have inhibited the vaccine response to RSV-F in Group 3. Lastly, Group 2 will have the lowest, or absent viral titers because the ai-mAb vaccine will elicit a superior neutralizing response that will be uninhibited by the presence of transferred antibodies as outlined above.

[0362] Multiple replicates will be performed in each assay and all binding experiments will be repeated at least twice. To account for differences in BCR repertories from individual donors, B cell sorting experiments will be performed on an equal mix of male and female donors. For the adoptive transfer experiments, comparable numbers of male and female mice in the same age range will be used. Immunizations will be conducted in groups of 5 mice and will be repeated three independent experiments to ensure reproducibility.

[0363] By using ai-mAb derived immunogens that are highly specific for unmutated BCRs pre-configured to neutralize RSV, a protective antibody response can be rapidly induced in an animal challenge model. This approach is particularly well suited to circumvent some of the hurdles that have hampered the development of a successful RSV vaccine, particularly in at-risk infant populations where the capacity to mount normal antibody responses is limited. This vaccine concept could also overcome additional challenges to infant immunization resulting from interference of de novo humoral responses by maternal antibodies.

[0364] Example 6 (Experimental). In a follow-up analysis, it was found that the ai-mAb could recognize VH3-21A / L1 -40 mAbs MxR and MPE8 that can cross-neutralize RSV and human metapneumovirus (HMPV) (FIG. 24A) (Corti et al., Nature. 2013; 501(7467): 439-43; and Caban etal., Nat Commun. 2023;14(1):798). It was also found that 5 of 46 unmutated VH3- 21 / VL1 -40 mAbs isolated from single cell sorting with the ai-mAb can cross-react with HMPV prefusion (preF). These mAbs show a weaker binding signal compared to MxR and MPE8 (FIG. 24B), both of which harbor somatic mutations. The RSV- neutralizing ADI-19425 mAb was include and weak cross-reactivity with HMPV preF (FIG. 24A) was also observed. Among 46 mAbs isolated from naive B cells sorted with the ai-mAb, 14 bound RSV preF, 4 bound preF from both HMPV and RSV, and one (MLR_48) only bound preF from HMPV. Therefore, the ai-mAb can engage genetically restricted B-cell receptors (BCRs) capable of cross-reacting with preF from RSV and HMPV including those that can cross-neutralize the two viruses.

[0365] Example 7 (Experimental). Mice lack VH3-21 and VL1-40 genes and thus knock-in (KI) mice were generated that express the ADI-19425 B cell receptor (BCR) (Goodwin et al., Immunity. 2018;48(2):339-49.e5). The KI strategy used CRISPR-Cas9 to introduce the re-arranged antibody (Ab) heavy chain (HC) and light chains (LCs) into the murine loci replacing the variable-joining and kappa light chain-joining gene clusters, respectively (FIGs. 25A and 25B) (Escolano et al., Cell. 2016; 166(6): 1445-58.e12). Binding of mouse B cells to DS-Cav1 tetramers (RSV preF (McLellan et al., Science. 2013;342(6158):592-8)) demonstrated a successful KI for both chains showing 65% of KI B cells staining with the tetramer while none from wild-type (WT) mice bound the tetramer (FIG. 25C). This data confirmed that the ADI-19425 BCR was successfully knocked in, but that allelic exclusion is incomplete such that not all B cells express the target BCR.

[0366] 5X105B cells harvested from KI mice were transferred into congenitally marked (CD45.1) mice. 24 hours later, the frequency of ADI-19425 B cells among splenic B cells was determined to be 0.06% by differential CD45.1 / CD45.2 staining (FIGs. 26A-26C). The frequency of B cells expressing VH3-21 / VL1-40 BCRs in humans is 0.144% (DeKosky et al., ProcNatl Acad Sci U S A. 2016;113(19):E2636-45; and Jaffe et a / ., Nature 611, 352-357, 2022) and the B cell sorting data indicates that 5% of the VH3-21 / VL1-40 B cells sorted with the anti-idiotypic monoclonal antibody (ai-mAb) are neutralizing (Scharffenberger et al., Cell Rep. 2024;43(10):114811). Therefore, it is estimated that a physiological frequency of neutralizing VH3-21 / VL1 -40 B cells is 0.0072%. Given that 65% KI B cells bind preF, it is estimated that under these adoptive transfer conditions, target B cells are present at 0.039% which is a reasonable approximation of the physiological frequency in humans.

[0367] To determine the immunogenicity of the bispecific ai-mAb in the adoptive transfer model, groups of mice were immunized with either 10 g of the bispecific ai-mAb or 10pg of an irrelevant control mAb formulated in saponin / monophosphoryl-lipid A (MPLA) nanoparticle (SMNP) adjuvant (Silva et al., Sci Immunol. 2021 ;6(66):eabf1152) 24h following adoptive transfer (FIG. 27A). Mice were bled every week for 4 weeks and serum was collected and used in respiratory syncytial virus (RSV)-A plaque reduction neutralization assays (FIGs. 27B-27F) (Scharffenberger et a / ., Cell Rep. 2024;43(10): 114811). All mice that received immunization with the bispecific ai-mAb generated RSV-A neutralizing serum titers that were maintained over the 4 week experiment, while the mice that received the irrelevant immunization did not produce neutralizing serum titers (FIGs. 27B-27F). This demonstrated the immunogenicity of the ai-mAb to activate target cells in the adoptive transfer model and to elicit an RSV-neutralizing serum response.

[0368] To determine whether the neutralizing serum responses seen in bispecific ai-mAb immunized animals was sufficient to protect against RSV infection, an immuni...

Claims

CLAIMSWhat is claimed is:

1. A multi-specific binding molecule comprising a first arm and a second arm, wherein the first arm comprises a heavy chain comprising the sequence of SEQ ID NO: 6 and a light chain comprising the sequence of SEQ ID NO: 7; and wherein the second arm comprises an scFv comprising the sequence of SEQ ID NO: 44 fused to a constant region comprising the sequence of SEQ ID NO: 43.

2. A multi-specific binding molecule comprising a first binding domain that binds a VH3-21 heavy chain and a second binding domain that binds a VL1-40 light chain, wherein(i) the first binding domain comprises a complementarity determining region (CDR) heavy (H)1 , CDRH2, and CDRH3 and a variable light chain comprising a CDR light (L)1 , CDRL2, and CDRL3 wherein the CDRH1 comprises the sequence of SEQ ID NO: 13, the CDRH2 comprises the sequence of SEQ ID NO: 14, and the CDRH3 comprises the sequence of SEQ ID NO: 15, and the CDRL1 comprises the sequence of SEQ ID NO: 16, the CDRL2 comprises the sequence of SEQ ID NO: 17, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 61, the CDRH2 comprises the sequence of SEQ ID NO: 62, and the CDRH3 comprises the sequence of SEQ ID NO: 15, and the CDRL1 comprises the sequence of SEQ ID NO: 16, the CDRL2 comprises the sequence of SEQ ID NO: 17, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 63, the CDRH2 comprises the sequence of SEQ ID NO: 64, and the CDRH3 comprises the sequence of SEQ ID NO: 65, and the CDRL1 comprises the sequence of SEQ ID NO: 66, the CDRL2 comprises the sequence YAS, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 67, the CDRH2 comprises the sequence of SEQ ID NO: 68, and the CDRH3 comprises the sequence of SEQ ID NO: 65, and the CDRL1 comprises the sequence of SEQ ID NO: 16, the CDRL2 comprises the sequence of SEQ ID NO: 69, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to North; or the CDRH1 comprises the sequence of SEQ ID NO: 70, the CDRH2 comprises the sequence of SEQ ID NO: 71, and the CDRH3 comprises the sequence of SEQ ID NO: 72, and the CDRL1 comprises the sequence of SEQ ID NO: 73, the CDRL2 comprises the sequence of SEQ ID NO: 74, and the CDRL3 comprises the sequence of SEQ ID NO: 75 according to Contact; and(ii) the second binding domain comprises a CDRH1 , CDRH2, and CDRH3 and a CDRL1 , CDRL2, and CDRL3 wherein: the CDRH1 comprises the sequence of SEQ ID NO: 19, the CDRH2 comprises the sequence of SEQ ID NO: 20, and the CDRH3 comprises the sequence of SEQ ID NO: 21 , and the CDRL1 comprises the sequence of SEQ ID NO: 22, the CDRL2 comprises the sequence of SEQ ID NO: 23, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 76, the CDRH2 comprises the sequence of SEQ ID NO: 77,and the CDRH3 comprises the sequence of SEQ ID NO: 21 , and the CDRL1 comprises the sequence of SEQ ID NO: 22, the CDRL2 comprises the sequence of SEQ ID NO: 23, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 78, the CDRH2 comprises the sequence of SEQ ID NO: 79, and the CDRH3 comprises the sequence of SEQ ID NO: 80, and the CDRL1 comprises the sequence of SEQ ID NO: 81, the CDRL2 comprises the sequence SAS, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 82, the CDRH2 comprises the sequence of SEQ ID NO: 83, and the CDRH3 comprises the sequence of SEQ ID NO: 80, and the CDRL1 comprises the sequence of SEQ ID NO: 22, the CDRL2 comprises the sequence of SEQ ID NO: 84, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to North; or the CDRH1 comprises the sequence of SEQ ID NO: 85, the CDRH2 comprises the sequence of SEQ ID NO: 86, and the CDRH3 comprises the sequence of SEQ ID NO: 87, and the CDRL1 comprises the sequence of SEQ ID NO: 88, the CDRL2 comprises the sequence of SEQ ID NO: 89, and the CDRL3 comprises the sequence of SEQ ID NO: 90 according to Contact.

3. The multi-specific binding molecule of claim 2, wherein the first binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity to SEQ ID NO: 1; and a variable light chain comprising the sequence of SEQ ID NO: 2 or a sequence having at least 95% sequence identity to SEQ ID NO: 2.

4. The multi-specific binding molecule of claim 2, wherein the first binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 93 or a sequence having at least 95% sequence identity to SEQ ID NO: 93; and a variable light chain encoded by the sequence of SEQ ID NO: 94 or a sequence having at least 95% sequence identity to SEQ ID NO: 94.

5. The multi-specific binding molecule of claim 2, wherein the second binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity to SEQ ID NO: 3; and a variable light chain comprising the sequence of SEQ ID NO: 4 or a sequence having at least 95% sequence identity to SEQ ID NO: 4.

6. The multi-specific binding molecule of claim 2, wherein the second binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 95 or a sequence having at least 95% sequence identity to SEQ ID NO: 95; and variable light chain encoded by the sequence of SEQ ID NO: 96 or a sequence having at least 95% sequence identity to SEQ ID NO: 96.

7. The multi-specific binding molecule of claim 2, wherein the multi-specific binding molecule comprises a first arm and a second arm.

8. The multi-specific binding molecule of claim 7, wherein the first arm comprises a heavy chain and a light chain; and the second arm comprises the second binding domain and an IgG Fc constant region.

9. The multi-specific binding molecule of claim 8, wherein the heavy chain comprises the variable heavy chain of the first binding domain and a heavy chain constant region.

10. The multi-specific binding molecule of claim 9, wherein the heavy chain constant region comprises the sequence of SEQ ID NO: 42 or a sequence having at least 95% sequence identity to SEQ ID NO: 42.

11. The multi-specific binding molecule of claim 8, wherein the heavy chain comprises the sequence of SEQ ID NO: 6 or a sequence having at least 95% sequence identity to SEQ ID NO: 6.

12. The multi-specific binding molecule of claim 8, wherein the light chain comprises the variable light chain of the first binding domain and a light chain constant region.

13. The multi-specific binding molecule of claim 12, wherein the light chain constant region comprises the sequence of SEQ ID NO: 41 or a sequence having at least 95% sequence identity to SEQ ID NO: 41.

14. The multi-specific binding molecule of claim 8, wherein the light chain comprise the sequence of SEQ ID NO: 7 or a sequence having at least 95% sequence identity to SEQ ID NO: 7.

15. The multi-specific binding molecule of claim 8, wherein the second binding domain is an scFv.

16. The multi-specific binding molecule of claim 15, wherein the scFv comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 45 or a sequence having at least 95% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45.

17. The multi-specific binding molecule of claim 8, wherein the second arm comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 8 or a sequence having at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 8.

18. The multi-specific binding molecule of claim 8, wherein the second arm is encoded by the sequence of SEQ ID NO: 97 or a sequence having at least 95% sequence identity to SEQ ID NO: 97.

19. The multi-specific binding molecule of claim 8, wherein the first arm comprises a heavy chain comprising the sequence of SEQ ID NO: 6 and a light chain comprising the sequence of SEQ ID NO: 7; and the second arm comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.

20. The multi-specific binding molecule of claim 8, wherein the first arm comprises a heavy chain encoded by the sequence of SEQ ID NO: 98 and a light chain encoded by the sequence of SEQ ID NO: 99; and the second arm is encoded by the sequence of SEQ ID NO : 97.

21. The multi-specific binding molecule of claim 7, wherein the first arm comprises a heavy chain and a light chain; and the second arm comprises the first binding domain and an IgG Fc constant region.

22. The multi-specific binding molecule of claim 21, wherein the heavy chain comprises the variable heavy chain of the second binding domain and a heavy chain constant region.

23. The multi-specific binding molecule of claim 22, wherein the heavy chain constant region comprises the sequence of SEQ ID NO: 42 or a sequence having at least 95% sequence identity to SEQ ID NO: 42.

24. The multi-specific binding molecule of claim 21 , wherein the heavy chain comprises the sequence of SEQ ID NO: 11 or a sequence having at least 95% sequence identity to SEQ ID NO: 11.

25. The multi-specific binding molecule of claim 21 , wherein the light chain comprises the variable light chain of the second binding domain and a light chain constant region.

26. The multi-specific binding molecule of claim 25, wherein the light chain constant region comprises the sequence of SEQ ID NO: 41 or a sequence having at least 95% sequence identity to SEQ ID NO: 41.

27. The multi -specific binding molecule of claim 21 , wherein the light chain comprise the sequence of SEQ ID NO: 12 or a sequence having at least 95% sequence identity to SEQ ID NO: 12.

28. The multi-specific binding molecule of claim 21 , wherein the first binding domain is an scFv.

29. The multi-specific binding molecule of claim 28, wherein the scFv comprises the sequence of SEQ ID NO: 46 or SEQ ID NO: 47 or a sequence having at least 95% sequence identity to SEQ ID NO: 46 or SEQ ID NO: 47.

30. The multi-specific binding molecule of claim 21, wherein the second arm comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.

31. The multi-specific binding molecule of claim 21, wherein the first arm comprises a heavy chain comprising the sequence of SEQ ID NO: 11 and a light chain comprising the sequence of SEQ ID NO: 12; and the second arm comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

32. The multi -specific binding molecule of claim 7, wherein the first arm comprises a first Fc region or fragment thereof and the second arm comprises a second Fc region or fragment thereof.

33. The multi-specific binding molecule of claim 32, wherein the first Fc region or fragment thereof comprises mutations that lower the isoelectric point.

34. The multi-specific binding molecule of claim 32, wherein the second Fc region or fragment thereof comprises mutations that lower the isoelectric point.

35. The multi-specific binding molecule of claim 32, wherein the first Fc region or fragment thereof comprises Fc silencing mutations.

36. The multi-specific binding molecule of claim 35, wherein the Fc silencing mutations comprise E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.

37. The multi-specific binding molecule of claim 32, wherein the second Fc region or fragment thereof comprises Fc silencing mutations.

38. The multi-specific binding molecule of claim 37, wherein the Fc silencing mutations comprise E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.

39. A binding domain that binds a VH3-21 heavy chain, wherein the binding domain comprises a complementarity determining region (CDR) heavy (H)1 , CDRH2, and CDRH3 and a variable light chain comprising a CDR light (L)1 , CDRL2, and CDRL3; wherein the CDRH1 comprises the sequence of SEQ ID NO: 13, the CDRH2 comprises the sequence of SEQ ID NO: 14, and the CDRH3 comprises the sequence of SEQ ID NO: 15, and the CDRL1 comprises the sequence of SEQ ID NO: 16, the CDRL2 comprises the sequence of SEQ ID NO: 17, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to Kabat; wherein the CORFU comprises the sequence of SEQ ID NO: 61, the CDRH2 comprises the sequence of SEQ ID NO: 62, and the CDRH3 comprises the sequence of SEQ ID NO: 15, and the CDRL1 comprises the sequence of SEQ ID NO: 16, the CDRL2 comprises the sequence of SEQ ID NO: 17, and the CDRL3 comprises the sequence of SEQID NO: 18 according to Chothia; wherein the CDRH1 comprises the sequence of SEQ ID NO: 63, the CDRH2 comprises the sequence of SEQ ID NO: 64, and the CDRH3 comprises the sequence of SEQ ID NO: 65, and the CDRL1 comprises the sequence of SEQ ID NO: 66, the CDRL2 comprises the sequence YAS, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to IMGT; wherein the CDRH1 comprises the sequence of SEQ ID NO: 67, the CDRH2 comprises the sequence of SEQ ID NO: 68, and the CDRH3 comprises the sequence of SEQ ID NO: 65, and the CDRL1 comprises the sequence of SEQ ID NO: 16, the CDRL2 comprises the sequence of SEQ ID NO: 69, and the CDRL3 comprises the sequence of SEQ ID NO: 18 according to North; or wherein the CDRH1 comprises the sequence of SEQ ID NO: 70, the CDRH2 comprises the sequence of SEQ ID NO: 71, and the CDRH3 comprises the sequence of SEQ ID NO: 72, and the CDRL1 comprises the sequence of SEQ ID NO: 73, the CDRL2 comprises the sequence of SEQ ID NO: 74, and the CDRL3 comprises the sequence of SEQ ID NO: 75 according to Contact.

40. The binding domain of claim 39, wherein the binding domain comprises a variable heavy chain having at least 95% sequence identity to the sequence of SEQ ID NO: 1 and a variable light chain having at least 95% sequence identity to the sequence of SEQ ID NO: 2.

41. The binding domain of claim 39, wherein the binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 1 and a variable light chain comprising the sequence of SEQ ID NO: 2.

42. The binding domain of claim 39, wherein the binding domain comprises a variable heavy chain encoded by the sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.

43. The binding domain of claim 39, wherein the binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence of SEQ ID NO: 94.

44. The binding domain of claims 39, wherein the binding domain is an scFv.

45. The binding domain of claim 44, wherein scFv comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 46 or SEQ ID NO:47.

46. The binding domain of claim 44, wherein scFv comprises the sequence of SEQ ID NO: 46 or SEQ ID NO:47.

47. The binding domain of claim 44, wherein the scFv is attached to a fragment of a constant heavy chain.

48. The binding domain of claim 47, wherein the constant heavy chain comprises an lgG1 heavy chain, an lgG2 heavy chain, an lgG3 heavy chain, or an lgG4 heavy chain.

49. The binding domain of claim 47, wherein the constant heavy chain comprises an lgG1 heavy chain.

50. A binding domain that binds a VL1-40 light chain, wherein the binding domain comprises a complementarity determining region (CDR) heavy (H)1 , CDRH2, and CDRH3 and a variable light chain comprising a CDR light (L)1 , CDRL2, and CDRL3; wherein the CDRH1 comprises the sequence of SEQ ID NO: 19, the CDRH2 comprises the sequence of SEQ ID NO: 20, and the CDRH3 comprises the sequence of SEQ ID NO: 21 , and the CDRL1 comprises the sequence of SEQ ID NO:22, the CDRL2 comprises the sequence of SEQ ID NO: 23, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 76, the CDRH2 comprises the sequence of SEQ ID NO: 77, and the CDRH3 comprises the sequence of SEQ ID NO: 21 , and the CDRL1 comprises the sequence of SEQ ID NO: 22, the CDRL2 comprises the sequence of SEQ ID NO: 23, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 78, the CDRH2 comprises the sequence of SEQ ID NO: 79, and the CDRH3 comprises the sequence of SEQ ID NO: 80, and the CDRL1 comprises the sequence of SEQ ID NO: 81, the CDRL2 comprises the sequence SAS, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 82, the CDRH2 comprises the sequence of SEQ ID NO: 83, and the CDRH3 comprises the sequence of SEQ ID NO: 80, and the CDRL1 comprises the sequence of SEQ ID NO: 22, the CDRL2 comprises the sequence of SEQ ID NO: 84, and the CDRL3 comprises the sequence of SEQ ID NO: 24 according to North; or the CDRH1 comprises the sequence of SEQ ID NO: 85, the CDRH2 comprises the sequence of SEQ ID NO: 86, and the CDRH3 comprises the sequence of SEQ ID NO: 87, and the CDRL1 comprises the sequence of SEQ ID NO: 88, the CDRL2 comprises the sequence of SEQ ID NO: 89, and the CDRL3 comprises the sequence of SEQ ID NO: 90 according to Contact.

51. The binding domain of claim 50, wherein the binding domain comprises a variable heavy chain having at least 95% sequence identity to SEQ ID NO: 3 and a variable light chain having at least 95% sequence identity to SEQ ID NO: 4.

52. The binding domain of claim 50, wherein the binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 3 and a variable light chain comprising the sequence of SEQ ID NO: 4.

53. The binding domain of claim 50, wherein the binding domain comprises a variable heavy chain encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 95 and a variable light chain encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 96.

54. The binding domain of claim 50, wherein the binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and a variable light chain encoded by the sequence of SEQ ID NO: 96.

55. The binding domain of claim 50, wherein the binding domain is an scFv.

56. The binding domain of claim 55, wherein the scFv comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

57. The binding domain of claim 55, wherein the scFv comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

58. The binding domain of claim 55, wherein the scFv is attached to a fragment of a constant heavy chain.

59. The binding domain of claim 58, wherein the constant heavy chain comprises an lgG1 heavy chain, an lgG2 heavy chain, an lgG3 heavy chain, or an lgG4 heavy chain.

60. The binding domain of claim 58, wherein the constant heavy chain comprises an lgG1 heavy chain.

61. A binding molecule comprising a binding domain that binds a VH3-21 / VL1-40 B cell receptors (BCRs).

62. The binding molecule of claim 61 , wherein the binding molecule is an anti-idiotypic antibody.

63. The binding molecule of claim 61 , wherein the binding molecule is a multi-specific binding molecule.

64. The binding molecule of claim 63, wherein the multi-specific binding molecule is a bispecific antibody.

65. The binding molecule of claim 63, wherein the multi-specific binding molecule comprises a binding domain that binds a VH3-21 and a binding domain that binds VL1-40.

66. The binding molecule of claim 65, wherein the binding domain that binds VH3-21 comprises the binding domain of claim 39.

67. The binding molecule of claim 65, wherein the binding domain that binds VL1-40 comprises the binding domain of claim 50.

68. The binding molecule of claim 61 , further comprising a first arm and a second arm.

69. The binding molecule of claim 68, wherein the first arm comprises a variable light chain, a constant light chain, a variable heavy chain, and a constant heavy chain; and wherein the second arm comprises an scFv fused to a portion of an Fc region.

70. The binding molecule of claim 69, wherein the variable light chain and variable heavy chain of the first arm comprise the binding domain that binds VH3-21; and the scFv of the second arm comprises the binding domain that binds VL1-40.

71. The binding molecule of claim 69, wherein the variable light chain and variable heavy chain of the first arm comprise the binding domain that binds VL1-40; and the scFv of the second arm comprises the binding domain that binds VH3-21.

72. The binding molecule of claim 69, wherein the constant light chain of the first arm comprises a kappa light chain or lambda light chain.

73. The binding molecule of claim 69, wherein the constant light chain of the first arm comprises a kappa light chain.

74. The binding molecule of claim 69, wherein the constant light chain comprises the sequence of SEQ ID NO: 41 or a sequence having at least 95% sequence identity to SEQ ID NO: 41.

75. The binding molecule of claim 69, wherein the constant heavy chain of the first arm comprises an IgG heavy chain.

76. The binding molecule of claim 75, wherein the IgG heavy chain comprises mutations that lower the isoelectric point.

77. The binding molecule of claim 76, wherein the mutations that lower the isoelectric point comprise N208D, Q295E, N384D, Q418E, and N421D in reference to SEQ ID NO: 27 with position numbering of SEQ ID NO: 27 starting at 118.

78. The binding molecule of claim 75, wherein the IgG heavy chain comprises heterodimer mutations.

79. The binding molecule of claim 78, wherein the heterodimer mutations comprise L368D and K370S in reference to SEQ ID NO: 27 with position numbering of SEQ ID NO: 27 starting at 118.

80. The binding molecule of claim 75, wherein the IgG heavy chain comprises Fc silencing mutations.

81. The binding molecule of claim 80, wherein the Fc silencing mutations comprise E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.

82. The binding molecule of claim 69, wherein the constant heavy chain of the first arm comprises the sequence of SEQ ID NO: 42 or a sequence having at least 95% sequence identity to SEQ ID NO: 42.

83. The binding molecule of claim 69, wherein the portion of the Fc region of the second arm comprises a fragment of an lgG1 antibody, fragment of an lgG2 antibody, fragment of an lgG3 antibody, or fragment of an lgG4 antibody.

84. The binding molecule of claim 69, wherein the portion of the Fc region of the second arm comprises a fragment of an lgG1 antibody.

85. The binding molecule of claim 84, wherein the fragment of the IgG 1 antibody comprises the sequence of SEQ ID NO: 43 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 43.

86. The binding molecule of claim 69, wherein the portion of the Fc region of the second arm comprises mutations C220S, E357Q, and S364K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.

87. The binding molecule of claim 69, wherein the portion of the Fc region of the second arm comprises Fc silencing mutations.

88. The binding molecule of claim 87, wherein the Fc silencing mutations comprise E233P, L234V, L235A, G236del, and S267K in reference to SEQ ID NO: 103 with position numbering of SEQ ID NO: 103 starting at 216.

89. The binding molecule of claim 61, comprising (i) a first arm comprising variable light chain that binds VH3-21, a constant light chain, variable heavy chain that binds VH3-21 , and a constant heavy chain; and (ii) a second arm comprising an scFv that binds VL1-40 fused to a portion of an Fc region.

90. The binding molecule of claim 89, wherein the first arm comprises a variable and constant heavy chain comprising the sequence of SEQ ID NO: 6 and a variable and constant light chain comprising the sequence of SEQ ID NO: 7; and the second arm comprising an scFv-Fc comprising the sequence of SEQ ID NO: 5.

91. The binding molecule of claim 89, wherein the first arm comprises a variable and constant heavy chain comprising the sequence of SEQ ID NO: 6 and a variable and constant light chain comprising the sequence of SEQ ID NO: 7; and the second arm comprising an scFv-Fc comprising the sequence of SEQ ID NO: 8.

92. The binding molecule of claim 89, wherein the first arm comprises a variable and constant heavy chain encoded by the sequence of SEQ ID NO: 98, a variable and constant light chain encoded by the sequence of SEQ ID NO: 99, and the second arm comprises an scFv-Fc encoded by the sequence of SEQ ID NO: 97.

93. The binding molecule of claim 61, comprising (i) a first arm comprising variable light chain that binds VL1-40, a constant light chain, a variable heavy chain that binds VL1-40, a constant heavy chain; and (ii) a second arm comprising an scFv that binds VH3-21 fused to a portion of an Fc region.

94. The binding molecule of claim 93, wherein the first arm comprises a variable and constant heavy chain comprising the sequence of SEQ ID NO: 11 and a variable and constant light chain comprising the sequence of SEQ ID NO: 12; and the second arm comprises an scFv-Fc comprising the sequence of SEQ ID NO: 9.

95. The binding molecule of claim 93, comprising a variable and constant heavy chain comprising the sequence of SEQ ID NO: 11 and a variable and constant light chain comprising the sequence of SEQ ID NO: 12; and the second arm comprises an scFv-Fc comprising the sequence of SEQ ID NO: 10.

96. A composition comprising the multi-specific binding molecule of claim 2 or the binding molecule of claim 61 and a pharmaceutically acceptable carrier.

97. The composition of claim 96, further comprising one or more adjuvants.

98. The composition of claim 97, wherein the one or more adjuvants are selected from alum, a squalene-based adjuvant, a STING agonist, or a liposome-based adjuvant.

99. The composition of claim 96, wherein the composition is a vaccine.

100. A nucleic acid sequence encoding the multi-specific binding molecule of claim 2, the binding molecule of claim 61 , or the binding domain of claims 39 or 50.

101. The nucleic acid sequence of claim 100, comprising a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.

102. The nucleic acid sequence of claim 100, comprising a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence of SEQ ID NO: 94.

103. The nucleic acid sequence of claim 100, comprising a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 95 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 96.

104. The nucleic acid sequence of claim 100, comprising a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and a variable light chain encoded by the sequence of SEQ ID NO: 96.

105. The nucleic acid sequence of claim 100, comprising an scFv fused to an Fc region or fragment thereof encoded a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 97.

106. The nucleic acid sequence of claim 100, comprising an scFv fused to an Fc region or fragment thereof encoded the sequence of SEQ ID NO: 97.

107. The nucleic acid sequence of claim 100, comprising a heavy chain encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 98.

108. The nucleic acid sequence of claim 100, comprising a heavy chain encoded by the sequence of SEQ ID NO: 98.

109. The nucleic acid sequence of claim 100, comprising a light chain encoded by a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 99.

110. The nucleic acid sequence of claim 100, comprising a light chain encoded by the sequence of SEQ ID NO: 99.

111. A vector comprising the nucleic acid sequence of claim 100.

112. A cell genetically modified to express the multi-specific binding molecule of claim 2 or the binding molecule of claim 61.

113. The cell of claim 112, wherein the cell is a 293E cell.

114. A method of stimulating a respiratory syncytial virus (RSV) or human metapneumovirus (HMPV) immune response in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a binding molecule that binds VH3-21 / L1-40 B cell receptors (BCRs), thereby stimulating an RSV or HMPV immune response in the subject.

115. The method of claim 114, wherein the binding molecule comprises an anti-idiotypic antibody.

116. The method of claim 115, wherein the binding molecule comprises the multi-specific binding molecule of claim 2 or the binding molecule of claim 61.

117. The method of claim 114, wherein the binding molecule comprises a multi-specific antibody comprising a first arm comprising a first binding domain that binds a VH3-21 heavy chain and a second arm comprising a second binding domain that binds a VL1-40 light chain.

118. The method of claim 117, wherein the first binding domain comprises a variable heavy chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1 and variable light chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2.

119. The method of claim 117, wherein the first binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 1 and variable light chain comprising the sequence of SEQ ID NO: 2.

120. The method of claim 117, wherein the first binding domain comprises a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.

121. The method of claim 117, wherein the first binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and a variable light chain encoded by the sequence of SEQ ID NO: 94.

122. The method of claim 117, wherein the first arm comprises a full heavy chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6 and a full light chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 7.

123. The method of claim 117, wherein the first arm comprises a full heavy chain comprising the sequence of SEQ ID NO: 6 and a full light chain comprising the sequence of SEQ ID NO: 7.

124. The method of claim 117, wherein the first arm comprises a full heavy chain encoded by a sequence having at least 95% sequence identity to sequence of SEQ ID NO: 98 and a full light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 99.

125. The method of claim 117, wherein the first arm comprises a full heavy chain encoded by the sequence of SEQ ID NO: 98 and a full light chain encoded by the sequence of SEQ ID NO: 99.

126. The method of claim 117, wherein the second binding domain comprises an scFv comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

127. The method of claim 117, wherein the second binding domain comprises an scFv comprising the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

128. The method of claim 117, wherein the second binding domain comprises an scFv comprising a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 95 and variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 96.

129. The method of claim 117, wherein the second binding domain comprises an scFv comprising a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and variable light chain encoded by the sequence of SEQ ID NO: 96.

130. The method of claim 117, wherein the second arm comprises an scFv and fragment of an Fc region.

131. The method of claim 117, wherein the second arm comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.

132. The method of claim 117, wherein the second arm comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.

133. The method of claim 117, wherein the second arm is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 97.

134. The method of claim 117, wherein the second arm is encoded by the sequence of SEQ ID NO: 97.

135. The method of claim 115, wherein the subject is a pediatric patient.

136. The method of claim 135, wherein the pediatric patient is 5 years of age or under.

137. The method of claim 135, wherein the pediatric patient is 1 year of age or under.

138. The method of claim 114, wherein the subject has RSV.

139. The method of claim 114, wherein the subject has HMPV.

140. The method of claim 114, wherein the subject does not have RSV, and wherein the therapeutically effective amount reduces the likelihood of the subject becoming infected with RSV.

141. The method of claim 114, wherein the subject does not have HMPV, and wherein the therapeutically effective amount reduces the likelihood of the subject becoming infected with HMPV.

142. The method of claim 114, wherein the subject does not have RSV or HMPV, and wherein the therapeutically effective amount reduces the likelihood of the subject becoming infected with RSV or HMPV.

143. The method of claim 114, wherein the therapeutically effective amount provides a prophylactic or therapeutic treatment against RSV or HMPV.

144. The method of claim 114, wherein the therapeutically effective amount provides a prophylactic or therapeutic treatment against RSV and HMPV.

145. The method of claim 114, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

146. The method of claim 114, wherein the administering precedes or follows administration of a different RSV vaccination protocol.

147. The method of claim 114, wherein the administering precedes or follows administration of a different HMPV vaccination protocol.

148. A method of enhancing production of RSV-neutralizing antibodies or HMPV-neutralizing antibodies comprising contacting the multi-specific binding molecule of claim 2 or the binding molecule of claim 61 with a population of cells comprising a VH3-21 / VL1 -40-expressing B cell.

149. The method of claim 148, wherein the method enhances production of RSV-neutralizing antibodies and HMPV- neutralizing antibodies.

150. A method of binding VH3-21 / L1-40 neutralizing B cell receptors (BCRs) comprising administering a binding molecule that binds VH3-21 / L1-40 to a population of cells comprising a VH3-21 / L1 -40-expressing B cell.

151. The method of claim 150, wherein the binding molecule comprises an anti-idiotypic antibody.

152. The method of claim 150, wherein the binding molecule comprises the multi-specific binding molecule of claim 2 or the binding molecule of claim 61.

153. The method of claim 150, wherein the binding molecule comprises a multi-specific antibody comprising a first arm comprising a first binding domain that binds a VH3-21 heavy chain and a second arm comprising a second binding domain that binds a VL1-40 light chain.

154. The method of claim 153, wherein the first binding domain comprises a variable heavy chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1 and variable light chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2.

155. The method of claim 153, wherein the first binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 1 and variable light chain comprising the sequence of SEQ ID NO: 2.

156. The method of claim 153, wherein the first binding domain comprises a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 93 and variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 94.

157. The method of claim 153, wherein the first binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 93 and variable light chain encoded by the sequence of SEQ ID NO: 94.

158. The method of claim 153, wherein the first arm comprises a full heavy chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6 and a full light chain comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 7.

159. The method of claim 153, wherein the first arm comprises a full heavy chain comprising the sequence of SEQ ID NO: 6 and a full light chain comprising the sequence of SEQ ID NO: 7.

160. The method of claim 153, wherein the first arm comprises a full heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 98 and a full light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 99.

161. The method of claim 153, wherein the first arm comprises a full heavy chain encoded by the sequence of SEQ ID NO: 98 and a full light chain encoded by the sequence of SEQ ID NO: 99.

162. The method of claim 153, wherein the second binding domain comprises an scFv comprising a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

163. The method of claim 153, wherein the second binding domain comprises an scFv comprising the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

164. The method of claim 153, wherein the second binding domain comprises an scFv comprising a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 95 and variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 96.

165. The method of claim 153, wherein the second binding domain comprises an scFv comprising a variable heavy chain encoded by the sequence of SEQ ID NO: 95 and variable light chain encoded by the sequence of SEQ ID NO: 96.

166. The method of claim 153, wherein the second arm comprises an scFv and fragment of an Fc region.

167. The method of claim 153, wherein the second arm comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.

168. The method of claim 153, wherein the second arm comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 8.

169. The method of claim 153, wherein the second arm is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 97.

170. The method of claim 153, wherein the second arm is encoded by the sequence of SEQ ID NO: 97.

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