Multimeric Anti-respiratory syncytial virus binding molecule

Abstract

The present disclosure provides, among other things, multimeric anti-RSV binding molecules and compositions useful for treating and preventing RSV infections. Methods of making the multimeric anti-RSV binding molecules are also disclosed, along with nucleotides, vectors, and host cells useful in making the multimeric anti-RSV binding molecules.

Description

MULTIMERIC ANTI-RESPIRATORY SYNCYTIAL VIRUS BINDING MOLECULECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 603,065, filed 27 November 2023, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on 14 November 2024, is named 071WOl-Sequence-Listing.xml and is 59 kilobytes in size.BACKGROUND

[0003] Antibodies and antibody-like molecules that can multimerize, such as IgA and IgM antibodies, have emerged as promising drug candidates, e.g., in the fields of immunooncology 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, 9,938,347, 10,351,631, 10,400,038, 10,570,191, 10,604,559, 10,618,978, 10,689,449, 10,787,520, 10,899,935, 11,401,337, 11,555,075, and 11,639,389 and U.S. Patent Application Publication Nos. US 2019-0185570, US 2019-0330360, US 2019-0330374, US 2019-0338040, US 2019-0338041, US 2020-0392239, and US 2022-0403009, the contents of which are incorporated herein by reference in their entireties.

[0004] Respiratory syncytial virus (RSV) belongs to the paramyxoviral family of enveloped, negative- strand RNA viruses. Diseases associated with this family include measles and mumps, as well as a variety of respiratory tract infections, such as those caused by RSV, human metapneumovirus (hMPV), and parainfluenza viruses (PIV). RSV virions are filamentous strands or spheres with a diameter of -150 nm. The RSV genome is linear and -15 kb long. The RSV envelope contains two major glycoproteins, the fusion (F) protein and the attachment (G) protein, both of which are the target of neutralizingantibodies in infected individuals. See, e.g., McLellan el al., Curr. Top. Microbiol. Immunol, 372:83-104 (2014).

[0005] Various monoclonal antibody therapies have been developed to address RSV disease. Nirsevimab (BEYFORTUS®) is a monoclonal antibody targeting the RSV F protein and was recently approved in the United States for preventing RSV-related lung disease in newborns and infants (see, for example, Prescribing Information for BEYFORTUS®, Highlights of Prescribing Information revised 08 / 2024; Full Prescribing Information, February 2024 and Patient Information for BEYFORTUS®, February 2024). Similarly, palivizumab (SYNAGIS®) is a monoclonal antibody targeting the RSV F protein and is prescribed to the prevent RSV infection in certain high risk populations (see, e.g., Prescribing Information for SYNAGIS®, Highlights of Prescribing Information revised 11 / 2021; Full Prescribing Information, 11 / 2020 and Patient Information for SYNAGIS®, revised 11 / 2021. See also, e.g., SYNAGIS® brochure, 8 / 23). The FDA did not approve an affinity-optimized version of palivizumab — motavizumab — because motavizumab showed only marginal efficacy over palivizumab.

[0006] There remains an urgent need for therapeutics to treat and / or prevent human respiratory diseases caused by RSV.SUMMARY

[0007] In one aspect, provided herein is a multimeric binding molecule comprising two to six bivalent binding units, wherein each binding unit comprises two IgM or IgA heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain, wherein three to twelve of the binding domains are identical and specifically bind to an RSV glycoprotein G. In some embodiments, the binding molecule can neutralize RSV more potently than a bivalent reference IgG antibody comprising two of the same binding domains comprised in the multimeric binding molecule that specifically bind to the RSV glycoprotein G.

[0008] In some embodiments, the multimeric binding molecule is a hexameric binding molecule comprising six bivalent IgM binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an antigen binding domain, and wherein the IgM heavy chain constant regions each comprise a Cp4 domain and an IgM tail-piece (tp) domain. In some embodiments, the multimeric binding molecule is a pentameric binding moleculecomprising five bivalent IgM binding units and a J chain or functional fragment or variant thereof, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof each associated with an antigen binding domain, and wherein the IgM heavy chain constant regions each comprise a Cp4 domain and an IgM tail-piece (tp) domain. In some embodiments, the IgM heavy chain constant regions or multimerizing fragments or variants thereof are human IgM constant regions or multimerizing fragments or variants thereof.

[0009] In some embodiments, the multimeric binding molecule is a dimeric or tetrameric binding molecule comprising two or four bivalent IgA binding units and a J chain or functional fragment or variant thereof, wherein each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an antigen binding domain, and wherein the IgA heavy chain constant regions each comprise a Ca3 domain and an IgA tail -piece (tp) domain. In some embodiments, the IgA heavy chain constant regions or multimerizing fragments or variants thereof are human IgA constant regions or multimerizing fragments or variants thereof.

[0010] In some further embodiments, each binding unit of the multimeric binding molecule comprises two heavy chains each comprising a heavy chain variable (VH) domain situated amino terminal to the constant region, or multimerizing fragments or variants thereof. By way of non-limiting example, the VH may comprise three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region (VL) may comprise three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences comprise, respectively: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; or SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. In some embodiments, the J chain or the functional fragment or variant of the J chain in the multimeric binding molecule is a human J chain (e.g., SEQ ID NO: 55) or a functional fragment or variant of the human J chain. In some embodiments, the multimeric binding molecule is for use in a method of treating or preventing RSV infection.

[0011] In yet some additional embodiments, the RSV neutralization potency of the multimeric binding molecule is measured as 50% inhibition concentration (ICso) in cellculture. In some further embodiments, the RSV neutralization potency is measured as 50% effective dose (ED50) in an animal model of infection.

[0012] In yet another aspect, provided herein is a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of a multimeric binding molecule as described herein. In some embodiments, the polynucleotide comprises part of a vector (e.g., a replication vector, a transfection vector, or an expression vector). Therefore, also provided herein is a vector comprising a polynucleotide as described herein.

[0013] Also provided herein is a host cell. In some embodiments, the host cell comprises a polynucleotide or vector as described herein, such that the host cell can express a multimeric binding molecule as provided herein.

[0014] In yet another aspect, disclosed herein is a method for treating or preventing RSV infection in a subject in need of treatment. The method comprises administering to the subject an effective amount of the multimeric binding molecule described herein. In some embodiments of the method, the subject is human. In some additional embodiments of the method, the multimeric binding molecule is administered via intravenous, subcutaneous, intramuscular, intranasal, and / or an inhalation route. In yet a further particular embodiment, the method comprises administering the multimeric binding molecule intranasally and / or by inhalation.

[0015] In yet a further aspect, the disclosure relates to use of the multimeric binding molecule as described herein in the manufacture of a medicament for the treatment or prevention of RSV infection.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1A shows binding of various exemplary test antibodies in an ELISA to the G glycoprotein of RSV subtype A (rsbl734) as a function of antibody concentration. FIG. IB shows binding of various test antibodies in an ELISA to the G glycoprotein of RSV subtype B (Bl) as a function of antibody concentration.

[0017] FIG. 2A shows the ability of various exemplary test antibodies directed against the F protein of RSV to neutralize RSV-A infection of Vero cells in the absence of complement. FIG. 2B shows the ability of various exemplary test antibodies directed against the F protein of RSV to neutralize RSV-A infection of Vero cells in the presence of 10% complement. FIG. 2C shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-A infection of Vero cells in theabsence of complement. FIG. 2D shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-A infection of Vero cells in the presence of 10% complement.

[0018] FIG. 3A shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-A infection of A549 cells in the absence of complement. FIG. 3B shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-A infection of A549 cells in the presence of 10% complement.

[0019] FIG. 4A shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-B infection of Vero cells in the absence of complement. FIG. 4B shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-B infection of Vero cells in the presence of 10% complement. FIG. 4C shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-B infection of A549 cells in the absence of complement. FIG. 4D shows the ability of various exemplary test antibodies directed against the G protein of RSV to neutralize RSV-B infection of A549 cells in the presence of 10% complement.

[0020] FIGs. 5A-5E show the effects of different concentrations of complement on the ability of IgG-Gl and IgM-Gl to neutralize RSV-A infection of Vero cells. FIG. 5A shows data from cells infected in the absence of complement. FIG. 5B shows data from cells infected in the presence of 0.4% complement. FIG. 5C shows data from cells infected in the presence of 1.1% complement. FIG. 5D shows data from cells infected in the presence of 3.3% complement. FIG. 5E shows data from cells infected in the presence of 10% complement.

[0021] FIG. 6A shows the percent change in average body weight over time of RSV-A2- challenged mice treated with various anti-RSV antibodies or controls both two hours before and two days after challenge. FIG. 6B shows the average body weight of each cohort over time of RSV-A2-challenged mice treated with various anti-RSV antibodies or controls both two hours before and two days after challenge. Shapes corresponding to each experimental cohort mark the lines according to the legend shown between FIG. 6A and FIG. 6B where IN: intranasal delivery and IM: intramuscular delivery.

[0022] FIG. 7A shows RSV titers collected from the lungs of RSV-A2-challenged mice treated with various anti-RSV antibodies or controls on day 4 post-infection. FIG. 7B shows RSV titers collected from the noses of RSV-A2-challenged mice treated withvarious anti-RSV antibodies or controls on day 4 post-infection. The broken line at the bottom of each panel indicates the limit of detection. Two asterisks (**) indicate p-value < 0.01, while four asterisks (****) indicate p < 0.0001. On the x-axis, IN: intranasal delivery; IM: intramuscular delivery.DETAILED DESCRIPTIONDefinitions

[0023] As used herein, the term “a” or “an” entity refers to one or more of that entity. For example, “a binding molecule,” is understood to represent one or more binding molecules. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0024] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0025] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2d ed., 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0026] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various embodiments or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0027] As used herein, “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). “Polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and “polypeptide” can be used instead of any of these terms. “Polypeptide” is also intended to refer to the products of postexpression modifications of a polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. A polypeptide can be generated in any manner, including by chemical synthesis.

[0028] A polypeptide as disclosed herein can be of a size of about 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as “folded.” Polypeptides which do not possess a defined three-dimensional structure but rather can adopt many different conformations are referred to as “unfolded.”

[0029] As used herein, “glycoprotein” refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogencontaining side chain of an amino acid, e.g., a serine or an asparagine. Asparagine (bylinked glycans are described in more detail elsewhere in this disclosure.

[0030] An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0031] As used herein, a “non-naturally occurring polypeptide” or any grammatical variant thereof, is a conditional definition that explicitly excludes, but only excludes, thoseforms of the polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial body, to be “naturally-occurring.”

[0032] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. “Fragment,” “variant,” “derivative,” and “analog” — as used herein — include any polypeptides which retain at least some of the properties of the corresponding native antibody or polypeptide, for example, specific binding to an antigen. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of, e.g., a polypeptide include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants can be non- naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or nonconservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. As used herein, a “derivative” of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those polypeptides that contain one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5- hydroxylysine can be substituted for lysine; 3 -methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0033] A “conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). By way of non-limiting example, substituting phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in a polypeptide, binding molecule, and / or antibody of the present disclosure do not abrogate the binding of thepolypeptide, binding molecule, or antibody containing the amino acid sequence to the antigen to which the antibody binds.

[0034] “Polynucleotide” encompasses a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0035] An “isolated” nucleic acid or polynucleotide conveys any form of the nucleic acid or polynucleotide that is separated from its native environment. For example, a gel-purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.” Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.” Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not one that would be found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0036] As used herein, a “non-naturally occurring polynucleotide” (or any grammatical variants thereof) is a conditional definition that explicitly excludes, but only excludes, those forms of the nucleic acid or polynucleotide that are, or might be, determined or interpreted by a judge, or an administrative or judicial body, to be “naturally-occurring.”

[0037] As used herein, a “coding region” is a portion of a nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example, promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions,e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can include heterologous coding regions, either fused or unfused to another coding region. Heterologous coding regions include without limitation, those encoding specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0038] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

[0039] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit P-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0040] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0041] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0042] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein. Proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Polypeptides secreted by vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse P-glucuronidase.

[0043] As used herein, “binding molecule” refers in its broadest sense to a molecule that specifically binds to a receptor or target, e.g., an epitope or an antigenic determinant. As described further herein, a binding molecule can comprise one of more “binding domains,” e.g., “antigen-binding domains” described herein. A non-limiting example of a binding molecule is an antibody or antibody-like molecule as described in detail herein that retains antigen-specific binding. In certain embodiments, a “binding molecule” comprises an antibody or antibody-like or antibody-derived molecule as described in detail herein.

[0044] As used herein, “binding domain” or “antigen-binding domain” can be used interchangeably and refers to a region of a binding molecule, e.g. , an antibody or antibodylike molecule, that is necessary and sufficient to bind specifically to a target, e.g, an epitope, a polypeptide, a cell, or an organ. For example, an “Fv,” e.g., a heavy chain variable region and a light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is a “binding domain.” Other antigen-binding domains include, without limitation, a single domain heavy chain variable region (VHH)of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold. A “binding molecule,” e.g, an “antibody”, as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more “antigen-binding domains.”

[0045] “Antibody” and “immunoglobulin” can be used interchangeably herein. An antibody includes at least the variable domain of a heavy chain (e.g., from a camelid species) or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise stated, “antibody” encompasses anything ranging from a small antigen-binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two complete heavy chains and two complete light chains, a dimeric or tetrameric IgA antibody that includes four or eight complete heavy chains and four or eight complete light chains and includes a J chain and / or a secretory component, or a pentameric or hexameric IgM antibody or IgM-like antibody that includes ten or twelve complete heavy chains, respectively, and ten or twelve complete light chains, respectively, and optionally includes a J chain or functional fragment or variant thereof.

[0046] “Immunoglobulin” comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, p, a, 5, s) with some subclasses among them (e.g, yl-y4 or al-a2)). It is the nature of this chain that determines the “isotype” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. The immunoglobulin subclasses (subtypes) e.g., IgGi, IgG2, IgGs, IgG4, IgAi, IgA2, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.

[0047] Light chains are classified as either kappa or lambda (K, X). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tailpiece” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are expressed, e.g., by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of eachchain. The basic structure of certain antibodies, e.g., IgG antibodies, includes two heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a “Y” structure, also referred to herein as an “H2L2” structure, or a “binding unit.”

[0048] “Binding unit” is used herein to refer to the portion of a binding molecule, e.g., an antibody or antibody-like molecule, which corresponds to a standard immunoglobulin structure, e.g. an “H2L2” immunoglobulin structure of two heavy chains and two light chains, or, for certain heavy chain-only antibodies an “H2” immunoglobulin structure. In certain embodiments, e.g., where the binding molecule is a bivalent IgG antibody, the terms “binding molecule” and “binding unit” are equivalent. Such a binding molecule is also referred to herein as “monomeric.” In other embodiments, e.g., where the binding molecule is a “multimeric binding molecule,” e.g., a dimeric or tetrameric IgA antibody or IgA-like antibody or a pentameric or hexameric IgM antibody or IgM-like antibody, the binding molecule comprises two or more “binding units”, i.e., two in the case of an IgA dimer, four in the case of an IgA tetramer, five in the case of an IgM pentamer, or six in the case of an IgM hexamer. A binding unit need not include full-length antibody heavy and light chains, but will typically be bivalent, i.e., will include two “antigen-binding domains,” as defined above. As used herein, certain examples of a binding molecule provided in this disclosure are “dimeric,” and include two bivalent binding units that include IgA constant regions or multimerizing fragments thereof. Certain examples of a binding molecule provided in this disclosure are “pentameric” or “hexameric,” and include five or six bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof. A binding molecule, e.g., an antibody or antibody -like molecule comprising two or more, e.g., two, five, or six binding units, is referred to herein as “multimeric.”

[0049] “J chain” as used herein refers to the J chain of IgM or IgA antibodies of any animal species, any functional fragment and / or variant thereof. In certain embodiments, the J chain is a mature human J chain or functional fragment or variant thereof, the amino acid sequence of which is presented as SEQ ID NO: 55. As persons of ordinary skill in the art will recognize, “a functional fragment” or “a functional variant” includes those fragments and variants that can associate with IgM heavy chain constant regions to form a pentameric IgM antibody or can associate with IgA heavy chain constant regions to form a dimeric IgA antibody. Exemplary modified J chains can be found, e.g., in U.S. Patent Nos. 9,951,134, 10,400,038, 10,618,978, and 11,639,389, each of which is incorporated herein by reference in its entirety.

[0050] As used herein, “IgM-derived binding molecule” refers to a binding molecule that includes at least a multimerizing fragment or variant of an IgM heavy chain constant region and further comprises a non-antibody binding and / or functional domain instead of an antibody antigen binding domain or subunit thereof, and any fragment, e.g., multimerizing fragment, or variant thereof.

[0051] As used herein, “IgM-like antibody” refers generally to a binding molecule that includes at least a multimerizing fragment or variant of an IgM heavy chain constant region that retains the ability to form hexamers or pentamers, e.g., in association with a J chain. An IgM-like antibody typically includes at least the Cp4 and IgM tailpiece (tp) domains of an 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. An IgM- like antibody can likewise be an antibody fragment in which one or more constant region domains are deleted, as long as the IgM-like antibody is capable of multimerizing into a hexamer and / or a pentamer. Thus, an IgM-like antibody can be, e.g., a hybrid IgM / IgG antibody or can be a “multimerizing fragment” of an IgM antibody.

[0052] As used herein, “IgA-derived binding molecule” refers to a binding molecule that includes at least a portion of an IgA heavy chain constant region, and further comprises a non-antibody binding and / or functional domain instead of an antibody antigen binding domain or subunit thereof.

[0053] As used herein, the term “IgA-like antibody” refers generally to a binding molecule that includes at least a multimerizing fragment or variant of an IgA heavy chain constant region that still retains the ability to form multimers, e.g., dimers, trimers, tetramers, and / or pentamers e.g., in association with a J chain. An IgA-like antibody typically includes at least the Ca3 and IgA tailpiece (tp) domains of an IgA 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. An IgA-like antibody can likewise be an antibody fragment in which one or more constant region domains are deleted, as long as the IgA- like antibody can form multimers, e.g., dimers and / or tetramers. Thus, an IgA-like antibody can be, e.g., a hybrid IgA / IgG antibody or can be a “multimerizing fragment” of an IgA antibody.

[0054] The terms “valency,” “bivalent,” “multivalent” and grammatical equivalents refer to the number of binding domains, e.g., antigen-binding domains in given binding molecule, e.g., antibody or antibody-like molecule, or in a given binding unit. As such, “bivalent,” “tetravalent,” and “hexavalent” in reference to a given binding molecule, e.g.,an IgM antibody or an IgM-like antibody, denote the presence of two antigen-binding domains, four antigen-binding domains, and six antigen-binding domains, respectively. A typical IgM antibody or IgM-like antibody where each binding unit is bivalent, can have 10 or 12 valencies. A bivalent or multivalent binding molecule, e.g., antibody or antibodylike molecule, can be monospecific, z.e., all of the antigen-binding domains are the same, or can be bispecific or multispecific, z.e., where two or more antigen-binding domains are different, e.g., bind to different epitopes on the same antigen, or bind to entirely different antigens.

[0055] “Epitope” includes any molecular determinant capable of specifically binding to an antigen-binding domain of an antibody or antibody-like molecule. In certain embodiments, an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have three-dimensional structural characteristics, and / or specific charge characteristics. An epitope is a region of a target that is bound by an antigen-binding domain of an antibody.

[0056] “Target” is used in the broadest sense to include substances that can be bound by a binding molecule, e.g., an antibody or antibody-like molecule. A target can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule, or a minimal epitope on such molecule. Moreover, a “target” can, for example, be a cell, an organ, or an organism, e.g., an animal, plant, microbe, or virus, which comprises an epitope that can be bound by a binding molecule.

[0057] Both the light and heavy chains of antibodies or antibody-like molecules are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant region domains of the light chain (CL) and the heavy chain (e.g., CHI, CH2, CH3, or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 (or CH4, e.g., in the case of IgM) and CL domains comprise the carboxy -terminus of the heavy and light chains, respectively.

[0058] 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 Cpl), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4), and can further include an IgM tail-piece.

[0059] A “full length IgA 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 (CAI or Cal), an IgA hinge region, an antibody heavy chain constant domain 2 (CA2 or Ca2), and an antibody heavy chain constant domain 3 (CA3 or Ca3), and can further include an IgA tail-piece.

[0060] As indicated above, variable region(s) allow a binding molecule, e.g., an antibody or antibody-like molecule, to recognize selectively and bind specifically to epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs) of a binding molecule, e.g., an antibody or antibody-like molecule, combine to form the antigen-binding domain. More precisely, an antigenbinding domain can be defined by three complementarity determining regions (CDRs) on each of the VH and VL chains. Certain antibodies form larger structures. For example, IgA can form a molecule that includes two or four H2L2 binding units and a J chain covalently connected via disulfide bonds, which can be further associated with a secretory component, and IgM can form a pentameric molecule that includes five H2L2 binding units and a J chain, or hexameric molecule that includes six H2L2 binding units, each covalently connected via disulfide bonds.

[0061] The six “complementarity determining regions” or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, referred to as “framework” regions, show less inter- molecular variability. The framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health & Human Services, (1983); and Chothia & Lesk (1987) J. Mol. Biol. 196:901-17, which are incorporated herein by reference in their entireties).

[0062] In the case where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides.

[0063] These particular regions have been described, for example, by Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-17 (1987), which are incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of amino acids when compared against each other. Other overlapping CDR definitions can be found, e.g., in Al-Lazikani B. et al., J. Mol. Biol. 273:927-48 (1997); MacCallum et al., J. Mol. Biol. 262:732-45 (1996); Abhinandan and Martin, Mol. Immunol. 45:3832-39 (2008); Lefranc et al., Dev. Comp. Immunol. 27:55-77 (2003); and Honegger and Pliickthun, J. Mol. Biol. 309:657-70 (2001), which are incorporated herein by reference in their entireties. Antibody variable domains can also be analyzed, e.g., using the IMGT information system (imgt dot cines dot fr / ) (IMGT® / V-Quest) to identify variable region segments, including CDRs. (See, e.g., Brochet et al., NucL Acids Res. 36:W503- 08 (2008). Application of any particular definition (or other definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated. The appropriate amino acids which encompass the “Kabat” and “Chothia” CDRs as defined by the above cited references are set forth below in Table 1 as a comparison. The exact amino acid numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids comprise a particular CDR given the variable region amino acid sequence of the antibody.Table 1. CDR Definitions* Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below).

[0064] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health & Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure.

[0065] 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, P-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept, of Health & Human Services (1991). IgM constant regions can be numbered sequentially (z.e., amino acid #1 starting with the first amino acid of the constant region, or by using the Kabat numbering scheme. A comparison of the numbering of two alleles of the human IgM constant region sequentially (presented herein as SEQ ID NO: 49 (allele IGHM*03) and SEQ ID NO: 50 (allele IGHM*04)) and by the Kabat system is set out below. The underlined amino acid residues are not accounted for in the Kabat system (“X,” double underlined below, can be serine (S) (SEQ ID NO: 49) or glycine (G) (SEQ ID NO: 50)):“Sequence” Sequential (SEQ ID NO: 49 or SEQ ID NO: 50) / KABAT numbering key for IgM heavy chain1 / 127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF SWKYKNNSDI 51 / 176 SSTRGFPSVL RGGKYAATSQ VLLPSKDVMQ GTDEHWCKV QHPNGNKEKN101 / 226 VPLPVIAELP PKVSVFVPPR DGFFGNPRKS KLICQATGFS PRQIQVSWLR 151 / 274 EGKQVGSGVT TDQVQAEAKE SGPTTYKVTS TLTIKESDWL ^QSMFTCRVD 201 / 324 HRGLTFQQNA SSMCVPDQDT AIRVFAIPPS FASIFLTKST KLTCLVTDLT 251 / 374 TYDSVTISWT RQNGEAVKTH TNISESHPNA TFSAVGEASI CEDDWNSGER 301 / 424 FTCTVTHTDL PSPLKQTISR PKGVALHRPD VYLLPPAREQ LNLRESATIT 351 / 474 CLVTGFSPAD VFVQWMQRGQ PLSPEKYVTS APMPEPQAPG RYFAHSILTV 401 / 524 SEEEWNTGET YTCWAHEAL PNRVTERTVD KSTGKPTLYN VSLVMSDTAG 451 / 574 TCY

[0066] By “specifically binds,” it is generally meant that a binding molecule, e.g., an antibody or antibody-like molecule binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule, e.g., an antibody or antibodylike molecule is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain, more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain epitope. For example, binding molecule “A” can be deemed to have a higher specificity for a given epitope than binding molecule “B,” or binding molecule “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”

[0067] A binding molecule, e.g., an antibody as disclosed herein can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5* 10'2sec'1, 10'2sec'1, 5* 10' 3 sec'1, 10'3sec'1, 5>< 10'4sec'1, 10'4sec'1, 5>< 10'5sec'1, or 10'5sec'1, 5>< 10'6sec'1, 10'6sec'1, 5>< 10'7sec'1, or 10'7sec'1.

[0068] A binding molecule, e.g., an antibody or antibody-like molecule disclosed herein can be said to bind a target antigen with an on rate (k(on)) of greater than or equal to 103M'1sec'1, 5x l03M'1sec'1, 104M'1sec1, 5x lO4M'1sec1, 105M'1sec1, 5x l05M'1sec1, 106M'1sec'1, or 5x l06M'1sec'1, or 107M'1sec'1.

[0069] A binding molecule, e.g., an antibody or antibody-like molecule, is said to competitively inhibit binding of a reference antibody or antigen-binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen-binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example,competition ELISA assays. A binding molecule can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0070] As used herein, “affinity” refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, “avidity” refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual antigen-binding domains in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.

[0071] Binding molecules, e.g., antibodies or antibody-like molecules as disclosed herein, can also be described or specified in terms of their cross-reactivity. As used herein, “crossreactivity” refers to the ability of a binding molecule, e.g., an antibody or antibody-like molecule, specific for one antigen, to react with a second antigen; it is a measure of relatedness between two different antigenic substances. Thus, a binding molecule is cross reactive if it binds to an epitope other than the one that induced its formation. The cross- reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can fit better than the original.

[0072] A binding molecule, e.g., an antibody or antibody-like molecule, can also be described or specified in terms of its binding affinity to an antigen. For example, a binding molecule can bind to an antigen with a dissociation constant or KD no greater than 5* 10' 2 M, IO’2M, 5x l0’3M, IO’3M, 5x l0’4M, IO’4M, 5x l0’5M, 10’5M, 5x l0’6M, IO’6M, 5X 10-7M, 10-7M, 5X 10-8M, 10’8M, 5X 10’9M, 10’9M, 5X 10-10M, 10-10M, 5x l0-nM, 10’ 11M, 5x l0’12M, 10’12M, 5X 10’13M, IO’13M, 5x lO44M, 10’14M, 5x l0’15M, or 10’15M.

[0073] “Antigen-binding antibody fragments” including single-chain antibodies or other antigen-binding domains can exist alone or in combination with one or more of the following: a hinge region, CHI, CH2, CH3, or CH4 domains, a J chain, or a secretory component. Also included are antigen-binding fragments that can include any combinationof variable region(s) with one or more of a hinge region, CHI, CH2, CH3, or CH4 domains, a J chain, or a secretory component.

[0074] Binding molecules, e.g., antibodies or antibody-like molecules can be from any animal origin including birds and mammals. The binding molecule can be, e.g. of human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken origin. In another embodiment, the variable region can be condricthoid in origin (e.g., from sharks). As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins, and can, in some instances, express endogenous immunoglobulins and in some instances, not, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. According to embodiments of the present disclosure, an IgM antibody or IgM-like antibody as provided herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgM antibody or IgM-like antibody is able to form a multimer, e.g., a hexamer or a pentamer, and an IgA antibody or IgA-like antibody as provided herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgA antibody or IgA-like antibody is able to form a multimer, e.g., a dimer and / or a tetramer. Such a fragment collectively comprises a “multimerizing fragment.”

[0075] As used herein, “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain. A heavy chain subunit can include at least one of: a VH domain, a CHI domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof. For example, a heavy chain subunit can include without limitation, a VH domain, a CHI domain, a hinge, a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof. Further, a heavy chain subunit can lack certain constant region domains, e.g., all or part of a CH2 domain. A heavy chain subunit can be modified such that it varies in amino acid sequence from a wild-type heavy chain subunit. According to embodiments of the present disclosure, a heavy chain subunit of a multimeric antibody, e.g., an IgM antibody, an IgM-like antibody, an IgA antibody, or an IgA-like antibody as provided herein comprises sufficient portions of an IgM or IgA heavy chain constant region to allow multimeric antibody to form a multimer, e.g., a hexamer or a pentamer. As used herein such a fragment comprises a “multimerizing fragment.”

[0076] As used herein, “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain. A light chain subunit includes one or both of a VL a CL (e.g., CK or CX domain.

[0077] Antibodies or antibody-like molecules can be described or specified in terms of the epitope(s) or portion(s) of a target, e.g., a target antigen that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an “epitope,” or an “antigenic determinant.” A target antigen can comprise a single epitope or at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.

[0078] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain in IgG, IgA, and IgD heavy chains, and provides flexibility to the molecule.

[0079] As used herein, “disulfide bond” includes the covalent bond formed between two sulfur atoms, e.g., in cysteine residues of a polypeptide. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. Disulfide bonds can be “intra-chain,” z.e., linking to cysteine residues in a single polypeptide or polypeptide subunit, or can be “inter-chain,” z.e., linking two separate polypeptide subunits, e.g., an antibody heavy chain and an antibody light chain or an IgM or IgA antibody heavy chain constant region and a J chain.

[0080] As used herein, “reference antibody” refers to an antibody with function similar to a multimeric binding molecule provided by this disclosure, e.g., an antibody functionally interacting with the target protein of interest that comprises similar or identical antigenbinding domains. Reference antibodies may be monoclonal or polyclonal antibodies. In a particular embodiment, a “reference antibody” is a single-binding unit antibody with identical binding domains to a corresponding multimeric binding molecule as provided herein, e.g., a multimeric antibody with two, four, five, or six binding units.

[0081] As used herein, the term “chimeric antibody” refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial, or modified) is obtained from a second species. In some embodiments, the target binding region or site wifi be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0082] The terms, “multispecific antibody” or “bispecific antibody” refer to an antibody or antibody-like molecule that has antigen-binding domains for two or more different epitopes within a single antibody molecule. Other binding molecules in addition to thecanonical antibody structure can be constructed with two binding specificities. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strbhlein & Heiss (2() \ 0) Future Oncol. 6: 1387-94; Mabry & Snavely (2010) IDrugs. 13:543-49). A bispecific antibody can also be a diabody.

[0083] As used herein, the term “engineered antibody” refers to an antibody in which a variable domain, constant region, and / or J chain is altered by at least partial replacement, addition, or deletion of one or more amino acids. In certain embodiments, entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody. Although alternate CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, CDRs can also be derived from an antibody of a different class, e.g., from an antibody from a different species. An engineered antibody in which one or more “donor” CDRs from a non-human antibody of known specificity are grafted into a human heavy or light chain framework region is referred to herein as a “humanized antibody.” In certain embodiments not all of the CDRs are replaced with the complete CDRs from the donor variable region and yet the antigen-binding capacity of the donor can still be transferred to the recipient variable domains. Given the explanations set forth in, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, by carrying out routine experimentation to obtain a functional engineered or humanized antibody.

[0084] As used herein, “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).

[0085] As used herein, “linked,” “fused,” “fusion,” or other grammatical equivalents can be used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although thereading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the “fused” CDRs are co-translated as part of a continuous polypeptide.

[0086] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is “amino-terminal” or “N-terminal” to another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain. Similarly, a portion of a polypeptide that is “carboxy-terminal” or “C- terminal” to another portion of a polypeptide is that portion that comes later in the sequential polypeptide chain. For example, in a typical antibody, the variable domain is “N-terminal” to the constant region, and the constant region is “C-terminal” to the variable domain.

[0087] “Expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., mRNA, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., mRNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0088] “Neutralizing” or “neutralize” as used herein refers to the ability of a therapeutic, e.g., a therapeutic antibody, to reduce and / or prevent viral infectivity. “Infectivity” as used herein refers to the ability of a virus to do one or more of: attach to cells; enter cells; release its nucleic acid; replicate its nucleic acid; synthesize viral proteins; and package its nucleic acid into new virions that can be released from the infected cell. A virus can be neutralized,e.g., by a therapeutic antibody, via the antibody’s ability to bind specifically to the virion and inhibit its ability to attach to a host cell receptor, thereby preventing entry into the host cell.

[0089] RSV infection begins when the glycoprotein (G) binds host-cell surface receptors and attaches the virus to the host cell surface. Infection then progresses through the fusing of viral and cell membranes, mediated by the fusion (F) protein (Collins et al., Curr Top Microbiol Immunol. 372:3-38 (2013)). Fusion can occur at the plasma membrane, but there is also evidence of entry by clathrin-mediated endocytosis without endosomal acidification. Both of these pathways involve the same F-mediated fusion mechanism (Kolokoltsov et al., J Virol. 81(14):7786-800 (2007)). The G and F proteins are heavily glycosylated and can interact with heparan sulfate and other cell-surface proteoglycans indiscriminately (Collins etal, 2013), which has made it difficult to identify a more specific RSV receptor. A neutralizing therapeutic can arrest the infection process at any one of these steps, as well as at several other steps later in the infection process.

[0090] “Potent” or “potency” as used herein refers to the amount of a particular substance required to produce an effect, e.g., the amount of a binding molecule required to neutralize RSV infectivity. In some embodiments, the potency is measured as the 50% effective concentration (ECso) or 50% inhibitory concentration (ICso) that is able to neutralize or otherwise block viral, e.g., RSV infectivity (e.g., block attachment of the virus to the cellular receptor), or provide therapeutic protection of a subject infected with a virus, e.g., RSV, measured, e.g., as a 50% effective dose (EDso), or prophylactic protection of a subject susceptible to human infection, measured, e.g., as EDso. As used herein in the context of virus neutralization, “ECso” and “ICso” can be used interchangeably.

[0091] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “alleviation” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, lessen the severity of symptoms of, and / or halt or slow the progression of an existing diagnosed pathologic condition or disorder. Terms such as “prevent,” “prevention,” “avoid,” “deter,” “deterrence,” “prophylactic,” and the like refer to prophylactic or preventative measures that can prevent the development of, or can reduce the symptoms of, a targeted pathologic condition or disorder in a subject who has not yet contracted the targeted pathologic condition or disorder.

[0092] The terms “protect,” “protection,” “protective,” and other related terms, as used herein, refer to the ability of a therapeutic or prophylactic agent to confer a desirable effect on a subject diagnosed with or susceptible to an infectious disease. Protection can include,for example, alleviation of or a reduction in infection -related symptoms in a subject infected with a virus, e.g., RSV, such that, for example, the subject does not need to be hospitalized or put on a ventilator. As it applies to a therapeutic or prophylactic animal model, “protection” can include a lower ED50 among a group of animal subjects challenged with the therapeutic agent either before or after challenge with RSV. Data points that can be used to measure ED50 vary, e.g., with the animal model or the amount of virus used to challenge the animal subjects. Data points can include, e.g., measurement of the virus titer in the lungs of the animals, weight loss, death, or disease symptoms such as fever or difficulty breathing.

[0093] By “subject” or “individual” or “animal” or “patient” is meant any subject. In certain embodiments, the subject is a mammalian subject for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on. In some embodiments related to treatment or therapy, the mammalian subject is a human.

[0094] As used herein, the phrase, “a subject that would benefit from therapy” refers to a subset of subjects, from amongst all prospective subjects, which would benefit from administration of a given therapeutic agent, e.g., a binding molecule such as an antibody, comprising one or more antigen-binding domains. Such a binding molecule, e.g., an antibody, can be used, e.g., for a diagnostic procedure and / or for treatment or prevention of a disease.RSV glycoprotein structure

[0095] The fusion (F) and glycoprotein (G) proteins are the two principal surface proteins in the RSV virion that control the initial stages of RSV infection, including attachment to the host cell surface (Collins et al, 2013). Most neutralizing antibodies elicited during natural infection target one or the other of these two surface proteins (Collins et al, 2013).

[0096] The RSV F protein (exemplary A2 strain sequence provided as SEQ ID NO: 56) is relatively conserved among RSV strains, although variations exist among F proteins, especially between the RSV / A and RSV / B strains (see Hause et al., PLOS One doi: 10.1371 / journal. pone.0175792 (April 17, 2017)). During the course of the replication cycle, F protein exists in multiple conformational forms. When a new virion is assembled, the F protein exists in the prefusion (Pre-F) trimeric form and contains the major antigenic site “0,” which is the primary target of neutralizing serum antibodies (Coultas et al.,Thorax 74 (10):986-93 (2019)). Once bound to its surface receptor, PreF refolds, losing 0 and enabling the protein to insert itself into the host cell membrane to drive fusion of the viral and host cell membranes (Griffiths et al., Clin. Microbiol. Rev. 30(l):277-319 (2017)). Finally, once fusion is complete, the F protein adopts is elongated, post fusion (PostF) conformation. The F protein also activates toll-like receptor 4 (TLR4), which triggers the innate immune response (Collins et al, 2013).

[0097] The RSV G protein (exemplary strain A2 provided as SEQ ID NO: 57) is primarily responsible for viral attachment to host cells (Collins et al, 2013). The RSV G protein is a type II membrane protein containing two mucin-like regions that are highly variable among RSV strains. These mucin-like regions flank the “G central conserved” (Gcc) domain. The highly conserved amino acids of the Gcc (e.g., residues 155-197 of SEQ ID NO: 57) include 4 cysteines (residues 173, 176, 182, & 186 of SEQ ID NO: 57) that form two disulfide bonds to stabilize a cysteine noose motif (McLellan et al., Curr Top Microbiol Immunol. 372:83-104 (2013)). Gbinds to glycosaminoglycans, such as heparan sulfate, to bind the virus to the host cell surface. In contrast to F protein, G protein inhibits signaling from TLR4 (Collins et al, 2013).Multimeric anti-RSV binding molecule

[0098] Provided herein is a multimeric binding molecule comprising two to six bivalent binding units, where each binding unit comprises two IgA or IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain, where three to twelve of the binding domains are identical and specifically bind to respiratory syncytial virus (RSV). The provided binding molecule can be used to treat or prevent respiratory diseases caused by RSV infection. In certain embodiments, the binding units bind specifically to a surface protein of RSV, e.g., the G protein of RSV, such as, for example, the RSV G protein central conserved domain (GCC).

[0099] In some embodiments, the three to twelve identical binding domains that specifically bind to RSV comprise one or more heavy chain variable region (VH) and / or light chain variable region (VL) sequences or fragments thereof derived from an antibody with the capacity to neutralize one or more RSV strains.

[0100] In certain embodiments, heavy chain constant regions in the provided binding molecule are each associated with a binding domain, e.g., an antibody antigen-binding domain, e.g., an scFv, a VHH, or the VH subunit of an antibody antigen-binding domain.

[0101] In some embodiments, the provided multimeric binding molecule is multispecific, e.g., bispecific, trispecific, or tetraspecific, where two or more binding domains associated with the heavy chain constant regions of the binding molecule specifically bind to different targets. In some other embodiments, the binding domains of the multimeric binding molecule all specifically bind to RSV.

[0102] In certain embodiments, each binding unit comprises two heavy chains each comprising a VH situated amino terminal to the heavy chain constant region, and two immunoglobulin light chains each comprising a light chain variable domain (VL) situated amino terminal to an immunoglobulin light chain constant region, e.g., a kappa or lambda constant region. The provided VH and VL combine to form an antigen-binding domain that specifically binds to the target (e.g., a surface protein of RSV, e.g., the G protein of RSV, e.g., the Gcc). In certain embodiments, each antigen-binding domain of each binding molecule is identical.

[0103] In further embodiments, the three to twelve identical binding domains of the multimeric binding molecule bind to the Gcc domain and comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, such that the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences comprise, respectively: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; or SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

[0104] In certain embodiments, the three to twelve identical binding domains of the multimeric binding molecule specifically bind to the RSV Gcc domain, each comprising an antibody VH and a VL comprising the CDR regions in the immediately preceding paragraph, and further wherein the VH and VL comprise amino acid sequences at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 5; the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 13; or the VH of SEQ ID NO: 17 and the VL of SEQ ID NO: 21.

[0105] The multimeric binding molecule can be assessed by potency relative to a bivalent reference IgG antibody that can bind an identical epitope of RSV (e.g., an epitope in theGCC region of the RSV G protein). For example, where the multimeric binding molecule is an IgM antibody, the reference bivalent IgG antibody will have two identical binding domains (z.e., the same VH and VL domains) as the three, four, five, six, seven, eight, nine, ten, eleven, or twelve identical binding domains of the IgM antibody. In some embodiments, the bivalent reference IgG antibody is incapable of neutralizing RSV where the multimeric binding molecule can neutralize RSV. In some embodiments, the bivalent reference IgG antibody can neutralize RSV, while the multimeric binding molecule can neutralize RSV at a lower concentration, e.g., a concentration that is at least 10-fold, 50- fold, 100-fold, or even 500-fold lower than the concentration of the bivalent reference IgG antibody.

[0106] The ability of an antibody to neutralize RSV can readily be determined by one of skill in the art, such as by measuring infectivity in vitro using a viral or pseudoviral infectivity assay. For example, Ngwuta et al. (2015). Sci. Translat. Med. 7(309):309ral62 describe an assay for measuring RSV neutralization. Similarly, rodent models of therapeutic efficacy are known and useful for assessing the neutralization potency of a potential therapeutic (see, e.g., Cianci et al. (2004) Antimicrob Agents Chemother. 48(2):413-22).

[0107] In certain embodiments, the greater potency of the provided multimeric binding molecule relative to the reference IgG can be measured, e.g., as inhibition of RSV binding to its receptor, at a lower ECso or ICso than that of the bivalent reference IgG antibody. In certain embodiments, the provided multimeric binding molecule can inhibit RSV binding to its receptor under conditions where the bivalent reference IgG antibody cannot inhibit binding. In certain embodiments, the provided multimeric binding molecule can neutralize RSV under conditions where the bivalent reference IgG antibody cannot neutralize. In certain embodiments, the provided multimeric binding molecule can protect infected animals, or prevent infection in uninfected animals in a therapeutic animal model at a lower EDso than the bivalent reference IgG antibody. In certain embodiments, the provided multimeric binding molecule can protect infected animals, or prevent infection in uninfected animals in a therapeutic animal model under conditions where the bivalent reference IgG antibody cannot protect. In certain embodiments, the provided multimeric binding molecule can comprise any combination of the foregoing properties.

[0108] In certain embodiments, the provided multimeric binding molecule can neutralize infectivity of RSV at a lower EC50 than the bivalent reference IgG antibody. In certain embodiments, the EC50 of the provided multimeric binding molecule is at least two-fold,at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least 10,000-fold lower than the ECso of the bivalent reference IgG antibody. The ECso can be measured either as mass per volume (e.g., pg / mL) or as molecular concentration (e.g., mol. / L).

[0109] In some embodiments, the provided multimeric binding molecule can confer protection against RSV infection in a therapeutic or prophylactic animal model at a lower EDso than the bivalent reference IgG antibody. In some embodiments, the binding molecule can confer protection against RSV infection in a therapeutic or prophylactic animal model under conditions where the bivalent reference IgG antibody cannot protect. As used herein, measurements of “protection” against RSV infection in an animal model can include a reduced viral load in the subject animals (e.g., in the lungs or nasopharynx), survival of the subject animals from an otherwise lethal RSV infection, and / or a reduction of symptoms typical of the infection in the animal model, e.g., weight loss, fever, difficulty breathing.

[0110] In some embodiments, the multimeric binding molecule reduces, inhibits, orblocks RSV from binding to its receptor at a lower EC50 than does the bivalent reference IgG antibody. In some embodiments the multimeric binding molecule reduces, inhibits, or blocks receptor binding under conditions where the bivalent reference IgG antibody cannot reduce, inhibit, or block binding. In certain embodiments, the receptor is expressed on the surface of a cell, e.g., a cultured host cell, or a cell in a susceptible subject, e.g., a human subject. In some embodiments, the multimeric binding molecule reduces, inhibits, or blocks RSV binding to CX3C chemokine receptor 1 (CX3CR1) and / or heparan sulfate proteoglycans at a lower EC50 than does the bivalent reference IgG antibody. In some embodiments, the EC50 is at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least thirty -fold, at least forty-fold, or at least fifty-fold lower than the EC50 of the bivalent reference IgG antibody.IgM antibodies and IgM-like antibodies

[0111] IgM is the first immunoglobulin produced by B cells in response to stimulation by antigen. Naturally occurring IgM is naturally present at around 1.5 mg / mL in serum with a half-life of about 5 days. IgM is a pentameric or hexameric molecule and thus includes five or six binding units. An IgM binding unit typically includes two light and two heavy chains. While an IgG heavy chain constant region contains three heavy chain constant domains (CHI, CH2 and CH3), the heavy (p) constant region of IgM additionally containsa fourth constant domain (CH4) and includes a C-terminal IgM “tailpiece.” The human IgM constant region typically comprises the amino acid sequence SEQ ID NO: 49 (identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1, allele IGHM*03) or SEQ ID NO: 50 (identical to, e.g., GenBank Accession No. sp|P01871.4, allele IGHM*04). The human Cpl region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 49 or SEQ ID NO: 50; the human Cp2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 49 or SEQ ID NO: 50, the human Cp3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO: 49 or SEQ ID NO: 50, the Cp 4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO: 49 or SEQ ID NO: 50, and the IgM tail-piece ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO: 49 or SEQ ID NO: 50.

[0112] Other forms and alleles of the human IgM constant region with minor sequence variations exist, including, without limitation, GenBank Accession Nos. CAB37838.1, and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 49 or SEQ ID NO: 50 described and claimed elsewhere in this disclosure can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species.

[0113] Five IgM binding units can form a complex with an additional small polypeptide chain (the J chain), or a functional fragment or variant thereof, to form a pentameric IgM antibody or IgM-like antibody, as discussed elsewhere herein. The precursor form of the human J chain is presented as SEQ ID NO: 54. The signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 54, and the mature human J chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 54. The mature human J chain includes the amino acid sequence SEQ ID NO: 55.

[0114] Without the J chain, an IgM antibody or IgM-like antibody typically assembles into a hexamer, comprising up to twelve antigen-binding domains. With a J chain, an IgM antibody or IgM-like antibody typically assembles into a pentamer, comprising up to ten antigen-binding domains. The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody or IgM-like antibody is thought to involve the Cp4 and IgM tail-piece domains. See, e.g., Braathen et al. (2002) J. Biol. Chem. 277:42755-62. Accordingly, a pentameric or hexameric IgM antibody provided in this disclosure typically includes at least the Cp4 and IgM tail-piece domains (also referred to herein collectively as Cp4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thusincludes at least the Cp4 and tp domains. An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a Cpl domain or a fragment thereof, and / or other IgM heavy chain domains. In certain embodiments, an IgM antibody or IgM-like antibody as provided herein can include a complete IgM heavy (p) chain constant region, e.g., SEQ ID NO: 49 or SEQ ID NO: 50, or a multimerizing fragment or variant, e.g., as provided herein.

[0115] In some embodiments, the multimeric binding molecule as described above is pentameric and comprises five bivalent binding units. In some embodiments, the multimeric binding molecule further comprises a J chain or functional fragment or variant thereof as described herein. In some embodiments, each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof.

[0116] In some embodiments, the multimeric binding molecule as described above is hexameric and comprises six bivalent binding units. In some embodiments, where each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof.

[0117] In certain embodiments, the disclosure provides a multimeric binding molecule, e.g., a pentameric or hexameric binding molecule, where the binding molecule includes ten or twelve IgM- heavy chains, and where the IgM heavy chains comprise IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain that specifically binds to a target. In certain embodiments, the disclosure provides an IgM antibody or IgM-like antibody that includes five or six bivalent binding units, where each binding unit includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an antigen-binding domain or subunit thereof. In certain embodiments, the two IgM heavy chain constant regions included in each binding unit are human IgM heavy chain constant regions. In some embodiments, the heavy chains are glycosylated. In some embodiments, the heavy chains are mutated to affect glycosylation.

[0118] Where the IgM antibody or IgM-like antibody provided in this disclosure is pentameric, it typically further includes a J chain, or functional fragment or variant thereof.

[0119] In certain embodiments, the two IgM heavy chain constant regions or multimerizing fragments or variants thereof within an individual binding unit each comprise a Cp4 domain or fragment or variant thereof, an IgM tail-piece (tp) or multimerizing fragment or variant thereof, or a combination of a Cp4 domain and a tp or multimerizing fragment or variant thereof. In certain embodiments, the two IgM heavychain constant regions or multimerizing fragments or variants thereof within an individual binding unit each further comprise a Cp3 domain or fragment or variant thereof, a Cp2 domain or fragment or variant thereof, a Cpl domain or fragment or variant thereof, or any combination thereof.

[0120] In some embodiments, the binding units of the IgM antibody or IgM-like antibody each comprise two light chains. In some embodiments, the binding units of the IgM antibody or IgM-like antibody each comprise two fragments of light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments, the light chains are hybrid kappalambda light chains. In some further embodiments, each binding unit comprises two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region.IgA antibodies and IgA-like antibodies

[0121] IgA plays a critical role in mucosal immunity and comprises about 15% of total immunoglobulin produced. IgA can be monomeric or multimeric, forming primarily dimeric molecules, but can also assemble as trimers, tetramers, and / or pentamers. See, e.g., de Sousa-Pereira & Woof (2019) Antibodies 8:57. An IgA binding unit typically includes two light and two heavy chains. IgA contains three heavy chain constant region domains (Cal, Ca2, & Ca3), a hinge region between Cal and Ca2, and includes a C-terminal IgA “tailpiece.” Human IgA has two subtypes, IgAl and IgA2. The human IgAl constant region typically includes the amino acid sequence SEQ ID NO: 51. The human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 51; the human IgAl hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO: 51, the human Ca3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO: 51, and the IgA tail-piece extends from about amino acid 331 to about amino acid 352 of SEQ ID NO: 51. The human IgA2 constant region typically includes the amino acid sequence SEQ ID NO: 52. The human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 52; the human IgA2 hinge region extends from about amino acid 102 to about amino acid 111 of SEQ ID NO: 52, the human Ca2 domain extends from about amino acid 113 to about amino acid 206 of SEQ ID NO: 52, the human Ca3 domain extends from about amino acid 215 to about amino acid 317 of SEQ ID NO: 52, and the IgA tail-piece extends from about amino acid 318 to about amino acid 340 of SEQ ID NO: 52.

[0122] Two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., the mature human J chain of SEQ ID NO: 55) and the secretory component (precursor, SEQ ID NO: 53, mature: amino acids 19 to 603 of SEQ ID NO: 53) to form a secretory IgA (slgA) antibody. The assembly of IgA binding units into a dimeric slgA antibody is thought to involve the Ca3 and IgA tail-piece domains (also referred to herein collectively as the Ca3-tp domain). Accordingly, a dimeric slgA antibody provided in this disclosure typically includes IgA constant regions that include at least the Ca3 and IgA tail-piece domains. Four IgA binding units can likewise form a tetramer complex with a J chain. A slgA antibody can also form as a higher order multimer, e.g., a pentamer.

[0123] An IgA heavy chain constant region can additionally include a Ca2 domain or a fragment thereof, an IgA hinge region, a Cal domain or a fragment thereof, and / or other IgA heavy chain domains. In certain aspects, an IgA antibody or IgA-like binding molecule as provided herein can include a complete IgA heavy (a) chain constant domain (e.g., SEQ ID NO: 51 or SEQ ID NO: 52), or a multimerizing fragment or variant thereof. In some embodiments, the IgA heavy chain constant regions or multimerizing fragments or variants thereof are human IgA constant regions.

[0124] In some embodiments, the multimeric binding molecule as described above is dimeric and comprises two bivalent binding units. In some further embodiments, the multimeric binding molecule further comprises a J chain or functional fragment or variant thereof as described herein. In some embodiments, each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof.

[0125] In some embodiments, the multimeric binding molecule as described above is tetrameric and comprises four bivalent binding units. In some embodiments, the multimeric binding molecule further comprises a J chain or functional fragment or variant thereof as described herein. In some additional embodiments, each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof.

[0126] In some embodiments, each binding unit of an IgA antibody or IgA-like antibody comprises two light chains. In some embodiments, each binding unit of an IgA antibody or IgA-like antibody comprises two fragments of light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments the light chains are hybrid kappa-lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains eachcomprising a VL situated amino terminal to an immunoglobulin light chain constant region.J chains and functional fragments or variants thereof

[0127] In certain embodiments, the multimeric binding molecule provided herein comprises a J chain or functional fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein is pentameric and comprises a J chain or functional fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein is a dimeric IgA molecule or a pentameric IgM molecule and comprises a J chain or functional fragment or variant thereof. In some embodiments, the multimeric binding molecule comprises a naturally occurring J chain sequence, such as a mature human J chain sequence (e.g., SEQ ID NO: 55). Alternatively, in some embodiments, the multimeric binding molecule comprises a variant J chain sequence, such as, for example, a variant sequence described herein with reduced glycosylation or reduced binding to one or more polymeric Ig receptors (e.g., plgR, Fc alpha-mu receptor (FcapR), or Fc mu receptor (FcpR)). See, e.g., U.S. Patent No. 10,899,835, which is incorporated herein by reference in its entirety. In some embodiments, the multimeric binding molecule comprises a functional fragment of a naturally occurring or variant J chain. A “functional fragment” or a “functional variant” in this context includes those fragments and variants that can associate with binding units, e.g., IgM or IgA heavy chain constant regions, to form a pentameric IgM antibody or IgM-like antibody or a dimeric IgA antibody or IgA- like antibody, and / or can associate with certain immunoglobulin receptors, e.g., the polymeric immunoglobulin receptor (plgR).Variant IgM constant regions

[0128] IgM heavy chain constant regions of a multimeric binding molecule as provided herein can be engineered to confer certain desirable properties to the multimeric binding molecule. For example, in certain embodiments, an IgM antibody or IgM-like antibody as provided herein can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity to cells in the presence of complement, relative to a reference IgM antibody or IgM-like antibody with corresponding reference human IgM constant regions identical, except for the mutations conferring reduced CDC activity. By “corresponding reference human IgM constant region” is meant a human IgM constant region that is identical to the variant IgM constant region except for the modification or modifications in the constant region affecting CDC activity. In certain embodiments, the variant human IgM constantregion includes one or more amino acid substitutions, e.g., in the Cp3 domain, relative to a wild-type human IgM constant region as described, e.g., in U.S. Patent 11,401,337, which is incorporated herein by reference in its entirety. Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g., in U.S. Patent 11,401,337.

[0129] In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310, P311, P313, and / or K315 of SEQ ID NO: 49 (human IgM constant region allele IGHM*03) or SEQ ID NO: 50 (human IgM constant region allele IGHM*04). In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 49 or SEQ ID NO: 50. In other embodiments the variant IgM constant region as provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 49 or SEQ ID NO: 50. In other embodiments the variant IgM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at positions P311 of SEQ ID NO: 49 or SEQ ID NO: 50 and P313 of SEQ ID NO: 49 or SEQ ID NO: 50. These proline residues can be independently substituted with any amino acid, e.g., with alanine, serine, or glycine. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 49 or SEQ ID NO: 50. The lysine residue can be independently substituted with any amino acid, e.g., with alanine, serine, glycine, or aspartic acid. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes a K315D substitution in SEQ ID NO: 49 or SEQ ID NO: 50.

[0130] Human and certain non-human primate IgM constant regions typically include five (5) naturally occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif’ comprises or consists of the amino acid sequence N- Xi-S / T, where N is asparagine, Xi 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 & Taylor (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: 49 or SEQ ID NO: 50 starting at positions 46 (“Nl”), 209(“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in nonhuman primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Accordingly, in some embodiments, IgM heavy chain constant regions of a multimeric binding molecule as provided herein comprise 5 N- linked glycosylation motifs: Nl, N2, N3, N4, & N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., Nl, N2, & N3) on each IgM heavy chain constant region are occupied by a complex glycan.

[0131] In certain embodiments, at least one, at least two, at least three, or at least four of the N-Xi-S / T motifs include an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif. In certain embodiments, the IgM antibody or IgM-like antibody includes an amino acid insertion, deletion, or substitution at motif Nl, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs Nl, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif. In some embodiments, the IgM constant region comprises one or more substitutions relative to a wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 49 (human IgM constant region allele IGHM*03) or SEQ ID NO: 50 (human IgM constant region allele IGHM*04). See, e.g., U.S. Patent App. Pub. No. US 2022-0306760, which is incorporated herein by reference in its entirety.Polynucleotides and Vectors

[0132] In certain embodiments, this disclosure provides a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of a multimeric binding molecule described herein. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a heavy chain constant region or multimerizing fragment or variant thereof and at least an antibody VH portion of the RSV G protein-binding domain. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising the heavy chain of the multimeric binding molecule. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a human IgM constant region or multimerizing fragment or variant thereof fused to the C-terminal end of a VH comprising HCDR1, HCDR2, and HCDR3 regions where: HCDR1 is SEQ ID NO: 2, HCDR2 is SEQ ID NO: 3, and HCDR3 is SEQ ID NO: 4; HCDR1 is SEQ ID NO: 10, HCDR2 is SEQ ID NO: 11, and HCDR3 is SEQ ID NO: 12; or HCDR1 is SEQ ID NO: 18, HCDR2 is SEQ ID NO: 19, and HCDR3 is SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a human IgM constant region or multimerizing fragmentor variant thereof fused to the C-terminal end of a VH comprising a HCDR1, HCDR2, and HCDR3 group as provided above and wherein the encoded polypeptide subunit has: an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1; an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9; or an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 17.

[0133] In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a light chain constant region and an antibody VL portion of the RSV G proteinbinding domain of a multimeric binding molecule as described herein. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising the light chain of the multimeric binding molecule. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a human kappa or lambda light chain constant region or fragment thereof fused to the C-terminal end of a VL comprising LCDR1, LCDR2, and LCDR3 regions where: LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7, and LCDR3 is SEQ ID NO: 8; LCDR1 is SEQ ID NO: 14, LCDR2 is SEQ ID NO: 15, and LCDR3 is SEQ ID NO: 16; or LCDR1 is SEQ ID NO: 22, LCDR2 is SEQ ID NO: 23, and LCDR3 is SEQ ID NO: 24. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a human kappa or lambda light chain constant region or fragment thereof fused to the C-terminal end of a VL comprising an LCDR1, LCDR2, and LCDR3 group as noted above, and where the encoded polypeptide subunit comprises: an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5; an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13; or an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21.

[0134] In certain embodiments, this disclosure provides a vector comprising one or more polynucleotides described herein. In some embodiments, the vector further comprises apolynucleotide comprising a nucleic acid sequence that encodes a J chain or a functional fragment or variant thereof.

[0135] In some embodiments, the vector is a viral vector, such as an adenoviral or adeno- associated viral (AAV) vector. In some embodiments, provided is a viral particle comprising a viral vector as disclosed herein. In some embodiments, the viral particle is an adenoviral particle or an AAV particle.

[0136] In certain embodiments, provided is a composition comprising a first vector and a second vector, where: a) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule, b) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule, and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes a J chain or a functional fragment or variant thereof, c) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and a polynucleotide comprising a nucleic acid sequence that encodes a J chain or a functional fragment or variant thereof, and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule, or d) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule and a polynucleotide comprising a nucleic acid sequence that encodes a J chain or a functional fragment or variant thereof and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule. In other embodiments, provided is a composition comprising a first vector, a second vector, and a third vector, where the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule, the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule, and the third vector comprises a polynucleotide comprising a nucleic acid sequence that encodes a J chain or a functional fragment or variant thereof.Host cells

[0137] In certain embodiments, this disclosure provides a host cell that is capable of producing a multimeric binding molecule as provided herein. In certain embodiments, the host cell comprises one or more vectors as described herein, a composition comprising multiple vectors as described herein, or polynucleotides described herein. Also provided is a method of producing a multimeric binding molecule as provided herein, z.e., by culturing the provided host cell, and recovering the multimeric binding molecule.Methods of Use

[0138] Also provided herein is a method of treating a disease or disorder in a subject in need of treatment, where the method includes administering to the subject a therapeutically effective amount of a multimeric binding molecule as provided herein. By “therapeutically effective dose or amount” or “effective amount” is intended an amount of a multimeric binding molecule that when administered brings about a positive therapeutic response with respect to treatment of subject. Examples of positive therapeutic responses include, without limitation, prevention of respiratory tract colonization or infection by RSV, prevention of RSV attachment, penetration, and / or replication upon exposure to the virus, prevention of RSV symptoms, alleviation of RSV symptoms, reduction of the number of RSV symptoms, or reduction in the severity of RSV symptoms. “Symptoms” include, without limitation, one or more of fever, chills, aches, fatigue, headache, sore throat, coughing, shortness of breath, difficulty breathing, pneumonia, and congestion.

[0139] Effective doses of compositions for treatment of a disease or disorder vary depending upon many different factors, including the particular components of the composition to be administered, means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the subject is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0140] In certain embodiments, administration of a multimeric binding molecule as provided herein to a subject achieves greater potency, e.g., greater efficacy at an equivalent dose or the ability to administer a lower dose and achieve equivalent efficacy, than administration of an equivalent amount of a monomeric binding molecule, such as an IgG, binding to the same binding partner. By “efficacy” is meant the ability of the treatment to,for example, reduce symptoms in an infected subject, reduce the severity of symptoms in an infected subject, prevent symptoms in an infected but asymptomatic subject, reduce the need for auxiliary oxygen in an infected subject or reduce time on a ventilator, reduce the need or the dosage of concomitant medications, reduce the time in intensive care, spare hospital resources, or prevent or reduce transmission from an infected subject to noninfected persons. In certain embodiments, the monomeric binding molecule includes identical antigen-binding domains to the multimeric binding molecule as provided herein. By “an equivalent amount” is meant, e.g., an amount measured by molecular weight, e.g., in total milligrams, or alternatively, a molar equivalent, e.g., where equivalent numbers of molecules are administered.

[0141] The subject can be any animal, e.g., a mammal, in need of treatment or prevention, in certain embodiments, the subject is a human subject.

[0142] In its simplest form, a preparation or composition to be administered to a subject in need thereof comprises a multimeric binding molecule as provided herein administered in a conventional dosage form, where the preparation can, in some embodiments, further comprise one or more pharmaceutical excipients, carriers, or diluents as described elsewhere herein.

[0143] A multimeric binding molecule of the disclosure can be administered by any suitable method, e.g., parenterally, orally, by inhalation (spray or dry powder), topically, intranasally, buccally, via an implanted reservoir, or a combination thereof. The term “parenteral” as used herein includes without limitation subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0144] In certain embodiments the multimeric binding molecule is delivered intranasally, e.g., in an atomized form produced by a suitable spray delivery device. In certain embodiments, the multimeric binding molecule is delivered orally. In certain embodiments, the multimeric binding molecule is delivered intranasally and orally.

[0145] In certain embodiments the multimeric binding molecule is delivered via inhalation, e.g., in a nebulized form via nebulizer, via a metered dose inhaler (MDI), or in dry powder form via a dry powder inhaler (DPI).

[0146] In certain embodiments, the multimeric binding molecule is delivered intravenously. In some embodiments, the multimeric binding molecule is delivered intranasally and intravenously. In certain embodiments, the multimeric binding moleculeis delivered orally and intravenously. In certain other embodiments, the multimeric binding molecule is delivered intranasally, orally, and intravenously.Pharmaceutical Compositions and Administration Methods

[0147] The disclosure further provides a composition, e.g., a pharmaceutical composition, comprising a multimeric binding molecule, or two or more multimeric binding molecules, as provided herein. In certain embodiments the composition includes a combination of two or more different multimeric binding molecules as described herein that bind to different epitopes on the surface of an RSV virion. In some embodiments, the different epitopes are all on the G protein, e.g., on the G protein central conserved domain. A composition as provided herein can further include one or more pharmaceutically acceptable carriers and / or excipients or diluents and can be formulated so as to be suitable for a desired mode of administration.

[0148] Methods of preparing and administering a multimeric binding molecule as provided herein to a subject in need thereof can be determined by a skilled person in view of this disclosure. The route of administration of can be, for example, oral, parenteral, intranasally, or by inhalation (e.g., aerosol). While these forms of administration are contemplated as suitable forms, another example of a form for administration is a solution for injection, in particular, for intravenous or intraarterial injection or drip. A suitable pharmaceutical composition can include, for example, one or more of a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizing agent (e.g., human albumin or a natural or synthetic polymer), etc. Pharmaceutical excipients suitable for use in a composition as provided herein can be found in, for example, Handbook of Pharmaceutical Excipients, Ninth Ed., Sheskey, P.J., et al, Eds., Pharmaceutical Press (2020).

[0149] As discussed herein, a multimeric binding molecule as provided herein can be administered in a pharmaceutically effective amount for the treatment of a subject in need thereof. The disclosed multimeric binding molecule can be formulated to facilitate administration and promote stability of the multimeric binding molecule. Pharmaceutical compositions accordingly can include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives, and the like. A pharmaceutically effective amount of a multimeric binding molecule as provided herein means an amount sufficient to achieve effective binding to a target and to achieve atherapeutic benefit. Suitable formulations are described in Remington: The Science and Practice of Pharmacy (Elsevier Science) 23rd ed. (2020).

[0150] Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions, or solutions.

[0151] Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents. In some embodiments, the pharmaceutical composition is administered by nasal aerosol. In some embodiments, the pharmaceutical composition, such as a pharmaceutical composition for administration by nasal aerosol, comprises a pH adjuster, such as HC1; a buffer; an emulsifier, such as polysorbate or carbomer; a sugar or mono- or polyol, such as a monosaccharide (e.g., glucose, dextrose, or fructose), disaccharide (e.g., sucrose, lactose, or maltose), ribose, glycerin, sorbitol, xylitol, inositol, propylene glycol, galactose, mannose, xylose, rhamnose, glutaraldehyde, ethanol, mannitol, polyethylene glycol, glycerol, chitosal, phenylethyl alcohol; a preservative; cellulose, such as microcrystalline cellulose or carboxymethylcellulose; or mixtures thereof.

[0152] In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition, such as a pharmaceutical composition for administration by inhalation, is administered in the form of a dry powder, such as for administration via a dry powder inhaler, or is administered in the form of a liquid, such as for administration via a nebulizer, such as an aiijet-compressor nebulizer or a mesh-based nebulizer. In some embodiments, the pharmaceutical composition, such as a pharmaceutical composition for administration by inhalation, comprises a sugar or mono- or polyol, such as, for example, lactose, trehalose, mannitol, and / or sorbitol; a buffer, such as histidine, proline, or an arginine buffer; saline; polysorbate; or mixtures thereof.

[0153] The amount of a multimeric binding molecule is combined with carrier materials to produce a single dosage form will vary depending, e.g., upon the particular multimeric binding molecule, the subject to be treated, and the particular mode of administration, among other factors. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosing regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0154] In keeping with the scope of the present disclosure, a multimeric binding molecule as provided herein can be administered to a subject in need of therapy in an amount sufficient to produce a therapeutic effect or a prophylactic effect. A multimeric binding molecule as provided herein can be administered to the subject in a conventional dosage form prepared by combining the multimeric binding molecule of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.

[0155] This disclosure also provides for the use of a multimeric binding molecule as provided herein in the manufacture of a medicament for treating, preventing, or managing RSV infection.

[0156] In some embodiments, the compositions and methods provided herein can be used for the treatment of infections that have not been effectively treated using existing or established treatments.

[0157] This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Green & Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2nd ed. (IRL Press), Vol 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press); Mullis et al. U.S. Pat. No. 4,683,195; Hames & Higgins, eds. (1985) Nucleic Acid Hybridization (IRL Press); Hames & Higgins, eds. (1984) Transcription & Translation (IRL Press); Freshney (2016) Culture of Animal Cells, 7th ed. (Wiley -Blackwell); Woodward, Immobilized Cells and Enzymes (IRL Press) (1985); Perbal (1988) A Practical Guide To Molecular Cloning; 2nd ed. (Wiley-Interscience); Miller & Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells, (Cold Spring Harbor Laboratory); Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science); Methods in Enzymology, Vols. 151-155 (Academic Press, Inc., N.Y.); Mayer & Walker, eds. (1987) Immunochemical Methods in Cell & Molecular Biology (Academic Press, London); Weir & Blackwell, eds.; and in Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0158] General principles of antibody engineering are set forth, e.g., in Strohl & Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). General principles of protein engineering are set forth, e.g., in Park & Cochran, eds. (2009), Protein Engineering and Design (CDC Press). General principles of immunology are set forth, e.g., in: Abbas & Lichtman (2017) Cellular and Molecular Immunology 9th ed. (Elsevier). Additionally, standard methods in immunology known in the art can be followed, e.g., in Current Protocols in Immunology (Wiley Online Library); Wild (2013), The Immunoassay Handbook, 4th ed. (Elsevier Science); Greenfield, ed. (2013), Antibodies, a Laboratory Manual, 2nd ed. (Cold Spring Harbor Press); and Ossipow & Fischer, eds., (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press).

[0159] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0160] The following examples are offered by way of illustration and not by way of limitation.ExamplesExample 1 : Materials & Methods

[0161] Antibodies: SEQ ID NO: pairs : SEQ ID NO: 1 / SEQ ID NO: 5 (Gl), SEQ ID NO: 9 / SEQ ID NO: 13 (G2), and SEQ ID NO: 17 / SEQ ID NO: 21 (G3) form binding domains that bind specifically to the RSV G protein. SEQ ID NO: pairs SEQ ID NO: 33 / SEQ ID NO: 37 (F2) and SEQ ID NO: 41 / SEQ ID NO: 45 (F3) form binding domains that bind specifically to the RSV pre-F protein. IgG and IgM versions of antibodies comprising binding domains as set forth above for Gl, G2, G3, F2, and F3 were produced and purified using recombinant protein techniques.

[0162] Cells: Vero cells (ATCC, CCL-81) were maintained in cell culture medium including advanced DMEM / F-12, 2% FBS, 4 mM L-glutamine and 100 U / mL penicillinstreptomycin. A549 cells (ATCC, CCL-185) were maintained in cell culture medium including Opti-MEM, 5% FBS and 100 U / mL penicillin-streptomycin.

[0163] Viruses: All viruses were provided by ViraTree LLC, North Carolina, US. Recombinant RSV viruses expressing green fluorescent protein (GFP) were used in the following assays. Final titers for neutralization and complement assays were as follows:• RSV-A: 800 pfu / mL (RSV-GFP5, ViraTree Cat. No. R125)• RSV-B: 3200 pfu / mL (RSVB-GFP3, ViraTree Cat. No. R423)

[0164] Neutralization and complement assay: Cells were seeded at 1.5x 104cells / well with 100 pL cell culture medium in 96-well plates. IgG and IgM antibodies were prepared with 3 -fold serial dilutions and mixed with equal volumes of virus. For neutralization assays in the presence of complement, baby rabbit complement was included in the virus solution. Once the virus and antibodies (or antibodies + complement) were mixed, the mixture was incubated 1-2 hours at 37°C. After the incubation, 100 pL of virus / antibody (MOI as above) mixture was added to 100 pL cells in each well. The plate was incubated in a 5% CO2, 37°C incubator for 24-48 hours.

[0165] Because the viruses used in the various assays carry a GFP reporter, infection was read as a GFP signal. The GFP signal was acquired by a BioTek Cytation 7 imaging reader (Agilent), and the virus entry events were counted by Gen5 software. IC50 curves were generated using a non-linear regression curve fit [log(inhibitor) vs. normalized response - variable slope] in GraphPad Prism 9 software.

[0166] ELISA assay: The extracellular domain of G protein was provided by Sino Biological (RSV A, rsbl734, Cat. No. 11070-V08H; RSV Bl, Cat. No. 13029-V08H). PIERCE™ 96-well polystyrene plates (white opaque) were coated with 2 pg / mL of extracellular RSV G proteins in PBS at 4°C overnight. Plates were washed 3 times with wash buffer (Pierce™ 20* PBS Tween™20 Buffer diluted in deionized water), followed by blocking in 5% FBS-PBS at room temperature for 1 hour. Plates were washed once and then 100 pL of anti-RSV G antibodies (1 :3 serial dilution), or appropriate controls were added. After 2 hours incubation, plates were washed 5*, followed by addition of 100 pL HRP-conjugated anti-human IgG (Bethyl Laboratories, Inc. Cat. No. A80104P) or antihuman IgM (Bethyl Laboratories, Inc. Cat. No. A80100P) with working concentration of 10 ng / mL. Then the plates were washed 5*, and HRP activity was detected using QUANT ARED™ Enhanced Chemifluore scent HRP Substrate Kit for color development. Fluorescence signal was acquired by BioTek Cytation 7 plate reader, and the report was generated by Gen5 software. EC50 curves were generated using a nonlinear regression curve fit [log(agonist) vs. response - variable slope] in GraphPad Prism 9 software.Example 2: ELISA Binding Assays

[0167] IgG-Gl, IgM-Gl, IgG-G2, and IgM-G2 were tested for their ability to bind in ELISA to the G proteins of an RSV-A strain and an RSV-B strain. A mutant variety of IgM-Gl with reduced ability to activate complement that includes P311 A and P313 S point mutations in the IgM heavy chain constant region (“IgM-Gl PAPS,” see U.S. Patent No.11,401,337) was also tested in this ELISA assay. The ECso values from these binding assays are shown in Table 2 below and in FIG. 1. Against both A and B strain viruses, the IgG versions of the antibodies bound at equivalent or lower ECso values than did the IgM versions. Table 2: Binding to G protein of RSV-A & RSV-BExample 3 : Neutralization Assays

[0168] IgG-Gl, IgM-Gl, IgG-G2, IgM-G2, IgG-F2, IgM-F2, IgG-F3, and IgM-F3 were tested for their ability to neutralize the ability of RSV-A to infect Vero cells in the presence or absence of 10% complement. IgG-G3 and IgM-G3 were also tested, but only in the presence of 10% complement. The ICso values from these neutralization assays are shown in Table 3 below and the results are shown in FIG. 2.Table 3: Neutralization of RSV-A on Vero

[0169] Among the anti-F antibodies, the IgG version of each antibody neutralized at a lower IC50 value than its corresponding IgM version. By contrast, among the anti-G antibodies, the IgM versions neutralized at lower IC50 values than the corresponding Ig-G versions. IgG-Gl and IgG-G2 were unable to neutralize infection (denoted in Table 3 and in the following tables by “X”) of Vero cells in the absence of complement, and IgG-Gl was unable to neutralize even in the presence of 10% complement. IgG-G3 was only able to achieve weak inhibition, even in the presence of complement (data not shown). The IgM PAPS mutants were unable to neutralize in the presence of complement (data not shown for IgM-Gl, FIG. 2B for IgM-Fl).

[0170] The anti-G IgG and IgM antibodies were also tested for their ability to neutralize the ability of RSV-A to infect A549 cells in the presence or absence of 10% complement. The IC50 values from these neutralization assays are shown in Table 4 below and the results in FIG. 3Table 4: Neutralization of RSV-A on A549

[0171] IgG-Gl and IgG-G2 were unable to neutralize infection of A549 cells in the absence of complement, where IgM-Gl and IgM-G2 neutralized both in the presence or absence of complement, although more potently in the presence of complement.

[0172] IgG-Gl, IgM-Gl, IgG-G2, and IgM-G2 were tested for their ability to neutralize the ability of RSV-B to infect Vero cells and A549 cells in the presence or absence of 10% complement. IgG-G3 and IgM-G3 were also tested, but only on Vero cells in the presenceof 10% complement. The IC50 values from these neutralization assays are shown in Table 5 below and the results are provided in FIG. 4.Table 5: Neutralization of RSV-B

[0173] IgG-Gl, IgG-G2, and IgM-G2 were unable to neutralize infection of Vero cells in the absence of complement. IgG-G3 was only able to achieve weak inhibition, even in the presence of complement, while the corresponding IgM-G3 neutralized RSV-B infection of Vero cells with an ICso of 12.39 ng / mL in the presence of complement. IgG-Gl and IgG-G2 were unable to neutralize infection of A549 cells in the absence of complement. On the other hand, IgM-Gl neutralized RSV-B infection in both cell lines both in the presence or absence of complement.

[0174] Finally, IgG-Gl and IgM-Gl were tested for the ability to neutralize the ability of RSV-A to infect Vero cells in the presence of varying concentrations of complement. The ICso values from these neutralization assays are shown in Table 6 below and the results are shown in FIG. 5.Table 6: Neutralization of RSV-A on Vero

[0175] As provided above, IgM-Gl was able to neutralize RSV-A infection with or without the addition of complement, while IgG-Gl was unable to neutralize infection in the absence of complement, but also was unable to neutralize in the presence of 0.4% and 1.1% complement.Example 4: In vivo RSV Challenge

[0176] Cohorts of 6-week-old, female, BALB / c mice (4 mice each) were pre-treated (two hours before infection) with 80 pg of IgG-Gl, IgM-Gl, or IgG-F3, followed by intranasal infection with 105pfu of RSV-A2. The mice also received another dose of antibody on day 2 post-infection. A control cohort was mock-infected and treated with vehicle alone. One cohort of the IgG-F3 mice received the antibody by intranasal delivery, while another cohort received intramuscular injection. All cohorts were weighed daily. On day 4 postinfection the mice were sacrificed, and their lungs and noses were harvested for weighing. Swabs were also collected from the lungs and noses, and titered on Vero cells for RSV plaques.

[0177] FIG. 6A shows the percent change in average body weight over time, while FIG. 6B shows the average body weight of each cohort over time. A decrease in body weight is well-recognized as a correlate of RSV pathogenesis (see, e.g., van Erp et al. (2019) Viruses 11(6):508). As can be seen, regardless of how the data are presented, the average body weights of the mice treated with IgM-Gl were equivalent to those of the mock infected mice, while all other cohorts show a more pronounced weight loss over time.

[0178] FIG. 7A shows RSV titers collected from the lung on day 4 post-infection from the various cohorts. FIG. 7B shows RSV titers collected from the nose on day 4 postinfection from the various cohorts. In both the nose and the lung, titers collected from the mock-infected cohort and from the IgM-Gl -treated cohort were below the limit of detection. There were no statistically significant differences among any of the cohorts with regard to either nose weight or lung weight on day 4 post-infection (data not shown).Table 7: Sequences

Claims

WHAT IS CLAIMED IS:

1. A multimeric binding molecule comprising two to six bivalent binding units, wherein each binding unit comprises two IgM or IgA heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain, wherein three to twelve of the binding domains are identical and specifically bind to a respiratory syncytial virus (RSV) glycoprotein G, and wherein the binding molecule can neutralize RSV more potently than a bivalent reference IgG antibody comprising two of the binding domains that specifically bind to the RSV glycoprotein G.

2. The multimeric binding molecule of claim 1, which is a hexameric binding molecule comprising six bivalent IgM binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof each associated with an antigen binding domain, and wherein the IgM heavy chain constant regions each comprise a Cp4 domain and an IgM tail-piece (tp) domain.

3. The multimeric binding molecule of claim 1, which is a pentameric binding molecule comprising five bivalent IgM binding units and a J chain or functional fragment or variant thereof, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof each associated with an antigen binding domain, and wherein the IgM heavy chain constant regions each comprise a Cp4 domain and an IgM tail-piece (tp) domain.

4. The multimeric binding molecule of claim 2 or claim 3, wherein the IgM heavy chain constant regions or multimerizing fragments or variants thereof are human IgM constant regions or multimerizing fragments or variants thereof.

5. The multimeric binding molecule of claim 1, which is a dimeric or tetrameric binding molecule comprising two or four bivalent IgA binding units and a J chain or functional fragment or variant thereof, wherein each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof each associated with an antigen binding domain, and wherein the IgA heavy chain constant regions each comprise a Ca3 domain and an IgA tail-piece (tp) domain.

6. The multimeric binding molecule of claim 5, wherein the IgA heavy chain constant regions or multimerizing fragments or variants thereof are human IgA constant regions or multimerizing fragments or variants thereof.

7. The multimeric binding molecule of any one of claims 1 to 6, wherein each binding unit comprises two heavy chains each comprising a heavy chain variable (VH) domain situated amino terminal to the constant region or multimerizing fragments or variants thereof.

8. The multimeric binding molecule of claim 7, wherein the VH comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences comprise, respectively: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; or SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

9. The multimeric binding molecule of any one of claims 3 to 8 wherein the J chain or functional fragment or variant thereof is a human J chain or functional fragment or variant thereof.

310. The multimeric binding molecule of any one of claims 1 to 9, wherein RSV neutralization potency is measured as 50% inhibition concentration (ICso) in cell culture.

11. The multimeric binding molecule of any one of claims 1 to 9, wherein RSV neutralization potency is measured as 50% effective dose (EDso) in an animal model of infection.

12. A polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of the multimeric binding molecule of any one of claims 1 to 9.

13. A vector comprising the polynucleotide of claim 12.

14. A host cell comprising the polynucleotide of claim 12, wherein the host cell can express a multimeric binding molecule comprising two to six bivalent binding units, wherein each binding unit comprises two IgM or IgA heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain, wherein three to twelve of the binding domains are identical and specifically bind to an RSV glycoprotein G.

15. A method for treating or preventing RSV infection in a subject in need of treatment, comprising administering to the subject an effective amount of the multimeric binding molecule of any one of claims 1 to 9.

16. The method of claim 15, wherein the subject is human.

17. The method of claim 15 or claim 16, wherein administering comprises intravenous, subcutaneous, intramuscular, intranasal, and / or inhalation administration.

18. The method of claim 17, wherein the administration is intranasal, and / or via inhalation.

19. Use of the multimeric binding molecule of any one of claims 1 to 11 in the manufacture of a medicament for the treatment or prevention of RSV infection.

20. The multimeric binding molecule of any one of claims 1 to 11, for use in a method of treating or preventing RSV infection.

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