Laminin-igg hybrid synthetic agents

Abstract

Described herein are engineered Fc-domain linked heterotrimeric synthetic agents. These engineered Fc-domain linked heterotrimeric synthetic agents may include a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, in which each Fc domain is linked to a heterotrimeric complex comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands. The entire synthetic agent (including the heterotrimeric complexes) may self-assemble. In some variations the alpha, beta and / or gamma strands of each heterotrimeric complex may be linked via an amino acid linker to a binding domain, such as a pair of antigen binding (Fab) domains.

Description

Attorney Docket No. 5470.976.WOLAMININ-IgG HYBRID SYNTHETIC AGENTSCLAIM OF PRIORITY

[0001] This patent application claims priority to U.S. provisional patent application no. 63 / 760,817, filed February 20, 2025, titled “LAMININ-IgG HYBRID SYNTHETIC AGENTS,” and herein incorporated by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. HD101344 and HD101562 awarded by National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] A Sequence Listing in XML format, submitted under 37 C.F.R. § 1.821, entitled 5470-976WO_ST26.xml, 37,001 bytes in size, generated on February 20, 2026, and filed herewith, is hereby incorporated by reference into the specification for its disclosures.BACKGROUND

[0004] Heterotrimeric protein complexes are well-known in eukaryotic systems and may be used as signaling molecules, that may help regulate transmembrane signaling, often by coupling to cell surface receptors. Such heterotrimeric complexes are known, for example, laminin heterotrimeric proteins are essential components of the basement membrane and the extracellular matrix that provide structural support, mediate cell adhesion, and are involved in critical biological processes such as tissue development, wound healing, and cellular signaling. Their interactions with cellsurface receptors, like integrins and dystroglycans, regulate processes ranging from cell migration to angiogenesis. Disruptions in laminin function are associated with various genetic disorders, as well as cancer progression and tissue degeneration.

[0005] Laminin-511 is an essential component of the basement membrane, playing a key role in cell adhesion, migration, differentiation, and tissue development. A synthetic agent mimicking laminin-511 could be used to promote tissue regeneration in injured or damaged tissues, especially for skin, nerves, and muscles. A synthetic heterotrimeric agent could be designed to target specificAttorney Docket No. 5470.976.WOcells or tissues (e.g., epithelial or endothelial cells) in disease conditions, including those in which the extracellular matrix (ECM) has been damaged or needs reinforcement. Such agents could also serve as a scaffold for targeted drug delivery, including in therapies targeting the ECM, cell migration, or immune responses. These synthetic agents may be incorporated into biomimetic materials.

[0006] It would be particularly beneficial to provide synthetic agents derived from laminin heterotrimeric domains. Such synthetic agents, particularly when stably linked together to provide multiple copies of the heterotrimeric domains, may have biomedical applications, including as agents by themselves, or when stably linked to each other, acting as the core of other synthetic agents, as they may serve as a stable and self-assembling core from which one or more binding domains of virtually may be linked. Synthetic agents including laminin-derived heterotrimeric domains may be therapeutically useful in the fields of immunology, tissue engineering, regenerative medicine, and targeted therapies.

[0007] Synthetic agents derived from laminin heterotrimeric domains, particularly those that may reliably and controllably self-assemble, may be used as therapeutic agents either by themselves or in combination with one or more, and preferably multiple, binding domains.SUMMARY OF THE DISCLOSURE

[0008] Described herein are compositions and methods including engineered Fc-domain linked heterotrimeric synthetic agents based on human laminin-511. These agents may be used with or without the addition of one, or preferably multiple, binding domains linked to some or all of the heterotrimeric domains.

[0009] In general, an engineered Fc-domain linked heterotrimeric synthetic agent may include: a pair of human or humanized Immunoglobulin fragment crystallizable (Fc) domains linked to at least one strand of a heterotrimeric complex comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0010] In any of these synthetic agents, two or three of the alpha, beta and gamma strands may be linked to copies of an antigen binding domain. For example, each of the alpha, beta and gamma strands may be linked to copies of one or more antigen binding domains. The one or more antigen binding domains may be the same antigen binding domain, or may be different antigen binding domains (e g., different antigen binding domains). Thus, in some examples, the antigen bindingAttorney Docket No. 5470.976.WOdomains are different antigen binding domains. The antigen binding domains may comprise a pair of antigen binding (Fab) domains. An antigen binding domain may be equivalently described as a protein binding domain herein.

[0011] Any appropriate binding domain (e.g., binding site) may be linked to the synthetic agent, in particular, binding domains formed by protein sequences that bind to another molecule with specificity. The binding partner may be referred to as a ligand or antigen and may include other proteins (resulting in a protein-protein interaction), enzyme substrates, second messengers, hormones, and / or allosteric modulators. The binding domain may be antigen-specific (e.g., FAB domains, MHC domains, etc.) or may be more general binding domains, including protein-binding domains (e.g., SH2, PH domains, etc.).

[0012] For example, the binding domains described herein may be FAB binding domains derived from an antibody. Other specific antigen-recognition domains (also referred to as binding domains herein) may include T Cell Receptor (TCR) antigen-recognition domains that may be formed, e.g., by the variable (V) regions of an a (alpha) and P (beta) chains (or y / 8 in some T cells). These V regions contain Complementarity Determining Regions (CDRs) similar to those in antibody Fab domains, which interact with peptide antigens presented by Maj or Histocompatibility Complex (MHC) molecules. The binding domain may be a Major Histocompatibility Complex (MHC) antigen-recognition domain, e.g., an MHC Class I and II molecules, including a peptide-binding groove, e.g., a binding groove formed by the al and a2 domains (or for MHC Class II, formed by the al and i domains). The binding domain may be a B Cell Receptor (BCR) antigenrecognition domain (which may also contain a Fab domain with variable regions that bind specific antigens, e.g., BCR’s light and heavy chains). The binding domain may be a Natural Killer (NK) Cell Receptor antigen-recognition domain (e.g., Killer-cell Immunoglobulin-like Receptors (KIRs), NKG2D, etc.). The binding domain may be a Pattern Recognition Receptors (PRRs) antigen-recognition domain (e.g., Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), etc.). The binding domain may be a Leukocyte Receptor Complex (LRC) Family Proteins antigen-recognition domain (e.g., Killer-cell Immunoglobulin-like Receptors (KIRs), Leukocyte Immunoglobulin-like Receptors (LILRs), etc ). These protein domains, like Fab domains, may be highly specific in recognizing particular antigens or molecular patterns and may be linked to copies of one or more of the alpha, beta and gamma strands of the Fc-domain linked heterotrimeric synthetic agents described herein.Attorney Docket No. 5470.976.WO

[0013] In some cases, the binding domain may be an active site (e.g., enzymatically active). The binding site may be a single- or a multi-chain binding site. Protein-binding domains may be structural units within proteins that enable them to interact with other proteins, nucleic acids, or other biomolecules. These domains are highly conserved. Examples of binding domains that may be linked to the synthetic agents described herein may include, but are not limited to SH2 Domain (Src Homology 2 Domain), SH3 Domain (Src Homology 3 Domain), PDZ Domain (Post-synaptic density protein, Drosophila disc large tumor suppressor, Zonula occludens-1 protein), WW Domain, FHA Domain (Forkhead-Associated Domain), PH Domain (Pleckstrin Homology Domain), Leucine Zipper Domain, Zinc Finger Domain, Death Domain, Bromodomain, Chromodomain, 14-3-3 Domain, Ankyrin Repeat Domain, WD40 Repeat Domain, TIR Domain (Toll / Interleukin-1 Receptor Domain), EF-Hand Domain, SAM Domain (Sterile Alpha Motif Domain).

[0014] In particular, the engineered Fc-domain linked heterotrimeric synthetic agents described herein may include any appropriate Fc domain. For example, the human or humanized Fc domain may be at least 80% identical (or 85%, or 90%, or 95% or 99%, or completely identical) with a human Immunoglobulin G (IgG) Fc domain, a human Immunoglobulin M (IgM) Fc domain, a human Immunoglobulin D (IgD) Fc domain, a human Immunoglobulin E (IgE) Fc domain, a human Immunoglobulin Al (IgAl) Fc domain, or a human Immunoglobulin A2 (IgA2) Fc domain. Homology may be determined at the protein (amino acid) sequence. Any of the human or humanized Fc domains may include known / common Fc mutations. For example, human IgG Fc domain may include common Fc mutations such as the LALA mutation (e.g., the L234A / L235A mutation to reduce binding to FcyRI and FcyRIIa receptors), the D265A and / or P331S mutations (to reduce binding to FcyR and / or promote aggregation), the L234F / L235E / P331S mutation (extended half-life and lack immune receptor binding), N297A, N297Q, and / or N297G mutations (create aglycosylated forms), PFc29 and PFc41 mutations (prolong the circulating half-lives of IgG antibodies), etc.

[0015] The Fc domain may be linked to any of the trimer domains. For example, in some cases the Fc domain is linked to the alpha strand; the Fc domain may be linked to the beta strand; the Fc domain may be linked to the gamma strand. The alpha strand(s) of the engineered Fc-domain linked heterotrimeric synthetic agents described herein may have a sequence that is 80% or more identical (e.g., 85% or more, 90% or more 95% or more, etc.) with the sequence of the humanAttorney Docket No. 5470.976.WOlaminin-511 alpha strand. The beta strand(s) of the engineered Fc-domain linked heterotrimeric synthetic agents described herein may have a sequence that is 80% or more identical (e.g., 85% or more, 90% or more 95% or more, etc.) with the sequence of the human laminin-511 beta strand. The gamma strand(s) of the engineered Fc-domain linked heterotrimeric synthetic agents described herein may have a sequence that is 80% or more identical (e.g., 85% or more, 90% or more 95% or more, etc.) with the sequence of the human laminin-511 gamma strand. These sequences are provided in the attached sequence listing.

[0016] In variations in which one or more Fab domains are linked to the alpha, beta, and / or gamma strands of the engineered Fc-domain linked heterotrimeric synthetic agents, the Fab domains may each comprise: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences of any of those listed in the attached sequence listing, and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence of any of those listed in the attached sequence listing.

[0017] Each of the Fc domains in the pair of Fc domains of the engineered Fc-domain linked heterotrimeric synthetic agents may be linked to a separate heterotrimeric complex each comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands. The heterotrimeric complexes may have identical sequences or may be different. For example, the Fc domains in the pair of Fc domains of the engineered Fc-domain linked heterotrimeric synthetic agent may be linked to copies of one or more heterotrimeric complexes having sequences for the alpha, beta and gamma strands, although they may all be at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0018] Any appropriate linker may be used to link the Fc domain to the alpha, beta and / or gamma strands. The Fc domain may be linked by an amino acid linker to the N-terminal and / or C-terminal end of the Fc domain to the strand (alpha, beta or gamma) of the heterotrimeric complex. In some cases the linker is an IgG hinge region. Example sequences of linkers that may be used to link the Fc domain to the strand (alpha, beta or gamma) are included in the attached sequence listing. For example, the linker may be 6xGGGGS (SEQ ID NO:9).

[0019] In any of these compositions, the heterotrimeric complex may be stabilized by disulfide bonds between the alpha, beta, and gamma strands. Multiple disulfide bonds may be included. The method of fabricating these proteins may include the formation of disulfide bonds.Attorney Docket No. 5470.976.WO

[0020] For example, described herein are synthetic agents (e.g., engineered Fc-domain linked heterotrimeric synthetic agents) comprising: a pair of human or humanized Immunoglobulin fragment crystallizable (Fc) domains, wherein each Fc domain is linked at the N-terminal end and / or C-terminal end to a different heterotrimeric complex each including an alpha strand, a beta strand or a gamma strand, wherein the alpha, beta and gamma strands are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0021] In any of these engineered Fc-domain linked heterotrimeric synthetic agents, two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes may be linked to copies of an antigen binding domain (e.g., protein binding domain). In some cases each of the alpha, beta and gamma strands of each of the heterotrimeric complexes may be linked to copies of an antigen binding domain (e.g., protein binding domain). The antigen binding domains may be the same antigen binding domain. The antigen binding domains may be different antigen binding domains. As mentioned, the antigen binding domains may comprise a pair of antigen binding (Fab) domains.

[0022] As mentioned above, the human or humanized Fc domain may be at least 80% identical with a human Immunoglobulin G (IgG) Fc domain, a human Immunoglobulin M (IgM) Fc domain, a human Immunoglobulin D (IgD) Fc domain, a human Immunoglobulin E (IgE) Fc domain, a human Immunoglobulin Al (IgAl) Fc domain, or a human Immunoglobulin A2 (IgA2) Fc domain. The Fc domain may be linked to the alpha strand, the beta strand, or the gamma strand.

[0023] The Fab domains may each comprise: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences (as listed in the attached sequence listing examples) and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence as listed in the attached sequence listing examples.

[0024] As mentioned, any of these engineered Fc-domain linked heterotrimeric synthetic agents may be linked to each heterotrimeric complex by a linker, e.g., an IgG hinge region a 6xGGGGS (SEQ ID NO:9) linker, etc.

[0025] In any of these compositions, the heterotrimeric complex may comprise a DHT03 structure that assemble specifically into a heterotrimer, meaning the three chains come together without forming dimers or other alternative complexes. These heterotrimeric complexes may be building blocks for larger protein structures described herein, and may be resolved using X-rayAttorney Docket No. 5470.976.WOcrystallography. As discussed and shown in the examples below, these engineered Fc-domain linked heterotrimeric synthetic agents, including the heterotrimeric complexes, may be expressed in any appropriate cell expression line, including but not limited to E. colt.

[0026] The engineered Fc-domain linked heterotrimeric synthetic agents described herein may include between one and four heterotrimeric complexes, and may be linked to either or both of the C- and N-terminal end of the Fc domains, e.g., via a linker. For example, the engineered Fc-domain linked heterotrimeric synthetic agents may include four heterotrimeric complexes.

[0027] An engineered Fc-domain linked heterotrimeric synthetic agents (e.g., a synthetic agent) may include: a pair of human or humanized IgG immunoglobulin fragment crystallizable (Fc) domains having the sequence of any of the Fc domains shown in the attached sequence listing, wherein each of the Fc domains are linked at the N-terminal end and / or the C-terminal end to an alpha strand of a self-assembled heterotrimeric complex comprising the alpha strand, a beta strand and a gamma strand, wherein the alpha strand has sequence that is at least 80% identical to human laminin-511 alpha strand, the beta strand has a sequence that is at least 80% identical to human laminin-511 beta strand and the beta strand has a sequence that is at least 80% identical to human laminin-511 gamma strand.

[0028] As discussed above, any of these engineered Fc-domain linked heterotrimeric synthetic agents may include one or more protein binding domains, such as but not limited to Fab domains. For example, at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes may be linked to a protein binding domain (e.g., an antigen binding domain). The antigen binding domains may be the same antigen binding domain or different antigen binding domains. In some cases, the antigen binding domains comprise a pair of antigen binding (Fab) domains, which may include the sequences of any of the FAB domains included in the attached sequence listing. For example, the Fab domains may each comprise: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences of those listed as HC in the attached sequence listing, and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence of those listed as LC in the attached sequence listing.

[0029] In general, the protein-binding domains may be linked to the heteromeric domains (e.g., an alpha, beta, or gamma strand) directly or via a linker. Any appropriate linker may be used,Attorney Docket No. 5470.976.WOincluding an amino acid linker or hinge regions (e.g., an IgG hinge region 6xGGGGS (SEQ TD NO:9) linker, etc.).

[0030] For example, a synthetic binding agent (e.g., an engineered Fc-domain linked heterotrimeric synthetic agent) may include a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to at least one copy of an antigen binding domain (e.g., protein binding domain), further wherein the antigen binding domain specifically binds to an epitope of a target, so that the synthetic binding agent binds to the target with high affinity, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0031] The antigen binding domain may comprise a pair of antigen binding (Fab) domains. The antigen binding domain on each of the alpha, the beta and / or the gamma strands may be the same antigen binding domain. Alternatively, different antigen-binding domains may be used. In some cases, different antigen-binding domains may be used that are each directed to different binding sites on the same antigen. In some cases, the different antigen-binding domains may be directed to different antigens. The antigen binding domains may comprise one or more antigen binding (Fab) domains, including any of those listed in the attached sequence listing.

[0032] For example, an engineered Fc-domain linked heterotrimeric synthetic agents (e.g., a synthetic binding agent) may include: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains having a sequence selected from those listed in the attached sequence listing, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein two or more of the alpha strand, the beta strand, and the gamma strand of the self-assembled heterotrimeric complex are each linked via amino acid linkers to a pair of antigen-binding (Fab) domains, wherein the alpha strand is at least 80% identical to the human laminin-511 alpha having the sequence listed in the attached sequence listing, wherein the beta strand is at least 80% identical to the human laminin-511 beta having the sequence of the sequence listed in the attached sequence listing, and wherein the gamma strand is at least 80% identical to the human laminin-511 gamma having the sequence of the sequence listed in the attached sequence listing.Attorney Docket No. 5470.976.WO

[0033] In some cases, the engineered Fc-domain linked heterotrimeric synthetic agents are specifically configured to target sperm. An engineered Fc-domain linked heterotrimeric synthetic agents having one or more protein binding sites targeting a sperm surface protein (e.g., an antisperm binding domain) may be used as a contraceptive. Including a topical contraceptive applied to a mucosal surface (e.g., vaginal mucosa).

[0034] For example, a synthetic binding agent (e.g., an engineered Fc-domain linked heterotrimeric synthetic agent) may include: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to an anti-sperm antigen binding domain, further wherein the anti-sperm antigen binding domain specifically binds to an epitope of a sperm, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0035] An engineered Fc-domain linked heterotrimeric synthetic agent may include: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains having a sequence selected from: those for Fc domains (e.g., for IgG, IgM, IgD, IgE, IgAl, and / or IgA2O as provided in the attached sequence listing, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein each of the alpha strand, beta strand, and gamma strand of the self-assembled heterotrimeric complex are linked via amino acid linkers to an anti-sperm antigen binding domain, further wherein the anti -sperm antigen binding domain specifically binds to an epitope of a sperm, wherein the alpha strand is at least 80% identical to the human laminin-511 alpha having the sequence provided in the attached sequence listing, wherein the beta strand is at least 80% identical to the human laminin-511 beta having the sequence provided in the attached sequence listing, and wherein the gamma strand is at least 80% identical to the human laminin-511 gamma having the sequence provided in the attached sequence listing.

[0036] In general, also described herein are methods of treating a patient, the method comprising delivering an engineered Fc-domain linked heterotrimeric synthetic agent to a patient in need thereof.

[0037] In some examples these methods and agents may be for treating a pathogen that is transmitted by respiration, such as coronaviruses (e.g., SARS-CoV-2, SARS-CoV, MERS-CoV,Attorney Docket No. 5470.976.WOHCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKUl, etc.), rhinoviruses (cold viruses), influenza viruses (e.g., Influenza A, e.g., H1N1, H3N2, Influenza B, Influenza C, Influenza D, etc.), Respiratory Syncytial Virus (RSV), Parainfluenza Viruses (e.g., HPIV-1, HPIV-2, HPIV-3, HPIV-4, etc.), Adenoviruses, Human Metapneumovirus (hMPV), Enteroviruses (e.g., Enterovirus D68, Coxsackievirus, etc.).

[0038] An engineered Fc-domain linked heterotrimeric synthetic agents may be configured to bind to RSV and may be used to treat and / or prevent RSV. For example, a synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to an anti-RSV antigen binding domain, further wherein the anti-RSV antigen binding domain specifically binds to an epitope of RSV, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0039] A synthetic binding agent may include: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains having a sequence selected from any of the Fc domains listed in the attached sequence listing, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein each of the alpha strand, beta strand, and gamma strand of the self-assembled heterotrimeric complex are linked via amino acid linkers to an anti-RSV antigen binding domain, further wherein the anti-RSV antigen binding domain specifically binds to an epitope of RSV, wherein the alpha strand is at least 80% identical to the human laminin-511 alpha having the sequence listed in the attached sequence listing, wherein the beta strand is at least 80% identical to the human laminin-511 beta having the sequence listed in the attached sequence listing, and wherein the gamma strand is at least 80% identical to the human laminin-511 gamma having the sequence listed in the attached sequence listing.

[0040] Also described herein are methods of treating, reducing infection or preventing RSV infection in a patient by delivering the synthetic binding agent to a patient in need thereof.

[0041] In general, the compositions (e.g., synthetic agents) and methods of making and using them described herein may be formed by obligate heterotrimerization. For example, any of these compsoitions may include a, 0, and y strands that are configured to self-assemble exclusively into a heterotrimer and not homotrimers or dimers. In some cases, these synthetic agents mayAttorney Docket No. 5470.976.WOinclude disulfide bond dependent stabilization. For example, any of these synthetic agent may include at least one disulfide bond formed between non identical strands (e.g., a-0, p-y, or a-y).

[0042] Although the synthetic agents described herein may include strands (a, P, and y strands) from any appropriate origin, it may be particularly useful to use a, P, and y strands having a laminin 511 E8 domain origin. For example, each of the a, P, and y strands may comprise an E8 laminin 511 trimerization region.

[0043] The synthetic agents described herein may be configured to have a desired spatial configuration. For example, in any of these synthetic agents the Fc domains may extend outward from the heterotrimeric complex such that the antigen binding domains are radially accessible.

[0044] For example, described herein are synthetic agents comprising: (a) a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains; and (b) at least one selfassembled heterotrimeric complex comprising an a strand, a P strand, and a y strand, (i) each of the a, P, and y strands comprising a laminin-511-derived trimerization region, (ii) the a, P, and y strands assembling exclusively as an obligate a / p / y heterotrimer and not as homotrimers or heterodimers, and (iii) the heterotrimer stabilized by at least one inter-strand disulfide bond between non-identical strands; wherein each Fc domain is linked via an amino acid linker to at least one of the a, P, or y strands; and wherein at least two of the a, P, and y strands are each linked to one or more binding domains.

[0045] A synthetic agent may comprise: a pair of human or humanized Fc domains, each comprising an IgG, IgM, IgA, IgD, or IgE Fc region; a self-assembled heterotrimeric laminin-511-derived complex comprising a, P, and y strands, each strand comprising a laminin-511 E8 trimerization region, the a, P, and y strands assembling exclusively into an obligate heterotrimer, and the complex stabilized by at least one disulfide bond between non-identical strands; each Fc domain linked through an IgG hinge or GGGGS-type linker to at least one heterotrimeric complex; and at least two of the a, p, or y strands each linked to one or more binding domains independently selected from Fab, scFv, VHH (variable region of camelid heavy-chain-only antibody), TCR, MHC, NK, PRR, SH2 (src Homology 2 domain), SH3 (src Homology 3 domain), or PDZ domains.

[0046] The binding domain may be independently selected from Fab, scFv, VHH, singledomain antibody, T-cell receptor (TCR) variable regions, MHC binding domains, NK-cellAttorney Docket No. 5470.976.WOreceptor domains (including KIR), pattern-recognition receptor domains, SH2, SH3, and PDZ domains.

[0047] The binding domains on the a, , and y strands may each be a Fab domain.

[0048] Each Fc domain may be an IgG, IgM, IgAl, IgA2, IgD, or IgE Fc domain, optionally comprising one or more Fc mutations selected from L234A / L235A (LALA), D265A, P331S, L234F / L235E / P331S, N297A, N297Q, N297G, PFc29, and PFc41. The amino acid linker may comprise an IgG hinge sequence (SEQ ID NO:7 or SEQ ID NO:8) or a (GGGGS)n linker (SEQ ID NO:9) or (GSSSS)n (SEQ ID NO: 10), wherein n is 1-10. Each Fc domain may be linked at its N-terminus, C-terminus, or both, to one heterotrimeric complex. The synthetic agent may include in total 1 to 4 heterotrimeric complexes per agent. The a strand may have at least 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2, the 0 strand may have at least 80% sequence identity to SEQ ID NO:3 or SEQ ID NO:4, and the y strand may have at least 80% sequence identity to SEQ ID NO:5 or SEQ ID NO:6.

[0049] The a strand may be SEQ ID NO: 1 or SEQ ID NO:2, the 0 strand may be SEQ ID NO:3 or SEQ ID NO:4, and the y strand may be SEQ ID NO:5 or SEQ ID NO:6.

[0050] The one or more binding domains may comprise a pair of Fab domains on at least two of the a, 0, and y strands. The binding domains on the a, 0, and y strands may be the same binding domain or different binding domains.

[0051] In general, the synthetic agent, at 37°C and at a concentration <10 pg / mL, may reduce progressively motile (PM) sperm by at least 90% within 90 seconds, as measured by CASA. The synthetic agent may immobilize at least 50% of virions in cervicovaginal mucus at a concentration <1 pg / mL, as quantified by multiple particle tracking.

[0052] In general at least 30% of Fc glycoforms may comprise a biantennary core glycan Manal 6(Manal 3)Man01 4GlcNAc01 4GlcNAc with terminal N acetylglucosamine on each branch and lacking fucose and galactose, thereby enhancing mucin crosslinking once the agent is bound to a target.

[0053] The synthetic agent may remain monomeric by SEC MALS with >90% monomer by area after incubation for 1 hour at pH 4.0 and 37°C.

[0054] The linker between Fab and the laminin trimerization region may comprise an IgG upper hinge (SEQ ID NO:7), conferring greater agglutination potency at 37°C than an otherwise identical construct comprising a (G4S)6 linker.Attorney Docket No. 5470.976.WO

[0055] The binding domains of each synthetic agent may comprise a heavy chain variable region and / or light chain variable region selected from SEQ ID NOs:21-34.

[0056] In general, the % identity to SEQ ID NOs: 1-6 may be calculated by BLASTP with default parameters and a gap-open penalty of 11 and gap-extension penalty of 1 across the full-length trimerization region.

[0057] The one or more binding domains may specifically bind a glycoform of CD52 (CD52g) on sperm.

[0058] The one or more binding domains may specifically bind a respiratory syncytial virus (RSV) epitope.

[0059] For example, a synthetic binding agent may comprise: (a) a pair of human or humanized IgG Fc domains selected from SEQ ID NOs: 11-15; and (b) two self-assembled heterotrimeric laminin 511-derived complexes as in claim 1, wherein one heterotrimeric complex is linked to the N-terminus of each Fc domain and one heterotrimeric complex is linked to the C-terminus of each Fc domain, and wherein at least two of the a, P, and y strands in each complex are linked to Fab domains. The synthetic binding agent may comprise, in total, 10 Fab domains per molecule. In some cases, the synthetic binding agent comprises, in total, 12 Fab domains per molecule. The Fab domains may comprise an anti-sperm Fab comprising the variable regions of SEQ ID NO:21 and SEQ ID NO:22. The Fab domains may comprise an anti-RSV Fab.

[0060] Also described herein are pharmaceutical compositions comprising any of these synthetic agents and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for mucosal delivery selected from vaginal, intranasal, buccal, ocular, pulmonary, or rectal administration. The composition may be a lyophilized powder, solution, gel, film, aerosol, spray, foam, suppository, ovule, or tablet.

[0061] For example, described herein are fast-dissolving antibody tablet (FDAT) comprising: (a) the synthetic agent of any of claims 1-25; (b) a cryo / lyoprotectant comprising trehalose; (c) a disintegrant / binder system comprising erythritol and microcrystalline cellulose; and (d) an effervescent couple comprising sodium bicarbonate and citric acid, wherein the tablet dissolves in cervicovaginal mucus in <120 seconds (e.g., 100 seconds or less, 90 seconds or less 80 seconds or less 70 seconds or less, 60 seconds or less, 45 seconds or less, 30 seconds or less, etc.) and, upon reconstitution, the synthetic agent retains sperm agglutination potency equivalentAttorney Docket No. 5470.976.WOto the pre-lyophilized agent. In some examples 1 mg of the synthetic agent per tablet yields > 90% (e.g., >92.5%, > 95%, > 96%, >97%, >98%, 99%, etc.) reduction of PM sperm in a sheep surrogate PCT within 2 minutes post semen instillation.

[0062] An intravaginal device may include a reservoir or polymer matrix loaded with any of the synthetic agents described herein and may be configured to release the agent at a rate of 1 pg / day to 10 mg / day (e.g., between 10 pg / day to 10 mg / day, between 50 pg / day to 10 mg / day, between 100 pg / day to 10 mg / day, between 500 pg / day to 10 mg / day, greater than about 1 pg / day, greater than about 10 pg / day, greater than about 50 pg / day, greater than about 100 pg / day, greater than about 100 pg / day, greater than about 500 pg / day, etc.).

[0063] Also described herein are kits comprising: any of the FDATs described herein; and instructions for reducing progressively motile sperm in vivo.

[0064] Also described herein are nucleic acids encoding each of: (i) an a strand polypeptide comprising a laminin 511 trimerization region and optionally a binding domain, (ii) a P strand polypeptide comprising a laminin 511 trimerization region and optionally a binding domain, (iii) a y strand polypeptide comprising a laminin 511 trimerization region and optionally a binding domain, and (iv) an Fc containing polypeptide; wherein at least one of (i)-(iii) or (iv) comprises an amino acid linker as described herein.

[0065] Also described are expression vectors comprising any of the nucleic acids described herein. Also described are host cells comprising any of these expression vectors, wherein the host cell is a mammalian cell, e.g., selected from CHO and HEK293 cells, or a plant or microbial production system.

[0066] Also described herein are methods of making any of these synthetic agents. For example, a method of making may include co-expressing in a host cell nucleic acids encoding the a, P, and y strands and an Fc containing polypeptide, and isolating the assembled agent (e g., by protein A affinity chromatography or other means).

[0067] Also described are methods of contraception comprising administering to a subject in need thereof an effective amount of any of these synthetic agents to the female reproductive tract, thereby reducing progressively motile sperm. Administering may be intravaginal administration of a solution or an FDAT and the dose of the synthetic agent is between, e.g., 1 pg and 50 mg, 1 pg and 40 mg, 1 pg and 30 mg, 1 pg and 20 mg, 2.5 pg and 50 mg, 2.5 pg and 25 mg, 2.5 pg and 20 mg, 3 pg and 50 mg, 3 pg and 40 mg, 3 pg and 30 mg, 3 pg and 20Attorney Docket No. 5470.976.WOmg, 3.3 pg and 100 mg, 3.3 pg and 50 mg, 3.3 pg and 30 mg, 3.3 pg and 20 mg, etc ). The synthetic agent may comprise anti CD52g Fab domains and may achieve >85% (e.g., 90% or more, 91% or more 92% or more, 93% or more, 94% or more 95% or more, 96% or more 97% or more 98% or more, 99% or more, etc.) reduction of PM sperm within about 90 seconds (within 80 seconds, 70 seconds, 60 seconds, etc.) at 37°C.

[0068] For example, a method of inhibiting transmission of a respiratory pathogen may comprise administering to a subject an effective amount of any of the synthetic agents described herein to a mucosal surface, thereby immobilizing the pathogen in mucus. The pathogen may be RSV and the synthetic agent may comprise an anti RSV binding domain.

[0069] Any of the synthetic agents described herein, after incubation at 37°C for 7-28 days in histidine / arginine buffer (pH 6.5), may retain agglutination potency and may be > 80% (e.g., 82.5% or more, 85% or more, 87.5% or more 90% or more, 92.5% or more, 95% or more, etc.) monomer by SEC MALS. At least two of the a, 0, and y strands may each be linked to a pair of Fab domains and the third may be optionally linked to a pair of Fab domains. Each heterotrimeric complex may provide three Fab display sites that are sterically accessible and radially distributed about the Fc domain.

[0070] The Fc domains and the heterotrimeric complexes may be arranged such that the unbound agent diffuses through mucus substantially unhindered yet, once bound to target, exhibits enhanced mucin crosslinking.

[0071] Any of the synthetic agents may be free of non covalent Fc mediated self hexamerization. As mentioned, the a, 0, and y strands may be derived from laminin 511 E8 domains. The strand strand disulfide bonds may be engineered by introducing cysteine residues at positions corresponding to SEQ ID NOs:2, 4, and 6. The agent may exhibit higher agglutination potency at 37°C than an otherwise identical construct comprising a non covalent IgG hexamerization platform.

[0072] The binding domains may target different epitopes on a same antigen or different antigens.

[0073] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.Attorney Docket No. 5470.976.WOBRIEF DESCRIPTION OF DRAWINGS

[0074] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0075] FIG. 1 is a schematic illustration of binding agents.

[0076] FIGS. 2A-2C are graphs showing sperm agglutination with antibodies assessed by agglutination kinetics assay against purified semen at various temperatures based on three unique semen samples.

[0077] FIGS. 3A-3B are graphs showing binding affinity assessed by whole-sperm ELISA at room temperature at 37°C, respectively.

[0078] FIG. 3C is a graph showing a size exclusion chromatography (SEC) profile for RGY mAb hexamer and monomer populations as a function of concentration and pH.

[0079] FIG. 4 is a schematic illustration of laminin-IgG multivalent hybrids.

[0080] FIG. 5A is an SDS-PAGE analysis of various LamH / mAbs in either non-reduced (NR) or reduced (R) conditions.

[0081] FIG. 5B is a graph showing relative monomer content for each LamH / i mAb.

[0082] FIG. 5C is a plot showing relative reduction in PM sperm fractions with LamH12 using a hinge linker and a flexible (G4S)e linker between the HC4 Fab and laminin trimerization domains, with controls.

[0083] FIG. 5D is a graph showing binding affinity of various mAbs assessed by wholesperm ELISA at 37°C.

[0084] FIG. 5E is a graph showing stability of various LamH / / Abs as assessed by melting temperatures (Tm).

[0085] FIG. 5F is a graph showing stability of various LamHw Abs as assessed by aggregation temperatures (Tagg).

[0086] FIG. 6A is a series of plots showing sperm agglutination kinetics of various IgG, RGY, and laminin multivalent hybrids.

[0087] FIG. 6B is a series of plots showing sperm agglutination kinetics of LamHIO compared to RGY and parent IgG when conducted using whole semen.

[0088] FIG. 6C is a plot showing sperm agglutination of parent IgG at 37°C at high concentration against purified sperm.Attorney Docket No. 5470.976.WO

[0089] FTG. 6D is a plot showing independent site evaluation of agglutination potency of LamHIO compared to parent IgG against purified sperm.

[0090] FIG. 7A depicts representative traces of semen in native cervicovaginal mucus (CVM) or CVM treated with HC4-IgG or LamHIO.

[0091] FIGS. 7B-7D are graph-plots depicting average, velocity, curvilinear velocity, straight-line velocity, respectively, of tracked sperm across control IgG, IgG, and LamHIO.

[0092] FIG. 7E is a graph-plot depicting percentage of sperm classified as PM.

[0093] FIG. 8A is a schematic of a study design for LamHIO and sperm agglutination in sheep.

[0094] FIG. 8B is a graph-plot showing a reduction in PM sperm compared to PBS control for LamHIO dosed at different concentrations.

[0095] FIG. 8C is a LamHIO Fast-Dissolving Ab Table (FDAT).

[0096] FIG. 8D is an illustration of the dissolution kinetics of FDAT in CVM.

[0097] FIG. 8E is a plot showing sperm agglutination potency of LamHIO in solution comparted to potency after reconstitution from lyophilized powder or FDAT.

[0098] FIG. 8F is a graph showing reduction in PM sperm compared to PBS control for placebo vs. LamHIO FDAT.

[0099] FIG. 8G is a schematic of a study design evaluating the effectiveness of LamHIO in agglutinating sperm within 5 minutes of vaginal dosing in sheep.

[0100] FIG. 9 is a table showing motility parameters of Hamilton-Thorne Ceros 12.3.

[0101] FIG. 10A is a graph screening trimerized Ab using the homotrimer domain from Collagen XV via SEC-MALS.

[0102] FIG. 10B is a graph screening trimerized Ab using the homotrimer domain from Collagen XVIII via SEC-MALS.

[0103] FIG. 10C is a graph screening a construct with a heterotrimer domain from Collagen IV via SEC-MALS.

[0104] FIG. 10D is a graph screening a construct with a computationally designed obligate heterotrimer DHT03 via SEC-MALS.

[0105] FIG. 11A is a graph showing SEC-MALS UV and Scattering Data of parent IgG.

[0106] FIG. 1 IB is a graph showing SEC-MALS UV and Scattering Data of LamH6.

[0107] FIG. 11C is a graph showing SEC-MALS UV and Scattering Data of LamHIO.Attorney Docket No. 5470.976.WO

[0108] FTG. 1 ID is a graph showing SEC-MALS UV and Scattering Data of LamH12.

[0109] FIG. 12A is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 10 pg / mL added to purified semen.

[0110] FIG. 12B is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 2.50 pg / mL added to purified semen.

[0111] FIG. 12C is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.625 pg / mL added to purified semen.

[0112] FIG. 12D is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.3125 pg / mL added to purified semen.

[0113] FIG. 12E is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.156 pg / mL added to purified semen.

[0114] FIG. 12F is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 40 pg / mL added to whole semen.

[0115] FIG. 12G is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 10 pg / mL added to whole semen.

[0116] FIG. 12H is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 2.5 pg / mL added to whole semen.

[0117] FIG. 121 is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 1.25 pg / mL added to whole semen.

[0118] FIG. 12 J is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 0.625 pg / mL added to whole semen.

[0119] FIG. 13A is a plot showing fraction of PM sperm compared to no mAb control at decreasing concentrations of HC4-IgG, HC4-RGY and LamHIO.

[0120] FIG. 13B is a line plot showing average reduction in escaped PM sperm across mAb concentrations.

[0121] FIGS. 13C-13E are lines plot showing comparisons of reduction in sperm escape at RT vs. 37°C for HC4-IgG, HC4-RGY, and LamHIO, respectively.

[0122] FIG. 14A shows agglutination potency in whole semen of IgG and LamHIO after incubation in whole human CVM.

[0123] FIG. 14B is a graph showing agglutination potency of LamHIO during long-term exposure to 37°C conditions.Attorney Docket No. 5470.976.WO

[0124] FTG. 14C is a graph showing monomer content of LamHIO after incubation at 37°C as measured by SEC-MALs.

[0125] FIG. 14D is a graph showing stability of LamHIO at 5 mg / mL and 0.5 mg / mL at pH7, and 5 mg / mL at pH4, as measured by comparison of UV-signal during size SEC.DETAILED DESCRIPTION

[0126] Described herein are novel synthetic agents, referred to equivalently as engineered Fc-domain linked heterotrimeric synthetic agents, comprising a pair of human or humanized Immunoglobulin fragment crystallizable (Fc) domains linked to at least one strand of a heterotrimeric complex comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands. These synthetic binding agents may have value by themselves, or may serve as an extremely flexible and easy to use highly multivalent platform, based on linking some or all of the alpha, beta and gamma domains to a binding domain, including but not limited to a Fab domain, that allows for precise tuning of Fab valency.

[0127] The engineered Fc-domain linked heterotrimeric synthetic agents and methods of making and using them are explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0128] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intendedAttorney Docket No. 5470.976.WOthat any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0130] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulation of nucleic acid sequences, and production of transformed cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0131] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.

[0132] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0133] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0134] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0135] The term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0136] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximationsAttorney Docket No. 5470.976.WOthat can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0137] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0138] The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel character! stic(s) of the claimed invention.

[0139] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0140] A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide), but is preferably either single or double stranded DNA sequences.

[0141] "Homology" or "identity" or "similarity," or grammatical variations thereof, refer to sequence similarity between two proteins or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical (e g., homologous) at that position. A degree of homology between sequences is a function of the number of matching or identical positions shared by the sequences. An "unrelated,” “non-identical” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with a sequence herein. Percent (%) identity may be determined using BLASTP with default settings or an equivalent alignment algorithm using a gap-open penalty of 11 and gap-extension penalty of 1. For example, “% identity” between polypeptide sequences may be determined using BLASTP (NCBI) with default parameters: substitution matrix BLOSUM62, word size 3, gap open penalty 11, gap extension penalty 1, compositional adjustments on, and with the full-length trimerization region as the comparison window. Alignments are performed pairwise against SEQ ID NOs:l-6 as applicable.Attorney Docket No. 5470.976.WO

[0142] A protein or protein region (or a nucleotide or nucleotide region) having a certain percentage (for example, at least about 80%, 85%, 90%, 95%, 98% or 99%, or any range derivable therein) of "sequence identity" or "sequence homology" to another sequence means that, when aligned, that percentage of amino acids (or bases) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.

[0143] As used herein, an “isolated” protein means a protein separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the protein. The term also encompasses protein that have been prepared synthetically.

[0144] By the terms “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and / or there is a delay in the progression of the condition.

[0145] As used herein, the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and / or reduces the symptoms associated with the condition after onset.

[0146] An “effective,” “prophylactically effective,” or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an “effective,” “prophylactically effective,” or “therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the effects need not be complete or curative, as long as some benefit is provided to the subject.

[0147] The engineered Fc-domain linked heterotrimeric synthetic agents described herein may be configured to trap or enhance trapping of an antigen (e.g., sperm, pathogen, including but not limited to virus, bacteria, etc.) in mucus. As used herein, the term “trapping potency” refers to the ability of a protein that specially binds to a target pathogen or sperm to inhibit movement of theAttorney Docket No. 5470.976.WOpathogen or sperm through mucus. Trapping potency can be measured by methods known in the art and as disclosed herein. Trapping potency can be quantitated, e.g., as the amount of antibody (e.g., concentration of antibody in mucus) needed to reduce the mobility of at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, etc.) of the pathogen or sperm within the mucus gel to at least one-tenth of its native mobility in solution (e.g., saline) and / or in mucus. Mobility in mucus can be measured using techniques well known in the art and described herein. Alternatively, trapping potency can be quantitated as the reduction in percentage of pathogens or sperm that penetrate mucus.

[0148] The term “enhances trapping potency” refers to enhancement compared to an antibody or other protein having the same antigen-binding domain. Further, any of the engineered Fc-domain linked heterotrimeric synthetic agents described herein may be selected or further configured to enhance mucin-crosslinking by including a glycosylation pattern comprising the biantennary core glycan structure Manal-6(Manal-3)Manpi-4GlcNAcpi-4GlcNAcpi with terminal N-acetylglucosamine on each branch. This glycosylation pattern may be on the Fc region(s) of the engineered Fc-domain linked heterotrimeric synthetic agents. Alternatively or additionally, a composition of the engineered Fc-domain linked heterotrimeric synthetic agents having multiple binding domains (e.g., Fab domains) described herein may be selected or configured such that at least x% of the engineered Fc-domain linked heterotrimeric synthetic agents have a glycosylation pattern comprising the biantennary core glycan structure Manotl-6(Manal-3)Manpi-4GlcNAcpi-4GlcNAcpi with terminal N-acetylglucosamine on each branch, where x% is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially all). A composition in which, for example, greater than 40% of the engineered Fc-domain linked heterotrimeric synthetic agents described herein (to enhance agglutination potency) while also possessing an oligosaccharide that provides increased mucin crosslinking, may be particularly beneficial for mucosal trapping once bound to the engineered Fc-domain linked heterotrimeric synthetic agents compared to antibodies (e.g., IgG) having the same antigen-binding domains as found in nature prior to any modification and / or selection.

[0149] As used herein, the term “bind specifically” or “specifically binds” means that the engineered Fc-domain linked heterotrimeric synthetic agents of the invention will bind with an epitope (including one or more epitopes) of a target pathogen or sperm, but does not substantially bind to other unrelated epitopes or molecules. In certain embodiments, the term refers to anAttorney Docket No. 5470.976.WOengineered Fc-domain linked heterotri meric synthetic agents that exhibits at least about 60% binding, e.g., at least about 70%, 80%, 90%, or 95% binding, to the target epitope relative to binding to other unrelated epitopes or molecules.

[0150] The compositions of engineered Fc-domain linked heterotrimeric synthetic agents and methods described herein may include methods for inhibiting and / or treating pathogen infection, eliminating pathogen from a mucosal surface, and providing contraception. In particular, the presently-disclosed subject matter relates to synthetic agents and compositions of these that are capable of trapping pathogens and sperm in mucus, thereby inhibiting transport of pathogens or sperm across or through mucus secretions, which may lead to the destruction of these pathogens and / or sperm.

[0151] The engineered Fc-domain linked heterotrimeric synthetic agents described herein may generally diffuse rapidly through the CVM, slowed only slightly by weak, transient adhesive interactions with mucins within the mucus. This rapid diffusion allows the engineered Fc-domain linked heterotrimeric synthetic agents configured to bind to a pathogen or sperm to accumulate rapidly on pathogen or sperm surfaces and to trap the bound pathogen or sperm in the mucus, thereby preventing infection / providing contraception. Pathogens or sperm trapped in CVM cannot reach their target cells in the mucosal surface, and will instead be shed with post-coital discharge and / or inactivated by spontaneous thermal degradation as well as additional protective factors in mucus, such as defensins. This pathogen trapping activity provides for protection without neutralization, and can effectively inhibit infection at sub-neutralization doses and / or using antibodies to non-neutralizing epitopes of a pathogen.

[0152] The engineered Fc-domain linked heterotrimeric synthetic agents described herein may include an oligosaccharide at a glycosylation site, the oligosaccharide comprising, consisting essentially of, or consisting of a pattern correlating with (providing) enhanced trapping potency of the antibody in mucus, and wherein the antibody specifically binds an epitope of a target (e.g., pathogen or sperm). The unique glycosylation pattern / unique oligosaccharide component of the protein, and in particular the Fc region, may maximize trapping potency of the synthetic binding agent once a synthetic binding agent forms a complex with one or more target (e.g., pathogen or sperm), without unduly hindering the ability of the unbound synthetic binding agent to diffuse readily through mucus to rapidly bind a target.Attorney Docket No. 5470.976.WO

[0153] In some embodiments, the engineered Fc-domain linked heterotrimeric synthetic agents described herein may include an oligosaccharide component that is bound to an N-linked glycosylation site in an Fc region of the synthetic agent (e.g., the core IgG portion of the engineered Fc-domain linked heterotrimeric synthetic agent). The N-linked glycosylation site can be an asparagine residue on the Fc region of the core, for example, the Asn 297 asparagine residue. The amino acid numbering is with respect to the standard amino acid structure of a human IgG molecule.

[0154] The N-glycan structure may be G0 / G0F form, or a pure GnGn form (e.g., with terminal N-acetylglucosamine on each branch without terminal galactose or sialic acid). In some embodiments, the oligosaccharide component, i.e., the glycan, consists essentially of, or consists of a core structure without any fucose residue. In other embodiments, the glycan does not contain any galactose residues. In some embodiments the glycan does not include galactose.

[0155] A composition of engineered Fc-domain linked heterotrimeric synthetic agents described herein may include different oligosaccharide components. In some embodiments, the composition is a mixture that comprises at least about 30% engineered Fc-domain linked heterotrimeric synthetic agents having multiple Fab domains described herein having the G0 / G0F core glycan structure (e.g., with or without the fucose residue), e.g., at least about 40%, 50%, 60%, 70%, 80%, 90% or more.

[0156] In some embodiments, the engineered Fc-domain linked heterotrimeric synthetic agents described herein are generated in a human cell line, e g., a 293 cell line, e.g., a 293T cell line, other mammalian cell lines (e.g. CHO), in plants (e.g. Nicotiana), or in other microorganisms (e.g. Tnchoderma).

[0157] The engineered Fc-domain linked heterotrimeric synthetic agents may be useful for binding target to trap the target in mucus to inhibit infection or impregnation by the target. In variations in which the target is a pathogen, the synthetic agent having can be directed to any pathogen that can infect a subject through a mucus membrane. Pathogens can be in the categories of algae, bacteria, fungi, parasites (helminths, protozoa), viruses, and subviral agents. Target pathogens further include synthetic systems comprising an antigen having an epitope, for example particles or particulates (e.g., polystyrene beads) comprising attached proteins, e.g., as might be used for bioterrorism. Pathogens include those that cause sexually-transmitted diseases (listed with the diseases caused by such pathogens), including, without limitation, Neisseria gonorrhoeaeAttorney Docket No. 5470.976.WO(gonorrhea); Chlamydia trachomatis (chlamydia, lymphogranuloma venereum); Treponema pallidum (syphilis); Haemophilus ducreyi (chancroid); Klebsiella granulomatis or Calymmatobacterium granulomatis (donovanosis), Mycoplasma genitalium, Ureaplasma urealyticum (mycoplasmas); human immunodeficiency virus HIV-1 and HIV-2 (HIV, AIDS); HTLV-1 (T-lymphotrophic virus type 1); herpes simplex virus type 1 andtype2 (HSV-1 andHSV-2); Epstein-Barr virus; cytomegalovirus; human herpesvirus 6; varicella-zoster virus; human papillomaviruses (genital warts); hepatitis A virus, hepatitis B virus, hepatitis C virus (viral hepatitis); molluscum contagiosum virus (MCV); Trichomona vaginalis (trichomoniasis); and yeasts, such as Candida albicans (vulvovaginal candidiasis). The engineered Fc-domain linked heterotrimeric synthetic agents may also be active against other diseases that are transmitted by contact with bodily fluids that may also be transmissible by sexual contact and are capable of being prevented by administration of the compositions according to this invention. Accordingly, the phrase, “sexually transmitted diseases (STDs),” is to be interpreted herein as including any disease that is capable of being transmitted in the course of sexual contact, whether or not the genital organs are the site of the resulting pathology. Pathogens also include those that cause respiratory diseases, including, without limitation, influenza (including influenza A, B, and C); severe acute respiratory syndrome (SARS); respiratory syncytial virus (RSV); parainfluenza; adenovirus; human rhinovirus; coronavirus; and norovirus. Other pathogens include, without limitation, Salmonella and Escherichia coli.

[0158] Pathogens may include those that affect non-human animals, such as livestock, e.g., swine (e.g., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotavirus, classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), encephalomyocarditis virus (EMCV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), pseudorabies virus (PRV), Japanese encephalitis virus (JEV), Brucella, Leptospira, Salmonella, and Lawsonia intracellular is, Pasteurella multocida, Brachyspira hyodysenteriae, Mycoplasma hyopneumoniae), ruminants (e.g., bovine virus diarrhoea virus (BVDV), border disease virus (BDV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), Pasteurella haemolytica, Pasteurella multocida, Haemophilus somnus, Haemophilus agnii, Moraxella bovis, Mycoplasma mycoides, Theileria annulata, Mycobacterium avium paratuberculosis'), ungulates (e.g., Brucella abortus, Mycobacterium bovis, Theileria parva, Rift Valley fever virus, foot-and-mouth disease virus, lumpy skin disease virus),Attorney Docket No. 5470.976.WOhorses (e.g., Rhodococcus equi, Salmonella choleraesuis, Pasteurella multocida, equine herpesvirus- 1, equine herpesvirus-4, equine influenza virus, Streptococcus equi), poultry (e.g., fowl pox virus, Newcastle disease virus, Marek's disease virus, avian influenza virus, infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV)), and the like.

[0159] The terms “virus” and “viral pathogen” are used interchangeably herein, and further refer to various strains of virus, e.g., influenza is inclusive of new strains of influenza, which would be readily identifiable to one of ordinary skill in the art. The terms “bacterium,” “bacteria,” and “bacterial pathogen” are used interchangeably herein, and further refer to antibiotic-resistant or multidrug resistant strains of bacterial pathogens. As used herein when referring to a bacterial pathogen, the term “antibiotic-resistant strain” or “multidrug resistant strain” refers to a bacterial pathogen that is capable of withstanding an effect of an antibiotic or drug used in the art to treat the bacterial pathogen (i.e., a non-resistant strain of the bacterial pathogen).

[0160] In some embodiments, it is contemplated that an engineered Fc-domain linked heterotrimeric synthetic agent is capable of broadly binding to viruses containing lipid envelopes, which are not necessarily specific to one virus.

[0161] In variations when the engineered Fc-domain linked heterotrimeric synthetic agents specifically binds a neutralizing epitope of the target pathogen, a sub-neutralization dose can be used. A sub-neutralization doses is a dose below that which would be needed to achieve effective neutralization. For example, in the case of an engineered Fc-domain linked heterotrimeric synthetic agents including a binding domain based on a Fab domain derived from an anti-HSV IgG antibody targeting HSV, an effective neutralization dose may be approximately 5 pg / ml. However, effective trapping using the engineered Fc-domain linked heterotrimeric synthetic agents can be achieved at a dose below 5 pg / ml, and even below a dose of 1 pg / ml.

[0162] As will be recognized by one of skill in the art, doses appropriate for trapping bacterial pathogens can be higher in some embodiments than the doses appropriate for trapping viral pathogens. It will further be recognized that appropriate doses may differ between pathogens, between mucosal surfaces, and also between individuals. It will also be recognized that different subjects and different mucosal surfaces may have different optimal glycan patterns and optimal antibody-mucin affinities, contributing to different optimal doses.

[0163] It is further proposed herein that engineered Fc-domain linked heterotrimeric synthetic agents that selectively bind non-neutralizing epitopes of a target pathogen can be used toAttorney Docket No. 5470.976.WOeffectively trap the target pathogen in mucus. As such, in some embodiments, the engineered Fc-domain linked heterotrimeric synthetic agents may specifically bind a non-neutralizing epitope, e.g., one or more non-neutralizing epitopes.

[0164] The presently-disclosed subject matter further includes engineered Fc-domain linked heterotrimeric synthetic agents that may selectively bind a conserved epitope of a target. A benefit of targeting a conserved epitope would be to preserve efficacy of the engineered Fc-domain linked heterotrimeric synthetic agents as against new strains of the pathogen. Targeting such epitopes has been avoided at times in the past because they were viewed as being ineffective targets; however, in view of the disclosure herein such epitopes can serve as effective targets.

[0165] The engineered Fc-domain linked heterotrimeric synthetic agents described herein may be particularly useful for binding sperm to trap the sperm in mucus to inhibit fertilization of an egg by the sperm. Sperm specific antigens that can be used as antibody targets are known in the art. See, e.g., U.S. Pat. Nos. 8,211,666, 8,137,918, 8,110,668, 8,012,932, 7,339,029, 7,230,073, and 7,125,550, each incorporated by reference in its entirety. As will be described herein one particular epitope region for human sperm may include the N-linked glycan of sperm CD52 glycoform. See also U.S. Pat. Nos. 5,227,160 and 6,355,235, herein incorporated by reference in their entirety.

[0166] The low-affinity binding interactions that the engineered Fc-domain linked heterotrimeric synthetic agents described herein forms with mucins may be influenced by glycosylation, and may also be Fc-dependent. As such, the engineered Fc-domain linked heterotrimeric synthetic agent may have a preserved and / or engineered Fc region in a core IgG region. Such synthetic binding agents may be one or more subclasses of IgG, e.g., IgGi, IgG2, IgGi, IgG4, or any combination thereof.

[0167] The following discussion is presented as a general overview of the techniques available for the production of engineered Fc-domain linked heterotrimeric synthetic agents; however, one of skill in the art will recognize that many variations upon the following methods are known.

[0168] The term “antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody can be monoclonal or polyclonal and can be of any species of origin, including (for example) mouse, rat, rabbit, horse, goat, sheep, camel, or human, or can be a chimeric or humanized antibody. See, e.g., Walker et al., Molec. Immunol. 26:403 (1989). The antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No.Attorney Docket No. 5470.976.WO4,816,567, The antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980.

[0169] Any of the engineered Fc-domain linked heterotrimeric synthetic agents described herein may include links to one or more binding domains. In some cases, the binding domains may be derived from an antibody, including Fab antibody fragments. Antibody fragments included within the scope of the present invention include, for example, Fab, Fab', F(ab)2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Such fragments can be produced by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 254:1275 (1989)). In some embodiments, the term “antibody fragment” as used herein may also include any protein construct that is capable of binding a target.

[0170] The Fab domains used as binding domains and linked to the engineered Fc-domain linked heterotrimeric synthetic agents described herein may be humanized or camelized. Humanized forms of non-human (e.g., murine) antibodies (or binding domains derived therefrom) may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constantAttorney Docket No. 5470.976.WOregion (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593 (1992)).

[0171] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues (e.g., all of the CDRs or a portion thereof) and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0172] The engineered Fc-domain linked heterotrimeric synthetic agents described herein may be formed recombinantly. For example, recombinant polypeptides may be expressed in eukaryotic or prokaryotic cells and purified using standard techniques.

[0173] Engineered Fc-domain linked heterotrimeric synthetic agents as described herein can be labeled. Labeling may include joining, either covalently or noncovalently, a substance which provides a detectable signal to the engineered Fc-domain linked heterotrimeric synthetic agent. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moi eties, chemiluminescent moieties, magnetic particles, and the like, engineered Fc-domain linked heterotrimeric synthetic agents as described herein may be useful for detecting or diagnosing the presence of a target on which an antigen is found.

[0174] Method of making engineered Fc-domain linked heterotrimeric synthetic agents (including with a glycosylation pattern of interest) can be achieved by any method known to those or skill in the art. For example, in some embodiments, mammalian cells can be used, such as, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and NS0- and SP2 / 0-mouse myeloma cells, to produce engineered Fc-domain linked heterotrimeric synthetic agents having the desired glycosylation pattern. In certain embodiments, human cell lines can be used, e.g., 293Attorney Docket No. 5470.976.WOcells. In some embodiments, non-mammalian cells can be used. The cell line can be genetically engineered to produce the antibodies with the desired oligosaccharide. Such cell lines can have altered expression, for example, of one or more enzymes affecting glycosylation patterns, e.g., glycosyltransferases. Glycosyltransferases include, without limitation, a galactosyltransferase, a fucosyltransferase, a glucosyltransferase, an N-acetylgalactosaminyltransferase, an N-acetylglucosaminyltransferase, a glucuronyltransferase, a sialyltransferase, a mannosyltransferase, a glucuronic acid transferase, a galacturonic acid transferase, an oligosaccharyltransferase, or any combination thereof. Specific examples include, without limitation, oligosaccharyltransferase, UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GDP -fucose protein :O-fucosyltransferase 1, GDP-fucose proteimO-fucosyltransferase 2, proteimO-glucosyltransferase, UDP-N-acetylglucosamine:peptide N-aeetylglucosaminyltransferase, protein:O-mannosyltransferase, pi,4 galactosyltransferase, and any combination thereof. Enzymes involved in glycosylation of proteins are well known in the art and can be manipulated using routine techniques. See, for example, U.S. Pat. Nos. 8,383,106, 8,367,374, 8,080,415, 8,025,879, 8,021,856, 7,906,329, and 7,846,434, each incorporated herein by reference in its entirety.

[0175] As would be recognized by one skilled in the art, the engineered Fc-domain linked heterotrimeric synthetic agents described herein can also be formed into suitable compositions, e.g., pharmaceutical compositions for administration to a subject in order to act as a contraceptive and / or to treat or prevent an infection caused by a target pathogen or a disease or disorder caused by infection by a target pathogen. A composition may comprise, consist essentially of, or consist of an engineered Fc-domain linked heterotrimeric synthetic agents in a prophylactically or therapeutically effective amount and a pharmaceutically-acceptable carrier.

[0176] Pharmaceutical compositions containing the engineered Fc-domain linked heterotrimeric synthetic agents described herein can be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such conventional materials for this purpose, e.g., saline, dextrose, water, glycerol, ethanol, and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the subject and the subject's condition, and a variety of modes of administration would be suitable for the compositions of the engineered Fc-domain linked heterotrimeric synthetic agents described herein. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are notAttorney Docket No. 5470.976.WOlimited to, topical, oral, intranasal, buccal, inhalation, anal, and vaginal administration, wherein such administration achieves delivery of the engineered Fc-domain linked heterotrimeric synthetic agents to a mucus membrane of interest.

[0177] The composition can be any type of composition suitable for delivering a synthetic agent (e.g., an engineered Fc-domain linked heterotrimeric synthetic agents) as described herein to a mucosal surface and can be in various forms known in the art, including solid, semisolid, or liquid form or in lotion form, either oil-in-water or water-in-oil emulsions, in aqueous gel compositions. Compositions include, without limitation, gel, paste, suppository, douche, ovule, foam, film, spray, ointment, pessary, capsule, tablet, jelly, cream, milk, dispersion, liposomes, powder / talc or other solid, suspension, solution, emulsion, microemulsion, nanoemulsion, liquid, aerosol, microcapsules, time-release capsules, controlled release formulation, sustained release formulation or bioadhesive gel (e.g., a mucoadhesive thermogelling composition) or in other forms embedded in a matrix for the slow or controlled release of the antibody to the surface onto which it has been applied or in contact.

[0178] If topical administration is desired, the composition may be formulated as needed in a suitable form, e.g., an ointment, cream, gel, lotion, drops (such as eye drops and ear drops), or solution (such as mouthwash). The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol. Other formulations for administration, including intranasal administration, etc., are contemplated for use in connection with the presently-disclosed subject matter. All formulations, devices, and methods known to one of skill in the art which are appropriate for delivering the engineered Fc-domain linked heterotrimeric synthetic agents or a composition containing the engineered Fc-domain linked heterotrimeric synthetic agents to one or more mucus membranes of a subject can be used in connection with the presently-disclosed subject matter.

[0179] Any of the compositions described herein may include mixtures of the engineered Fc-domain linked heterotrimeric synthetic agents described herein, including mixtures having different numbers of protein binding domains and / or different numbers of heterotrimeric complexes.

[0180] The compositions used in the methods described herein may include other agents that do not negatively impact or otherwise affect the inhibitory and / or contraceptive effectiveness ofAttorney Docket No. 5470.976.WOthe components of the composition, including antibodies, antimicrobial agents, and / or spermfunction inhibitors. For example, solid, liquid or a mixture of solid and liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions. Suitable physiologically acceptable, substantially inert carriers include water, a polyethylene glycol, mineral oil or petrolatum, propylene glycol, hydroxyethylcellulose, carboxymethyl cellulose, cellulosic derivatives, polycarboxylic acids, linked polyacrylic acids, such as carbopols; and other polymers such as poly(lysine), poly(glutamic acid), poly(maleic acid), polylactic acid), thermal polyaspartate, and aliphatic-aromatic resin; glycerin, starch, lactose, calcium sulphate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, stearic acid, syrup, peanut oil, olive oil, saline solution, and the like.

[0181] The pharmaceutical compositions described herein useful in the methods of the present invention may further include diluents, fdlers, binding agents, colorants, stabilizers, perfumes, gelling agents, antioxidants, moisturizing agents, preservatives, acids, and other elements known to those skilled in the art. For example, suitable preservatives are well known in the art, and include, for example, methyl paraben, propyl paraben, butyl paraben, benzoic acid and benzyl alcohol.

[0182] For injection, the carrier may typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL® (BASF, Parsippany, NJ). For other methods of administration, the carrier can be either solid or liquid.

[0183] For oral administration, the engineered Fc-domain linked heterotrimeric synthetic agents described herein can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Compositions can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or fdm coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-Attorney Docket No. 5470.976.WOcoated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0184] Compositions suitable for buccal (sub-lingual) administration include tablets or lozenges comprising the antibody in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the antibody in an inert base such as gelatin and glycerin or sucrose and acacia. The composition can comprise an orally dissolvable or degradable composition. Alternately, the composition can comprise a powder or an aerosolized or atomized solution or suspension comprising the antibody. Such powdered, aerosolized, or atomized compositions, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers.

[0185] Compositions of the engineered Fc-domain linked heterotrimeric synthetic agents described herein that are suitable for parenteral administration comprise sterile aqueous and nonaqueous injection solutions of the engineered Fc-domain linked heterotrimeric synthetic agents, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The compositions can be presented in unit / dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0186] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect, there is provided an injectable, stable, sterile composition comprising the engineered Fc-domain linked heterotrimeric synthetic agents described herein, in a unit dosage form in a sealed container. The engineered Fc-domain linked heterotrimeric synthetic agents may be provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.

[0187] Compositions suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by admixing the engineered Fc-domain linked heterotrimeric synthetic agents described herein with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.Attorney Docket No. 5470.976.WO

[0188] The engineered Fc-domain linked heterotri meric synthetic agents can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the engineered Fc-domain linked heterotrimeric synthetic agents, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract Ellis Harwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. l ^i (1992). Aerosols of liquid particles comprising the engineered Fc-domain linked heterotrimeric synthetic agents can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the engineered Fc-domain linked heterotrimeric synthetic agents can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0189] Alternatively, one can administer the engineered Fc-domain linked heterotrimeric synthetic agents in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0190] The engineered Fc-domain linked heterotrimeric synthetic agents herein may be coated or impregnated on a device (or a composition including the engineered Fc-domain linked heterotrimeric synthetic agents may be coated or impregnated). The device can be for delivery of the engineered Fc-domain linked heterotrimeric synthetic agents and compositions of the synthetic agent to a mucus membrane, e.g., to the vagina or uterus. In one embodiment, a device includes a solid support adapted to be inserted into the vagina. The support can be impregnated with or coated with a composition of the engineered Fc-domain linked heterotrimeric synthetic agents described herein. The release of synthetic agent from the devices may be controlled by the material composing these devices, such as silicone elastomers, ethylene vinyl acetate and polyurethane polymers. Devices, such as cervicovaginal and rectal devices, include, without limitation, a ring, rod, applicator, sponge, cervical cap, tampon, diaphragm, or intrauterine device. Applicators canAttorney Docket No. 5470.976.WObe those currently used commercially to deliver spermicidal gels or anti-yeast compounds and include, without limitation, plunger-type applicators, pessaries, sprays, squeezable tubes, vaginal rings, cervical rings, sponges, and the like. All such means for delivery are intended to be encompassed by the present invention.

[0191] As noted, engineered Fc-domain linked heterotrimeric synthetic agents may be capable of diffusing through mucus when it is unbound, to allow the engineered Fc-domain linked heterotrimeric synthetic agents to bind a target (e.g., pathogen or sperm) at a desirable rate. It is also desirable that, when the engineered Fc-domain linked heterotrimeric synthetic agents described herein are bound to the target, the cumulative effect of the antibody-mucin interactions effectively traps the pathogen or sperm in the mucus and / or agglutinates the target. To facilitate this goal, in some embodiments, it can be desirable to provide a composition that includes more than one engineered Fc-domain linked heterotrimeric synthetic agent, wherein each synthetic agent specifically binds a different epitope of the pathogen or sperm. Such a composition may provide the ability for an increased number of engineered Fc-domain linked heterotrimeric synthetic agents to become bound to the pathogen or sperm, thereby strengthening the antibody-mucin interactions that serve to trap the pathogen or sperm in the mucus.

[0192] In some embodiments, a composition includes a first engineered Fc-domain linked heterotrimeric synthetic agents and a second engineered Fc-domain linked heterotrimeric synthetic agents, wherein the first synthetic agent specifically binds a first epitope of the target and the second agent specifically binds a second epitope of the target, wherein the first epitope is distinct from the second epitope. In certain embodiments, the composition includes three or more different engineered Fc-domain linked heterotrimeric synthetic agents, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different engineered Fc-domain linked heterotrimeric synthetic agents, wherein each synthetic agent specifically binds a different epitope of the target.

[0193] It is also desirable to provide a composition that can provide treatment or prevention of infection due to more than one target pathogen. In some embodiments of the presently-disclosed subject matter, a composition includes a first engineered Fc-domain linked heterotrimeric synthetic agents and a second engineered Fc-domain linked heterotrimeric synthetic agents, wherein the first synthetic agent specifically binds an epitope of a first target pathogen and the second synthetic agent specifically binds an epitope of second target pathogen. In certain embodiments, the composition includes three or more different engineered Fc-domain linked heterotrimeric syntheticAttorney Docket No. 5470.976.WOagents, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different synthetic agents, wherein each synthetic agent specifically binds an epitope of a different target. As discussed above, in some variations the target may be the same, but the engineered Fc-domain linked heterotrimeric synthetic agents may have different numbers of Fab s.

[0194] In other embodiments, a composition provides both contraception and treatment or prevention of infection by one or more target pathogens. In some embodiments, a composition includes a first engineered Fc-domain linked heterotrimeric synthetic agents and a second engineered Fc-domain linked heterotrimeric synthetic agents, wherein the first synthetic binding agent specifically binds an epitope of sperm and the second synthetic binding agent specifically binds an epitope of a target pathogen. In certain embodiments, the composition includes three or more different engineered Fc-domain linked heterotrimeric synthetic agents described herein, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more different engineered Fc-domain linked heterotrimeric synthetic agents, wherein one or more engineered Fc-domain linked heterotrimeric synthetic agents bind different epitopes of sperm and one or more synthetic binding agent having multiple Fabs specifically binds an epitope of a target pathogen or multiple target pathogens.

[0195] In some embodiments, the pharmaceutical composition can further include an additional active agent, e.g., a prophylactic or therapeutic agent. For example, the additional active agent can be an antimicrobial agent, as would be known to one of skill in the art. The antimicrobial agent may be active against algae, bacteria, fungi, parasites (helminths, protozoa), viruses, and subviral agents. Accordingly, the antimicrobial agent may be an antibacterial, antifungal, antiviral, antiparasitic, or antiprotozoal agent. The antimicrobial agent is preferably active against infectious diseases. Suitable antiviral agents include, for example, virus-inactivating agents such as nonionic, anionic and cationic surfactants, and C31G (amine oxide and alkyl betaine), polybiguanides, docosanol, acylcarnitine analogs, octyl glycerol, and antimicrobial peptides such as magainins, gramicidins, protegrins, and retrocyclins. Mild surfactants, e.g., sorbitan monolaurate, may advantageously be used as antiviral agents in the compositions described herein. Other antiviral agents that may advantageously be utilized in the compositions described herein include nucleotide or nucleoside analogs, such astenofovir, acyclovir, amantadine, didanosine, foscamet, ganciclovir, ribavirin, vidarabine, zalcitabine, and zidovudine. Further antiviral agents that may be used include non-nucleoside reverse transcriptase inhibitors, such as UC-781 (thiocarboxanilide), pyridinones, TIBO, nevaripine, delavirdine, calanolide A, capravirine and efavirenz. From these reverseAttorney Docket No. 5470.976.WOtranscriptase inhibitors, agents and their analogs that have shown poor oral bioavailability are especially suitable for administration to mucosal tissue, in combination with antibodies and compositions of the invention, to prevent sexual transmission of HIV. Other antiviral agents that may be used are those in the category of HIV entry blockers, such as cyanovirin-N, cyclodextrins, carregeenans, sulfated or sulfonated polymers, mandelic acid condensation polymers, monoclonal antibodies, chemokine receptor antagonists such as TAK-779, SCH-C / D, and AMD-3100, and fusion inhibitors such as T-20 and 1249.

[0196] Suitable antibacterial agents include antibiotics, such as aminoglycosides, cephalosporins, including first, second and third generation cephalosporins; macrolides, including erythromycins, penicillins, including natural penicillins, penicillinase-resistant penicillins, aminopenicillins, extended spectrum penicillins; sulfonamides, tetracyclines, fluoroquinolones, metronidazole and urinary tract antiseptics.

[0197] Suitable antifungal agents include amphotericin B, nystatin, griseofulvin, flucytosine, fluconazole, potassium iodide, intraconazole, clortrimazole, miconazole, ketoconazole, and tolnaftate. Suitable antiprotozoal agents include antimalarial agents, such as chloroquine, primaquine, pyrimethamine, quinine, fansidar, and mefloquine; amebicides, such as dioloxamide, emetine, iodoquinol, metronidazole, paromomycine and quinacrine; pentamidine isethionate, atovaquone, and eflornithine.

[0198] In certain embodiments, the additional active agent can be a sperm-function inhibitor, e g., an agent that has the ability to inhibit the function of sperm, to otherwise inhibit fertilization of an egg by sperm and / or to otherwise prevent pregnancy, such as by killing and / or functionally inactivating sperm or by other effects on the activity of the sperm. In some embodiments, the active agent may have at least dual functions, such as acting as a sperm-function inhibitor and as an antimicrobial agent.

[0199] Sperm-function inhibitors include, without limitation, surfactants, including nonionic surfactants, cationic surfactants, and anionic surfactants; spermicides, such as nonoxynol-9 (a-(4-Nonylphenyl)-a)-hydroxynona(oxyethylene); other sperm-inactivators such as sulfated or sulfonated polymers such as polystyrene sulfonate, mandelic acid condensation polymers, cyclodextrins; antimicrobial peptides such as gramicidins, magainins, indolicidin, and melittin; and acid-buffering compositions, such as BufferGel and AcidForm. Nonionic surfactants include, for example, sorbitan monolaurate, nonylphenoxypolyethoxy ethanol, p-Attorney Docket No. 5470.976.WOdiisobutyphenoxypolyethoxy ethanol, polyoxyethylene (10) oleyl ether and onyx-ol. Suitable anionic surfactants include, without limitation, sodium alkyl sulfonates and the sodium alkylbenzene sulfonates. Cationic surfactants include, for example, the quaternary ammonium surfactants, such as cetyl pyrimidinium chloride and benzalkonium chlorides. Zwitterionic surfactants such as acylcarnitine analogs and C31G are especially suitable for their mild skin and mucosal irritation properties.

[0200] The presently-disclosed subject matter further includes a kit including any of the engineered Fc-domain linked heterotrimeric synthetic agents described herein or a composition comprising the engineered Fc-domain linked heterotrimeric synthetic agents as described herein; and optionally a device for administering the synthetic agent or composition. In some embodiments, the kit can include multiple engineered Fc-domain linked heterotrimeric synthetic agents and / or compositions containing such synthetic agents. In some embodiments, each of the multiple synthetic agents provided in such a kit can specifically bind to a different epitope of the target, e.g., pathogen or sperm. In other embodiments, each of the multiple engineered Fc-domain linked heterotrimeric synthetic agents as described herein provided in such a kit can specifically bind to an epitope of a different target pathogen or to an epitope of sperm. In some embodiments, the kit can further include an additional active agent, e.g., antimicrobial, such as an antibiotic, an antiviral, or other antimicrobial, or a sperm-function inhibitor as would be known to one of skill in the art.ENGINEERED FC-DOMAIN LINKED HETEROTRIMERIC SYNTHETIC AGENTS

[0201] In general, the engineered Fc-domain linked heterotrimeric synthetic agents described herein may include a pair of human or humanized Immunoglobulin fragment crystallizable (Fc) domains linked to one strand of a heterotrimeric complex comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0202] These synthetic agents may be synth etized as described herein and may be used in cell culture and / or in treatment. Optionally and in some cases these synthetic agents may be modified as described above and in greater detail below, to include one preferably a plurality of binding domains. When linked to copies of one or more binding domains, such as Fab domains, these synthetic agents may be used to specifically bind the target of the binding domain.Attorney Docket No. 5470.976.WO

[0203] The binding domains may be linked to the end (e.g., the C-terminus or N-terminus) of one or more of the alpha, beta and gamma strands of the heterotrimeric complex(es) that are not linked to the Fc domain of the synthetic agent. In some cases, the synthetic agent may be engineered to include between 2-12 binding domains per molecule. As compared to other multivalent constructs (see, e.g., PCT / US2019 / 052396, filed 9 / 23 / 2019), the engineered Fc-domain linked heterotrimeric synthetic agents described herein may retain picomolar potencies at physiological temperatures (e.g., > 30 degrees C) while maintaining virtually all of the desirable stability properties of conventional IgGs. Preliminary testing using one example of an engineered Fc-domain linked heterotrimeric synthetic agents, referred to herein as a “LamHIO” construct which is described in greater detail below, including an anti-sperm binding domain, showed that this LamHIO synthetic agent reduces progressively motile sperm in the sheep vagina by >99% even at just 33 pg dosed per sheep, and can be formulated into rapidly dissolving tablets for instant, on-demand contraception, achieving 100% sperm agglutination within just minutes of vaginal dosing.

[0204] In general, the engineered Fc-domain linked heterotrimeric synthetic agents is a laminin-511 hybrid platform and may provide not only the most potent biologic for nonhormonal contraception to date, but also represents a promising platform for engineering potent multivalent binding agents for other biomedical applications.EXAMPLES

[0205] As described herein, any of the engineered Fc-domain linked heterotrimeric synthetic agents described herein may be used to form a synthetic binding agent in which the heterotrimeric complex (comprising an alpha, beta and gamma strand that is at least 80% identical to human laminin-511 alpha, beta and gamma strands) is linked to copies of one or more binding domains. For example, a engineered Fc-domain linked heterotrimeric synthetic agent may comprise a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand, wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to copies of one or more antigen binding domains, further wherein the antigen binding domains specifically bind to an epitope of a target, so that the syntheticAttorney Docket No. 5470.976.WObinding agent binds to the target with high affinity, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0206] These engineered Fc-domain linked heterotrimeric synthetic agents, when including one or more binding domains may be referred to as simply synthetic binding agents, may include heterotrimeric complexes that are at least 80% identical to the human laminin-511. These engineered Fc-domain linked heterotrimeric synthetic agents and may enhance agglutination and / or muco-trapping of the target. In some cases, the methods described herein may include enhancing agglutination and / or muco-trapping of a target using any of these synthetic binding agents. The target typically has one or more epitopes, and may be a virus, bacteria, fungus, sperm or parasite, as described above. The synthetic binding agents described herein may be multimeric, having multiple epitope-binding regions. Any of these epitope-binding regions may be immunoglobulin fragment antigen binding (Fab) regions or fragments. The Fab fragments / domains may be directed to the same epitope to recognize the foreign body. Thus, the synthetic binding agents for enhancing agglutination and / or muco-trapping described herein may include one or more additional Fab domains, wherein the one or more additional Fab domains all specifically bind to the target epitope with high affinity, and may reduce the mobility of the target in mucus to less than about 50% relative to its native mobility in mucus. The synthetic binding agents may be recombinant (e.g., engineered), as described above. Any of the synthetic binding agents described herein may be further configured (or selected) to enhance mucin crosslinking once bound to the target, but may otherwise be relatively free to diffuse through mucus (e.g., have a low affinity for mucins). As used herein the term “native mobility” refers to the mobility of the target (e.g., sperm, virus, bacteria, etc.) in the same environment (e.g., mucus, saline, etc.) in the absence of a synthetic binding agent.

[0207] Also described herein are also methods and compositions (including compositions of the heterotrimeric engineered / synthetic binding agents) that could provide bactericidal and / or microbicidal effect by more effectively clumping together pathogens that undergo cell division, which leads to a chain of bacteria and / or other pathogens, and potentially inhibiting replication, triggering cell death, e.g., forming aggregations (including in some variations multi -pathogen aggregations) and / or preventing the spread of the infection through either agglutination or enchained growth.

[0208] PathogensAttorney Docket No. 5470.976.WO

[0209] In some variations, the target may be a pathogen, e g., all of the one or more binding domains (e g., Fab domains) may specifically bind to a pathogen. For example, the pathogen may be one (or more) of: Acinetobacter baumannii; Bacteroides fragilis; Burkholderia cepacia; Clostridium difficile; Clostridium sordellii; Carbapenem-resistant Enterobacteriaceae; Enterococcus faecalis; Escherichia coli; Hepatitis A; Hepatitis B; Hepatitis C; human immunodeficiency virus HIV-1 and HIV-2 (HIV, AIDS); Influenza; Klebsiella pneumonia; Methicillin-resistant Staphylococcus aureus; Morganella morganii; Mycobacterium abscessus; Norovirus; Psuedomonas aeruginosa; Staphylococcus aureus; Stenotrophomonas maltophilia; Mycobacterium tuberculosis; Vancomy in-resistant Staphylococcus aureus; Vancomycin-resistant Enterococci; Neisseria gonorrhoeae (gonorrhea); Chlamydia trachomatis (chlamydia, lymphogranuloma venereum); Treponema pallidum (syphilis); Haemophilus ducreyi (chancroid); Klebsiella granulomatis or Calymmatobacterium granulomatis (donovanosis), Mycoplasma genitalium, Ureaplasma urealyticum (mycoplasmas); HTLV-1 (T-lymphotrophic virus type 1); herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2); Epstein-Barr virus; cytomegalovirus; human herpesvirus 6; varicella-zoster virus; human papillomaviruses (genital warts); hepatitis A virus, hepatitis B virus, hepatitis C virus (viral hepatitis); molluscum contagiosum virus (MCV); Trichomona vaginalis (trichomoniasis); and yeasts, such as Candida albicans (vulvovaginal candidiasis). In some variations, the pathogen includes a fungus, such as Aspergillus .

[0210] Administering may comprise administering by any appropriate route, or more than one route. For example, administering may comprise administering to the subject vaginally (e.g., from an intravaginal ring, IVR). Administering may comprise administering systemically to the subject. Administering may comprise administering to the subject as a vaginal film. Administering may comprise administering from a nebulizer. Administering may comprise administering by inhalation. Administering may comprise an eye drop.

[0211] Administering may comprise an oral capsule or pill. Administering may comprise a mouth wash. In some variations, administering comprises delivering between 0.01 mg / day and 100 mg / day of the synthetic binding agent. Administering may comprise administering in an amount sufficient to agglutinate or form enchainment of the target while maintaining or enhancing muco-trapping, with the overall net effect of reducing the permeability of the target through mucus, and / or reducing the growth or presence of the target.Attorney Docket No. 5470.976.WO

[0212] The synthetic agents described herein may be configured to form heterotrimeric complexes targeting one or more viruses, including, but not limited to: coronaviruses (e.g., SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKUl, etc ), rhinoviruses (cold viruses), influenza viruses (e.g., Influenza A, e.g., H1N1, H3N2, Influenza B, Influenza C, Influenza D, etc.), Respiratory Syncytial Virus (RSV), Parainfluenza Viruses (e.g., HPIV-1, HPIV-2, HPIV-3, HPIV-4, etc.), Adenoviruses, Human Metapneumovirus (hMPV), Enteroviruses (e.g., Enterovirus D68, Coxsackievirus, etc.), Human Bocavirus (HBoV), Measles Virus, Human Herpesvirus 3 (Varicella-Zoster Virus), Human Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hantaviruses, etc.

[0213] For example, the synthetic agents described herein are methods of making and using them may be directed to respiratory viruses such as RSV. For example, described herein are synthetic binding agent, the synthetic binding agent comprising a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to an anti-RSV antigen binding domain, further wherein the anti-RSV antigen binding domain specifically binds to an epitope of RSV, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0214] Contraceptive Use

[0215] In some variations, and particularly contraceptive methods, target may be sperm. In some variations, the antigen is CD52g. The mobility of the sperm in mucus may be slowed down by at least 50% compared to the native mobility of the sperm in the mucus. Unintended pregnancy is associated with poor maternal and child health outcomes and represents a public health burden of nearly $21 billion in the US alone as of 2010. Despite the widespread availability of contraceptive methods, nearly half of all pregnancies in the U.S. remain unintended or unwanted. A key factor in limited contraceptive use is the hormonal nature of the vast majority of contraceptive options available; although generally safe and effective, many women simply prefer non-hormonal options due to real or perceived side-effects. Substantial fractions also have medical contraindications. The only long-acting, reversible non-hormonal contraceptive available is the copper IUD, which is often discontinued due to side effects such as dysmenorrhea and heavy menstrual bleeding. The currently available on-demand, female-initiated non-hormonalAttorney Docket No. 5470.976.WOcontraceptives either increase the risk of STI transmission, are messy to administer (gels, cream), and / or are non-discrete. Thus, there is a major unmet need for alternative non-hormonal contraceptives.

[0216] Compared to hormonal contraceptives, non-hormonal contraceptive options remain limited, leading to substantial dissatisfaction among many women and consequently high rates of unintended pregnancies. As shown by some women with immune infertility, anti-sperm antibodies (ASAs) can block sperm from swimming through mucus and reaching the egg, thus enabling highly effective non-hormonal contraception. Problematically, current IgG-based ASAs possess limited potencies at physiological temperature, and most multivalent monoclonal antibody (mAb) formats are ill-suited to the vaginal environments. What is needed is a multivalent mAb platform that retains potency at physiological temperatures and properties of conventional IgGs.

[0217] In general, the synthetic agents described herein may be configured to form heterotrimeric complexes targeting sperm, in order to increase agglutination of sperm in vaginal mucosa. For example, described herein are synthetic binding agents comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a selfassembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to an anti-sperm antigen binding domain, further wherein the anti-sperm antigen binding domain specifically binds to an epitope of a sperm, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

[0218] Among the different potential therapeutic classes, anti-sperm antibodies (ASAs), the basis of immune-infertility in many women, may offer the most promising means of safe and effective non-hormonal contraception. ASA can preclude otherwise motile sperm from reaching the egg, either by clumping sperm into clusters too large to swim through mucus (i.e. agglutination), or by crosslinking sperm to mucins via multiple low-affinity Fc-mucin bonds (i.e. muco-trapping). One such ASA (H6-3C423, or ‘HC4’) has been extensively investigated for this purpose. Originally an IgM ASA isolated from an immune infertile woman, HC4 binds a unique glycoform of CD52 (CD52g) found ubiquitously only on cells in the male reproductive tract and are generally absent in other tissues in women. To facilitate bioprocessing for clinical development, HC4 has been reformatted as IgG monoclonal antibody (mAb), and recently testedAttorney Docket No. 5470.976.WOas a vaginal film in an exploratory clinical study. Since IgG is generally a poor agglutinating molecule compared to polymeric species such as IgM, there is considerable interests in advancing higher valency mAh formats that possess much greater potencies than IgG while retaining the stability and manufacturing ease of IgG.

[0219] In engineering more potent ASA for non-hormonal contraception, it has been found that HC4-IgG quickly unbinds from CD52g on sperm at physiological temperature (i.e., 37°C). As the original IgM is known to cause immune infertility in vivo, increased Fab valency may generate sufficient binding avidity to overcome the temperature-induced Fab unbinding from sperm. Certain multivalent mAb platforms are found to be ill-suited for vaginal applications.

[0220] A novel multivalent mAb platform described herein offers advantages for vaginal application and stability at physiological temperature. This platform may be based on linking laminin-511 trimerization domains to IgGl-Fc and co-expressed with other Fab-trimerization domain conjugates. Development of such a laminin-IgG hybrid platform, as well as in vitro and in vivo studies supporting the lead candidate, LamHIO, as an exceptionally promising mAb for non-hormonal contraception are described herein.

[0221] Sufficient multivalency is required to maintain sperm agglutination potency at physiological temperature. The vast majority of sperm motility studies are performed at room temperature.

[0222] FIG. 1 is a schematic illustration of binding agents. Schematic illustration of IgG 102, FFIFF 104, and RGY 106 mAb formats is shown. Components of mAbs include HC4 Fab region 108 having variable light chain 109 VL, constant light chain 110 CL, variable heavy chain 111 VH, constant heavy chain 112, Fc (fragment crystallization region) 113, and RGY mutations 114.

[0223] FIGS. 2A-2C are graphs showing sperm agglutination with antibodies assessed by agglutination kinetics assay against purified semen at various temperatures based on three unique semen samples. Sperm agglutination with Ab at 10 pg / mL as assessed by agglutination kinetics assay against purified sperm at final concentration of 20* 106sperm / mL, performed atRT 201 and at 37°C 203 (n=3 semen samples from unique donors). As shown in FIGS. 2A, 2B, and 2C, the difference between percentage reduction in progressively motile (PM) sperm 206 approaches 0 over time 208.Attorney Docket No. 5470.976.WO

[0224] Via a carefully thermally-calibrated microscopy setup, HC4-IgG is able to effectively agglutinate purified PM sperm at 10 pg / mL at room temperature, consistent with FIGS. 2A-2C.In contrast, at 37°C 204, HC4-IgG loses almost all ability to agglutinate.

[0225] FIGS. 3A-3B are graphs showing binding affinity assessed by whole-sperm ELISA at room temperature 302 at 37°C 303, respectively. Samples were plated in triplicate and averaged.

[0226] The loss of sperm binding is reproduced via temperature-controlled ELISA assays, with HC4-IgG exhibiting an ICso of ~5nM at RT 301 (FIG.3A) and binding essentially below the limit of detection at 37°C 303 (FIG.3B).

[0227] As the Fab variable region was derived from an IgM sequence isolated from an immune-infertile woman, the loss in sperm binding and agglutination observed for HC4-IgG may be resolved with a suitable multivalent HC4 mAb. Experiments have used a decaval ent molecule comprised of ten total Fabs and one Fc, where additional Fabs are fused serially to the N- and C-terminus of the heavy chains of HC4-IgG. The molecule termed FFIFF 304 (see FIG. 1) in reflection of its molecular arrangement, possesses at least 16-fold greater sperm agglutination potency than the parent HC4-IgG 302. Unfortunately, while capable of mediating more stable binding and sperm agglutination than HC4-IgG 302, its overall potency was still nearly halved when the temperature is raised to at 37°C 303. The reduced binding is also confirmed via temperature-controlled ELISA assays. At RT 301, the ECso of FFIFF was 4.4 nM (FIG. 3A), which increased to beyond the measurement range at 37°C (FIG. 3B).

[0228] Due to the Fab arrangements, most Fab domains on FFIFF 304 are likely to experience substantial steric hindrance near the N-terminus of the heavy chain, potentially contributing to reduced binding valency and occupancy of available Fabs. This contrasts sharply with conventional IgM, where the 10-12 Fab domains in each IgM are free from any such steric obstruction. Other multivalent IgG with structural formats similar to IgM have been explored, specifically focused on IgGs with Fc mutations known as RGY 306 (see FIG. 1) that allows self-hexamerization via non-covalent associations between different IgG-Fc. The resulting HC4-IgG-RGY hexamer (i.e., HC4-RGY) exhibits superior sperm-agglutination (FIG. 2A) and sperm binding (FIG. 3A) at RT comparable to FFIFF. HC4-RGY also exhibited comparable sperm agglutination potency at 37°C as RT, and a markedly less reduction in ECso, which was ~1.1 nM at RT 302 (FIG. 3A) and -7.3 nM at 37°C 304 (FIG. 3B). These results underscore that suitablyAttorney Docket No. 5470.976.WOengineered multivalent IgGs can overcome the increased HC4-Fab unbinding at elevated temperatures.

[0229] FIG. 3C is a graph showing a size exclusion chromatography profile for RGY mAb hexamer and monomer populations as a function of concentration and pH. P-values were calculated by two-way ANOVA with post-hoc Holm-Sidak test. ****p < 0.0001. Lines and error bars indicate arithmetic mean values and standard deviation.

[0230] Despite the excellent potency at 37°C, there are critical shortcomings with the RGY-platform that limits its use for vaginal applications. Specifically, RGY-mediated assembly is non-covalent, and thus the ratio of RGY hexamers 330 and monomers 332 represents an equilibrium that can be altered by the environment. Indeed, at neutral pH, HC4-RGY shifts from -10% monomer at 5 mg / mL 320 to -25% monomer at 0.5 mg / mL 322. Similarly, after incubation at pH 4 324 for 1 hr, the monomer 332 fraction was increased to -62% of the mAb. These results make HC4-RGY incompatible with delivery at low concentrations to the vagina, where pH typically ranges between 3-5 due to acidification by resident Lactobacilli.

[0231] Design and characterization of highly multivalent mAbs with unencumbered Fabs using laminin trimerization domains

[0232] Based on the excellent sperm agglutination potency observed with HC4-RGY and the strong biophysical stability observed with FFIFF, a multivalent IgG format is developed that does not require fusing additional Fabs along the N-terminus of the heavy chains, while also avoiding non-covalent self-assembly of multiple IgG molecules. Trimerization domains present in various human proteins are introduced, which could be fused between the C-terminus of a Fab and the N-terminus of the Fc region. Additional binding domains can then be incorporated by expressing additional conjugates of Fab fused to a trimerization domain.

[0233] Homotrimeric domains present in collagen XV and collagen XVIII, which have been used previously to display VHH and scFV without an Fc domain, are screened. This results in distinct populations with multiple peaks on chromatography (see FIGS. 8D-8E), likely due to limited control over the assembly. Three heterotrimeric domains are screened, two of which produced poorly and assembled mixed populations after fusion into the IgG (see FIGS. 8F-8G).In contrast, molecules utilizing the heterotrimerization domain from laminin-511, composed of unique alpha, gamma, and beta domains, which is further stabilized by disulfide bonds when complexed, yield a single peak consistent with the expected size of a properly assembled molecule.Attorney Docket No. 5470.976.WO

[0234] FTG. 4 is a schematic illustration of laminin-IgG multivalent hybrids. As shown here, there is a panel of laminin-511 IgG hybrids with different number of HC4-binding domains (LamH / z), including either 6 (LamH6 402), 10 (LamHIO 404), or 12 (LamH12 406) HC4-Fab domains 408, having variable light chain region 409, constant light chain region 410. Also shown is IgGl Fc region 413 with variable heavy chain 411 and constant heavy chain 412. The sub-units of heterotrimer including alpha-5 subunit 401, beta-1 subunit 403, and gamma- 1 subunit 405. In some of these examples, each Fc domain may be linked to two heterotrimeric complexes, e.g., one at the N-terminal and one at the C-terminal end.

[0235] FIG. 5A is an SDS-PAGE analysis of various LamHzz mAbs in either non-reduced (NR) 512 or reduced (R) 514 conditions. All three LamHn constructs 502 / 504 / 506 demonstrated a clean monomer band, and are reducible to correctly sized individual protein chains in an SDS-PAGE.

[0236] FIG. 5B is a graph showing relative monomer content for each LamH / z mAb. Correct size of the monomer and overall homogeneity was further confirmed by SEC-MALS, with all constructs demonstrating similar homogeneity to the parent IgG (see also FIGS. 11A-11D).

[0237] FIG.5C is a plot showing relative reduction in PM sperm fractions with LamH12 using a hinge linker and a flexible (G4S)e linker between the HC4 Fab and laminin trimerization domains, with controls. Notably, the choice of linker is critical to effective sperm agglutination at 37°C. Even with 12 Fabs, LamH12 506 constructs utilizing glycine-serine linkers between the Fab and trimerization domains (LamH12-GS 507) exhibited less than half of the sperm agglutination potency as essentially the same LamH12506 format utilizing linkers composed of the upper-hinge region of a native IgGl 501 (LamH12506).

[0238] FIG. 5D is a graph showing binding affinity of various mAbs assessed by whole-sperm ELISA at 37°C. Samples were plated in triplicate and averaged. Binding affinities, as measured by ELISA at 37°C 513, were found to be 3.7nM, 2.3nM, and 11.2nM for LamH6 502, LamHIO 504 and LamH12506, respectively.

[0239] FIG. 5E is a graph showing stability of various LamH / z Abs as assessed by melting temperatures (Tm) 515. All 3 constructs 502 / 504 / 506 exhibited similar thermal stability properties to the parent IgG, with melting temperatures 508 Tml > 71.2°C and Tm2 > 80.3°C.

[0240] Stability of various LamHn mAbs are assessed by (FIG. 5E) melting temperatures (Tm) and (FIG. 5F) aggregation temperatures (Tagg) measured by nanoDSF. Experiment wasAttorney Docket No. 5470.976.WOperformed in triplicate and averaged. P-values were calculated using one-way ANOVA with post-hoc Dunnett’s multiple comparisons test. ****p < 0.0001. Lines and error bars indicate arithmetic mean values and standard deviation.

[0241] FIG. 5F is a graph showing stability of various LamH / Abs as assessed by aggregation temperatures (Tagg). Aggregation temperatures Tagg 517 include temperatures 508 > 80°C.

[0242] LamHIO rapidly agglutinates both purified motile sperm and sperm in whole semen.

[0243] Sperm is thought capable of ascending to the endocervix as quickly as 90 seconds post coitus; thus, rapid sperm agglutination is likely an important correlate for contraceptive efficacy.

[0244] FIG. 6A is a series of plots showing sperm agglutination kinetics of various IgG, RGY, and laminin multivalent hybrids. The kinetics of sperm agglutination were quantified by measuring the reduction in PM sperm 650 over time immediately following mixing of sperm and various mAbs via Computer-Assisted Sperm Analysis (CASA), with the key metric defined as >90% reduction in PM sperm 652 within 90s. Potency was tested against purified (washed) sperm at a concentration 625 of 20xl06sperm / mL. At 37°C, the parent IgG reduced PM sperm 650 by less than 10% on average after 90s at the highest concentration tested (10 pg / mL) across 6 semen samples (see also FIGS. 12A-12E), and had no observable effect at lower concentrations.

[0245] FIG. 6B is a series of plots showing sperm agglutination kinetics of LamHIO compared to RGY and parent IgG when conducted using whole semen. In order to agglutinate consistently at 37°C, the concentration 625 of IgG had to be increased 100-fold to 1 mg / mL. In contrast, RGY induced nearly 100% reduction in PM sperm 651 within 30s at 10 pg / mL, achieved >90% reduction in PM sperm 651 within 90s in 5 of 6 semen samples tested at 2.5 pg / mL, and in 3 of 6 semen samples tested at 0.625 pg / mL (see also FIGS. 12A-12E).

[0246] Despite only 6 HC4 Fab domains, the sperm-agglutination potency of LamH6602 was nearly on par with HC4-RGY with 12 Fabs per molecule. LamHIO 604 exhibited considerably greater sperm agglutination and was the most potent sperm agglutinator, mediating highly effective sperm agglutination in all 6 semen samples tested at 0.625 pg / mL. Indeed, in all semen samples tested, LamHIO 604 captured >90% of sperm within just 30s at 10 pg / mL, within 60s at 2.5 pg / mL, and within 90s at 0.625 pg / mL. The difference in sperm agglutination was statistically significant over LamH6 602 at 2.5 pg / mL, and over both HC4-RGY and LamH6 602 at 0.625 pg / mL (see also FIGS. 12A-12E). Interestingly, LamH12 606 was slightly less effective than LamHIO 604Attorney Docket No. 5470.976.WOdespite the 2 additional Fabs, capturing 90% of sperm within 90s in only 4 out of 6 samples at 0.625 pg / mL. Based on these results, LamHIO 604 may serve as the best candidate in future assays.

[0247] FIG. 6C is a plot showing sperm agglutination of parent IgG at 37°C at high concentration against purified sperm. The sperm agglutination kinetics of LamHIO is assessed against whole semen. Notably, the concentration of sperm in whole semen is variable, ranging from 20-100 million / mL in samples examined, and also contains substantial extracellular vesicles with CD52g 36 in the seminal plasma. Not surprisingly, the parent HC4-IgG 600 was unable to agglutinate meaning fractions of sperm at 37°C in whole semen, even at the highest tested concentration 625 of 40 pg / mL (see also FIGS. 12F-12J). HC4-RGY captured at least 90% of PM sperm within 30s at 40 pg / mL in all 6 samples, 90% within 90s in 5 out of 6 samples at 10 pg / mL, but was almost entirely ineffective at 2.5 pg / mL (see also FIGS. 12F-12J). LamHIO remained more effective than HC4-RGY in whole semen, able to capture nearly 100% of sperm within 30s at both 40 pg / mL and 10 pg / mL in all samples, and at least 90% capture within 90s in 5 out of 6 samples down to 2.5 pg / mL in whole semen (see also FIGS. 12F-12J). The ~4-fold shift in mAb concentrations necessary to support potent sperm agglutination in whole semen compared to purified sperm is consistent with findings regarding FFIFF.

[0248] FIG. 6D is a plot showing independent site evaluation of agglutination potency of LamHIO compared to parent IgG against purified sperm. The stability of LamHIO 604 after exposure to whole, undiluted CVM at native pH overnight at 37°C was further assessed. The supernatant of the sample was then centrifuged to remove mucins, and the remaining mAb in the supernatant was assessed for agglutination potency against whole semen. Although there was a modest reduction in the agglutination potency, LamHIO 604 was at least as stable as the parent IgG 600 in CVM (see also FIG. 14A), indicating that the laminin trimerization domains are not prone to acidic or enzymatic cleavage from CVM. Additionally, LamHIO 604 remained stable and effective in solution after being held at 37°C for at least 2 weeks (see FIGS. 14B-14D). SEC analysis of LamHIO 604 at 5 mg / mL, 0.5 mg / mL and at pH 4 further showed that LamHIO 604 does not undergo concentration and pH-dependent dissociation as does RGY (see FIG. 14E).

[0249] LamHIO more effectively reduces sperm escape from ejaculate than IgG and RGY Hexamer.

[0250] Generally, bulk mixing of semen and cervicovaginal mucus (CVM) is limited due to their viscoelastic rheological properties; sperm must swim out of the ejaculate and towards theAttorney Docket No. 5470.976.WOcervical os. As a separate independent assessment of sperm agglutination, a modified sperm escape assay was utilized to measure the quantity of sperm which escape the ejaculate when exposed to a layer of media containing ASAs. The parent IgG exhibited an average of only -26% reduction in escaped PM sperm at 40 pg / mL compared to no Ab control, and on average no observable reduction at lower concentrations across 6 semen samples tested (see also FIGS. 13A-13B).Consistent with sperm agglutination kinetics study, RGY reduced escaped PM sperm by -97% at 40 pg / mL, by -77% at 10 pg / mL and by only -16% at 0.625 pg / mL. LamHIO again offered the most effective sperm agglutination among all mAbs tested, achieving 100% reduction in escaped PM sperm at 40 pg / mL, -97% reduction at 10 pg / mL, and still -50% reduction at 0.625 pg / mL across the 6 semen samples tested. The reduction in efficacy at preventing sperm escape was again compared at 37°C and at RT. Although LamHIO does exhibit some minor loss in sperm agglutination potency at elevated temperature, it retains the sperm agglutination activity measured at RT to a greater extent than either the parent HC4-IgG or HC4-RGY (see FIG. 14C).

[0251] LamHIO preserves muco-trapping Fc-effector function.

[0252] The “shaking phenomenon” has been observed, in which ASAs can trap individual sperm in mucus despite the vigorous motion of the sperm flagellum. This phenomenon is dependent on the Fc portion of Ab forming adhesive crosslinks with mucins and is consistent with prior observations in which Ab can immobilize viruses in mucus by low-affinity crosslinks between the Fc domain and mucins. It was determined whether laminin-based IgG hybrid molecules can retain the muco-trapping potencies against individual spermatozoa, given that the additional Fabs trimerized via the laminin-511 domain to the C-terminus of Fc likely poses steric hindrance to Fc access.

[0253] FIG. 7A depicts representative traces of semen in native CVM or CVM treated with HC4-IgG or LamHIO. High spatiotemporal video microscopy can be utilized to quantify the motility of hundreds of individual spermatozoa in different conditions.

[0254] FIGS.7B-7D are graph-plots depicting average, velocity, curvilinear velocity, straight-line velocity, respectively, of tracked sperm across control IgG, IgG, and LamHIO.

[0255] FIG. 7E is a graph-plot depicting percentage of sperm classified as PM.

[0256] Both parent IgG 700 and LamHIO 704 reduced the velocity of the average path (FIG.7B) and curvilinear velocity (FIG.7C) ~2-fold compared to control IgG, and reduced the straight-line velocity (FIG. 7D) and the overall % PM sperm (FIG. 7E) at least 3 -fold. These resultsAttorney Docket No. 5470.976.WOsuggest LamHIO 704 possesses comparable muco-trapping potencies as TgGmAbs 700 despite its considerably greater wingspan.

[0257] LamHIO effectively reduces PM sperm in the sheep vagina.

[0258] FIG.8A is a schematic of a study design for LamHIO and sperm agglutination in sheep. As the unique glycoform of CD52g targeted by H6-3C4 is found only in human and chimpanzee sperm, there is no suitable animal model available to directly assess contraceptive efficacy. Instead, the in vivo effectiveness of LamHIO was evaluated using a sheep model of human post-coital test (PCT), which have proven effective predictors of eventual contraceptive efficacy. Sheep is typically used for assessing vaginal products due to physiological and geometrical similarity of the female reproductive tract to humans. Either PBS or defined quantities of LamHIO were instilled into the sheep vagina 860, followed by simulated intercourse with a vaginal dilater (15 strokes) 862, instillation of whole human semen 864, and further brief simulated intercourse (5 strokes) 866. Finally, the sample was recovered from the sheep vagina 2 mins after instillation of the semen and immediately assessed for sperm motility 868.

[0259] FIG. 8B is a graph-plot showing a reduction in PM sperm compared to PBS control for LamHIO dosed at different concentrations. Across the 6 animals tested in a blinded fashion, both 333 pg and 33 pg of LamHIO agglutinated >99% of PM sperm, while 3.3 pg agglutinated on average -90% of PM sperm. These results confirm LamHIO possesses markedly greater potencies than HCA IgG and also the decavalent FFIFF, underscoring the potentially very strong contraceptive potencies of LamHIO in humans.

[0260] FIG. 8C is a LamHIO Fast-Dissolving Ab Tablet (FDAT). LamHIO is stable after lyophilization and formulation into FDAT for on-demand non-hormonal contraception.

[0261] FIG. 8D is an illustration of the dissolution kinetics of FDAT in CVM. The suitability of LamHIO for formulation into a tablet as a means of on-demand contraceptive delivery was assessed. LamHIO was lyophilized in the presence of trehalose, then pressed into a 6-mm round FDAT with a hydraulic press. The FDAT rapidly dissolves upon exposure to moisture, taking approximately a bit more than 2 minutes (120s+ mix 869) to dissolve upon exposure to CVM.

[0262] FIG. 8E is a plot showing sperm agglutination potency of LamHIO in solution comparted to potency after reconstitution from lyophilized powder or FDAT. LamHIO maintained agglutination potency after reconstitution either from lyophilized powder 871 or FDAT 872,Attorney Docket No. 5470.976.WOevaluated by comparing the agglutination potency against purified sperm to the original LamHIO solution 870.

[0263] FIG. 8F is a graph showing reduction in PM sperm compared to PBS control for placebo vs. LamHIO FD AT. The same sheep pseudo-PCT model from FIG.8A was used to assess the potency of LamHIO-FDAT. Here, sheep received either PBS, a placebo FDAT 880, or a LamHIO FDAT 881.

[0264] FIG. 8G is a schematic of a study design evaluating the effectiveness of LamHIO in agglutinating sperm within 5 minutes of vaginal dosing in sheep. After receiving either placebo FDAT or LamHIO FDAT 890, the sheep underwent a brief simulated intercourse 892, followed by semen inoculation 5 minutes after the initial FDAT insertion 894, and PM sperm fractions were assessed 2 minutes later 896. Studies were once again executed in a blinded fashion. Compared to PBS, placebo FDAT slightly reduced PM sperm by -34%. In contrast, LamHIO-FDAT reduced PM sperm by 100% across all tested sheep.

[0265] Generation of trimerized constructs for screening.

[0266] The variable light (VL) and variable heavy (VH) nucleotide sequences of the published sequence of H6-3C4 were used in all constructs. Each protein chain was cloned into the paH mammalian expression vector as the backbone. The initially screened constructs were analogous to LamH6, where it was intended to display 6 Fabs, 3 trimerized on each arm of the IgG. The linker used between all globular domains was 6xGGGGS (SEQ ID NO:9). Two homotrimer domains from collagen XV and XVIII were screened. Each construct required a light chain (LC), a midlength chain (MC) containing the trimerization domain, and a HC containing a trimerization domain and the Fc region of the Ab. The LC was constructed in the format VL-C domains. The MC was expressed in the format Vu-CHi-Linker-trimer domain. The HCs were expressed in the formats Vn-CHi-Linker-trimer domain-CH2-CH3. We additionally screened a heterotrimer domain from the NCI portion of collagen IV. This trimer is composed of two al chains and one a2 chain. This construct required plasmids for an LC, an MC containing the al trimerization domain, and an HC containing the a2 trimerization domain and Fc region of the Ab. The LC was constructed in the format VL-CX domains. The MC was expressed in the format Vu-CHi-Linker-al trimer domain. The HC was expressed in the format Vn-CHi-Linker-a2 trimer domain-CH2-CH3. The last trimer domain screened was a computational designed trimer known as DHT03, which requires 3 unique A, B, and C trimer domains. This construct required plasmids for an LC, an MCAttorney Docket No. 5470.976.WOcontaining the A trimerization domain, an MC containing the B trimerization domain and an HC containing the C trimerization domain and Fc region of the Ab. The LC was constructed in the format VL-CA domains. The MCs were expressed in the formats Vn-CHi-Linker-A trimer domain and Vn-CHi-Linker-B trimer domain. The HC was expressed in the format Vn-CHi-Linker-C trimer domain-CH2-CH3.

[0267] Stability in CVM assay.

[0268] Either LamHIO or the parent IgG was diluted into 100 pl whole, native CVM to a final concentration of 160 pg / mL. The native pH was measured via micro pH meter and recorded; native pH ranged from 3.8-4.2. Aliquots were incubated for 18h overnight at either 37°C or 4°C. The following day, the CVM- Ab mixtures were diluted with 100 pl PBS and mixed thoroughly by vortexing. The mucins were then spun out by centrifugation at 15,000g for 10 min, and the supernatant harvested. The supernatant was treated as having an initial concentration of 80 pg / mL of Ab. These were then assessed for agglutination activity by the agglutination kinetics assay against whole semen.

[0269] Long-term temperature stability assay.

[0270] LamHIO was prepared at 5 mg / mL concentration in 20mM Histidine, lOOmM Arginine, pH 6.5. The Ab was split into 10 0.2 mL aliquots. Half of the aliquots were stored in 4°C as controls, and the other half was stored in a 37°C incubator for 28 days. At each time point: Day 0, Day 7, Day 14, Day 21, and Day 28, one aliquot was taken out from both 4°C and 37°C for the characterization using SEC-MALS, SDS-PAGE, and Agglutination Kinetics assay against purified sperm.

[0271] FIG. 9 is a table showing motility parameters of Hamilton-Thorne Ceros 12.3.

[0272] FIG. 10A is a graph screening trimerized Ab using the homotrimer domain from Collagen XV via SEC-MALS.

[0273] FIG. 10B is a graph screening trimerized Ab using the homotrimer domain from Collagen XVIII via SEC-MALS.

[0274] Both constructs using a homotrimer domain were able to assemble in two distinct populations, with only the smaller population corresponding to the correct size of the desired 6-Fab display format.

[0275] FIG. 10C is a graph screening a construct with a heterotrimer domain from Collagen IV via SEC-MALS.Attorney Docket No. 5470.976.WO

[0276] FTG. 10D is a graph screening a construct with a computationally designed obligate heterotrimer DHT03 via SEC-MALS.

[0277] Both heterotrimers expressed a primary population too small to be the assembled 6-Fab display format.

[0278] FIG. 11A is a graph showing SEC-MALS UV and Scattering Data of parent IgG.

[0279] FIG. 1 IB is a graph showing SEC-MALS UV and Scattering Data of LamH6.

[0280] FIG. 11C is a graph showing SEC-MALS UV and Scattering Data of LamHIO.

[0281] FIG. 11D is a graph showing SEC-MALS UV and Scattering Data of LamH12.

[0282] FIG. 12A is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 10 pg / mL added to purified semen.

[0283] FIG. 12B is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 2.50 pg / mL added to purified semen.

[0284] FIG. 12C is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.625 pg / mL added to purified semen.

[0285] FIG. 12D is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.3125 pg / mL added to purified semen.

[0286] FIG. 12E is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.156 pg / mL added to purified semen.

[0287] The kinetics of sperm agglutination across mAb concentrations were determined by the relative reduction in PM sperm fractions at 30 second intervals following addition of mAbs to purified sperm, compared to sperm -handling media negative control. Data represents n=6 independent experiments with semen samples from unique donors. Measurements in each sample were performed in duplicate and averaged.

[0288] FIG. 12F is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 40 pg / mL added to whole semen.

[0289] FIG. 12G is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 10 pg / mL added to whole semen.

[0290] FIG. 12H is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 2.5 pg / mL added to whole semen.

[0291] FIG. 121 is a line graph showing the kinetics of sperm agglutination at a mAb concentration of 1.25 pg / mL added to whole semen.Attorney Docket No. 5470.976.WO

[0292] FTG. 12J is a line graph showing the kinetics of sperm agglutination at a mAh concentration of 0.625 pg / mL added to whole semen.

[0293] The kinetics of sperm agglutination across mAb concentrations were determined by the relative reduction in PM sperm fractions at 30 second intervals following addition of mAbs to whole semen, compared to sperm-handling media negative control. Data represents n=6 independent experiments with semen samples from unique donors. Measurements in each sample were performed in duplicate and averaged.

[0294] FIG. 13A is a plot showing fraction of PM sperm compared to no mAb control at decreasing concentrations of HC4-IgG, HC4-RGY and LamHIO. Data represents n=6 independent experiments from semen samples from unique donors. Lines represent the median value in FIG.13A.

[0295] FIG. 13B is a line plot showing average reduction in escaped PM sperm across mAb concentrations.

[0296] FIGS. 13C-13E are lines plot showing comparisons of reduction in sperm escape at RT vs. 37°C for HC4-IgG, HC4-RGY, and LamHIO, respectively. Data represents n=5 independent experiments of semen samples from unique donors. Experiments in each sample were performed in duplicate and averaged. P-values were obtained by two-way ANOVA with post-hoc Tukey’s multiple comparisons test. **P<0.01, ***P<0.001, ****P < 0.0001. Lines and error bars indicate arithmetic mean values and standard deviation in FIGS. 13B-13C.

[0297] FIG. 14A shows agglutination potency in whole semen of IgG and LamHIO after incubation in whole human CVM, at either 37°C or 4°C. Data obtained from n=3 unique CVM samples, each of which were paired with at least 2 unique semen samples.

[0298] FIG. 14B is a graph showing agglutination potency of LamHIO during long-term exposure to 37°C conditions, at 7-day intervals up to 28 days.

[0299] FIG. 14C is a graph showing monomer content of LamHIO after incubation at 37°C as measured by SEC-MALs, at 7-day intervals to 28 days.

[0300] FIG. 14D is a graph showing stability of LamHIO at 5 mg / mL and 0.5 mg / mL at pH7, and 5 mg / mL at pH4, as measured by comparison of UV-signal during SEC. P-values were obtained by two-way ANOVA with post-hoc Dunnett’s test. *P<0.05, ****P<0.001. Lines in FIG.14A represent the median. Lines and error bars in FIG. 14B indicate arithmetic mean values and standard deviation. As used herein, “obligate heterotrimer” may refer to an a / / y complex that selfAttorney Docket No. 5470.976.WOassembles with >90% heterotrimer and <10% heterodimer / homotrimer by SEC-MALS at 4-8 °C in PBS (pH 7.4), without added crosslinkers. “Monomer content” may refer to the main molecular species quantified by area under the UV trace in SEC-MALS, excluding high-molecular-weight and low-molecular-weight shoulders. “Rapid agglutination” may refer to > 80% (e.g., 85% or more, 90% or more etc.) reduction of progressively motile sperm within 90 seconds at 37 °C, quantified by CASA (parameters per Fig. 9).

[0301] In general, mAb-mediated potent non-hormonal contraception has been a long-sought goal for decades. Motivated by the discovery of immune infertility, the first attempts were based on contraceptive vaccines, which suffered from variable effectiveness and risk of permanent infertility. As mAb bioprocessing began to mature, the efforts then transitioned to harnessing antisperm IgG mAbs to enable transient contraception. Given the limited agglutination potencies of conventional IgGs, there is intense interests in engineering more potent mAbs. The latest and most iteration of engineered mAbs for non-hormonal contraception Fis described herein, with LamHIO capable of achieving over 100-fold greater agglutination potencies than the predicate HC4-IgG, while maintaining exceptional stability at low pH and low concentrations. These properties make the LamH platform promising for the development of monospecific multivalent Abs where agglutination is a critical effector function. The controlled assembly of up to 3 unique protein chains in the trimer also allows for multivalent and multi-specific applications, if single-domain binders are used.

[0302] The improved performance of LamH / / constructs over FFIFF and RGY at 37°C highlights essential features on multivalent AS As, including stability, Fab presentation and valency, as well as linker selection. For instance, LamH6 is similar to RGY in performance, despite halving the number of Fab domains per molecule. This is likely due to the covalent stabilization of the laminin trimerization domains, which minimizes concentration and pH-dependent disassembly of the multivalent molecules. All LamH / 7 constructs as well as RGY outperform FFIFF, which appears to confirm our hypothesis that the fusion of the Fab at the N-terminus in FFIFF is detrimental to the binding of the H6-C34 Fab. This is further supported by the slightly lower performance of LamH12 compared to LamHIO despite two additional Fab: in LamH12, the additional Fabs can only be added by fusing to the C-terminus of the alpha domain located to the C-terminus of the Fc, and thus occupy the N-terminus of the additional Fabs. Thus, LamH12 actually contains fewer available Fab per pg of protein compared to LamHIO. Finally, theAttorney Docket No. 5470.976.WOenhanced performance of LamH constructs using a hinge linker compared to a glycine serine linker underscores the importance of selecting suitable linkers in maximizing multivalent binding. We speculate this is due to an entropic penalty from the higher flexibility of glycine-serine linkers; after a Fab unbinds, the more flexible GS linkers likely allow the Fab domain to more quickly diffuse away from the previously bound epitope, thus reducing the probability of rapid rebinding. This in turn decreases the net number of bonds that GS-containing molecules can make with sperm at steady state, making it easier for sperm to escape agglutinates.

[0303] For example, the disclosed agents may employ a laminin-511-derived a / p / y heterotrimeric core that assembles obligately into a covalently stabilized heterotrimer via inter-strand disulfide bonds (e.g., in SEQ ID NOs:2, 4, and 6). Unlike non-covalent IgG hexamer platforms (e.g., RGY) whose oligomer ratio varies with pH and concentration, the laminin heterotrimer remains monodisperse by SEC-MALS (>90% monomer) after incubation at pH 4.0 and 37 °C. In contrast to homotrimer collagen motifs that give mixed species, laminin-511 E8 trimerization yields a single, designable a / p / y stoichiometry suitable for precise Fab valency control.

[0304] In addition to the orders of magnitude improvement in the sperm agglutination potency of LamHIO, the exceptionally low dose (tens of micrograms) needed to achieve complete agglutination in sheep also likely reflects the superior biodistribution with direct vaginal delivery that makes the entire dose available at the site of action. Systemic delivery dilutes the dose into a large blood volume, which distributes most of the dose to non-target tissues, and results in substantial delay before ASA can be distributed into the FRT. Instead, the low total volume of secretions within the FRT (~lmL or less) implies that even relatively small total amounts of mAb can achieve a high local concentration. Finally, local delivery also likely improves safety, both because mAbs delivered to mucosal surfaces like the FRT is poorly absorbed into systemic circulation, and also because vagina is a poorly immune inductive site with limited immune responses even when inoculating antigens in the presence of highly immunostimulatory adjuvants.

[0305] In the past, high costs of mAb production and focus on systemic delivery has limited the development of ASAs as a contraceptive strategy. Local delivery combined with declining costs due to advances in efficiency with CHO-based manufacturing over the past decades mean that ASAs may soon be a cost-effective contraceptive alternative. Multivalent formats such as LamHIO that can reduce the total dose needed will help further drive down costs. Additionally,Attorney Docket No. 5470.976.WOLamHIO provides the benefit of covalent assembly of the protein chains, improving stability and homogeneity compared to formats such as RGY.

[0306] Sperm must swim through mucus to ascend into the upper reproductive track and eventually reach the egg. Of -200 million total sperm in the ejaculate, only -1% of the spermatozoa enter the cervix, and only a few dozen might reach the egg. While low sperm count alone is not predictive of low fertility, both poor sperm motility in cervical mucus and reduced total sperm count contribute to an increased odds ratio for infertility. Human semen typically contains 45-65 million sperm / mL, while a concentration below 5 million sperm / mL is considered severely oligospermic and is associated with low fertility. Similarly, azoospermia and oligospermia induced by testosterone, which does not affect sperm morphology or in vitro fertilizing capacity, exhibits very low fertility rates at 0.8 conceptions per 100 women-years at concentrations below 1 million sperm / mL. Together, this suggests that substantial reduction of progressive sperm motility, even if incomplete, has the potential to provide effective contraception.

[0307] Further studies may directly demonstrate pregnancy prevention as the target antigen is present only on human and chimpanzee sperm, and chimpanzee studies are not possible in the US. Thus, an in-vivo sheep model closely mimicking the human PCT test was used as a proof-of-concept. Efficacy in humans will need to be rigorously evaluated in clinical studies. A recent Phase 1 clinical trial of the parent IgG has shown promising initial safety and reduction of progressively motile sperm when delivering a relatively high dose of 20mg in a vaginal film. Efficacy was promising when delivering a dose of Img via tablet in sheep as described in FIGS. 8A-8G, and delivery in solution suggests that LamHIO remains effective even at doses as low as 33pg. This may allow for sustained delivery of Ab in other formats such as a vaginal ring in future studies.

[0308] Materials and Methods.

[0309] Human semen and human cervicovaginal mucus (CVM) samples used in all in vitro studies were collected under a protocol approved by the Institutional Review Board (IRB) of the University of North Carolina (UNC) at Chapel Hill (IRB #10-1817). Informed written consent was obtained from all male and female subject prior to the collection of semen and CVM samples. Human semen samples used in the sheep surrogate PCT study were collected under a protocol approved by the IRB of the University of Texas Medical Branch (UTMB). All male volunteers provided informed written consent prior to sample collection. Sheep studies using six femaleAttorney Docket No. 5470.976.WOMerino crossbred sheep followed protocols approved by the UTMB Institutional Animal Care and Use Committee.

[0310] Semen collection and purification of motile sperm.

[0311] Health male subjects between 18-45 years old were asked to collect semen by masturbation into sterile 50 mb sample cups. Subjects were asked to refrain from sexual activity at least 48 hours prior to sample collection. Samples were incubated at least 15min after collection at room temperature (RT), or until liquefaction. Samples which did not liquefy were discarded. Density gradient centrifugation was used to separate the motile sperm fraction from liquefied ejaculate. A two-layer gradient was prepared by layering a 1 ,5mL of Isolate 50% density medium over a 1 ,5mL Isolate 90% density medium (Irvine Scientific, #99264). Up to 3mL of ejaculate was carefully layered on top of the gradient, then centrifuged at RT at 300xg for 20min. After centrifugation, the upper layers containing seminal plasma and dead cells was removed, taking care not to disturb the pellet of motile sperm at the bottom. The sperm pellet was then washed two times with sperm multipurpose handling medium (MHM) (Irvine Scientific, #90166). The washed pellet was then resuspended in MHM, and assessed for count and mobility using CASA. Samples were discarded if they did not meet the lower reference limit for total motility (40%) and sperm count (15 x 106 sperm / mL) indicated by World Health Organization guidelines.

[0312] CASA Sperm Count and Motility analysis.

[0313] Hamilton Thome CASA version 12.3 was used in all experiments unless stated otherwise. For each sample, 2 chambers of a 20pm depth sperm counting slide (Leja 4-Chamber slides) were filled with 3 pL of the sample. In each chamber, 10 randomly selected microscopic fields near the slide center were imaged and averaged for count and motility. The assessments of the 2 chambers were then averaged. The complete set of parameters used by the Hamilton Thorne Ceros 12.3 software are listed in FIG. 9. Sperm were defined as progressively motile if they had a VAP of at least 25pm / s and 80% STR.

[0314] CVM collection and processing.

[0315] CVM was collected as previously described. Briefly, undiluted CVM was self sampled from women of reproductive age, ranging from 20 to 33 years old using a disposable menstrual cup (Instead Softcup). Participants inserted the device into the vagina for at least 60s, twisted while removing, and placed the ring into a 50-mL conical tube. Samples were centrifuged at 500xg for 5min to collect the secretions off the ring. The samples averaged 0.3g per sample, ( / unprotectedAttorney Docket No. 5470.976.WOintercourse before providing the sample. All samples were collected a minimum of 72h after the reported end of the last menstrual cycle.

[0316] Construction of LamH Construct Plasmids.

[0317] The variable light (VL) and variable heavy (VH) nucleotide sequences of the published sequence of H6-3C424 were used in all constructs. Each protein chain was cloned into the paH mammalian expression vector as the backbone. The trimerization domains were obtained from the E8 domain of laminin-51130. Each construct required a light chain (LC), two mid-length chains (MC) containing [3 and y trimerization domains, and a heavy chain (HC) containing the a trimerization domain and Fc region of the antibody. The LC and two MCs were identical across constructs. The linker used was the upper hinge region of IgGl antibody, amino acid sequence EPKSCDKTHT (SEQ ID NO:7). The LC was constructed in the format VL-C domains. The chain was expressed in the format VH-CHl-Linker-0 trimer domain. The y chain was expressed in the format VH-CHl-Linker-y trimer domain. The HCs were expressed in the formats VH-CH1-Linker-a trimer domain-CH2-CH3, VH-CHl-Linker-a trimer domain-CH2-CH3 -Linker- a trimer domain, and VH-CHl-Linker-a trimer domain-CH2-CH3 -Linker- a trimer domain-Linker- VH-CH1 for LamH6, LamH 10, and LamH 12 respectively.

[0318] Expression and Purification of Ab.

[0319] Plasmids expressing LamH constructs or the parent IgG were co-transfected into Expi293F cells using ExpiFectamine293 transfection reagents (Gibco, Gaithersburg, MD). For IgG and RGY, HC and LC plasmid were co-transfected at a 1 : 1 ratio following the manufacturer’s protocol. For LamH6, LamHIO, and LamH12, a HC, 0 MC, y MC, and LC were co-transfected in a 1:2:2:5 ratio at 2 pg total DNA per 1 mL culture. Transfected cells were grown at 37° in a 5% CO2 incubator while shaken at 125 rpm for 3-5days, or until viability dropped below 60%. The culture supernatant was then harvested by centrifugation for lOmin at lOOOxg and passed through 0.22pm vacuum filters. Abs were then purified using protein A chromatography. 120mL of culture supernatant was mixed with 250pL of Pierce protein A plus resin (Thermofisher Scientific, #53142) then split into 50mL conical tubes and incubated at 4°C overnight with end-over-end mixing. The supernatant was then loaded into 20mL Econo-Pac® Chromatography Columns (Biorad, #732101) until all resin was collected. The resin was then washed three times with 5mL lx phosphate-buffered saline (PBS). Protein was then eluted from the column using 900pL Pierce IgG Elution Buffer (Thermofisher Scientific, #21004), which was immediately neutralized withAttorney Docket No. 5470.976.WO1 OOpL IM Tris-HCl, pH 8.0 (Therm ofisher Scientific, #15568025). Purified Ab was quantified by nanodrop using absorbance at 280nm, using the molecular weight and extinction coefficient of each protein. The molecular weight of LamH6, LamHIO, and LamH12 were calculated as 403kDa, 655kDa, and 753kDa respectively. The extinction coefficients were calculated as 606220 cm-1 M-1, 972240 cm-1 M-l, and 1142440 cm-1 M-l respectively.

[0320] Biophysical Characterization of Abs.

[0321] The relative size of unreduced purified constructs and the molecular weight of their component protein chains was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in reducing and non-reducing conditions. For each sample, 1-2 pg of protein was added to 3.75 pL NuPAGETM lithium dodecyl sulfate (LDS) sample buffer (Thermo Scientific, #NP0007). In reduced-condition aliquots, 1 pL of 0.5 M tris (2-carboxyethyl) phosphine (TCEP) (Thermo Scientific, #T2556) was added as the reducing agent. Aliquots were then filled with nuclease-free water to a final volume of 15 pL. Samples were then denatured by holding in a thermocycler at 70°C for lOmin. Samples were loaded into a NuPAGETM 3-12% Bis-Tris gel (Thermo Scientific, #NP0322), and NovexTM Sharp Pre-stained Protein Standard (Thermo Scientific, #LC5800) was used as the standard. The gel was run for 1 hr at 200V, then stained for 1 hr with Imperial Protein Stain (Thermo Scientific, #24615) before being destained overnight in double-distilled water.

[0322] Size exclusion chromatography with multi angle light scattering (SEC-MALS) was employed to evaluate the molecular weight of the unreduced purified Abs and the overall homogeneity of the Ab solutions. A Bio-Rad FPLC system with a GE Superdex 200 10 / 300 column was connected to a Wyatt miniDAWN MALS instrument. Experiments were run at 4°C with a flow rate of 0.25 mL / min. The column was equilibrated with sterile-filtered lx PBS pH 7.4, then lOOpL of each sample at a concentration of l-2mg / mL was injected onto the column. MALS data was analyzed using Wyatt ASTRA 8 software.

[0323] The Tm and Tagg of the purified Abs were determined via nano differential scanning fluorimetry (nanoDSF) using a Nanotemper Prometheus NT.48 system. Samples were diluted to 0.5mg / mL in IX PBS, pH7.4, then loaded into Prometheus NT.48 capillaries. Each sample was evaluated in triplicate. Thermal denaturation experiments were executed over the range of 20°C to 95°C increasing at l°C / min, while measuring intrinsic tryptophan fluorescence at 330nm and 350nm. The Tm was calculated automatically by the manufacturer’s software from the firstAttorney Docket No. 5470.976.WOderivative of the ratiometric measurement of these fluorescent signals as temperature increases. Similarly, the Tagg is calculated by the manufacturer’s software based on measurements of back-reflected light from a light beam passed through the sample at each temperature. As the Ab aggregates, more light is scattered and the back-reflection of light is reduced.

[0324] Whole sperm enzyme-linked immunosorbent assay (ELISA).

[0325] Binding affinity of each construct was evaluated by ELISA against whole sperm. Motile sperm were purified from whole semen as described above, then coated onto half-area high-binding polystyrene plates (CLS3690, Corning) at 2 x 105sperm per well in 50 pL of NaHCCh buffer, pH 9.6. Plates were incubated overnight at 4°C, then centrifuged at 300xg for 20min. The supernatant was decanted, then the plate air-dried for 1 hr at 45°C. Plates were washed 3x with PBS, then blocked with 150pL per well of 5% milk at RT for 1 hr. After an additional 3x wash with PBS, Abs were diluted in 1% milk at a starting concentration of lOOnM, then serially diluted at 5-fold intervals and plated in triplicate at 50 pL per well. Plates were incubated for 1 hr at either RT or 37°C. Plates were washed again 3x with PBS, which was either at RT or had been prewarmed to 37°C according to the incubation temperature of the prior step. Afterwards, plates were incubated for 1 hr at RT with 50pL per well of HRP-conjugated goat anti-human IgG Fc (Abeam, # ab98624) diluted 1:10,000 in 1% milk. The washing procedure was repeated and then the plate was developed by incubating 50 pL per well of 1 -Step Ultra TMB ELISA Substrate (Thermofisher Scientific, #34028) in the dark at RT for 15 min. The reaction was then quenched using 50 pL per well of IN HC1. The absorbance at 450 nm (signal) and 570 nm (background) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices).

[0326] Agglutination Kinetics Assay.

[0327] Purified sperm resuspended at an initial concentration of 40 * 106sperm / mL in sperm multi -handling media. A sperm counting assessment chamber slide (Leja, 4-chamber, 20pm depth) was pre-warmed on a MiniTherm® stage warmer set to 37°C. Sperm stock and antibody dilutions were held at 37°C in a water bath. Then, 6 pL of purified sperm was mixed 1:1 (working concentration 20 x 106sperm / mL) with 6 pL of Ab dilution in 0.2 mL PCR tubes, moving rapidly to minimize cooling time. A timer was started immediately upon initiating mixing, then 3 pL of sperm Ab-mixture transferred to the chamber slide. The Centerfield of the slide was then imaged and analyzed by CASA at 30, 60, and 90s. The reduction in percentage of PM sperm at each point was computed by normalizing to the PM sperm count in a control slide mixed with sperm handlingAttorney Docket No. 5470.976.WOmedium alone. Data was obtained from n=6 samples all from unique donors, and each sample was assessed in duplicate and the average of the two chambers taken. The assessment was also repeated for each donor using unaltered whole semen in place of purified sperm using the selected candidate Ab LamHIO. Assessments were started at 10 pg / mL when working with purified sperm or 40 pg / mL when using whole semen, and 4-fold dilutions assessed until complete failure of the LamHIO was observed.

[0328] Sperm escape assay.

[0329] Whole semen was placed into a PCR tube, and an equal volume of media containing the ASA layered gently on top. The sample was then held at a 45° angle at 37°C for 5 minutes. A sample was taken from the topmost section of media and evaluated via CASA for the quantity of PM sperm.

[0330] For each sample, 40 pL aliquots of whole semen were transferred to individual 0.2 mb PCR tubes. Abs were diluted in sperm handling medium, and subsequently 40 pL of either untreated medium or an Ab dilution was layered gently on top of the whole semen. The tubes were then held in a thermocycler propped at a 45° angle at 37°C for 5 min. Following this incubation, 6 pL was extracted from the top layer and transferred to a chamber slide held in a stage warmer. The number of PM sperm was summed over 10 fields of view in each condition, and the percentage decrease calculated by normalizing to the untreated control. Data represents 6 independent experiments from 6 unique sample donors. Each experimental condition was assessed in duplicate on each sample. The Abs were assessed starting at 40 pg / mL and working down with 4-fold serial dilutions.

[0331] Fluorescent sperm labeling.

[0332] SYBR14 dye, a membrane permanent nucleic acid stain, was used to stain live sperm. Motile sperm were isolated from whole semen as described above. The purified sperm was incubated with SYBR14 from a Live / Dead Sperm Viability kit (Thermo Scientific, #L7011) at a final concentration of 200nM for lOmin in a water bath at 37°C. The labelled sperm was then washed 3 times with sperm washing medium after centrifuging at 300xg for lOminutes to remove unbound fluorophores. The pellet was then resuspended in ImL of sperm handling medium and assessed for count and motility via CASA.

[0333] Muco-trapping Assessment.

[0334] The muco-trapping potency of each antibody was assessed as previouslyAttorney Docket No. 5470.976.WOdescribed. Briefly, multiple particle tracking of fluorescently labelled sperm was performed at subagglutinating concentrations of sperm. Fresh CVM was titrated to pH 6.8-7.1 using small volumes of 3N NaOH, then diluted 3-fold using sperm washing medium to mimic the dilution of CVM alkaline seminal fluid. Next, 100 pL of the diluted and pH-adjusted CVM was aliquoted into chambers of a CultreWell™ chamber slide (Invitrogen, Thermo Scientific #C37000). Next, Ab was added to a final concentration of 10 pg / mL and mixed well within the chamber using a wiretrol. Finally, sperm was added to a final concentration of 2*104sperm / mL. Translational motions of the fluorescently labeled sperm were recorded using an electron-multiplying charge-coupled-device camera (Evolve 512; Photometries, Tucson, AZ) mounted on an inverted epifluorescence microscope (AxioObserver DI; Zeiss, Thornwood, NY) equipped with an Alpha Plan- Apo 20 / 0.4 objective, environmental (temperature and CO2) control chamber, and lightemitting diode (LED) light source (Lumencor Light Engine DAPI / GFP / 543 / 623 / 690). Videos (512 x 512 pixels, 16-bit image depth) covering at least 10 fields of view and 50 total tracked sperm were captured for each condition using MetaMorph imaging software (Molecular Devices, Sunnyvale, CA) at a temporal resolution of 66.7 ms and spatial resolution of 50 nm (nominal pixel resolution, 0.78 pm / pixel) for 10s. Assessments of the parent IgG were done at room temperature to allow sufficient binding. Convolutional neural network-based tracking software was used to determine x, y coordinates of each sperm in each video frame. These coordinates were used to calculate quantitative metrics commonly used to describe sperm motion, such as velocity of the average path (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and straightness (STR). As sperm did not swim as quickly in CVM as in buffer or semen in which these characteristics are commonly assessed, VSL and STR were relied upon to classify progressive sperm in place of VAP and VCL. Based on visual inspection of classification success in a subset of videos, sperm were marked progressive if they exhibited at least 3pm / s VSL and 0.8 STR. Data represent 7 independent experiments on unique semen-CVM pairs, composed of 5 unique semen specimens and 4 unique CVM specimens.

[0335] Formulation of FDAT,

[0336] LamHIO was lyophilized in histidine buffer (50mM, pH 6.5) with 46% by weight trehalose. For each tablet, 1.92mg (Img Ab, 0.92mg trehalose content) of the lyophilized powder was then mixed with 5mg sodium bicarbonate, 5mg citric acid, 19.04 mg erythritol, and 19.04mg microcrystalline cellulose for a total of 50mg. This mixture was ground into a fine powder withAttorney Docket No. 5470.976.WOmortar and pestle, then loaded into a custom die and punch. A customized hydraulic press (Carver) set at 0.03t was used to press the powder together into 6-mm diameter tablets. LamHIO was evaluated for activity post- lyophilization and manufacture into tablets by reconstitution into PBS or vaginal fluid simulant then comparing agglutination via an agglutination kinetics assay as described above. Dissolution of the final tablet was assessed by placing a tablet into either ImL of vaginal fluid simulant or ImL pooled CVM, and imaging at 15s intervals.

[0337] In Vivo Surrogate Efficacy Studies.

[0338] On each test day, all sheep were dosed with the same semen mixture pooled from 3-5 donors, then received a randomized Ab treatment. Each Ab condition was assessed at least 3 times in the same group of sheep. Experiments were performed in 6 individual Merino crossbred sheep over 3 weeks, with 7 days between each treatment round. In the first two weeks, Ab was delivered in solution. Sheep received ImL of solution containing 333 pg, 33 pg, or 3.3 pg of LamHIO or PBS alone, which was instilled into the vagina followed by mixing using a vaginal dilator for 15 strokes. Then, ImL of pooled whole semen was pipetted into the sheep’s vagina, followed by simulated intercourse with a vaginal dilator for 5 strokes. At 2 minutes after semen was introduced, fluids were collected and from the sheep vagina and assessed for progressive sperm count in a hemocytometer (Bright-Line Hemacytometer) under a light microscope (Olympus 1X71) using a 20* objective with Thorlabs camera. During the last week, sheep received either a LamHIO tablet, a placebo tablet, or a PBS solution. The tablets were allowed to dissolve for 5 minutes, then the experiment proceeded following the same procedure as above. Treatments and quantifications were performed blinded.

[0339] Statistical Analysis.

[0340] All analyses were performed with GraphPad Prism software (v. 10.2.3). For multiple group comparisons involving two factors, P-values were calculated using two-way ANOVA with post-hoc Tukey’s test (FIG. IB, FIG. 3A, FIG. 4A, FIG. 5A). For multiple group comparisons involving one factor, p-values were calculated using one-way ANOVA with post-hoc Tukey’s test (FIG. 2B, FIG. 7F). For the muco-trapping analysis, repeated-measures ANOVA was used with post-hoc Holm-Sidak test (FIG. 6B). In all analyses, a = 0.05 was the threshold for statistical significance. The values for N, P, and the specific statistical test performed for each experiment are included in the appropriate figure legends. All data are presented as the mean ± SD.Attorney Docket No. 5470.976.WO

[0341] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, it should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0342] Although illustrated as separate elements, the method steps described and / or illustrated herein may represent portions of a single application. In addition, in some embodiments one or more of these steps may represent or correspond to one or more software applications or programs that, when executed by a computing device, may cause the computing device to perform one or more tasks, such as the method step.

[0343] A person of ordinary skill in the art will recognize that any process or method disclosed herein can be modified in many ways. The process parameters and sequence of the steps described and / or illustrated herein are given by way of example only and can be varied as desired. For example, while the steps illustrated and / or described herein may be shown or discussed in a particular order, these steps do not necessarily need to be performed in the order illustrated or discussed.

[0344] The various exemplary methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein or comprise additional steps in addition to those disclosed. Further, a step of any method as disclosed herein can be combined with any one or more steps of any other method as disclosed herein.

[0345] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described orAttorney Docket No. 5470.976.WOshown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0346] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0347] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under”, or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0348] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.Attorney Docket No. 5470.976.WO

[0349] In general, any of the compounds and methods described herein should be understood to be inclusive, but all or a sub-set of the components and / or steps may alternatively be exclusive and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps.

[0350] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all subranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0351] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is providedAttorney Docket No. 5470.976.WOprimarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0352] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.SEQUENCE LISTINGSEQ ID NO:1Alpha strand of Human Laminin (Laminin subunit alpha-5) ENVERWQGQYEGLRGQDLGQAVLDAGHSVSTLEKTLPQLLAKLSILENRGVHNASLAL SASIGRVRELCAQARGAASKVKV SEQ ID NO:2Modified alpha strand of Human Laminin (Laminin subunit alpha-5), where x may any amino acid residue.ENVERWQGQYEGLRGQDLGQAVLDAGHSVSTLEKTLPQLLAKLSILENRGVHxASLAL SASIGRVRELCAQARGAASKVKV SEQ ID NO:3Beta strand of Human Laminin (Laminin subunit beta **) DARRKAEMLQNEAKTLLAQANSKLQLLKDLERKYEDNQRYLEDKAQELARLEGEVRS LLKDISQKVAVYSTCL SEQ ID NO:4Modified beta strand of Human Laminin (Laminin subunit beta **), where x may be any amino acid residue.Attorney Docket No. 5470.976.WODARRKAEMLQNEAKTLLAQANSKLQLLKDLERKYEDNQRYLEDKAQELARLEGEVRS LLKDISQKVAVYSxCxSEQ ID NO:5Gamma strand of Human Laminin (Laminin subunit gamma **) DTVDLNKLNE1EGTLNKAKDEMKVSDLDRKVSDLENEAKKQEAA1MDYNRD1EE1MKC1 RNLEDIRKTLP SGCFNTP SIEKP SEQ ID NO:6Modified gamma strand of Human Laminin (Laminin subunit gamma **), where x may be any amino acid residue.DTVDLNKLNEIEGTLNKAKDEMKVSDLDRKVSDLENEAKKQEAAIMDYNRDIEEIMKCI RNLEDIRKTLPSxCxxTPSIEKPSEQ ID NO:7IgG hinge region (linker region)EPKSCDKTHT SEQ ID NO:8Linker region (linker region) remove the cysteine,EPKSSDKTHT SEQ ID NO:9Linker region (linker region) can be repeated lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, etc.GGGGS SEQ ID NO:10Linker region (linker region) can be repeated lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, etc.GSSSS SEQ ID NO: 11IgGFc domain CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12IgGl Fc domain ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13IgG2 Fc domainAttorney Docket No. 5470.976.WOPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTI TIFITLFLLSVCYSATITFFKVKWIFSSVVDLKQTIVPDYRNMIRQGA SEQ ID NO: 14IgG3 Fc domain PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTK PREEQ YNSTFRVVSVLTVLHQDWLNGKEYKCKVSNK ALP APIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK LTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPELQLEESCAEAQDGELDGLWTTI TIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIIPDYRNMIGQGA SEQ ID NO:15IgG4 Fc domain EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLELQLEESCAEAQDGELDGLWTTI TIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIVPDYRNMIRQGA SEQ ID NO: 16IgM Fc domain VPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQVWSLREGKQVGSGVTTD QVQAEAKESGPTTYKVTSTLTIKESDWLGESMFTCRVDHRGLTFQQNASSMCVPDQDT AIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTREENGAVKTHTNISESHPNATF SAVGEASICEDDDWSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLN LRESATITCLVTGFSPADVFVQWMQRGEPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSE EEWNTGQTYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY SEQ ID NO:17IgD Fc domain FTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGT S VTCTLNHP SLPPQRLMALREP AAQ AP VKL SLNLL AS SDPPEAAS WLLCEVSGF SPPNILL MWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTL LNASRSLEVSYVTDHGPM SEQ ID NO:18IgE Fc domain CADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTR KEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVY AFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPR SEQ ID NO: 19IgAl Fc domainAttorney Docket No. 5470.976.WOPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYS VSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNE LVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAE DWKKGDTFSCMVGHEALPLAFTQKTIDRLADWQMPPPYVVLDLPQETLEEETPGANLW PTTITFLTLFLLSLFYSTALTVTSVRGPSGNREGPQY SEQ ID NO:20IgA2 Fc domain PRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVEGPPERDLCGCYS VSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNE LVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTYAVTSILRVAAE DWKKGETFSCMVGHEALPLAFTQKTIDRLAGKPTHINVSVVMAEADGTCY SEQ ID NO:21Anti-Sperm Fab HC QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTN YNPSLRSRVTISVDTSKNQFSLKLRSVTAADTAVYYCARGFMVRGIMWNYYYMDVWG KGTTVTVSPASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTEP KSCDKTHTENVERWQGQYEGLRGQDLGQAVLDAGHSVSTLEKTLPQLLAKLSILENRG VHNASLALSASIGRVRELCAQARGAASKVKVEPKSCDKTHT SEQ ID NO:22Anti-Sperm Fab LC SSELTQDPVVSVALGQTVRITCQGDSLRTYHASWYQQKPRQAPVLVIYDENNRPSGIPD RFSGSTSGNTASLTITGAQAEDEADYYCNSRDSSGNRLVFGGGTKLTVLGQPKAAPSVT LFPP S SEELQ ANK ATL VCLI SDF YPGA VT V AWK AD S SP VK AGVETTTP SKQ SNNK Y A A S SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO:23Anti-Gonorrhea Fab VH:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMEWVRQAPGQGLEWMGVINPNNR FTSYNQNFRGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRWYQYDYWGQGTLV TVSS SEQ ID NO:24Anti-Gonorrhea Fab VL:Q AVVTQEP SLT VRS STGAVTT SNYANWFQKPGQ APRTLIYGINNRAPWTP ARF SGSLLG GKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL SEQ ID NO:25Anti-E.Coli Fab HC EVKLVESGGGLVQPGGSLSLSCAASGFTFSDYYMTWVRQAPGKAPEWLALIRNKRNGD TAEYSASVKGRFT1SRDYSRSILHLQMNALRTEDSATYYCVRQGRGYTLDYWGQGTSV TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAV LQSGLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPAttorney Docket No. 5470.976.WOSEQ ID NO:26Anti-E.Coli Fab LC DIQMNQSPSSLSASLGDTISITCRASQNINIWLSWYQQKPGNVPKLLIYKASNLHTGVPSR FSGSGSGTDFTLIISSLQPEDIATYYCLQGQSYPRTFGGGTKLEIKRGDAAPTVSIFPPSSEQ LTSGGASVVCFLNNFYPKD1NVKWK1DGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERHNSYTCEATHKTSTSPIVKSFNRG SEQ ID NO:27Anti-HIV HC ADLVQSGAVVKKPGDSVRISCEAQGYRFPDYIIHWIRRAPGQGPEWMGWMNPMGGQV NIPWKFQGRVSMTRDTSIETAFLDLRGLKSDDTAVYYCVRDRSNGSGKRFESSNWFLDL WGRGTAVTIQSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP A VLQ S SGLYSL S SWT VPS S SLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHT SEQ ID NO:28Anti HIV LC QSALTQPRSVSASPGQSVTISCTGTHNLVSWCQHQPGRAPKLLIYDFNKRPSGVPDRFSG SGSGGTASLTITGLQDDDDAEYFCWAYEAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ ANKATLVCLTSDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO:29Anti-Siglecl5 Fab HC EVQLVESGGGLVQPGGSLRLSCAASGYAFTNYLIEWVRKAPGKGLEWVGVINPGSGGS NYNEKFKGRATISADNSKNTLYLQMNSLRAEDTAVYYCARSGGFYFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ S SGL YSLS S VVT VP S S SLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHT SEQ ID NO:30Anti-Siglecl5 Fab LC DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQRKPGKAPKLLISGATSLETGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSSPLTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:31Anti-CD3 Fab HC EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGV STYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQS SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT SEQ ID NO:32Anti-CD3 Fab LCAttorney Docket No. 5470.976.WODIQMTQSPSSLSASVGDRVTTTCRASQDTRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:33Anti-TGFbeta Fab HC EVQLVESGGGLVQPGGSLRLSCAASGYAFTNYLIEWVRQAPGKGLEWVGVINPGSGGS NYNEKFKGRATISADNSKNTLYLQMNSLRAEDTAVYYCARSGGFYFDYWGQGTLVTV SSASTLGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT SEQ ID NO:34Anti-TGFbeta Fab LC RIQMTQSPSSLSASVGDRVTITCRASQSVLYSSNQKNYLAWYQDKPGKAPKLLIYWAST RESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYLSSDTFGQGTKVEIKRTVAAPSV FIFPPSDKQLKSGTARVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

Attorney Docket No. 5470.976.WOWHAT IS CLAIMED IS:

1. A synthetic agent comprising: a pair of human or humanized Immunoglobulin fragment crystallizable (Fc) domains linked to at least one strand of a heterotrimeric complex comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

2. The synthetic agent of claim 1, wherein two or three of the alpha, beta and gamma strands are linked to copies of an antigen binding domain.

3. The synthetic agent of claim 1, wherein each of the alpha, beta and gamma strands are linked to copies of one or more antigen binding domains.

4. The synthetic agent of claim 2 or 3, wherein the one or more antigen binding domains are copies of the same antigen binding domain.

5. The synthetic agent of claim 2 or 3, wherein the antigen binding domains are copies of different antigen binding domains.

6. The synthetic agent of claim 2 or 3, wherein the antigen binding domains comprise antigen binding (Fab) domains.

7. The synthetic agent of any one of claims 1-6, wherein the human or humanized Fc domain is at least 80% identical with a human Immunoglobulin G (IgG) Fc domain, a human Immunoglobulin M (IgM) Fc domain, a human Immunoglobulin D (IgD) Fc domain, a human Immunoglobulin E (IgE) Fc domain, a human Immunoglobulin Al (IgAl) Fc domain, or a human Immunoglobulin A2 (IgA2) Fc domain.

8. The synthetic agent of any one of claims 1-7, wherein the Fc domain is linked to the alpha strand.

9. The synthetic agent of any one of claims 1-7, wherein the Fc domain is linked to the beta strand.Attorney Docket No. 5470.976.WO10. The synthetic agent of any one of claims 1-7, wherein the Fc domain is linked to the gamma strand.

11. The synthetic agent of any one of claims 1-10, wherein the human alpha strand has a sequence having at least 80% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, wherein the human beta strand has a sequence having at least 80% sequence identity to SEQ ID NO:3 or SEQ ID NO:4, and wherein the human gamma strand has a sequence having at least 80% sequence identity to SEQ ID NO:5 or SEQ ID NO:6.

12. The synthetic agent of any one of claims 1-11, wherein the human alpha strand has a sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the human beta strand has a sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein the human gamma strand has a sequence of SEQ ID NO:5 or SEQ ID NO:

613. The synthetic binding agent of claim 6, wherein the Fab domains each comprise: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences comprising one or more of: SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence comprising one or more of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:3414. The synthetic binding agent of any one of claims 1-13, wherein the Fc domain is linked by an amino acid linker at the N-terminal and / or C-terminal end of the Fc domain to the at least one strand of the heterotrimeric complex.

15. The synthetic binding agent of claim 14, wherein the linker comprises an IgG hinge region.

16. The synthetic binding agent of claim 14, wherein the linker is the sequence of one of SEQ IDNOs:7-10.Attorney Docket No. 5470.976.WO17. The synthetic binding agent of any one of claims 1-16, wherein the heterotrimeric complex is stabilized by disulfide bonds between the alpha, beta, and gamma strands.

18. The synthetic binding agent of any one of claims 1-17, wherein each of the Fc domains in the pair of Fc domains are linked to a different heterotrimeric complex each comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

19. The synthetic binding agent of any one of claims 1-17, wherein one of the Fc domains in the pair of Fc domains are linked to a different heterotrimeric complex each comprising alpha, beta and gamma strands that are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

20. A synthetic binding agent comprising: a pair of human or humanized Immunoglobulin fragment crystallizable (Fc) domains, wherein each Fc domain is linked at the N-terminal end and / or C-terminal end to a different heterotrimeric complex each including an alpha strand, a beta strand or a gamma strand, wherein the alpha, beta and gamma strands are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

21. The synthetic binding agent of claim 20, wherein two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to copies of one or more antigen binding domains.

22. The synthetic binding agent of claim 20, wherein each of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to copies of one or more antigen binding domains.

23. The synthetic binding agent of claim 21 or 22, wherein the antigen binding domains are copies of the same antigen binding domain.

24. The synthetic binding agent of claim 21 or 22, wherein the antigen binding domains are copies of different antigen binding domains.

25. The synthetic binding agent of claim 21 or 22, wherein the antigen binding domains comprise antigen binding (Fab) domains.Attorney Docket No. 5470.976.WO26. The synthetic binding agent of any one of claims 20-25, wherein the human or humanized Fc domain is at least 80% identical with a human Immunoglobulin G (IgG) Fc domain, a human Immunoglobulin M (IgM) Fc domain, a human Immunoglobulin D (IgD) Fc domain, a human Immunoglobulin E (IgE) Fc domain, a human Immunoglobulin Al (IgAl) Fc domain, or a human Immunoglobulin A2 (IgA2) Fc domain.

27. The synthetic binding agent of any one of claims 20-26, wherein the Fc domain is linked to the alpha strand.

28. The synthetic binding agent of any one of claims 20-26, wherein the Fc domain is linked to the beta strand.

29. The synthetic binding agent of any one of claims 20-26, wherein the Fc domain is linked to the gamma strand.

30. The synthetic binding agent of claim 25, wherein the Fab domain comprises: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences comprising one or more of: SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence comprising one or more of: SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:3431. The synthetic binding agent of any one of claims 20-30, wherein the Fc domain is linked to each heterotrimeric complex by a linker.

32. The synthetic binding agent of claim 31, wherein the linker is an IgG hinge region.

33. The synthetic binding agent of claim 31, wherein the linker is the sequence of one of SEQ ID NOs:7-1034. The synthetic binding agent of any one of claims 20-33, wherein the heterotrimeric complex is stabilized by disulfide bonds between the alpha, beta, and gamma strands.Attorney Docket No. 5470.976.WO35. The synthetic binding agent of any one of claims 20-34, comprising four heterotrimeric complexes.

36. A synthetic binding agent comprising: a pair of human or humanized IgG immunoglobulin fragment crystallizable (Fc) domains having the sequence of one of SEQ ID NOs:ll-20, wherein each of the Fc domains are linked at the N-terminal end and / or the C-terminal end to an alpha strand of a self-assembled heterotrimeric complex comprising the alpha strand, a beta strand and a gamma strand, wherein the alpha strand has sequence that is at least 80% identical to SEQ ID NO:1 or SEQ ID NO:2, the beta strand has a sequence that is at least 80% identical to SEQ ID NO:3 or SEQ ID NO:4 and the gamma strand has a sequence that is at least 80% identical to SEQ ID NO:5 or SEQ ID NO:6.

37. The synthetic binding agent of claim 36, wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to copies of one or more antigen binding domains.

38. The synthetic binding agent of claim 37, wherein the antigen binding domains are copies of the same antigen binding domain.

39. The synthetic binding agent of claim 37, wherein the antigen binding domains are copies of different antigen binding domains.

40. The synthetic binding agent of any one of claims 37-39, wherein the antigen binding domains comprise antigen binding (Fab) domains.

41. The synthetic binding agent of claim40 wherein the Fab domains each comprise: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences of one of: SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence of one of:SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34Attorney Docket No. 5470.976.WO42. The synthetic binding agent of any one of claims 36-41, wherein the Fc domain is linked to each heterotrimeric complex by an amino acid linker.

43. The synthetic binding agent of claim 42, wherein the linker is an IgG hinge region.

44. The synthetic binding agent of claim 42, wherein the linker is the sequence of one of SEQ ID NOs:7-1045. The synthetic binding agent of any one of claims 36-44, wherein the heterotrimeric complex is stabilized by disulfide bonds between the alpha, beta, and gamma strands.

46. The synthetic binding agent of any one of claims 36-45, comprising four heterotrimeric complexes.

47. A synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a selfassembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to copies of one or more antigen binding domains, further wherein the antigen binding domains specifically bind to an epitope of a target, so that the synthetic binding agent binds to the target with high affinity, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

48. The synthetic binding agent of claim 47, wherein the antigen binding domains linked to each of the alpha, the beta and / or the gamma strands are copies of the same antigen binding domain.

49. The synthetic binding agent of claim 47, wherein the antigen binding domains linked to the alpha, the beta and / or the gamma strands are copies of different antigen binding domains.

50. The synthetic binding agent of any one of claims 47-49, wherein the antigen binding domains comprise antigen binding (Fab) domains.Attorney Docket No. 5470.976.WO51. The synthetic binding agent of claim 50, wherein the Fab domains each comprise: (i) a heavy chain (HC) with a variable region (VH) comprising complementarity determining regions (CDRs) having the amino acid sequences of one of: SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; and / or (ii) a light chain (LC) with a variable region (VL) comprising complementarity determining regions (CDRs) having the amino acid sequence of one of:SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:3452. The synthetic binding agent of any one of claims 47-51, wherein the Fc domain is linked to each heterotrimeric complex by an amino acid linker.

53. The synthetic binding agent of claim 52, wherein the linker is an IgG hinge region.

54. The synthetic binding agent of claim 52, wherein the linker is the sequence of one of SEQ ID NOs:7-1055. The synthetic binding agent of claim 48, wherein the Fc domain is linked to the alpha strand of the heteromeric complex.

56. The synthetic binding agent of any one of claims 47-55, wherein the heterotrimeric complex is stabilized by disulfide bonds between the alpha, beta, and gamma strands.

57. The synthetic binding agent of any one of claims 47-56, comprising four heterotrimeric complexes.

58. The synthetic binding agent of any one of claims 47-57, wherein the alpha, the beta and the gamma strands of each of the heterotrimeric complexes are linked to copies of one or more an antigen binding domains.

59. A synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains having a sequence selected from: SEQ ID NOs:ll-20, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein two or more of the alpha strand, the beta strand, and the gamma strand of the self-assembledAttorney Docket No. 5470.976.WOheterotrimeric complex are each linked via amino acid linkers to a pair of antigen-binding (Fab) domains, wherein the alpha strand is at least 80% identical to the human laminin- 511 alpha having the sequence of SEQ ID NO: 1 or SEQ ID NO:2, wherein the beta strand is at least 80% identical to the human laminin-511 beta having the sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein the gamma strand is at least 80% identical to the human laminin-511 gamma having the sequence of SEQ ID NO:5 or SEQ ID NO:

660. A synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a selfassembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to an anti-sperm antigen binding domain, further wherein the anti-sperm antigen binding domain specifically binds to an epitope of a sperm, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

61. A synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains having a sequence selected from: SEQ ID NOs: 11-20, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein each of the alpha strand, beta strand, and gamma strand of the self-assembled heterotrimeric complex are linked via amino acid linkers to an anti-sperm antigen binding domain, further wherein the anti-sperm antigen binding domain specifically binds to an epitope of a sperm, wherein the alpha strand is at least 80% identical to the human laminin-511 alpha having the sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the beta strand is at least 80% identical to the human laminin-511 beta having the sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein the gamma strand is at least 80% identical to the human laminin-511 gamma having the sequence of SEQ ID NO:5 or SEQ ID NO:6.

62. A method of treating a patient, the method comprising delivering the synthetic binding agent of claim 60 or 61 to a patient in need thereof.Attorney Docket No. 5470.976.WO63. A synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains, each linked to a selfassembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein at least two or three of the alpha, beta and gamma strands of each of the heterotrimeric complexes are linked to an anti-RSV antigen binding domain, further wherein the anti-RSV antigen binding domain specifically binds to an epitope of RSV, wherein the alpha strand, beta strand and gamma strand are at least 80% identical to the human laminin-511 alpha, beta and gamma strands.

64. A synthetic binding agent, the synthetic binding agent comprising: a pair of human or humanized immunoglobulin fragment crystallizable (Fc) domains having a sequence selected from: SEQ ID NOs:ll-20, each linked to a self-assembled heterotrimeric complex comprising an alpha strand, a beta strand and a gamma strand; wherein each of the alpha strand, beta strand, and gamma strand of the self-assembled heterotrimeric complex are linked via amino acid linkers to an anti-RSV antigen binding domain, further wherein the anti-RSV antigen binding domain specifically binds to an epitope of RSV, wherein the alpha strand is at least 80% identical to the human laminin-511 alpha having the sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the beta strand is at least 80% identical to the human laminin-511 beta having the sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein the gamma strand is at least 80% identical to the human laminin-511 gamma having the sequence of SEQ ID NO:5 or SEQ ID NO:6.

65. A method of treating a patient, the method comprising delivering the synthetic binding agent of claim 63 or 64 to a patient in need thereof.

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