Multimeric coronavirus binding molecules and uses thereof

Abstract

This disclosure provides multimeric binding molecules that bind to a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2. This disclosure also provides compositions comprising the multimeric binding molecules, polynucleotides that encode the multimeric binding molecules, and host cells that can produce the binding molecules. Further this disclosure provides methods of using the multimeric binding molecules, including methods for treating and preventing human sarbecovirus disease, e.g., coronavirus disease 2019 (COVID-19).

Description

IGM Ref. No.002-056WO2 MULTIMERIC CORONAVIRUS BINDING MOLECULES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 499,679,filed 2 May 2023; and 63 / 509,678,filed 22 June 2023, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on 1 May 2024, is named 056WO2-Sequence-Listing.xml and is 51 kilobytes in size. BACKGROUND

[0003] Antibodies and antibody-like molecules that can multimerize, such as IgA and IgM antibodies, have emerged as promising drug candidates, e.g., in the fields of immuno- oncology and infectious diseases, allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos.9,951,134, 9,938,347, 10,351,631, 10,400,038, 10,570,191, 10,604,559, 10,618,978, 10,689,449, 10,787,520, 10,899,935, 11,401,337, 11,555,075, & 11,639,389, and U.S. Patent Application Publication Nos. US 2019-0330374, US 2019-0330360, US 2019-0338040, US 2019-0338041, & US 2020-0392239, the contents of which are incorporated herein by reference in their entireties.

[0004] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single stranded, positive sense enveloped RNA virus. SARS-CoV-2 causes Coronavirus Disease 2019 (COVID-19), which is currently causing a global pandemic. COVID-19 is highly contagious and commonly causes fever, cough, and shortness of breath, and can lead to pneumonia, blood clots, organ failure, and death. The four main structural proteins of the SARS-CoV-2 include spike (S), envelope (E), membrane (M), and nucleic capsid (N).

[0005] The trimeric S protein binds the angiotensin-converting enzyme 2 (ACE2) receptor, alters its conformation to a fusogenic protein, which facilitates fusion of the cellular and viral membranes, and thereby enables SARS-CoV-2 to enter cells. The SIGM Ref. No.002-056WO2 protein comprises two units: S1 and S2, with the S1 domain comprising the receptor- binding domain (RBD). See, e.g., Lan, J., et al., Nature 581:215-220 (2020). The S protein and specifically the RBD are required for entry into cells, which has made the RBD a favored target of potential therapeutic monoclonal antibodies. Unfortunately, variants of the SARS-CoV-2 S protein continue to emerge, and the virus evades treatments on two fronts—increased transmissibility and immune evasion. See, e.g., Fang, F., and Shi, P-Y, Emerg. Microbes Infect.11: 208-211 (2022). In particular, various versions of the Omicron variant have virtually supplanted all earlier strains of SARS- CoV-2 worldwide in the last year, leading the World Health Organization (WHO) to name it as a Variant of Concern. See, e.g., who.int / en / activities / tracking-SARS-CoV-2- variants / (last visited, August 5, 2022). The Omicron variants are largely resistant to most of the monoclonal antibody therapies that had proven effective against earlier strains of SARS-CoV-2. See, e.g., Strohl, WR, et al., BioDrugs 36:231-323 (2022).

[0006] There remains an urgent need for therapeutics to treat and / or prevent human coronavirus infections, e.g., SARS and COVID-19. SUMMARY

[0007] This disclosure provides a multimeric binding molecule that includes five or six bivalent binding units, where each binding unit includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding polypeptide, where three to twelve of the binding polypeptides are identical and include a human angiotensin-converting enzyme 2 (ACE2) ectodomain, and where the ACE2 ectodomain consists essentially of an amino acid sequence at least 90% identical to amino acids 19 to X of SEQ ID NO: 14, where X is an integer greater than or equal to 610 and less than 740. In certain embodiments, X is an integer greater than or equal to 615 and less than 740. In certain embodiments, X is equal to 615. In certain embodiments, the ACE2 ectodomain consists essentially of an amino acid sequence at least 95% identical to amino acids 19 to X of SEQ ID NO: 14. In certain embodiments, the ACE2 ectodomain consists essentially of amino acids 19 to 615 of SEQ ID NO: 14. In certain embodiments, the ACE2 ectodomain consists essentially of amino acids 18 to 615 of SEQ ID NO: 14.

[0008] In certain embodiments, the ACE2 ectodomain comprises a substitution of an amino acid corresponding to R237, H345, H378, or E375 of SEQ ID NO: 14.IGM Ref. No.002-056WO2

[0009] In certain embodiments, the ACE2 ectodomain comprises: (a) a glutamine (Q), a leucine (L), a lysine (K), or an alanine (A) substitution of the amino acid corresponding to R237 of SEQ ID NO: 14, (b) an L, glycine (G), or proline (P) substitution of the amino acid corresponding to H345 of SEQ ID NO: 14, (c) an L, G, or asparagine (N) substitution of the amino acid corresponding to H378 of SEQ ID NO: 14, or 3(d) an L, A, or Q substitution of the amino acid corresponding to E375 of SEQ ID NO: 14. In some embodiments, the multimeric binding molecule comprises an amino acid sequence comprising amino acids 18 to 870 or amino acids 19 to 870 of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32

[0010] In certain embodiments, each binding unit of the provided multimeric binding molecule includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including an IgM Cµ4 domain and an IgM tailpiece domain. In certain embodiments, each IgM heavy chain constant region or multimerizing fragment or variant thereof further includes an IgM Cµ3 domain situated N-terminal to the IgM Cµ4 and IgM tailpiece domains. In certain embodiments, each IgM heavy chain constant region or multimerizing fragment or variant thereof further includes an IgM Cµ2 domain situated N-terminal to the IgM Cµ3 domain.

[0011] In certain embodiments, each IgM heavy chain constant region is a human IgM constant region with the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, or a multimerizing fragment or variant thereof.

[0012] In certain embodiments, each IgM heavy chain is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ3, Cµ4, and tailpiece domains, where each variant multimerizing fragment consists essentially of amino acids 217 to 453 of SEQ ID NO: 35, where the multimeric binding molecule has reduced complement- dependent cytotoxicity (CDC) activity relative to a corresponding binding molecule including the wild type multimerizing fragment of the human IgM constant region consisting essentially of amino acids 217 to 453 of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the variant multimerizing fragment further includes a variant human IgG1 hinge region fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, where the variant human IgG1 hinge region consists essentially of the amino acid sequence SEQ ID NO: 5. In one embodiment, the multimeric binding molecule includes amino acids 18 to 870 or amino acids 19 to 870 of SEQ ID NO: 21. In certain embodiments, the variant multimerizing fragment furtherIGM Ref. No.002-056WO2 includes a (G4S)4linker (SEQ ID NO: 12) fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, where the (G4S)4linker (SEQ ID NO: 12) consists essentially of the amino acid sequence SEQ ID NO: 12. In one embodiment, the multimeric binding molecule includes amino acids 18 to 872 or amino acids 19 to 872 of SEQ ID NO: 25. In certain embodiments, the variant multimerizing fragment further includes a camelid linker fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, where the camelid linker consists essentially of the amino acid sequence SEQ ID NO: 18 or SEQ ID NO: 34. In one embodiment, the multimeric binding molecule includes amino acids 18 to 887 or amino acids 19 to 887 of SEQ ID NO: 29.

[0013] In certain embodiments, each IgM heavy chain is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ2, Cµ3, Cµ4, and tailpiece domains, where each variant multimerizing fragment consists essentially of amino acids 105 to 453 of SEQ ID NO: 35, where the multimeric binding molecule has reduced complement-dependent cytotoxicity (CDC) activity relative to a corresponding binding molecule including the wild type multimerizing fragment of the human IgM constant region consisting essentially of amino acids 105 to 453 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the multimeric binding molecule includes amino acids 18 to 964 or amino acids 19 to 964 of SEQ ID NO: 22.

[0014] The disclosure further provides a multimeric binding molecule that includes five or six bivalent binding units, where each binding unit includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each connected C- terminal to a binding polypeptide via a linker, where three to twelve of the binding polypeptides are identical and include a human angiotensin-converting enzyme 2 (ACE2) ectodomain, and where the ACE2 ectodomain includes an amino acid sequence at least 90% identical to amino acids 19 to 740 of SEQ ID NO: 14, and where the linker consists essentially of (G4S)4 (SEQ ID NO: 12), a camelid linker consisting essentially of amino acids 1 to 27 of SEQ ID NO: 18 or 2 to 28 of SEQ ID NO: 18 (SEQ ID NO: 34), or a camelid linker consisting essentially of SEQ ID NO: 18 or SEQ ID NO: 34. In certain embodiments, the ACE2 ectodomain includes amino acids 19 to 740 of SEQ ID NO: 14. In certain embodiments, the ACE2 ectodomain includes amino acids 18 to 615 or amino acids 19 to 615 of SEQ ID NO: 14. In certain embodiments, each IgM heavy chain constant region is a human IgM constant region comprising SEQ ID NO: 1 or SEQ IDIGM Ref. No.002-056WO2 NO: 2, or a multimerizing fragment or variant thereof. In certain embodiments, each IgM heavy chain is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ3, Cµ4, and tailpiece domains, wherein each variant multimerizing fragment consists essentially of amino acids 217 to 453 of SEQ ID NO: 35, wherein the multimeric binding molecule has reduced complement-dependent cytotoxicity (CDC) activity relative to a corresponding binding molecule comprising the wild type multimerizing fragment of the human IgM constant region consisting essentially of amino acids 217 to 453 of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the linker consists essentially of (G4S)4(SEQ ID NO: 12), and the multimeric binding molecule includes amino acids 18 to 997 or amino acids 19 to 997 of SEQ ID NO: 26. In certain embodiments, the linker consists essentially of amino acids 1 to 27 of SEQ ID NO: 18, 2 to 28 of SEQ ID NO: 18 (SEQ ID NO: 34), SEQ ID NO: 18, or SEQ ID NO: 34 and the multimeric binding molecule includes amino acids 18 to 1005 or amino acids 19 to 1005 of SEQ ID NO: 27.

[0015] In certain embodiments, the provided multimeric binding molecule is pentameric and includes five bivalent binding units, and further includes a J-chain or functional fragment or variant thereof. In certain embodiments, the J-chain or functional fragment thereof is a human J-chain comprising the amino acid sequence SEQ ID NO: 7 or a functional fragment thereof. In certain embodiments, the J-chain or functional fragment or variant thereof is a variant J-chain including one or more single amino acid substitutions, deletions, or insertions relative to a wild-type J-chain that can affect the serum half-life of the multimeric binding molecule; and where the multimeric binding molecule exhibits an increased serum half-life upon administration to an animal relative to a reference multimeric binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions, and is administered in the same way to the same animal species. In certain embodiments, the J-chain or functional fragment thereof includes an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J-chain (SEQ ID NO: 7). In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 7 is substituted with alanine (A), serine (S), or arginine (R). In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 7 is substituted with alanine (A). In certain embodiments, the J-chain is a variant human J-chain and includes the amino acid sequence SEQ ID NO: 8.IGM Ref. No.002-056WO2

[0016] In certain embodiments, the J-chain or functional fragment or variant thereof is a modified J-chain and further includes a heterologous polypeptide, where the heterologous polypeptide is directly or indirectly fused to the J-chain or functional fragment or variant thereof. In certain embodiments, the heterologous polypeptide is fused to the J-chain or fragment thereof via a peptide linker, which can consist of the amino acid sequence GGGGS (SEQ ID NO: 9), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 13). In certain embodiments, the heterologous polypeptide is fused to the N-terminus of the J-chain or fragment or variant thereof, the C-terminus of the J-chain or fragment or variant thereof, or to both the N-terminus and C-terminus of the J-chain or fragment or variant thereof.

[0017] In certain embodiments, a multimeric binding molecule as provided by this disclosure includes ten identical binding polypeptides. In certain embodiments, a multimeric binding molecule as provided by this disclosure is hexameric and includes six bivalent binding units and twelve identical binding polypeptides.

[0018] In certain embodiments, a multimeric binding molecule as provided by this disclosure can neutralize infectivity of a human sarbecovirus at a lower 50% effective concentration (EC50) than a reference binding molecule, where the reference binding molecule includes the ACE2 ectodomain and a constant domain from an IgG antibody. In certain embodiments, the reference binding molecule includes the amino acid sequence SEQ ID NO: 23 or SEQ ID NO: 24. In certain embodiments, the EC50 is at least two- fold, at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500- fold, or at least 1000-fold lower than the EC50 of the reference binding molecule.

[0019] In certain embodiments, a multimeric binding molecule as provided by this disclosure can inhibit binding of the human sarbecovirus to its receptor at a lower 50% inhibitory concentration (IC50) than the reference binding molecule. In certain embodiments, the reference binding molecule includes the amino acid sequence SEQ ID NO: 23 or SEQ ID NO: 24. In certain embodiments, the IC50 is at least two-fold, at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500-fold, or at least 1000-fold lower than the IC50 of the reference binding molecule.

[0020] In certain embodiments, the human sarbecovirus is SARS-CoV or SARS-CoV-2.

[0021] This disclosure further provides a composition that includes the provided multimeric binding molecule. In certain embodiments, the composition may furtherIGM Ref. No.002-056WO2 comprise a surfactant, such as a fatty acid ester of polyoxyethylene sorbitan (a “PS surfactant”). Suitable PS surfactants include, by way of non-limiting example, polyoxyethylene (80) sorbitan monolaurate (PS80) and PS20. In certain embodiments, the composition has a neutral pH and further comprises an amino acid, NaCl, and a sugar. By way of non-limiting example, the composition has a pH of 6.5 and comprises 20 mM histidine, 300 mM NaCl, 2%(w / v) sucrose, and 0.2%(w / v) PS20.

[0022] This disclosure further provides a polynucleotide that includes a nucleic acid sequence that encodes the provided multimeric binding molecule or a subunit thereof. Also provided is a vector that includes the provided polynucleotide, and a host cell that includes the provided polynucleotide or the provided vector, where the host cell can express the provided multimeric binding molecule.

[0023] The disclosure further provides a method for treating or preventing a human sarbecovirus disease in a subject in need of treatment where the method includes administering to the subject an effective amount of the provided multimeric binding molecule, where the multimeric binding molecule exhibits greater potency than a reference binding molecule. In certain embodiments, the human sarbecovirus disease is severe acute respiratory syndrome (SARS) or coronavirus disease 2019 (COVID-19). In certain embodiments, the subject is a human subject. In certain embodiments, the multimeric binding molecule can be administered intravenously, subcutaneously, intramuscularly, intranasally, and / or via inhalation. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0024] FIG.1 illustrates some of the ACE2-IgG1 and ACE2-IgM chimeric constructs of the present disclosure. FIG.1A illustrates the manner in which the 615 amino acid ACE2 ectodomain and the 740 amino acid ACE2 ectodomain were incorporated into pentameric and hexameric IgM scaffolds. FIG.1B depicts a 615 amino acid ACE2 ectodomain fused to an IgG1 constant region that includes the IgG1 hinge, Cγ2, and Cγ3 domains. FIG.1C depicts a 615 amino acid ACE2 ectodomain fused to the IgM constant region Cµ3 and Cµ4 domains via a mutated IgG1 hinge region. FIG.1D depicts a 740 amino acid ACE2 ectodomain fused to an IgG1 constant region that includes the IgG1 hinge, Cγ2, and Cγ3 domains. FIG.1E depicts a 740 amino acid ACE2 ectodomain fused to the IgM constant region Cµ3 and Cµ4 domains via a mutated IgG1 hinge region.IGM Ref. No.002-056WO2

[0025] FIG.2 illustrates particular ACE2-IgG1 and ACE2-IgM chimeric constructs. FIG. 2A depicts ACE2-G2 (SEQ ID NO:23). FIG.2B depicts ACE2-G1 (SEQ ID NO:24). FIG.2C depicts ACE2-M4 (amino acids 18 or 19 to 995 of SEQ ID NO:19). FIG.2D depicts ACE2-M3 (amino acids 18 or 19 to 870 of SEQ ID NO:21). FIG.2E depicts ACE2-M2 (amino acids 18 or 19 to 1089 of SEQ ID NO:20). FIG.2F depicts ACE2-M1 (amino acids 18 or 19 to 964 of SEQ ID NO:22).

[0026] FIG.3 shows binding of ACE2-IgM and ACE2-IgG chimeras to the receptor binding domain (RBD) in S-protein from SARS-CoV strains. FIG.3A depicts results from an ELISA with B.1.617, Delta Variant RBD. FIG.3B depicts results from an ELISA with BA.1, Omicron Variant RBD.

[0027] FIG.4 shows binding of ACE2-IgM and ACE2-IgG chimeras to the RBD in S- protein from SARS-CoV strains. FIG.4A depicts results from an ELISA with BA.2, Omicron Variant RBD. FIG.4B depicts results from an ELISA with BA.3, Omicron Variant RBD.

[0028] FIG.5 shows results of pseudovirus neutralization assays. FIG.5A depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 Wuhan-Hu-1 / D614G. FIG. 5B depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 BA.2, Omicron Variant.

[0029] FIG.6 shows results of pseudovirus neutralization assays from a pseudovirus expressing the S protein of SARS-CoV-2 Wuhan-Hu-1 / D614G.

[0030] FIG.7 shows results of pseudovirus neutralization assays. FIG.7A depicts results from an empty pseudovirus vector. FIG.7B depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 Wuhan-Hu-1 / D614G. FIG.7C depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 Alpha Variant. FIG.7D depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 Beta Variant. FIG. 7E depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 Gamma Variant. FIG.7F depicts results from a pseudovirus expressing the S protein of SARS- CoV-2 Delta Variant. FIG.7G depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 BA.1, Omicron Variant. FIG.7H depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 BA.2, Omicron Variant. FIG.7I depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 BA.2.12.1, Omicron Variant. FIG.7J depicts results from a pseudovirus expressing the S protein of SARS-CoV-2 BA.2.75, Omicron Variant. FIG.7K depicts results from a pseudovirusIGM Ref. No.002-056WO2 expressing the S protein of SARS-CoV-2 BA.4 / 5, Omicron Variant. FIG.7L depicts results from a pseudovirus expressing the S protein of SARS-CoV-1 Urbani Variant. FIG.7M presents a dot-plot of various pseudoviruses neutralized with ACE2-M3 or ACE2-G1.

[0031] FIG.8 shows results of SARS-CoV-2 virus neutralization assays. FIG.8A depicts results from Wuhan-Hu-1 / D614G strain. FIG.8B depicts results from Omicron Variant BA.1. FIG.8C depicts results from Omicron Variant BA.2.

[0032] FIG.9A depicts the average weights of mice—control and ACE2-M3-treated— over time following infection with Omicron BA.2 SARS-CoV-2. FIG.9B depicts the survival of those cohorts of mice over time. FIG.9C depicts the average weights of mice—control and ACE2-M3-treated—over time following infection with Wuhan-Hu- 1 / D614G SARS-CoV-2.

[0033] FIG.10A depicts the average weights of two cohorts of mice—control and ACE2- M3-treated—over time. FIG.10B depicts the survival of those cohorts of mice over time.

[0034] FIG.11A depicts SARS-CoV-2 titers from lung tissues (N=4 per cohort) collected at day 4 post challenge from animals pretreated with ACE2-M3 and then infected with Omicron BA.2 SARS-CoV-2. FIG.11B depicts SARS-CoV-2 titers from lung tissues (N=4 per cohort) collected at day 4 post challenge from animals treated with ACE2-M3 post-infection with Omicron BA.2 SARS-CoV-2. FIG.11C depicts SARS-CoV-2 titers from lung tissues (N=4 per cohort) collected at day 4 post challenge from animals pretreated with ACE2-M3 and then infected with Wuhan-Hu-1 / D614G SARS-CoV-2.

[0035] FIG.12A shows a dynamic light scattering (DLS) plot of a 1 mg / mL solution of ACE2-M3 in 10 mM sodium acetate, 5%(w / v) sorbitol, 0.02%(w / v) PS20, pH 4.0. FIG. 12B shows a DSL plot of the same solution post-atomization. FIG.12C shows a DSL plot of the same solution post-nebulization.

[0036] FIG.13A shows a DSL plot of a 1 mg / mL solution of ACE2-M3 in10 mM sodium acetate, 5%(w / v) sorbitol, 0.01%(w / v) PS20, pH 6.5. FIG.13B shows a DSL plot of the same solution post-atomization. FIG.13C shows a DSL plot of the same solution post- nebulization.

[0037] FIG.14A shows a DSL plot of a 1.0 mg / mL solution of ACE2-M3 in 10 mM sodium acetate, 5%(w / v) sorbitol, 0.02%(w / v) PS20, pH 6.5. FIG.14B shows a DSL plot of the same solution post-atomization. FIG.14C shows a DSL plot of the same solution post-nebulization.IGM Ref. No.002-056WO2

[0038] FIG.15A shows a DSL plot overlay of a 1 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.2%(w / v) PS20, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line). FIG.15B shows a DSL plot overlay of a 5 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.2%(w / v) PS20, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line).

[0039] FIG.16A shows a DSL plot overlay of a 5 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.05%(w / v) PS20, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line). FIG.16B shows a DSL plot overlay of a 5 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.10%(w / v) PS20, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line). FIG.16C shows a DSL plot overlay of a 5 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.15%(w / v) PS20, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line).

[0040] FIG.17A shows a DSL plot overlay of a 1 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.04%(w / v) PS80, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line). FIG.17B shows a DSL plot overlay of a 1 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.1%(w / v) PS80, pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line). FIG.17C shows a DSL plot overlay of a 1 mg / mL solution of ACE2-M3 in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.1% w / v poloxamer 188 (P188), pH 6.5 pre-nebulization (solid line) and post-nebulization (dashed line).

[0041] FIG.18 shows DSL plots of 1 mg / mL solutions in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.2%(w / v) PS20, pH 6.5. FIG.18A (pre-nebulization) and FIG.18B (post-nebulization) show DSL plots of ACE2-M3. FIG.18C (pre-nebulization) and FIG. 18D (post-nebulization) show DSL plots of ACE2-M12. FIG.18E (pre-nebulization) and FIG.18F (post-nebulization) show DSL plots of ACE2-M10. FIG.18G (pre- nebulization) and FIG.18H (post-nebulization) show DSL plots of ACE2-M11.

[0042] FIG.19A shows plot of ACE2-M3 aggregation particle size in pH 4.0 buffer as a function of temperature. FIG.19B shows plot of ACE2-M3 aggregation particle size in pH 6.5 buffer as a function of temperature.IGM Ref. No.002-056WO2 DETAILED DESCRIPTION Definitions

[0043] As used herein, the term "a" or "an" entity refers to one or more of that entity; for example, "a binding molecule," is understood to represent one or more binding molecules. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0044] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0045] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, P-S (ed.), 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, Lacki, J.M. (ed.) 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Cammack, R., et al., (eds.) 2d ed., 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0046] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various embodiments or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0047] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides,IGM Ref. No.002-056WO2 oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0048] A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt many different conformations and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid, e.g., a serine or an asparagine. Asparagine (N)-linked glycans are described in more detail elsewhere in this disclosure.

[0049] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0050] As used herein, the term “a non-naturally occurring polypeptide” or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial body, to be “naturally-occurring.”IGM Ref. No.002-056WO2

[0051] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" as disclosed herein include any polypeptides which retain at least some of the properties of the corresponding native polypeptide. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of, e.g., a polypeptide include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants can be non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. As used herein a "derivative" of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those polypeptides that contain one or more derivatives of the twenty standard amino acids. For example, 4- hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0052] A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides, binding molecules, and antibodies of the present disclosure do not abrogate the binding of a binding molecule containing the amino acid sequence to a binding partner to which the binding molecule binds. Methods of identifying nucleotide and amino acid conservativeIGM Ref. No.002-056WO2 substitutions which do not eliminate binding are well-known in the art (see, e.g., Brummell et al., Biochem.32: 1180-1187 (1993); Kobayashi et al., Protein Eng.12:879- 884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0053] The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The terms "nucleic acid" or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0054] By an "isolated" nucleic acid or polynucleotide is intended any form of the nucleic acid or polynucleotide that is separated from its native environment. For example, gel- purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.” Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.” Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not one that would be found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0055] As used herein, the term “a non-naturally occurring polynucleotide” or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the nucleic acid or polynucleotide that are, or might be, determined or interpreted by a judge, or an administrative or judicial body, to be “naturally-occurring.”

[0056] As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more codingIGM Ref. No.002-056WO2 regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can include heterologous coding regions, either fused or unfused to another coding region. Heterologous coding regions include without limitation, those encoding specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0057] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

[0058] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin,IGM Ref. No.002-056WO2 heat shock protein, bovine growth hormone and rabbit ß-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0059] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0060] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0061] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ß-glucuronidase.

[0062] Disclosed herein are certain binding molecules, or binding-partner-binding fragments, variants, or derivatives thereof. As used herein, the term “binding molecule” refers in its broadest sense to a molecule that includes a “binding polypeptide,” or two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve “binding polypeptides” that specifically binds to a “binding partner” target or molecular determinant, or two or more “binding partner” targets or molecular determinants. As described further herein, aIGM Ref. No.002-056WO2 binding molecule can include one or more “binding polypeptides” or a fragment thereof, as described herein. According to certain embodiments of the present disclosure, the one or more “binding polypeptides” of the binding molecule are not antibodies or antigen- binding domains derived from antibodies. That is, the binding polypeptides do not include antigen-binding domains, e.g., a VH and / or a VL, of an antibody molecule.

[0063] As used herein, the term “binding polypeptide” refers to a region of a binding molecule, situated N-terminal to an IgM or IgA constant region or multimerizing fragment thereof, that is sufficient to specifically bind to a binding partner, e.g., a receptor-binding protein expressed on the surface of a virus or a cell, or where the binding polypeptide includes a portion of the polypeptide that is sufficient to specifically bind to a binding partner. For example, an extracellular portion of the human angiotensin- converting enzyme-2 (ACE2) that is sufficient to bind to the receptor binding domain (RBD) of the spike protein of a sarbecovirus, e.g., SARS-CoV-2, is a “binding polypeptide” as defined herein, whereas the RBD, is the “binding partner,” expressed on a virus particle or an infected cell, that the ACE2 polypeptide binds to.

[0064] The term "immunoglobulin" as used herein refers to polypeptide that is, or is derived from, an immunoglobulin molecule, and includes a portion of a binding molecule as provided herein. This disclosure provides binding molecules that are not traditional “antibodies,” in that they do not include the typical antibody antigen-binding domains of an antibody but do include immunoglobulin constant region domains that allow the binding molecules provided herein to readily multimerize into pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are relatively well understood. (See, e.g., Greenfield, E.A. (ed.), Antibodies: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 2014).

[0065] As will be discussed in more detail below, the term “immunoglobulin” includes various broad classes of polypeptides that can be distinguished biochemically. Only a subset of immunoglobulin polypeptides have the ability to multimerize. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, µ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4 or α1-α2)). It is the nature of this chain that determines the "isotype" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (subtypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readilyIGM Ref. No.002-056WO2 discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure. In certain embodiments, this disclosure provides modified human IgM constant regions.

[0066] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be, but is not required to be, bound with either a kappa or lambda light chain. In some embodiments, multimeric binding molecules as provided herein do not include light chains. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are expressed, e.g., by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C- terminus at the bottom of each chain. The basic structure of certain antibodies, e.g., IgG antibodies, includes two heavy chain subunits and optionally two light chain subunits covalently connected via disulfide bonds to form a “Y” structure, or a “binding unit.”

[0067] The term “binding unit” is used herein to refer to the portion of a binding molecule which corresponds to a standard Y-shaped immunoglobulin structure, i.e., two heavy chains or fragments thereof and optionally, two light chains. In certain embodiments, e.g., where the binding molecule is a bivalent IgG-derived binding molecule, the terms “binding molecule” and “binding unit” are equivalent. Such binding molecules are also referred to herein as “monomeric.” In other embodiments, e.g., where the binding molecule is a “multimeric binding molecule,” e.g., a dimeric or tetrameric IgA-derived binding molecule or , a pentameric or hexameric IgM-derived binding molecule, the binding molecule comprises two or more “binding units.” Two in the case of an IgA- derived dimer, four in the case of an IgA-derived tetramer, or five or six in the case of an IgM-derived pentamer or hexamer, respectively. In certain embodiments, a binding unit will be bivalent, i.e., will include two “binding polypeptides,” as defined above. As used herein, certain binding molecules are “dimeric,” and include two bivalent binding units that include IgA constant regions or multimerizing fragments thereof. Certain binding molecules provided in this disclosure are “pentameric” or “hexameric,” and include five or six bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof. A binding molecule comprising two or more, e.g., two, five, or six binding units, is referred to herein as “multimeric.”IGM Ref. No.002-056WO2

[0068] The term “J-chain” as used herein refers to the J-chain of IgM or IgA antibodies of any animal species, any functional fragment thereof, derivative thereof, and / or variant thereof, including a mature human J-chain, the amino acid sequence of which is presented as SEQ ID NO: 7. Various J-chain variants and modified J-chain derivatives are available, e.g., in U.S. Patent No.10,899,835, and / or are disclosed herein. “A functional fragment” or “a functional variant” includes those fragments and variants that can associate with IgM heavy chain constant regions to form a pentameric IgM antibody or can associate with IgA heavy chain constant regions to form a dimeric IgA antibody.

[0069] The term “modified J-chain” is used herein to refer to a derivative of a J-chain polypeptide comprising a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding polypeptide or functional domain introduced into or attached to the J- chain sequence. The introduction can be achieved by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety or by attachment through a peptide or chemical linker. The term “modified human J-chain” encompasses, without limitation, the human J-chain comprising the amino acid sequence of SEQ ID NO: 7 or functional fragment thereof, or functional variant thereof, modified by the introduction of a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding polypeptide. In certain embodiments, the heterologous moiety does not interfere with efficient polymerization of IgM into a pentamer or IgA into a multimer, e.g., a dimer or tetramer, and binding of such polymers to a target. Exemplary modified J-chains can be found, e.g., in U.S. Patent Nos.9,951,134, 10,400,038, 10,618,978, and 11,639,389, each of which is incorporated herein by reference in its entirety.

[0070] As used herein, the term “IgM-derived binding molecule” refers to IgM-derived binding molecules comprising non-antibody binding polypeptides instead of an antibody antigen binding domain or subunit thereof.

[0071] The terms “valency,” “bivalent,” “multivalent” and grammatical equivalents, refer to the number of binding polypeptides in given binding molecule, or in a given binding unit. As such, the terms “monovalent,” “bivalent”, “tetravalent”, and “hexavalent” in reference to a given binding molecule denote the presence of one binding polypeptide, two binding polypeptides, four binding polypeptides, and six binding polypeptides, respectively. An IgM-derived binding molecule, where each binding unit is bivalent, can have 10 or 12 valencies. A bivalent or multivalent binding molecule can be monospecific, i.e., all of the binding polypeptides are the same, or can be bispecific or multispecific,IGM Ref. No.002-056WO2 e.g., where two or more binding polypeptides are different, e.g., bind to different regions on the same binding partner, or bind to entirely different binding partners.

[0072] The term “binding partner” is used in the broadest sense to be a target of a binding polypeptide as provided herein and includes substances that can be bound by a binding molecule as provided herein. A binding partner can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule. In certain embodiments, the binding partner is a receptor or other moiety expressed or present on the surface of a virus or a virus- infected cell. Moreover, a “binding partner” can, for example, be a cell, an organ, or an organism, e.g., an animal, plant, microbe, or virus, that can be bound by a binding molecule or binding polypeptide as provided herein. In certain embodiments, a “binding partner” for a binding molecule as provided herein is a sarbecovirus, e.g., a SARS-CoV or SARS-CoV-2 virus, the spike (S) protein expressed by the virus, or the receptor binding domain (RBD) of the S protein. In certain embodiments, the binding partner can be the S protein or the RBD of the S protein expressed on the surface of a recombinant or virus- infected cell.

[0073] Both the light and heavy chains of immunoglobulins are divided into regions or “domains” of structural and functional homology. For example, the constant region domains of an IgM heavy chain (e.g., CH1 or Cµ1, CH2 or Cµ2, CH3 or Cµ3, CH4 or Cµ4, or tailpiece) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, ability to multimerize, and the like. By convention the numbering of the constant region domains increases as they become more distal from the amino-terminus of the typical immunoglobulin.

[0074] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure.

[0075] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell SurfaceIGM Ref. No.002-056WO2 Antigens, β-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, α-2 Macroglobulins, and Other Related Proteins,” U.S. Dept. of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region, or by using the Kabat numbering scheme. A comparison of the numbering of two alleles of the human IgM constant region sequentially (presented herein as SEQ ID NO: 1 (allele IGHM*03) and SEQ ID NO: 2 (allele IGHM*04)) and by the Kabat system is set out below. The underlined amino acid residues are not accounted for in the Kabat system (“X,” double underlined below, can be serine (S) (SEQ ID NO: 1) or glycine (G) (SEQ ID NO: 2)): Sequential (SEQ ID NO: 1 or SEQ ID NO: 2) / KABAT numbering key for IgM heavy chain 1 / 127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF SWKYKNNSDI 51 / 176 SSTRGFPSVL RGGKYAATSQ VLLPSKDVMQ GTDEHVVCKV QHPNGNKEKN 101 / 226 VPLPVIAELP PKVSVFVPPR DGFFGNPRKS KLICQATGFS PRQIQVSWLR 151 / 274 EGKQVGSGVT TDQVQAEAKE SGPTTYKVTS TLTIKESDWL XQSMFTCRVD 201 / 324 HRGLTFQQNA SSMCVPDQDT AIRVFAIPPS FASIFLTKST KLTCLVTDLT 251 / 374 TYDSVTISWT RQNGEAVKTH TNISESHPNA TFSAVGEASI CEDDWNSGER 301 / 424 FTCTVTHTDL PSPLKQTISR PKGVALHRPD VYLLPPAREQ LNLRESATIT 351 / 474 CLVTGFSPAD VFVQWMQRGQ PLSPEKYVTS APMPEPQAPG RYFAHSILTV 401 / 524 SEEEWNTGET YTCVVAHEAL PNRVTERTVD KSTGKPTLYN VSLVMSDTAG 451 / 574 TCY

[0076] By "specifically binds," it is generally meant that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof binds to a binding partner via its binding polypeptide, and that the binding entails some complementarity between the binding polypeptide and the binding partner. According to this definition, a binding molecule is said to "specifically bind" to an binding partner when it binds to that binding partner via its binding polypeptide more readily than it would bind to a random, unrelated binding partner. The term "specificity" is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain binding partner. For example, binding molecule "A" can be deemed to have a higher specificity for a given binding partner than binding molecule "B," or binding molecule "A" can be said to bind to binding partner "C" with a higher specificity than it has for related binding partner "D."

[0077] A binding molecule disclosed herein can be said to bind a binding partner with an off rate (k(off)) of less than or equal to 5 X 10-2sec-1, 10-2sec-1, 5 X 10-3sec-1, 10-3sec-1, 5 X 10-4sec-1, 10-4sec-1, 5 X 10-5sec-1, or 10-5sec-15 X 10-6sec-1, 10-6sec-1, 5 X 10-7sec-1or 10-7sec-1.IGM Ref. No.002-056WO2

[0078] A binding molecule disclosed herein can be said to bind a binding partner with an on rate (k(on)) of greater than or equal to 103M-1sec-1, 5 X 103M-1sec-1, 104M-1sec-1, 5 X 104M-1sec-1, 105M-1sec-1, 5 X 105M-1sec-1, 106M-1sec-1, or 5 X 106M-1sec-1or 107M-1sec-1.

[0079] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual binding partner with one or more binding polypeptides. See, e.g., Greenfield, E.A. (ed.), Antibodies: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 2014. As used herein, the term "avidity" refers to the overall stability of the complex between a population of binding polypeptides and one or more binding partners. Avidity is related to both the affinity of individual binding polypeptides in the population with specific binding partners, and also the valencies of the binding molecule and the binding partner. For example, the interaction between a multimeric binding molecule with a binding partner present at a high density on the surface of a virus particle would be of high avidity. Various binding partners disclosed herein, e.g., SARS- CoV-2 spike protein RBDs from different wild-type and or variant viruses, including variants of concern, may have differing affinities for the extracellular domain of ACE2 based on the sequence and structure of the wild-type or variant RBD.

[0080] A binding molecule can also be described or specified in terms of its binding affinity to a binding partner. For example, a binding molecule can bind to a binding partner with a dissociation constant or KD no greater than 5 x 10-2M, 10-2M, 5 x 10-3M, 10-3M, 5 x 10-4M, 10-4M, 5 x 10-5M, 10-5M, 5 x 10-6M, 10-6M, 5 x 10-7M, 10-7M, 5 x 10-8M, 10-8M, 5 x 10-9M, 10-9M, 5 x 10-10M, 10-10M, 5 x 10-11M, 10-11M, 5 x 10-12M, 10-12M, 5 x 10-13M, 10-13M, 5 x 10-14M, 10-14M, 5 x 10-15M, or 10-15M.

[0081] As used herein, the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain. A binding molecule comprising a heavy chain subunit can include at least one of: a binding polypeptide, a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof. In certain embodiments, a binding molecule can include, in addition to a binding polypeptide, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, a tailpiece, and a J-chain. Further, a binding molecule for use in the disclosure can lack certain constant region portions, e.g., all or part of a CH2 domain. It will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain subunit) can be modified such that they vary in amino acidIGM Ref. No.002-056WO2 sequence from the original immunoglobulin molecule. According to embodiments of the present disclosure, an IgM-derived binding molecule as provided herein comprises sufficient portions of an IgM heavy chain constant region to allow the IgM-derived binding molecule to form a multimer, e.g., a hexamer or a pentamer. As used herein such a fragment comprises a “multimerizing fragment.”

[0082] As used herein, the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain. The light chain subunit includes a CL (e.g., Cκ or Cλ) domain.

[0083] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain in IgG, IgA, and IgD heavy chains, and provides flexibility to the molecule. Certain multimeric binding molecules as provided herein include a modified IgG hinge region or a camelid hinge region fused to a multimerizing fragment of an IgM heavy chain constant region, e.g., the Cµ3, Cµ4, and tailpiece domains.

[0084] As used herein, the term “disulfide bond” includes the covalent bond formed between two sulfur atoms, e.g., in cysteine residues of a polypeptide. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. Disulfide bonds can be “intra-chain,” i.e., linking to cysteine residues in a single polypeptide or polypeptide subunit, or can be “inter-chain,” i.e., linking two separate polypeptide subunits.

[0085] As used herein, the term “reference binding molecule” refers to a binding molecule with function similar to a multimeric binding molecule provided by this disclosure, e.g., a monomeric binding molecule comprising an extracellular domain of the ACE2 protein. In a particular embodiment, a “reference binding molecule” is a monovalent or divalent monomeric, single-binding unit binding molecule with one or two binding polypeptides identical to those of a corresponding multimeric binding molecule as provided herein. In certain embodiments, the reference binding molecule is a monovalent or divalent fusion protein comprising an extracellular region of angiotensin-converting enzyme 2 (ACE2) fused to the Fc region of an IgG antibody to produce a monovalent and / or divalent single- binding unit reference binding molecule. Exemplary reference binding molecules are provided herein or are reported, e.g., in Arimori, T. et al., Trends Pharmacol. Sci. doi: 10.1016 / j.tips.2022.06.011 (2022) and Strohl, WR, et al., BioDrugs 36:231-323 (2022).IGM Ref. No.002-056WO2

[0086] As used herein, the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).

[0087] As used herein, the terms "linked," "fused" or "fusion" or other grammatical equivalents can be used interchangeably. These terms refer to the joining together of two or more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, an in-frame linker sequence.

[0088] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is “amino-terminal” or “N-terminal” to another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain. Similarly, a portion of a polypeptide that is “carboxy-terminal” or “C- terminal” to another portion of a polypeptide is that portion that comes later in the sequential polypeptide chain.

[0089] The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein furtherIGM Ref. No.002-056WO2 include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0090] The terms “neutralizing” or “neutralize” as used herein refer to the ability of a therapeutic, e.g., a therapeutic binding molecule, to reduce and / or prevent viral infectivity. The term “infectivity” as used herein refers to the ability of the virus to do one or more of attach to cells, enter cells, release its nucleic acid, replicate its nucleic acid, synthesize viral proteins, and package its nucleic acid into new virions that can be released from the infected cell. A virus can be neutralized, e.g., by a binding molecule as provided herein, via the binding molecule’s ability to specifically bind to the virion and inhibit its ability to attach to a host cell receptor, thereby preventing entry into the host cell.

[0091] With regard to pH, “neutral” refers to a pH from 6.4 to 7.6.

[0092] The terms “potent” or “potency” as used herein refer to the amount required to produce an effect, e.g., the amount of a binding molecule required to neutralize infectivity of a human sarbecovirus. In some embodiments, the potency is measured as the 50% effective concentration (EC50) or 50% inhibitory concentration (IC50) to neutralize or otherwise block infectivity (e.g., block attachment of the virus to the cellular receptor) of the human sarbecovirus, or therapeutic protection of a subject infected with a human sarbecovirus, measured, e.g., as a 50% effective dose (ED50), or prophylactic protection of a subject susceptible to human infection, measured, e.g., as a 50% effective dose (ED50). As used herein in the context of virus neutralization, the terms “50% effective concentration (EC50)” and “50% inhibitory concentration (IC50)” can be used interchangeably.

[0093] As used herein, a “human coronavirus” or the subset “human sarbecovirus” is a virus of the family Coronaviridae that is capable of infecting humans. Some coronaviruses can be traced to zoonotic sources, e.g., bats. In certain embodiments, the human coronavirus is within the genus betacoronaviridae. Certain betacoronaviruses of the lineage sarbecovirus derive from the bat reservoir and have jumped to humans (“human sarbecoviruses”). These human sarbecoviruses can cause severe respiratory syndromes in humans and include, without limitation, SARS-CoV and SARS-CoV-2.IGM Ref. No.002-056WO2 See, e.g., Sadia, A., and Basra, M.A.R., Drug Dev. Res. DOI: 10.1002 / ddr.21710 (2020). The terms “SARS-CoV” and “SARS-CoV-1” are used interchangeably herein.

[0094] The phrase “structural protein” of a virus as used herein refers to a protein that is a component of a mature assembled viral particle and includes naturally-occurring and / or engineered variants. The four main structural proteins of the SARS-CoV and SARS-CoV- 2 viruses include spike (S), envelope (E), membrane (M), and nucleic capsid (N). See, e.g., Sadia and Basra M.A.R., Drug Dev. Res. DOI: 10.1002 / ddr.21710 (2020).

[0095] The phrases “emerging variant”, “escape mutant” or “escape variant” as used herein refer to strains of a human coronavirus, e.g., a sarbecovirus, e.g., SARS-CoV-2 comprising one or more mutations, such as a substitution, addition, or deletion, that increases transmissibility of the virus and / or reduces or prevents neutralization by a treatment or prophylactic, such as an antibody treatment or prophylaxis. An exemplary escape variant is a variant of an initial strain of SARS-CoV-2 that arises following contact of the initial strain of SARS-CoV-2, or cells infected with the initial strain of SARS-CoV- 2, with an antibody capable of neutralizing the initial strain of SARS-CoV-2, where the escape variant is more resistant to neutralization by the antibody or is no longer capable of being neutralized by the antibody. SARS-CoV-2 escape variants can include one or more mutations, such as an amino acid substitutions, additions, or deletions, typically in the spike (S) protein, and typically in the receptor binding domain (RBD) of the S protein. Known SARS-CoV-2 escape variants that may be treated by compositions and methods described herein include, but are not limited to, 20I / 501Y.V1 VOC 202012 / 01 (also referred to as B.1.1.7), 20H / 501Y.V2 (also referred to as B.1.351), Delta variant (also referred to as B.1.617.2), Delta Plus, 20H, 20I, 21A (Delta) Delta Plus, 21B (Kappa), 21D (Eta), B.1.1.318 variants, P.1 variants and Omicron subvariants BA.1, BA.2, BA.3, BA.4, and BA.5.

[0096] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, lessen the severity of symptoms of, and / or halt or slow the progression of an existing diagnosed pathologic condition or disorder. Terms such as “prevent,” “prevention,” “avoid,” “deterrence,” “prophylactic,” and the like refer to prophylactic or preventative measures that can prevent the development of, or can reduce the symptoms of, a targeted pathologic condition or disorder in a subject who has not yet contracted the targeted pathologic condition or disorder. The targeted pathologic condition or disorder can be, for example,IGM Ref. No.002-056WO2 COVID-19. Thus, “those in need of treatment” can include those already infected with the human sarbecovirus, as well as those who wish to prevent infection, or reduce or alleviate symptoms associated with an infection should they become infected.

[0097] The terms “protect,” “protection,” “protective,” and other related terms, as used herein, refer to the ability of a therapeutic or prophylactic agent to confer a desirable effect on a subject diagnosed with or susceptible to an infectious disease resulting from a human sarbecovirus infection, such as COVID-19. Protection can include, for example, alleviation of or a reduction in infection-related symptoms in a subject infected with a human sarbecovirus, such as SARS-CoV-2, such that, for example, the subject does not need to be hospitalized or put on a ventilator. Protection can also include, for example, preventing healthcare workers, family members, or other contacts of infected patients from becoming infected with the human sarbecovirus, e.g., SARS-CoV-2, or if they do become infected, reducing the symptoms related to infection. As it applies to a therapeutic or prophylactic animal model, “protection” can include a lower 50% effective dose (ED50) among a group of animal subjects challenged with the therapeutic agent either before or after challenge with the human sarbecovirus. Data points that can be used to measure ED50 vary, e.g., with the animal model or the amount of human sarbecovirus used to challenge the animal subjects. Data points can include, e.g., measurement of the virus titer in the lungs of the animals, weight loss, death, or disease symptoms such as fever or difficulty breathing.

[0098] As used herein, the terms “serum half-life” or “plasma half-life” refer to the time it takes (e.g., in minutes, hours, or days) following administration for the serum or plasma concentration of a drug, e.g., a binding molecule described herein, to be reduced by 50%. Two half-lives can be described: the alpha half-life, α half-life, or t1 / 2α, which is the rate of decline in plasma concentrations due to the process of drug redistribution from the central compartment, e.g., the blood in the case of intravenous delivery, to a peripheral compartment (e.g., a tissue or organ), and the beta half-life, β half-life, or t1 / 2β, which is the rate of decline due to the processes of excretion or metabolism.

[0099] As used herein, the term “area under the plasma drug concentration-time curve” or “AUC” reflects the actual body exposure to drug after administration of a dose of the drug and is expressed in mg*h / L. This area under the curve can be measured, e.g., from time 0 (t0) to infinity (∞) and is dependent on the rate of elimination of the drug from the body and the dose administered.IGM Ref. No.002-056WO2

[0100] As used herein, the term “mean residence time” or “MRT” refers to the average length of time the drug remains in the body.

[0101] By "subject" or "individual" or "animal" or "patient" is meant any subject. In certain embodiments, the subject is a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.

[0102] As used herein, , the term “a subject that would benefit from therapy” refers to a subset of subjects, from amongst all prospective subjects, which would benefit from administration of a given therapeutic agent, e.g., a binding molecule as provided herein. Such binding molecules can be used, e.g., for a diagnostic procedure and / or for treatment or prevention of a disease. Human sarbecoviruses, e.g., SARS-CoV-1 and SARS-CoV-2 binding molecules

[0103] Provided herein are multimeric binding molecules comprising two to six bivalent binding units or variants or fragments thereof, where each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding polypeptide, where three to twelve of the binding polypeptides comprise a soluble extracellular domain of an angiotensin-converting enzyme 2 (ACE2), e.g., human ACE2. The provided binding molecules can be used to treat or prevent human severe respiratory diseases caused by human sarbecoviruses, e.g., Coronavirus Disease 2019 (COVID-19).

[0104] In some embodiments, the provided multimeric binding molecule is more potent at neutralizing infectivity of a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2, than a reference monovalent or bivalent monomeric binding molecule comprising a corresponding soluble extracellular domain of ACE2, e.g., human ACE2 fused to an IgG Fc domain (a “reference binding molecule”). In some embodiments, the reference binding molecule can bind SARS-CoV-2. In some embodiments, the reference binding molecule can bind more than one type of human sarbecovirus. In some embodiments, the reference binding molecule can bind both SARS-CoV-2 and SARS-CoV.

[0105] In some embodiments, the reference binding molecule can neutralize SARS-CoV- 2. In some embodiments, the reference binding molecule can neutralize SARS-CoV. In some embodiments, the reference binding molecule can neutralize more than one type ofIGM Ref. No.002-056WO2 human sarbecovirus. In some embodiments, the reference binding molecule can neutralize SARS-CoV and SARS-CoV-2.

[0106] In some embodiments, the provided binding molecule comprises five or six bivalent binding units and has greater avidity than the corresponding reference binding molecule.

[0107] The ability of binding molecule as provided herein to neutralize a human sarbecovirus can readily be determined by one of skill in the art, such as by measuring infectivity in vitro using a viral or pseudoviral infectivity assay, such as an assay adapted from Richman et al. (PNAS, 2003, 100(7): 4144-4149) or described in Yuan et al., Science10.1126 / science.abb7269; (2020) or Muruato et al. (2020, Nat Comm.11(1):4059. doi: 10.1038 / s41467-020-17892-0). In some embodiments, the neutralization is determined at a specific concentration or within a range of concentrations of a reference binding molecule, e.g., at 0.1 nM, 1 nM, or 0.1 nM to 10 nM.

[0108] In certain embodiments, the greater potency of the provided multimeric binding molecule relative to the reference binding molecule can be measured, e.g., as inhibition of binding of the human sarbecovirus to its receptor, e.g., the SARS-CoV or SARS-CoV-2 spike protein binding to angiotensin-converting enzyme 2 (ACE2), at a lower 50% effective concentration (EC50) or lower 50% inhibitory concentration (IC50) than that of the reference binding molecule. In certain embodiments, the provided multimeric binding molecule can inhibit binding of the human sarbecovirus to its receptor, e.g., SARS-CoV or SARS-CoV-2 spike protein binding to ACE2 under conditions where the reference binding molecule cannot inhibit binding. In certain embodiments, the provided multimeric binding molecule can neutralize the human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2, at a lower EC50than the reference binding molecule. In certain embodiments, the provided multimeric binding molecule can neutralize the human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2, under conditions where the reference binding molecule cannot neutralize the respective human sarbecovirus. In certain embodiments, the provided multimeric binding molecule can protect infected animals, or prevent infection in uninfected animals in a therapeutic animal model at a lower 50% effective dose (ED50) than the reference binding molecule. In certain embodiments, the provided multimeric binding molecule can protect infected animals, or prevent infection in uninfected animals in a therapeutic animal model under conditions where the referenceIGM Ref. No.002-056WO2 binding molecule cannot protect. In certain embodiments, the provided multimeric binding molecule can comprise any combination of the foregoing properties.

[0109] In certain embodiments, the provided multimeric binding molecule can neutralize infectivity of a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2, at a lower EC50 than the reference binding molecule. In certain embodiments, the provided multimeric binding molecule can neutralize infectivity of a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 under conditions where the reference binding molecule cannot neutralize. In certain embodiments, the EC50of the provided multimeric binding molecule is at least two-fold, at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500-fold, or at least 1000-fold, or at least 10,000-fold lower than the EC50 of the reference binding molecule. The EC50can be measured either as mass per volume, e.g., µg / ml, or as the number of molecules present, e.g., moles / liter. In certain embodiments, the conditions where the reference binding molecule cannot neutralize comprise neutralization of an antibody-resistant variant of a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2. In certain embodiments, the antibody resistant variant of the human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2, comprises an “escape mutant” of a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 that arose following contact with a reference binding molecule or other therapeutic agent. By “the human sarbecovirus, e.g., a SARS-CoV or SARS-CoV-2 virus that arose following contact with the reference binding molecule or other therapeutic agent” is meant a variant sarbecovirus that arises in response to selective pressure.

[0110] In some embodiments, the provided multimeric binding molecule can confer protection against a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 infection in a therapeutic or prophylactic animal model at a lower 50% effective dose (ED50) than the reference binding molecule, or the provided binding molecule can confer protection against the human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 infection in a therapeutic or prophylactic animal model under conditions where the reference binding molecule cannot protect. As used herein, measurements of “protection” against the human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 infection in an animal model can include a reduced viral load in the subject animals, e.g., in the animals’ lungs, survival of the subject animals from an otherwise lethal infection, and / or a reduction of symptoms typical of the human sarbecovirus infection in the animal model, e.g., weight loss, fever,IGM Ref. No.002-056WO2 difficulty breathing, or neurological symptoms. The ED50can be measured either as mass per volume, e.g., µg / ml, or as the number of molecules present, e.g., moles / liter.

[0111] In some embodiments, the provided multimeric binding molecule reduces, inhibits, or blocks the human sarbecovirus from binding to its receptor, ACE2, at a lower EC50 than the reference binding molecule or reduces, inhibits, or blocks the human sarbecovirus from binding to ACE2 under conditions where the reference binding molecule cannot reduce, inhibit, or block the human sarbecovirus from binding to ACE2. In certain embodiments, the receptor is expressed on the surface of a cell, e.g., a cultured host cell, e.g., a Vero cell, or a cell in a susceptible subject, e.g., a human subject. In some embodiments, the provided binding molecule inhibits human sarbecovirus binding to ACE2 expressed on cells at a lower 50% effective concentration (EC50) than the reference binding molecule. In some embodiments, the EC50is at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least thirty- fold, at least forty-fold, or at least fifty-fold lower than the EC50 of the reference binding molecule.

[0112] The multimeric binding molecule provided by this disclosure comprises an extracellular domain of ACE2, the cell surface receptor involved in the attachment of the human sarbecoviruses to a cell, a prerequisite for viral infection of the cell. In some embodiments, the human sarbecovirus that utilizes ACE2 as a receptor is SARS-CoV (also referred to herein as SARS-CoV1), SARS-CoV-2, or human coronavirus NL63 / HCoV-NL63, and the provided multimeric binding molecule reduces, inhibits, or blocks the human sarbecovirus from binding to ACE2 on the surface of cells, thereby blocking infectivity. An exemplary precursor human ACE2 amino acid sequence (UniprotKB Q9BYF1) is presented as SEQ ID NO: 14. The signal peptide includes amino acids 1 to 17 (UniprotKB ) or 1 to 18 (Zhu, J., et al., Front. Virol., doi: 10.3389 / fviro.2022.916700 (2022)) of SEQ ID NO: 14, the mature protein comprises amino acids 18 or 19 to 805 of SEQ ID NO: 14, the extracellular domain comprises amino acids 18 or 19 to 740 of SEQ ID NO: 14, the transmembrane domain comprises amino acids 741 to 761 of SEQ ID NO: 14, and the cytoplasmic domain comprises amino acids 762 to 805 of SEQ ID NO: 14. Amino acids 30-41, 82-84, and 353-357 of SEQ ID NO: 14 are reported to interact with the SARS-CoV spike protein. See Zhang, C., et al., EMBO J.24:1634-1643 (2005). The extracellular portion of ACE2 that is associated with enzymatic activity is the peptidase domain (PD) and is reported to comprise amino acidsIGM Ref. No.002-056WO2 18 or 19 to 615 of SEQ ID NO: 14. See, e.g., Zhu, J., et al., Front. Virol., doi: 10.3389 / fviro.2022.916700 (2022). In certain embodiments, the ACE2 domain comprises one or more mutations that reduce or ablate the enzymatic activity, e.g., mutations in residues R237, H345, E375, and / or H378. For example, ACE2 domains for use in the multimeric binding molecules disclosed herein may contain one or more mutations from among R237K, R237L, R237Q, H345G, H345L, H345P, E375A, E375L, E375Q, H378G, H378L, and H378N. In certain embodiments, the ACE2 domain has a primary sequence according to residues 18–615 of SEQ ID NO: 30.

[0113] ACE2 is expressed in cells as a homodimer. Interactions required for dimerization involve amino acids within the “neck” region of the ACE2 extracellular domain, extending from amino acid 616 to amino acid 740 of SEQ ID NO: 14 as determined by cryo-electron microscopy. See e.g., Yan, R., et al., Science 367:1444-1448 (2020). As persons of ordinary skill in the art will recognize, the amino acid coordinates associated with the various domains or functional regions of any protein are approximate, and can vary by, e.g., 1, 2, 3, 4, or 5 amino acids depending on how the regions are assessed by a given researcher.

[0114] In some embodiments, the multimeric binding molecule reduces, inhibits, or blocks the SARS-CoV or the SARS-CoV-2 S protein from binding to ACE2 at a lower EC50 than the reference binding molecule or reduces, inhibits, or blocks the SARS-CoV or the SARS-CoV-2 S protein from binding to ACE2 under conditions where the reference binding molecule cannot reduce, inhibit, or block the SARS-CoV or the SARS- CoV-2 S protein from binding to ACE2. In certain embodiments, the ACE2 is human ACE2, e.g., amino acids 18 or 19 to 805 of SEQ ID NO: 14. In certain embodiments, ACE2 is expressed on the surface of a cell, e.g., a cultured host cell, e.g., a Vero cell, or a cell in a susceptible subject, e.g., a human subject. In some embodiments, the binding molecule inhibits SARS-CoV-2 binding to its receptor, e.g., ACE2, at a lower 50% effective concentration (EC50) than the reference binding molecule. In some embodiments, the EC50is at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least thirty-fold, at least forty-fold, or at least fifty-fold lower than the EC50 of the reference binding molecule. Human sarbecoviruses are known to develop one or more mutations overtime that may alter the behavior of the virus. For example, SARS-CoV-2 has developed multiple spike mutationsIGM Ref. No.002-056WO2 over time that have made the virus more transmissible and / or more resistant to vaccines and established treatments than the original SARS-CoV-2 strain.

[0115] A reference precursor SARS-CoV S protein (UniProtKB - P59594 (SPIKE_SARS)) is presented herein as SEQ ID NO: 16. Myriad variant SARS CoV S proteins have been sequenced and are available in the literature but share the common structure of SEQ ID NO: 16. As reported in UniProtKB, the signal peptide of the precursor SARS-CoV S protein corresponds to amino acids 1 to 13 of SEQ ID NO: 16, the mature SARS-CoV S protein corresponds to amino acids 14 to 1255 of SEQ ID NO: 16, the S1 region of the SARS-CoV S protein corresponds to amino acids 14 to 667 of SEQ ID NO: 16, the S2 region of the SARS-CoV S protein corresponds to amino acids 668 to 1255 of SEQ ID NO: 16, the receptor binding domain (RBD) of the SARS-CoV S protein corresponds to amino acids 306 to 527 of SEQ ID NO: 16 (underlined below), the receptor binding motif of the SARS-CoV S protein corresponds to amino acids 424 to 494 of SEQ ID NO: 16, the extracellular domain of the SARS-CoV S protein corresponds to amino acids 14 to 1195 of SEQ ID NO: 16, the transmembrane domain of the SARS-CoV S protein corresponds to amino acids 1196 to 1216 of SEQ ID NO: 16, and the cytoplasmic domain of the SARS-CoV S protein corresponds to amino 1214 to 1255 of SEQ ID NO: 16. As persons of ordinary skill in the art will recognize, the RBD of various SARS-CoV S proteins present in the environment have mutated so an RBD that “corresponds” to amino acids 306 to 527 of SEQ ID NO: 16 may not be identical to amino acids 306 to 527 of SEQ ID NO: 16. The TMPRSS2 or furin cleavage site between the S1 and S2 subunits is between amino acids 667 and 668 of SEQ ID NO: 16. SEQ ID NO: 16: SARS-CoV Spike Protein, UniProt: P59594 1 MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGV YYPDEIFRSD TLYLTQDLFL 61 PFYSNVTGFH TINHTFGNPV IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS 121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT FEYISDAFSL DVSEKSGNFK 181 HLREFVFKNK DGFLYVYKGY QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP 241 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ NPLAELKCSV KSFEIDKGIY 301 QTSNFRVVPS GDVVRFPNIT NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF 361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG QTGVIADYNY KLPDDFMGCV 421 LAWNTRNIDA TSTGNYNYKY RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND 481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI KNQCVNFNFN GLTGTGVLTP 541 SSKRFQPFQQ FGRDVSDFTD SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD 601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE HVDTSYECDI PIGAGICASY 661 HTVSLLRSTS QKSIVAYTMS LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC 721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT REVFAQVKQM YKTPTLKYFG 781 GFNFSQILPD PLKPTKRSFI EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL 841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE 901 NQKQIANQFN KAISQIQESL TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN 961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK 1021 RVDFCGKGYH LMSFPQAAPH GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFNIGM Ref. No.002-056WO2 1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN 1141 HTSPDVDLGD ISGINASVVN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL 1201 GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV KLHYT

[0116] A reference precursor SARS-CoV-2 S protein (UniProtKB - P0DTC2 (SPIKE_SARS2)) is presented herein as SEQ ID NO: 17. Myriad variant SARS CoV-2 S proteins have been sequenced and are available in the literature but share the common structure of SEQ ID NO: 17. As reported in UniProtKB, the signal peptide of the precursor SARS-CoV-2 S protein corresponds to amino acids 1 to 12 of SEQ ID NO: 17, the mature SARS-CoV-2 S protein corresponds to amino acids 13 to 1273 of SEQ ID NO: 17, the S1 region of the SARS-CoV-2 S protein corresponds to amino acids 13 to 685 of SEQ ID NO: 17, the S2 region of the SARS-CoV-2 S protein corresponds to amino acids 686 to 1273 of SEQ ID NO: 17, the receptor binding domain (RBD) of the SARS-CoV-2 S protein corresponds to amino acids 319 to 541 of SEQ ID NO: 17 (underlined below, Yan, R. et al., Science 367:1444-1448 (2020)), the receptor binding motif of the SARS-CoV-2 S protein corresponds to amino acids 437 to 508 of SEQ ID NO: 17, the extracellular domain of the SARS-CoV-2 S protein corresponds to amino acids 13 to 1213 of SEQ ID NO: 17, the transmembrane domain of the SARS-CoV-2 S protein corresponds to amino acids 1214 to 1234 of SEQ ID NO: 17, and the cytoplasmic domain of the SARS-CoV-2 S protein corresponds to amino 1235 to 1273 of SEQ ID NO: 17. As persons of ordinary skill in the art will recognize, the RBD of various SARS- CoV-2 S proteins present in the environment have mutated so an RBD that “corresponds” to amino acids 319 to 541 of SEQ ID NO: 17 may not be identical to amino acids 319 to 541 of SEQ ID NO: 17. The TMPRSS2 or furin cleavage site between the S1 and S2 subunits is between amino acids 685 and 686 of SEQ ID NO: 17 (Hoffmann, M. et al., Cell 181:271-280 (2020)). SEQ ID NO: 17: SARS-CoV-2 Spike Protein, UniProt: P0DTC2 1 MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS 61 NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV 121 NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE 181 GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT 241 LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK 301 CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN 361 CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD 421 YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC 481 NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN 541 FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP 601 GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY 661 ECDIPIGAGI CASYQTQTNS PRRARSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI 721 SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE 781 VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDCIGM Ref. No.002-056WO2 841 LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM 901 QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN 961 TLVKQLSSNF GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA 1021 SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA 1081 ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP 1141 LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL 1201 QELGKYEQYI KWPWYIWLGF IAGLIAIVMV TIMLCCMTSC CSCLKGCCSC GSCCKFDEDD 1261 SEPVLKGVKL HYT

[0117] Human sarbecoviruses are known to develop one or more mutations, particularly in the receptor binding domain (RBD) of the spike (S) protein over time that may alter the behavior of the virus. The SARS-CoV-2 variants are identified, for example, by nomenclature referred to as “pango” lineages (Rambaut, A., et al., Nature Microbiol. 5:1403-1407 (2020)), and have been assigned Greek letter nomenclature by the World Health Organization (who.int / en / activities / tracking-SARS-CoV-2-variants / , last visited August 6, 2022). SARS CoV-2 variants of clinical relevance are cataloged by the Centers for Disease Control, e.g., at cdc.gov / coronavirus / 2019-ncov / variants / variant-info.html (last visited August 6, 2022). The CDC catalog is updated regularly. Those of skill in the art will understand that these lineages do not correspond to exact sequences and are generally characterized by the amino acid variations noted below, particularly in the RBD region, but may be mixtures and may include additional or fewer amino acid deletions, insertions, and / or substitutions relative to SEQ ID NO: 17.

[0118] For example, the pango lineage B.1.1.7 or WHO “Alpha” variant first identified in the UK includes an RBD substitution of tyrosine (Y) for asparagine (N) at a position corresponding to amino acid 501 in SEQ ID NO: 17, and can include additional spike protein alterations such as amino acid deletions at positions corresponding to amino acids 69, 70, and 144 of SEQ ID NO: 17, and amino acid substitutions A570D, D614G, P681H, T716I, S982A, D1118H corresponding to the indicated positions in SEQ ID NO: 17. By “an amino acid corresponding to amino acid 501 in SEQ ID NO: 17 (and other spike protein mutations described herein) is meant the amino acid in the sequence of any given SARS-CoV-2 spike protein, which is homologous to N501 in SEQ ID NO: 17. Variant viruses carrying this “N501Y” mutation have been shown to be more highly transmissible than the non-variant virus. See Leung, K., et al., Euro Surveill.26:2002106. doi: 10.2807 / 1560-7917.ES.2020.26.1.2002106 (2021).

[0119] The pango lineage B.1.351 or WHO “Beta” variant first identified in South Africa includes K417N, E484K and N501Y RBD substitutions corresponding to the indicated positions in SEQ ID NO: 17 and can include additional spike protein substitutions such asIGM Ref. No.002-056WO2 D80A, D215G, D614G, and A701V and amino acid deletions corresponding to amino acids 241-243 (all positions corresponding to SEQ ID NO: 17). This variant is likewise believed to be more highly transmissible. See Wibmer, CK et al., Nature Med.:27:622- 625, doi: 10.1038 / s41591-021-01285-x (2021).

[0120] The pango lineage P.1 or WHO “Gamma” variant first identified in Brazil includes K417T, E484K, and N501Y RBD substitutions corresponding to the indicated positions in SEQ ID NO: 17 and can include additional spike protein substitutions such as L18F, T20N, P26S, D138Y, R190S, D614G, H655Y, and T1027I (all positions corresponding to SEQ ID NO: 17). This variant again is believed to be more highly transmissible. See Faria, Nuno R., et al., Virological (2021) (available at icpcovid.com, visited February 19, 2021). Additionally, a SARS-CoV-2 variant with a D614G mutation is believed to have increased infectivity and transmissibility (Korber B., et al., Cell 182:812-827 (2020)).

[0121] The pango lineage B.1.525 or WHO “Eta” variant first identified in Nigeria includes an E484K RBD substitution corresponding to the indicated position in SEQ ID NO: 17 and can include additional spike protein alterations such as amino acid deletions at positions corresponding to amino acids 69, 70, and 144 of SEQ ID NO: 17, and spike protein substitutions Q52R, A67V, D614G, Q677H, F888L (all positions corresponding to SEQ ID NO: 17).

[0122] The pango lineage B.1.617.1 or WHO “Kappa” variant first identified in India includes L452R and E484Q RBD substitutions corresponding to the indicated positions in SEQ ID NO: 17 and can include spike protein substitutions such as G142D, E154K, D614G, P681R, Q1071H, and H1101D (all positions corresponding to SEQ ID NO: 17).

[0123] The pango lineage B.1.617.2 or WHO “Delta” variant first identified in India includes L452R and T478K RBD substitutions corresponding to the indicated positions in SEQ ID NO: 17 and can include additional spike protein substitutions such as T19R, G142D, D614G, P681R, and D950N (all positions corresponding to SEQ ID NO: 17). A newly-emerging Delta variant, designated “Delta Plus” or Pango lineage B.1.617.2 / AY.1 further includes a K417N substitution corresponding to SEQ ID NO: 17.

[0124] The pango lineage B.1.617.2.v2 variant first identified in India includes L452R and T478K RBD substitutions corresponding to the indicated positions in SEQ ID NO: 17 and can include additional spike protein alterations such as amino acid deletions at positions corresponding to amino acids 157 and 158 of SEQ ID NO: 17, and spike proteinIGM Ref. No.002-056WO2 substitutions such as T19R, G142D, E156G, D614G, P681R, and D950N (all positions corresponding to SEQ ID NO: 17).

[0125] The pango lineage B.1.618 variant first identified in India includes an E484K RBD substitution corresponding to the indicated position in SEQ ID NO: 17 and can include additional spike protein alterations such as amino acid deletions at positions corresponding to amino acids 145 and 146 of SEQ ID NO: 17, and spike protein substitutions such as H49Y and D614G (all positions corresponding to SEQ ID NO: 17).

[0126] The pango lineages B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 and BA.5, or WHO “Omicron” lineages were first identified in South Africa and include myriad spike protein and RBD substitutions corresponding to SEQ ID NO: 17 including, without limitation A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (all positions corresponding to SEQ ID NO: 17). The “Omicron” variants have supplanted all previous variants due to increased transmissibility. This has resulted in reduction of efficacy of previously effective monoclonal antibody treatments, and in reduction in the efficacy of vaccines.

[0127] Of particular concern is the potential development of escape mutations that can reduce or prevent neutralization by a therapy, such as an antibody therapy. The multimeric binding molecules disclosed herein may be able to maintain the ability to bind and neutralize strains of the human sarbecovirus that are escape mutants.

[0128] In some embodiments, the multimeric binding molecule reduces, inhibits, or blocks the human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 from binding to its receptor, e.g., angiotensin-converting enzyme 2 (ACE2). In some embodiments, the multimeric binding molecule reduces, inhibits, or blocks the human sarbecovirus binding to its receptor more than a reference binding molecule. In some embodiments, the binding molecule inhibits human sarbecovirus, e.g., SARS-CoV-2 binding to its receptor, e.g., ACE2, at a lower 50% inhibitory concentration (IC50) than the reference binding molecule.

[0129] In some embodiments, the multimeric binding molecule is pentameric and comprises five bivalent binding units and a J-chain or functional fragment or variant thereof as described herein.IGM Ref. No.002-056WO2

[0130] In some embodiments, the multimeric binding molecule is hexameric and comprises six bivalent binding units.

[0131] In certain embodiments, the three to twelve identical binding polypeptides of the multimeric binding molecule comprise an extracellular SARS-CoV or SARS-CoV-2 RBD-binding fragment of angiotensin-converting enzyme 2 (ACE2). In certain embodiments, the three to twelve human sarbecovirus binding polypeptides comprise an extracellular SARS-CoV or SARS-CoV-2 RBD-binding fragment of SEQ ID NO: 14. In certain embodiments, the three to twelve human sarbecovirus binding polypeptides, e.g., SARS-CoV-2-binding polypeptides, of the binding molecule comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to amino acids 18 to 740 or amino acids 19 to 740 of SEQ ID NO: 14. In certain embodiments, the three to twelve human sarbecovirus binding polypeptides, e.g., SARS-CoV-2-binding polypeptides, of the binding molecule comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to amino acids 18 to X or amino acids 19 to X of SEQ ID NO:14, wherein X is an integer greater than or equal to 610 and less than 740. In certain embodiments, the multimeric binding molecule is a pentameric or hexameric binding molecule comprising three to twelve IgM-derived heavy chains each comprising at least the Cμ3, Cμ4, and tailpiece (tp) domains corresponding to an IgM heavy chain, e.g., a human IgM heavy chain fused to an extracellular domain of ACE2, e.g., human ACE2, e.g., an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to amino acids 18 to 740 or amino acids 19 to 740 of SEQ ID NO: 14, or at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to amino acids 18 to X or amino acids 19 to X of SEQ ID NO: 14, wherein X is an integer greater than or equal to 610 and less than 740. In certain embodiments, the heavy chains comprise the Cμ3, Cμ4, and tailpiece (tp) domains corresponding to an IgM heavy chain, e.g., a human IgM heavy chain with a modified human IgG1 hinge region fused to the N-terminus where the cysteine at position 7 of the human IgG1 hinge region is substituted with serine, e.g., amino acids 724 to 741 of SEQ ID NO: 15 (VEPKSSD KTHTCPPCPA P (SEQ ID NO: 5)). In certain embodiments, the multimeric binding molecule comprises ten or twelve heavy chains comprising the amino acid sequence SEQ ID NO: 15. In certain embodiments, the heavy chains comprise the Cμ3, Cμ4, and tailpiece (tp) domains corresponding to an IgM heavy chain, e.g., a human IgM heavy chain, with a camelid hinge region fused to the N-terminus, e.g., SEQ ID NO: 18IGM Ref. No.002-056WO2 (DEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP) or SEQ ID NO: 34 (EPKIPQPQPKPQPQPQPQPKPQPKPEPE). In certain embodiments, the multimeric binding molecule comprises ten or twelve heavy chains comprising amino acids 18 or 19 to 1005 of SEQ ID NO: 27. In certain embodiments, the binding molecule is engineered to reduce or eliminate complement-mediated cytotoxicity, e.g., comprising amino acid substitutions P311A, P313S corresponding to the positions in SEQ ID NO: 1 and SEQ ID NO: 2, or comprising the amino acid substitution K315D corresponding to the position in SEQ ID NO: 1 and SEQ ID NO: 2. IgM-derived binding molecules

[0132] IgM is the first immunoglobulin produced by B cells in response to stimulation by antigen. Naturally occurring IgM is naturally present at around 1.5 mg / ml in serum with a half-life of about 5 days. IgM is a pentameric or hexameric molecule and thus includes five or six binding units. The human IgM constant region typically comprises the amino acid sequence SEQ ID NO: 1 (identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1, allele IGHM*03) or SEQ ID NO: 2 (identical to, e.g., GenBank Accession No. sp|P01871.4, allele IGHM*04). The human Cµ1 region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 1 or SEQ ID NO: 2; the human Cµ2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 1 or SEQ ID NO: 2, the human Cµ3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO: 1 or SEQ ID NO: 2, the Cµ 4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO: 1 or SEQ ID NO: 2, and the tailpiece ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0133] Other forms and alleles of the human IgM constant region with minor sequence variations exist, including, without limitation, GenBank Accession Nos. CAB37838.1, and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 described and claimed elsewhere in this disclosure can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species.

[0134] In a binding molecule as provided herein, each IgM heavy chain constant region can be associated with a binding polypeptide, e.g., an ACE-2 ectodomain.

[0135] Five IgM-derived binding units can form a complex with an additional small polypeptide chain (the J-chain), or a functional fragment, variant, or derivative thereof, toIGM Ref. No.002-056WO2 form a pentameric IgM-derived binding molecule. The precursor form of the human J- chain is presented as SEQ ID NO: 6. The signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 6, and the mature human J-chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 6. The mature human J-chain includes the amino acid sequence SEQ ID NO: 7.

[0136] Exemplary variant and modified J-chains are provided elsewhere herein. Without the J-chain, an IgM-derived binding molecule assembles into a hexamer, comprising up to twelve binding polypeptides. With a J-chain, an IgM-derived binding molecule assembles into a pentamer, comprising up to ten binding polypeptides. The assembly of five or six IgM binding units into a pentameric or hexameric IgM-derived binding molecule requires at least the Cµ4 and tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM-derived binding molecule as provided in this disclosure includes at least the Cµ4 and tailpiece domains (also referred to herein collectively as Cµ4-tp). A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cµ4-tp domains. An IgM heavy chain constant region can additionally include a Cµ3 domain or a fragment thereof, a Cµ2 domain or a fragment thereof, a Cµ1 domain or a fragment thereof, and / or other IgM heavy chain domains. In certain embodiments, an IgM-derived binding molecule as provided herein can include a complete IgM heavy (µ) chain constant domain, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or a variant, derivative, or analog thereof, e.g., as provided herein.

[0137] In certain embodiments, the disclosure provides a multimeric binding molecule, e.g., a pentameric or hexameric binding molecule, where the binding molecule includes ten or twelve IgM-derived heavy chains, and where the IgM-derived heavy chains comprise IgM heavy chain constant regions each associated with am ACE2 ectodomain that can, e.g., specifically bind to a human sarbecovirus S protein RBD. In certain embodiments, the disclosure provides an IgM-derived binding molecule that includes five or six bivalent binding units, where each binding unit includes two IgM or IgM-like heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an ACE2 ectodomain. In certain embodiments, the two IgM heavy chain constant regions included in each binding unit are human IgM heavy chain constant regions. In some embodiments, the heavy chains are glycosylated. In some embodiments, the heavy chains can be mutated to affect glycosylation.IGM Ref. No.002-056WO2

[0138] Where the IgM-derived binding molecule provided in this disclosure is pentameric, the IgM-derived binding molecule typically further includes a J-chain, or functional fragment or variant thereof. In certain embodiments, the J-chain is a modified J-chain or variant thereof that further comprises one or more heterologous moieties attached to the J- chain, as described elsewhere herein. In certain embodiments, the J-chain can be mutated to affect, e.g., enhance, the serum half-life of the IgM-derived binding molecule provided herein. In certain embodiments, the J-chain can be mutated to affect glycosylation.

[0139] An IgM heavy chain constant region can include one or more of a Cµ1 domain or fragment or variant thereof, a Cµ2 domain or fragment or variant thereof, a Cµ3 domain or fragment or variant thereof, a Cµ4 domain or fragment or variant thereof, and / or an IgM tailpiece, provided that the constant region can serve a desired function in the IgM- derived binding molecule, e.g., associate with second IgM constant region to form a binding unit, and / or associate with other binding units (and in the case of a pentamer, a J- chain) to form a hexamer or a pentamer. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a Cµ4 domain or fragment or variant thereof, a tailpiece (tp) or fragment or variant thereof, or a combination of a Cµ4 domain and a tp or fragment or variant thereof. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each further comprise a Cµ3 domain or fragment or variant thereof, a Cµ2 domain or fragment or variant thereof, a Cµ1 domain or fragment or variant thereof, or any combination thereof. Linker domains

[0140] In certain embodiments, the ACE2 ectodomain is connected to the IgM or IgG1 constant domain by one or more polypeptide linkers. Suitable linkers include, by way of non-limiting example, a mutated IgG1 “hinge” domain (SEQ ID NO: 5), a poly- glycine / serine domain (e.g., SEQ ID NO: 9, 10, 11, 12, or 13), or a “hinge” domain from a camelid immunoglobulin (e.g., SEQ ID NO: 18, or amino acids 2–28 of SEQ ID NO: 18). In certain embodiments, combinations of more than one type of linker (e.g., SEQ ID NO: 12, SEQ ID NO: 18, and / or SEQ ID NO: 34) can be used to connect the ACE2 ectodomain to the IgM or IgG1 constant domain. In certain embodiments, a linker for use in the making of the binding molecules of this disclosure can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orIGM Ref. No.002-056WO2 100% identity to at least 15 consecutive amino acids of any one or more of SEQ ID NOs: 5, 9–13, 18, and 34. ACE2-Immunoglobulin fusion proteins

[0141] Exemplary immunoglobulin fusion proteins are disclosed herein, suitable for making the pentameric and hexameric binding molecules described herein. Suitable immunoglobulin fusion proteins for making the pentameric and hexameric binding molecules include, without limitation, sequences comprising amino acids 18 or 19 to 995 of SEQ ID NO: 19, amino acids 18 or 19 to 1089 of 20, amino acids 18 or 19 to 870 of SEQ ID NO: 21, amino acids 18 or 19 to 964 of SEQ ID NO: 22, amino acids 18 or 19 to 872 of SEQ ID NO: 25, amino acids 18 or 19 to 997 of SEQ ID NO: 26, amino acids 18 or 19 to 1005 of SEQ ID NO: 27, amino acids 18 or 19 to 997 of SEQ ID NO: 28, amino acids 18 or 19 to 887 of SEQ ID NO: 29, amino acids 18 or 19 to 870 of SEQ ID NO: 30, amino acids 18 or 19 to 870 of SEQ ID NO: 31, amino acids 18 or 19 to 870 of SEQ ID NO: 32, and multimerizing fragments and variants thereof.

[0142] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 995 of SEQ ID NO: 19, or to a multimerizing fragment or variant thereof.

[0143] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 1089 of SEQ ID NO: 20, or to a multimerizing fragment or variant thereof.

[0144] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 870 of SEQ ID NO: 21, or to a multimerizing fragment or variant thereof.IGM Ref. No.002-056WO2

[0145] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 964 of SEQ ID NO: 22, or to a multimerizing fragment or variant thereof.

[0146] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 872 of SEQ ID NO: 25, or to a multimerizing fragment or variant thereof.

[0147] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 997 of SEQ ID NO: 26, or to a multimerizing fragment or variant thereof.

[0148] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 1005 of SEQ ID NO: 27, or to a multimerizing fragment or variant thereof.

[0149] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 997 of SEQ ID NO: 28, or to a multimerizing fragment or variant thereof.

[0150] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at leastIGM Ref. No.002-056WO2 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 887 of SEQ ID NO: 29, or to a multimerizing fragment or variant thereof.

[0151] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 1 to 870 of SEQ ID NO: 30, or to a multimerizing fragment or variant thereof. In one embodiment, the binding molecule is pentameric comprising ten immunoglobulin fusion proteins.

[0152] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 870 of SEQ ID NO: 31, or to a multimerizing fragment or variant thereof. In one embodiment, the binding molecule is pentameric comprising ten immunoglobulin fusion proteins.

[0153] In certain embodiments, the pentameric and hexameric binding molecules are composed of ten or twelve immunoglobulin fusion proteins, each comprising two immunoglobulin fusion proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 1 to 870 of SEQ ID NO: 32, or to a multimerizing fragment or variant thereof. In one embodiment, the binding molecule is pentameric comprising ten immunoglobulin fusion proteins. J-chains and functional fragments or variants thereof

[0154] In certain embodiments, the multimeric binding molecule provided herein comprises a J-chain or functional fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein is pentameric and comprises a J-chain or functional fragment or variant thereof. In some embodiments, the multimeric binding molecule can comprise a naturally occurring J-chain sequence, such as a mature human J- chain sequence (e.g., SEQ ID NO: 7). Alternatively, in some embodiments, the multimeric binding molecule can comprise a variant J-chain sequence, such as a variant sequence described herein with reduced glycosylation or reduced binding to one or moreIGM Ref. No.002-056WO2 polymeric Ig receptors (e.g., pIgR, Fc alpha-mu receptor (FcαµR), or Fc mu receptor (FcµR)). See, e.g., U.S. Patent No.10,899,835, which is incorporated herein by reference in its entirety. In some embodiments, the multimeric binding molecule can comprise a functional fragment of a naturally occurring or variant J-chain. As persons of ordinary skill in the art will recognize, “a functional fragment” or a “functional variant” in this context includes those fragments and variants that can associate with binding units, e.g., IgM heavy chain constant regions, to form a pentameric IgM-derived binding molecule, and / or can associate with certain immunoglobulin receptors, e.g., the polymeric immunoglobulin receptor (pIgR).

[0155] In certain embodiments, the J-chain can be modified, e.g., by introduction of a heterologous moiety, or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of binding molecule to assemble and bind to its binding target(s). See U.S. Patent Nos.9,951,134, 10,400,038, 10,618,978, and 11,630,389, each of which is incorporated herein by reference in its entirety.

[0156] Accordingly, a binding molecule provided by this disclosure can comprise a modified J-chain or functional fragment or variant thereof comprising a heterologous moiety, e.g., a heterologous polypeptide, introduced, e.g., fused or chemically conjugated, into the J-chain or fragment or variant thereof. In certain embodiments, the heterologous polypeptide can be fused to the N-terminus of the J-chain or functional fragment or variant thereof, the C-terminus of the J-chain or functional fragment or variant thereof, or to both the N-terminus and C-terminus of the J-chain or functional fragment or variant thereof. In certain embodiments, the heterologous polypeptide can be fused internally within the J-chain or functional fragment or variant thereof. In some embodiments, the heterologous polypeptide can be introduced into the J-chain at or near a glycosylation site. In some embodiments, the heterologous polypeptide can be introduced into the J- chain within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N-terminus. In certain embodiments, the heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 7 between cysteine residues 92 and 101 of SEQ ID NO: 7, or an equivalent location in a J-chain sequence, e.g., a J-chain variant or functional fragment of a J-chain. In a further embodiment, the heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 7 at or near a glycosylation site. In a further embodiment, the heterologous polypeptide can beIGM Ref. No.002-056WO2 introduced into the mature human J-chain of SEQ ID NO: 7 within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N-terminus.

[0157] In certain embodiments, the heterologous moiety can be a peptide or polypeptide sequence fused in frame to the J-chain or chemically conjugated to the J-chain or fragment or variant thereof. In certain embodiments, the heterologous polypeptide is fused to the J-chain or functional fragment thereof via a peptide linker. Any suitable linker can be used, for example the peptide linker can include at least 5 amino acids, at least ten amino acids, and least 20 amino acids, at least 30 amino acids or more, and so on. In certain embodiments, the peptide linker includes at least 5 amino acids, but no more than 25 amino acids. In certain embodiments, the peptide linker consists of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. In certain embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 9), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 13).

[0158] Heterologous moieties to be attached to a J-chain can include, without limitation, a binding moiety, e.g., an antibody or antigen-binding fragment thereof, e.g., a single chain Fv (scFv) molecule, e.g., an scFv that binds pIgR or mucin, a cytokine, e.g., IL-2 or IL-15 (see, e.g., U.S. Patent Application Publication No.2021 / 0380701, which is incorporated herein by reference in its entirety), a stabilizing peptide that can increase the half-life of the binding molecule, e.g., human serum albumin (HSA) or an HSA binding molecule, a pIgR or mucin binding molecule, or a heterologous chemical moiety such as a polymer. Variant J-chains that confer increased serum half-life

[0159] In certain embodiments, the J-chain is a functional variant J-chain that includes one or more single amino acid substitutions, deletions, or insertions relative to a reference J- chain identical to the variant J-chain except for the one or more single amino acid substitutions, deletions, or insertions. For example, certain amino acid substitutions, deletions, or insertions can result in the IgM-derived binding molecule exhibiting an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant J-chain, and is administered using the same method to the same animal species. In certain embodiments, the variant J-chain can include one, two, three, or four single amino acid substitutions, deletions, orIGM Ref. No.002-056WO2 insertions relative to the reference J-chain. Exemplary J-chains that confer increased serum half-life can be found, e.g., in U.S. Patent No.10,899,935, which is incorporated herein by reference in its entirety.

[0160] In certain embodiments, the variant J-chain comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 7). By “an amino acid corresponding to amino acid Y102 of the mature wild-type human J-chain” is meant the amino acid in the sequence of the J- chain, which is homologous to Y102 in the human J-chain. For example, see U.S. Patent No.10,899,835, which is incorporated herein by reference in its entirety. The position corresponding to Y102 in SEQ ID NO: 7 is conserved in the J-chain amino acid sequences of at least 43 other species. See FIG.4 of U.S. Patent No.9,951,134, which is incorporated by reference herein. Certain mutations at the position corresponding to Y102 of SEQ ID NO: 7 can inhibit the binding of IgM pentamers comprising the variant J-chain to certain immunoglobulin receptors, e.g., the human or murine Fcαµ receptor, the murine Fcµ receptor, and / or the human or murine polymeric Ig receptor (pIgR).

[0161] A multimeric binding molecule comprising a mutation at the amino acid corresponding to Y102 of SEQ ID NO: 7 has an improved serum half-life when administered to an animal than does a corresponding multimeric binding molecule that is identical except for the substitution, and which is administered to the same species in the same manner. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 7 can be substituted with any amino acid. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 7 can be substituted with alanine (A), serine (S) or arginine (R). In a particular embodiment, the amino acid corresponding to Y102 of SEQ ID NO: 7 can be substituted with alanine. In a particular embodiment the J-chain or functional fragment or variant thereof is a variant human J-chain referred to herein as “J*,” and comprises the amino acid sequence SEQ ID NO: 8.

[0162] Wild-type J-chains typically include one N-linked glycosylation site. In certain embodiments, a variant J-chain or functional fragment thereof of a multimeric binding molecule as provided herein includes a mutation within the asparagine(N)-linked glycosylation motif N-X1-S / T, e.g., starting at the amino acid position corresponding to amino acid 49 (motif N6) of the mature human J-chain (SEQ ID NO: 7) or J* (SEQ ID NO: 8), where N is asparagine, X1is any amino acid except proline, and S / T is serine or threonine, and where the mutation prevents glycosylation at that motif. As demonstratedIGM Ref. No.002-056WO2 in U.S. Patent No.10,899,835, mutations preventing glycosylation at this site can result in the multimeric binding molecule as provided herein, exhibiting an increased serum half- life upon administration to a subject animal relative to a reference multimeric binding molecule that is identical except for the mutation or mutations preventing glycosylation in the variant J-chain, and is administered in the same way to the same animal species.

[0163] For example, in certain embodiments, the variant J-chain or functional fragment thereof of a pentameric IgM-derived binding molecule as provided herein can include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 7 or SEQ ID NO: 8, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or where the variant J-chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 7 or SEQ ID NO: 8. In certain embodiments, the position corresponding to N49 of SEQ ID NO: 7 or SEQ ID NO: 8 is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular embodiment, the position corresponding to N49 of SEQ ID NO: 7 or SEQ ID NO: 8 can be substituted with alanine (A). In another particular embodiment, the position corresponding to N49 of SEQ ID NO: 7 or SEQ ID NO: 8 can be substituted with aspartic acid (D). In some embodiments, the position corresponding to S51 of SEQ ID NO: 7 or SEQ ID NO: 8 is substituted with alanine (A) or glycine (G). In some embodiments, the position corresponding to S51 of SEQ ID NO: 7 or SEQ ID NO: 8 is substituted with alanine (A). Variant IgM constant regions

[0164] IgM heavy chain constant regions of a multimeric binding molecule as provided herein can be engineered to confer certain desirable properties to the multimeric binding molecules provided herein. For example, in certain embodiments, IgM heavy chain constant regions can be engineered to confer enhanced serum half-life to multimeric binding molecules as provided herein. Exemplary IgM heavy chain constant region mutations that can enhance serum half-life of an IgM-derived binding molecule are disclosed in U.S. Patent No.10,899,835, which is incorporated by reference herein in its entirety. For example, a variant IgM heavy chain constant region of the IgM antibody, IgM-like antibody, or IgM-derived binding molecule as provided herein can include an amino acid substitution at a position corresponding to amino acid S401, E402, E403, R344, and / or E345 of a wild-type human IgM constant region (e.g., SEQ ID NO: 1 orIGM Ref. No.002-056WO2 SEQ ID NO: 2). By “an amino acid corresponding to amino acid S401, E402, E403, R344, and / or E345 of a wild-type human IgM constant region” is meant the amino acid in the sequence of the IgM constant region of any species which is homologous to S401, E402, E403, R344, and / or E345 in the human IgM constant region. In certain embodiments, the amino acid corresponding to S401, E402, E403, R344, and / or E345 of SEQ ID NO: 1 or SEQ ID NO: 2 can be substituted with any amino acid, e.g., alanine.

[0165] In certain embodiments, an IgM-derived binding molecule as provided herein can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity to cells in the presence of complement, relative to a reference IgM-derived binding molecule with corresponding reference human IgM constant regions that are identical, except for the mutations conferring reduced CDC activity. These CDC mutations can be combined with any of the mutations to confer increased serum half-life as provided herein. By “corresponding reference human IgM constant region” is meant a human IgM constant region that is identical to the variant IgM constant region except for the modification or modifications in the constant region affecting CDC activity. In certain embodiments, the variant human IgM constant region includes one or more amino acid substitutions, e.g., in the Cµ3 domain, relative to a wild-type human IgM constant region as described, e.g., in U.S. Patent 11,401,337, which is incorporated herein by reference in its entirety. Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g., in U.S. Patent 11,401,337.

[0166] In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310, P311, P313, and / or K315 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments the variant IgM constant region as provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments the variant IgM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at positions P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and P313 of SEQ ID NO: 1 or SEQ ID NO: 2. These proline residues can beIGM Ref. No.002-056WO2 independently substituted with any amino acid, e.g., with alanine, serine, or glycine. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2. The lysine residue can be independently substituted with any amino acid, e.g., with alanine, serine, glycine, or aspartic acid. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2 with aspartic acid.

[0167] Human and certain non-human primate IgM constant regions typically include five (5) naturally occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif” comprises or consists of the amino acid sequence N- X1-S / T, where N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 1 or SEQ ID NO: 2 starting at positions 46 (“N1”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Accordingly, in some embodiments, IgM heavy chain constant regions of a multimeric binding molecule as provided herein comprise 5 N-linked glycosylation motifs: N1, N2, N3, N4, and N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., N1, N2, and N3) on each IgM heavy chain constant region are occupied by a complex glycan.

[0168] In certain embodiments, at least one, at least two, at least three, or at least four of the N- X1-S / T motifs can include an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif. In certain embodiments, the IgM-derived multimeric binding molecule can include an amino acid insertion, deletion, or substitution at motif N1, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs N1, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif. In some embodiments, the IgM constant region comprises one or more substitutions relative to a wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 1 (human IgM constant regionIGM Ref. No.002-056WO2 allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). See, e.g., U.S. Patent Application No.2022 / 0306760, which is incorporated herein by reference in its entirety. Polynucleotides and Vectors

[0169] In certain embodiments, this disclosure provides a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of a multimeric binding molecule described herein. For example, in certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 995 of SEQ ID NO: 19, or to a multimerizing fragment or variant thereof.

[0170] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 1089 of SEQ ID NO: 20, or to a multimerizing fragment or variant thereof.

[0171] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 870 of SEQ ID NO: 21, or to a multimerizing fragment or variant thereof.

[0172] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 964 of SEQ ID NO: 22, or to a multimerizing fragment or variant thereof.

[0173] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 872 of SEQ ID NO: 25, or to a multimerizing fragment or variant thereof.

[0174] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 997 of SEQ ID NO: 26, or to a multimerizing fragment or variant thereof.

[0175] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at leastIGM Ref. No.002-056WO2 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 1005 of SEQ ID NO: 27, or to a multimerizing fragment or variant thereof.

[0176] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 997 of SEQ ID NO: 28, or to a multimerizing fragment or variant thereof.

[0177] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 887 of SEQ ID NO: 29, or to a multimerizing fragment or variant thereof.

[0178] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 870 of SEQ ID NO: 30, or to a multimerizing fragment or variant thereof.

[0179] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 870 of SEQ ID NO: 31, or to a multimerizing fragment or variant thereof.

[0180] In certain embodiments, the polynucleotide can encode an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 18 or 19 to 870 of SEQ ID NO:32, or to a multimerizing fragment or variant thereof.

[0181] In certain embodiments, this disclosure provides a vector comprising one or more polynucleotides described herein (e.g., a polynucleotide encoding an immunoglobulin fusion protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one or more of the mature amino acid sequence of SEQ ID NOs: 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31, 32, or to a multimerizing fragment or variant thereof.). In some embodiments, the vector further comprises a polynucleotide comprising a nucleic acid sequence that encodes a J- chain or a functional fragment or variant thereof, such as a J-chain, functional fragment or variant thereof described herein.

[0182] In some embodiments, the vector is a viral vector, such as an adenoviral or adeno- associated viral (AAV) vector. In some embodiments, this disclosure provides a viralIGM Ref. No.002-056WO2 particle comprising a viral vector disclosed herein. In some embodiments, the viral particle is an adenoviral particle or an AAV particle.

[0183] In certain embodiments, this disclosure provides a composition comprising a first vector and a second vector, where: the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes a J-chain or a functional fragment or variant thereof Host cells

[0184] In certain embodiments, this disclosure provides a host cell that is capable of producing the multimeric binding molecule as provided herein. In certain embodiments, the host cell comprises one or more vectors, a composition comprising multiple vectors, or polynucleotides disclosed herein. The disclosure also provides a method of producing the multimeric binding molecule as provided herein, where the method comprises culturing the provided host cell, and recovering the multimeric binding molecule. Methods of Use

[0185] The disclosure further provides a method of treating a disease or disorder in a subject in need of treatment, where the method includes administering to the subject a therapeutically effective amount of a multimeric binding molecule as provided herein. By "therapeutically effective dose or amount" or "effective amount" is intended an amount of a multimeric binding molecule that when administered brings about a positive therapeutic response with respect to treatment of subject. Examples of positive therapeutic responses include, without limitation, prevention of respiratory tract colonization or infection by a human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2, prevention of human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 attachment, penetration, and / or replication upon exposure to the virus, prevention of human sarbecovirus, e.g., SARS- CoV or SARS-CoV-2 symptoms, alleviation of human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 symptoms, reduction of the number of human sarbecovirus, e.g., SARS- CoV or SARS-CoV-2 symptoms, or reduction in the severity of human sarbecovirus, e.g., SARS-CoV or SARS-CoV-2 symptoms. “Symptoms” include, without limitation, one or more of fever, chills, muscle or body aches, fatigue, headache, sore throat, coughing, shortness of breath, difficulty breathing, loss of taste and / or the ability to smell, pneumonia, congestion, nausea, or diarrhea.IGM Ref. No.002-056WO2

[0186] Effective doses of compositions for treatment of a disease or disorder vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the subject is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0187] In certain embodiments, the disclosure provides a method for treating a human sarbecovirus disease, e.g., Corona Virus Disease 2019 (COVID-19) in a subject in need of treatment, where the method includes administering to the subject an effective amount of a multimeric binding molecule as provided herein. In certain embodiments, administration of a multimeric binding molecule as provided herein to a subject results in greater potency, e.g., greater efficacy at an equivalent dose or the ability to administer a lower dose and achieve equivalent efficacy, than administration of an equivalent amount of reference binding molecule binding to the same binding partner. By “efficacy” is meant the ability of the treatment to, for example, reduce symptoms in an infected subject, reduce the severity of symptoms in an infected subject, prevent symptoms in an infected but asymptomatic subject, reduce the need for auxiliary oxygen in an infected subject or reduce time on a ventilator, reduce the need or the dosage of concomitant medications, reduce the time in intensive care, spare hospital resources, or prevent or reduce transmission from an infected subject to non-infected persons. In certain embodiments, the multimeric binding molecule as provided herein can also treat the subject more safely, e.g., by preventing antibody-dependent enhancement of infection, and by effectively neutralizing “escape mutant” viruses. In certain embodiments, the reference binding molecule includes identical ACE2 ectodomains to the multimeric binding molecule as provided herein. By “an equivalent amount” is meant, e.g., an amount measured by molecular weight, e.g., in total milligrams, or alternatively, a molar equivalent, e.g., where equivalent numbers of molecules are administered.

[0188] In other embodiments, the disclosure provides a method for preventing a human sarbecovirus disease, e.g., Corona Virus Disease 2019 (COVID-19) in a subject in need thereof, e.g., a subject susceptible to human sarbecovirus, e.g., SARS-CoV-2 infection or a subject susceptible to more severe human sarbecovirus infection-associated disease or disorder, e.g.,COVID-19 symptoms due to proximity to human sarbecovirus infection-IGM Ref. No.002-056WO2 associated disease or disorder, e.g.,COVID-19 patients, e.g., healthcare providers and / or family members, or due to secondary conditions such as advanced age, diabetes, heart disease, or obesity, where the method includes administering to the subject an effective amount of a multimeric binding molecule as provided herein. In certain embodiments, administration of a multimeric binding molecule as provided herein to a subject results in greater potency, e.g., as noted above, than administration of an equivalent amount of a reference binding molecule binding to the same binding partner. In certain embodiments, the reference binding molecule includes identical ACE2 ectodomains to the multimeric binding molecule as provided herein. By “an equivalent amount” is meant, e.g., an amount measured by molecular weight, e.g., in total milligrams, or alternative, a molar equivalent, e.g., where equivalent numbers of molecules are administered.

[0189] The subject can be any animal, e.g., a mammal, in need of treatment or prevention, in certain embodiments, the subject is a human subject.

[0190] In its simplest form, a preparation to be administered to a subject is multimeric binding molecule as provided herein administered in a conventional dosage form, which can be combined with a pharmaceutical excipient, carrier or diluent as described elsewhere herein.

[0191] A multimeric binding molecule of the disclosure can be administered by any suitable method, e.g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, intranasally, buccally, vaginally, via an implanted reservoir, or a combination thereof. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0192] In certain embodiments, the multimeric binding molecule is delivered intranasally, e.g., in an atomized form produced by a suitable spray delivery device, e.g., a MAD NASAL™ Intranasal Mucosal Atomization Device, produced by Teleflex. In certain embodiments, the multimeric binding molecule is delivered orally, e.g., in an atomized form produced by a suitable spray delivery device, e.g., a MAD NASAL™ Intranasal Mucosal Atomization Device, produced by Teleflex. In certain embodiments, the multimeric binding molecule is delivered intranasally and orally, e.g., in an atomized form produced by a suitable spray delivery device, e.g., a MAD NASAL™ Intranasal Mucosal Atomization Device, produced by Teleflex.IGM Ref. No.002-056WO2

[0193] In certain embodiments, the multimeric binding molecule is delivered via inhalation, e.g., in a nebulized form.

[0194] In certain embodiments, the multimeric binding molecule is delivered intranasally, e.g., in an atomized form produced by a suitable spray delivery device, e.g., a MAD NASAL™ Intranasal Mucosal Atomization Device, produced by Teleflex. In certain embodiments, the multimeric binding molecule is delivered orally, e.g., in an atomized form produced by a suitable spray delivery device. In certain embodiments, the multimeric binding molecule is delivered intranasally and orally. In some embodiments, the multimeric binding molecule is delivered by inhalation to the lung using a suitable pulmonary delivery devise, such as a nebulizer, a metered dose inhaler, or a dry powder inhaler. Pharmaceutical Compositions and Administration Methods

[0195] The disclosure further provides a composition, e.g., a pharmaceutical composition, comprising a multimeric binding molecule, or two or more multimeric binding molecules, as provided herein. In certain embodiments, the composition includes a cocktail of two or more different multimeric binding molecules as described herein. A composition as provided herein can further include a pharmaceutically acceptable carrier and / or excipient and can be formulated so as to be suitable for a desired mode of administration.

[0196] Suitable excipients that can be included in the compositions include surfactants. By way of non-limiting example, the compositions may comprise 0.10 to 0.50%(w / v) of fatty acid esters of polyoxyethylene sorbitan (a “PS surfactant”), such as polyoxyethylene (20) sorbitan monolaurate (PS20) or PS80. Suitable surfactant concentrations include, for example, 0.10% (w / v), 0.15% (w / v), 0.20% (w / v), 0.25% (w / v), 0.30% (w / v), 0.35% (w / v), 0.40% (w / v), 0.45% (w / v), and 0.50% (w / v).

[0197] Additional suitable excipients include amino acids, salts, and sugars or sugar alcohols. Where amino acids are included (e.g., arginine, glycine, histidine, etc.), they can be present in concentrations from 5 mM to 50 mM, e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. Where salts are included (e.g., NaCl, KCl, etc.), they can be present in concentrations from 100 mM to 500 mM, e.g., 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 400 mM, 450 mM, or 500 mM. Where sugars (e.g., sucrose, glucose, fructose, xylose, etc.) or sugar alcohols (e.g. mannitol, sorbitol, etc.) are included, they can be present in concentrations from 0.50% (w / v) to 5.00% (w / v), e.g., 0.50% (w / v),IGM Ref. No.002-056WO2 1.00% (w / v), 1.50% (w / v), 2.00% (w / v), 2.50% (w / v), 3.00% (w / v), 3.50% (w / v), 4.00% (w / v), 4.50% (w / v), and 5.00% (w / v). In certain embodiments, the composition will have a neutral pH.

[0198] By way of non-limiting example, the composition may have a pH of 6.5 and comprise 20 mM histidine, 300 mM NaCl, 2%(w / v) sucrose, and 0.2%(w / v) PS20. In certain embodiments, the composition may consist of a multimeric binding molecule as described herein, water, 20 mM histidine, 300 mM NaCl, 2% (w / v) sucrose, and 0.2% (w / v) PS20.

[0199] Methods of preparing and administering a multimeric binding molecule as provided herein to a subject in need thereof can be determined by a skilled person in view of this disclosure. The route of administration of can be, for example, oral, parenteral, intranasal, by inhalation, or topical. In some embodiments, the multimeric binding molecule is administered in the form of an aerosol. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While these forms of administration are contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous administration, or for intraarterial injection or drip. A suitable pharmaceutical composition can include any of a number of excipients, such as, for example, a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e.g., human albumin), etc.

[0200] As discussed herein, a multimeric binding molecule as provided herein can be administered in a pharmaceutically effective amount for the treatment of a subject in need thereof. In this regard, it will be appreciated that the disclosed multimeric binding molecule can be formulated so as to facilitate administration and promote stability of the active agent. Pharmaceutical compositions accordingly can include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives, and the like. A pharmaceutically effective amount of a multimeric binding molecule as provided herein means an amount sufficient to achieve effective binding to a target and to achieve a therapeutic benefit. Suitable formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 23d ed. (2020).

[0201] Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions, powders, or solutions.IGM Ref. No.002-056WO2

[0202] Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents. In some embodiments, the pharmaceutical composition is administered by nasal aerosol. In some embodiments, the pharmaceutical composition, such as a pharmaceutical composition for administration by nasal aerosol, comprises a pH adjuster, such as HCl; a buffer; an emulsifier, such as polysorbate or carbomer; sugar or mono- or polyol, such as a monosaccharide (e.g., glucose, dextrose, or fructose), disaccharide (e.g., sucrose, lactose, or maltose), ribose, glycerin, sorbitol, xylitol, inositol, propylene glycol, galactose, mannose, xylose, rhamnose, glutaraldehyde, ethanol, mannitol, polyethylene glycol, glycerol, chitosal, phenylethyl alcohol; a preservative; cellulose, such as microcrystalline cellulose or carboxymethylcellulose; or mixtures thereof.

[0203] In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition, such as a pharmaceutical composition for administration by inhalation, is in the form of a dry powder, such as for use with a dry powder inhaler, or is in the form of a liquid, such as for use with a nebulizer, such as an airjet-compressor nebulizer or a mesh-based nebulizer. In some embodiments, the pharmaceutical composition, such as a pharmaceutical composition for administration by inhalation, comprises sugar or mono- or polyol, such as lactose, trelose, mannitol, sorbitol; buffer, such as histidine, proline, or arginine buffer; saline; polysorbate; or mixtures thereof.

[0204] The amount of a multimeric binding molecule that can be combined with carrier materials to produce a single dosage form will vary depending, e.g., upon the subject treated and the particular mode of administration. The composition can be administered as a single dose, or as multiple doses or over an established period of time, e.g., as an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0205] In keeping with the scope of the present disclosure, a multimeric binding molecule as provided herein can be administered to a subject in need of therapy in an amount sufficient to produce a therapeutic effect or a prophylactic effect. A multimeric binding molecule as provided herein can be administered to the subject in a conventional dosage form prepared by combining the multimeric binding molecule of the disclosure with aIGM Ref. No.002-056WO2 conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.

[0206] This disclosure also provides for the use of a multimeric binding molecule as provided herein in the manufacture of a medicament for treating, preventing, or managing a human sarbecovirus infection, e.g., COVID-19.

[0207] In some embodiments, the compositions and methods provided herein can be used for the treatment of infections that have not been effectively treated using existing or established treatments. Further Embodiments

[0208] Embodiment 1. A multimeric binding molecule comprising five or six bivalent binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding polypeptide, wherein three to twelve of the binding polypeptides are identical and comprise a human angiotensin-converting enzyme 2 (ACE2) ectodomain, and wherein the ACE2 ectodomain consists essentially of an amino acid sequence at least 90% identical to amino acids 19 to X of SEQ ID NO: 14, wherein X is an integer greater than or equal to 610 and less than 740.

[0209] Embodiment 2. The multimeric binding molecule of embodiment 1, wherein X is an integer greater than or equal to 615 and less that 740.

[0210] Embodiment 3. The multimeric binding molecule of embodiment 1 or embodiment 2, wherein X is 615.

[0211] Embodiment 4. The multimeric binding molecule of any one of embodiments 1 to 3, wherein the ACE2 ectodomain consists essentially of an amino acid sequence at least 95% identical to amino acids 19 to X of SEQ ID NO: 14.

[0212] Embodiment 5. The multimeric binding molecule of any one of embodiments 1 to 4, wherein the ACE2 ectodomain consists essentially of amino acids 19 to 615 of SEQ ID NO: 14.

[0213] Embodiment 6. The multimeric binding molecule of any one of embodiments 1 to 5, wherein the ACE2 ectodomain consists essentially of amino acids 18 or 19 to 615 of SEQ ID NO: 14.IGM Ref. No.002-056WO2

[0214] Embodiment 7. The multimeric binding molecule of any one of embodiments 1 to 4, wherein the ACE2 ectodomain comprises a substitution of the amino acid corresponding to R237, H345, H378, or E375 of SEQ ID NO: 14.

[0215] Embodiment 8. The multimeric binding molecule of embodiment 7, wherein the ACE2 ectodomain comprises:

[0216] (a) a glutamine (Q), a leucine (L), a lysine (K), or an alanine (A) substitution of the amino acid corresponding to R237 of SEQ ID NO: 14,

[0217] (b) an L, glycine (G), or proline (P) substitution of the amino acid corresponding to H345 of SEQ ID NO: 14,

[0218] (c) an L, G, or asparagine (N) substitution of the amino acid corresponding to H378 of SEQ ID NO: 14, or

[0219] (d) an L, A, or Q substitution of the amino acid corresponding to E375 of SEQ ID NO: 14.

[0220] Embodiment 9. The multimeric binding molecule of any one of embodiments 1 to 8, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each comprising an IgM Cµ4 domain and an IgM tailpiece domain.

[0221] Embodiment 10. The multimeric binding molecule of embodiment 9, wherein each IgM heavy chain constant region or multimerizing fragment or variant thereof further comprises an IgM Cµ3 domain situated N-terminal to the IgM Cµ4 and IgM tailpiece domains.

[0222] Embodiment 11. The multimeric binding molecule of embodiment 10, wherein each IgM heavy chain constant region or multimerizing fragment or variant thereof further comprises an IgM Cµ2 domain situated N-terminal to the IgM Cµ3 domain.

[0223] Embodiment 12. The multimeric binding molecule of any one of embodiments 9 to 11, wherein each IgM heavy chain constant region is a human IgM constant region comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a multimerizing fragment or variant thereof.

[0224] Embodiment 13. The multimeric binding molecule of embodiment 12, wherein each IgM heavy chain is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ3, Cµ4, and tailpiece domains, wherein each variant multimerizing fragment consists essentially of amino acids 217 to 453 of SEQ ID NO: 35, wherein the multimeric binding molecule has reduced complement-dependentIGM Ref. No.002-056WO2 cytotoxicity (CDC) activity relative to a corresponding binding molecule comprising the wild type multimerizing fragment of the human IgM constant region consisting essentially of amino acids 217 to 453 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0225] Embodiment 14. The multimeric binding molecule of embodiment 13, comprising a variant human IgG1 hinge region fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, wherein the variant human IgG1 hinge region consists essentially of SEQ ID NO: 5.

[0226] Embodiment 15. The multimeric binding molecule of embodiment 14, comprising amino acids 18 or 19 to 870 of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0227] Embodiment 16. The multimeric binding molecule of embodiment 14, comprising amino acids 18 or 19 to 870 of SEQ ID NO: 21.

[0228] Embodiment 17. The multimeric binding molecule of embodiment 13, comprising a (G4S)4 linker fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, wherein the (G4S)4 linker consists essentially of SEQ ID NO: 12.

[0229] Embodiment 18. The multimeric binding molecule of embodiment 17, comprising amino acids 18 or 19 to 872 of SEQ ID NO: 25.

[0230] Embodiment 19. The multimeric binding molecule of embodiment 13, comprising a camelid linker fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, wherein the camelid linker consists essentially of the amino acid sequence SEQ ID NO: 18 or SEQ ID NO: 34.

[0231] Embodiment 20. The multimeric binding molecule of embodiment 19, comprising amino acids 18 or 19 to 887 of SEQ ID NO: 29.

[0232] Embodiment 21. The multimeric binding molecule of embodiment 12, wherein each IgM heavy chain is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ2, Cµ3, Cµ4, and tailpiece domains, wherein each variant multimerizing fragment consists essentially of amino acids 105 to 453 of SEQ ID NO: 35, wherein the multimeric binding molecule has reduced complement-dependent cytotoxicity (CDC) activity relative to a corresponding binding molecule comprising the wild type multimerizing fragment of the human IgM constant region consisting essentially of amino acids 105 to 453 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0233] Embodiment 22. The multimeric binding molecule of embodiment 21, comprising amino acids 18 or 19 to 964 of SEQ ID NO: 22.IGM Ref. No.002-056WO2

[0234] Embodiment 23. A multimeric binding molecule comprising five or six bivalent binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each connected C-terminal to a binding polypeptide via a linker, wherein three to twelve of the binding polypeptides are identical and comprise a human angiotensin-converting enzyme 2 (ACE2) ectodomain, and wherein the ACE2 ectodomain comprises an amino acid sequence at least 90% identical to amino acids 19 to 740 of SEQ ID NO: 14, and wherein the linker consists essentially of (G4S)4(SEQ ID NO: 12), a camelid linker consisting essentially of amino acids 1 to 27 of SEQ ID NO: 18, or a camelid linker consisting essentially of SEQ ID NO: 18 or SEQ ID NO: 34.

[0235] Embodiment 24. The multimeric binding molecule of embodiment 23, wherein the ACE2 ectodomain comprises amino acids 19 to 740 of SEQ ID NO: 14.

[0236] Embodiment 25. The multimeric binding molecule of embodiment 23, wherein the ACE2 ectodomain comprises amino acids 18 or 19 to 615 of SEQ ID NO: 14.

[0237] Embodiment 26. The multimeric binding molecule of any one of embodiments 23 to 25, wherein each IgM heavy chain constant region is a human IgM constant region comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a multimerizing fragment or variant thereof.

[0238] Embodiment 27. The multimeric binding molecule of embodiment 26, wherein each IgM heavy chain is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ3, Cµ4, and tailpiece domains, wherein each variant multimerizing fragment consists essentially of amino acids 217 to 453 of SEQ ID NO: 35, wherein the multimeric binding molecule has reduced complement-dependent cytotoxicity (CDC) activity relative to a corresponding binding molecule comprising the wild type multimerizing fragment of the human IgM constant region consisting essentially of amino acids 217 to 453 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0239] Embodiment 28. The multimeric binding molecule of embodiment 27, wherein the linker consists essentially of (G4S)4(SEQ ID NO: 12).

[0240] Embodiment 29. The multimeric binding molecule of embodiment 28, comprising amino acids 18 or 19 to 997 of SEQ ID NO: 26.

[0241] Embodiment 30. The multimeric binding molecule of embodiment 27, wherein the linker consists essentially of SEQ ID NO: 34 or amino acids 1 to 27 of SEQ ID NO: 18.IGM Ref. No.002-056WO2

[0242] Embodiment 31. The multimeric binding molecule of embodiment 30, comprising amino acids 18 or 19 to 1005 of SEQ ID NO: 27.

[0243] Embodiment 32. The multimeric binding molecule of any one of embodiments 1 to 31 comprising five bivalent binding units, and further comprises a J-chain or functional fragment or variant thereof.

[0244] Embodiment 33. The multimeric binding molecule of embodiment 32, wherein the J-chain or functional fragment thereof is a human J-chain comprising the amino acid sequence SEQ ID NO: 7 or a functional fragment thereof.

[0245] Embodiment 34. The multimeric binding molecule of embodiment 32, wherein the J-chain or functional fragment or variant thereof is a variant J-chain comprising one or more single amino acid substitutions, deletions, or insertions relative to a wild-type J- chain that can affect serum half-life of the multimeric binding molecule; and wherein the multimeric binding molecule exhibits an increased serum half-life upon administration to an animal relative to a reference multimeric binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions, and is administered in the same way to the same animal species.

[0246] Embodiment 35. The multimeric binding molecule of embodiment 34, wherein the J-chain or functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the wild-type mature human J-chain (SEQ ID NO: 7).

[0247] Embodiment 36. The multimeric binding molecule of embodiment 35, wherein the amino acid corresponding to Y102 of SEQ ID NO: 7 is substituted with alanine (A), serine (S), or arginine (R).

[0248] Embodiment 37. The multimeric binding molecule of embodiment 36, wherein the amino acid corresponding to Y102 of SEQ ID NO: 7 is substituted with alanine (A).

[0249] Embodiment 38. The multimeric binding molecule of embodiment 37, wherein the J-chain is a variant human J-chain and comprises the amino acid sequence SEQ ID NO: 8.

[0250] Embodiment 39. The multimeric binding molecule of any one of embodiments 32 to 38, wherein the J-chain or functional fragment or variant thereof is a modified J-chain, and further comprises a heterologous polypeptide, wherein the heterologous polypeptide is directly or indirectly fused to the J-chain or functional fragment or variant thereof.

[0251] Embodiment 40. The multimeric binding molecule of embodiment 39, wherein the heterologous polypeptide is fused to the J-chain or fragment thereof via a peptide linker.IGM Ref. No.002-056WO2

[0252] Embodiment 41. The multimeric binding molecule of embodiment 40, wherein the peptide linker consists of GGGGS (SEQ ID NO: 9), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 13).

[0253] Embodiment 42. The multimeric binding molecule of any one of embodiments 39 to 41, wherein the heterologous polypeptide is fused to the N-terminus of the J-chain or fragment or variant thereof, the C-terminus of the J-chain or fragment or variant thereof, or to both the N-terminus and C-terminus of the J-chain or fragment or variant thereof.

[0254] Embodiment 43. The multimeric binding molecule of any one of embodiments 1 to 42, comprising ten identical binding polypeptides.

[0255] Embodiment 44. The multimeric binding molecule of any one of embodiments 1 to 31, comprising six bivalent binding units, and comprising twelve identical binding polypeptides.

[0256] Embodiment 45. The multimeric binding molecule of any one of embodiments 1 to 44, which can neutralize infectivity of a human sarbecovirus at a lower 50% effective concentration (EC50) than a reference binding molecule, wherein the reference binding molecule comprises the ACE2 ectodomain and a constant domain from an IgG antibody.

[0257] Embodiment 46. The multimeric binding molecule of embodiment 45, wherein the reference binding molecule comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0258] Embodiment 47. The multimeric binding molecule of embodiment 45 or embodiment 46, wherein the EC50 is at least two-fold, at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500-fold, or at least 1000-fold lower than the EC50 of the reference binding molecule.

[0259] Embodiment 48. The multimeric binding molecule of any one of embodiments 1 to 47, which can inhibit binding of the human sarbecovirus to its receptor at a lower 50% inhibitory concentration (IC50) than the reference binding molecule.

[0260] Embodiment 49. The multimeric binding molecule of embodiment 48, wherein the reference binding molecule comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0261] Embodiment 50. The multimeric binding molecule of embodiment 48 or embodiment 49, wherein the IC50 is at least two-fold, at least five-fold, at least ten-fold,IGM Ref. No.002-056WO2 at least fifty-fold, at least 100-fold, at least 500-fold, or at least 1000-fold lower than the IC50 of the reference binding molecule.

[0262] Embodiment 51. The multimeric binding molecule of any one of embodiments 45 to 50, wherein the human sarbecovirus is SARS-CoV or SARS-CoV-2.

[0263] Embodiment 52. A composition comprising the multimeric binding molecule of any one of embodiments 1 to 51.

[0264] Embodiment 53. The composition of embodiment 52, wherein the composition further comprises 0.1 to 0.5%(w / v) of fatty acid esters of polyoxyethylene sorbitan (PS surfactant).

[0265] Embodiment 54. The composition of embodiment 53, wherein the PS surfactant is polyoxyethylene (20) sorbitan monolaurate (PS20).

[0266] Embodiment 55. The composition of embodiment 54, wherein the composition has a neutral pH and further comprises an amino acid, NaCl, and a sugar.

[0267] Embodiment 56. The composition of embodiment 55, wherein the composition has a pH of 6.5 and comprises 20 mM histidine, 300 mM NaCl, 2%(w / v) sucrose, and 0.2%(w / v) PS20.

[0268] Embodiment 57. The composition of embodiment 56, wherein the composition consists of the multimeric binding molecule, water, 20 mM histidine, 300 mM NaCl, 2%(w / v) sucrose, and 0.2%(w / v) PS20.

[0269] Embodiment 58. A polynucleotide comprising a nucleic acid sequence that encodes the multimeric binding molecule of any one of embodiments 1 to 51, or a subunit thereof.

[0270] Embodiment 59. A vector comprising the polynucleotide of embodiment 58.

[0271] Embodiment 60. A host cell comprising the polynucleotide of embodiment 58 or the vector of embodiment 59, wherein the host cell can express the multimeric binding molecule of any one of embodiments 1 to 51.

[0272] Embodiment 61. A method for treating or preventing a human sarbecovirus disease in a subject in need of treatment comprising administering to the subject an effective amount of the multimeric binding molecule of any one of embodiments 1 to 51, wherein the multimeric binding molecule exhibits greater potency than a reference binding molecule.IGM Ref. No.002-056WO2

[0273] Embodiment 62. The method of embodiment 61, wherein the human sarbecovirus disease is severe acute respiratory syndrome (SARS) or coronavirus disease 2019 (COVID-19).

[0274] Embodiment 63. The method of embodiment 61 or embodiment 62, wherein the subject is human.

[0275] Embodiment 64. The method of any one of embodiments 61 to 63, wherein the administering comprises intravenous, subcutaneous, intramuscular, intranasal, and / or inhalation (pulmonary) administration.

[0276] This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press); Mullis et al. U.S. Pat. No.4,683,195; Hames and Higgins, eds. (1985) Nucleic Acid Hybridization (IRL Press); Hames and Higgins, eds. (1984) Transcription And Translation (IRL Press); Freshney (2016) Culture Of Animal Cells, 7th Edition (Wiley-Blackwell); Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985); Perbal (1988) A Practical Guide To Molecular Cloning; 2d Edition (Wiley-Interscience); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); S.C. Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science); Methods in Enzymology, Vols.151-155 (Academic Press, Inc., N.Y.); Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Weir and Blackwell, eds.; and in Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0277] General principles of antibody engineering are set forth, e.g., in Strohl, W.R., and L.M. Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). General principles of protein engineering are set forth, e.g., in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). General principles of immunology are set forth, e.g., in: Abbas and Lichtman (2017) Cellular and Molecular Immunology 9th Edition (Elsevier). Additionally, standard methods in immunology known in the art canIGM Ref. No.002-056WO2 be followed, e.g., in Current Protocols in Immunology (Wiley Online Library); Wild, D. (2013), The Immunoassay Handbook 4th Edition (Elsevier Science); Greenfield, ed. (2013), Antibodies, a Laboratory Manual, 2d Edition (Cold Spring Harbor Press); and Ossipow and Fischer, eds., (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press).

[0278] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0279] The following examples are offered by way of illustration and not by way of limitation. Examples Example 1: Generation of Binding Molecules for SARS-CoV-1 and SARS-CoV-2

[0280] Two ACE2 constructs were designed (FIG.1A): a 615 amino acid version of the ACE2 ectodomain (“615, mACE2” in FIG.1A) that lacks the residues necessary for dimerization; and a 740 amino acid version that forms ACE2 homodimers (“740, ACE2” in FIG.1A). Both constructs were incorporated into IgM and IgG1 scaffolds according to standard cloning protocols. The 615 amino acid ACE2 ectodomain is shown, joined to an IgG1 scaffold in FIGs.1B & 2B, and the 740 amino acid ACE2 ectodomain in FIGs.1D & 2A. A first IgM scaffold joins the ACE2 ectodomain directly to the IgM constant region (ACE2-M1, shown in FIG.2F and ACE2-M2, shown in FIG.2E). A second IgM scaffold connects the ACE2 ectodomain to the IgM constant region by means of a mutated IgG1 hinge region (SEQ ID NO: 5). ACE2-M3, shown in FIGs.1C & 2D and ACE2-M4, shown in FIGs.1E & 2C include this second IgM scaffold design. FIG.1C illustrates the arrangement of the IgM constant domains, the IgG1 hinge (“γHinge” in FIG.1C), and the ACE2 ectodomain. Because FIG.1C exemplifies a pentameric IgM chimera, the molecule also contains a J-chain, as indicated. A third scaffold connects the ACE2 ectodomain to the IgM constant region by means of a poly-glycine / serine linker (SEQ ID NO: 12). ACE2-M5, ACE2-M6, and ACE2-M8 include this third IgM scaffold design. A fourth scaffold design connects the ACE2 ectodomain to the IgM constant region by means of a linker derived from camel immunoglobulins (e.g., SEQ ID NO: 18 or SEQ ID NO: 34). In addition to the poly-glycine / serine linker, ACE2-M8 comprises three point mutations in the ACE2 ectodomain that affect angiotensin converting activity. The ACE2-M7 makes use of a truncated version of this camelid linker that contains onlyIGM Ref. No.002-056WO2 amino acids 2–28 of SEQ ID NO: 18 (SEQ ID NO: 34) to connect the 740 amino acid ACE2 to the IgM constant region, while ACE2-M9 uses the full SEQ ID NO: 18 linker to connect the 615 amino acid ACE2 to the IgM constant region. The correspondence between these constructs and their respective SEQ ID NOs are shown in Table 1. Table 1. Constructs Exemplified

[0281] The ACE2-IgM chimeric constructs were expressed, some in cells with an exemplary J-chain (SEQ ID NO: 7) and some in cells without the J-chain. The ACE2- IgM chimeras expressed with J-chains were able to form pentameric IgM molecules, while those without a J-chain formed hexameric IgM molecules (FIG.1A). The IgM and IgG antibody constructs were expressed and purified according to standard methods, e.g., as described in Keyt, BA, et al., Antibodies DOI: 3390 / antib9040053 (2020).IGM Ref. No.002-056WO2 Example 2: Binding Measured by ELISA

[0282] ELISA was used to determine how the IgM and IgG1 formats affect binding to SARS-CoV-2 RBD. ELISA assays used 96-well white polystyrene ELISA plates (Pierce 15042) coated with 100 µL per well of 0.5 µg / mL recombinant SARS-CoV-2 Delta strain RBD, Omicron BA.1 strain RBD, Omicron BA.2 strain RBD, and Omicron BA.3 strain RBD overnight at 4°C. Plates were then washed 5 times with 0.05% PBS-Tween and blocked with 2% BSA-PBS. After blocking, 100 µL of serial dilutions of pentameric ACE2-M1 IgM molecules, pentameric ACE2-M2 IgM molecules, pentameric ACE2-M3 IgM molecules, pentameric ACE2-M4 IgM molecules, hexameric ACE2-M4 IgM molecules, ACE2-G1 IgG molecules, ACE2-G2 IgG molecules, or an isotype control IgM molecule were added to the wells and incubated at room temperature for 2 hours. The plates were then washed 10 times and incubated with HRP conjugated mouse anti-human kappa (Southern Biotech, 9230-05; 1:6000 diluted in 2% BSA-PBS) for 30 min. After 10 final washes using 0.05% PBS-Tween, the plates were read out using SuperSignal chemiluminescent substrate (ThermoFisher, 37070). Luminescent data were collected on an EnVision plate reader (Perkin-Elmer).

[0283] While the control showed no detectable binding, all of the ACE2-G series and ACE2-M series molecules showed similar binding. Binding of a sample of these molecules to Delta strain RBD is shown in FIG.3A, to BA.1 RBD in FIG.3B, to BA.2 RBD in FIG.4A, and binding to BA.3 RBD is shown in FIG.4B. The EC50 values for the binding of the four ACE2 constructs to the RBD of each of the WT D614G, the Omicron BA.1, and the Omicron BA.2 strains are shown in Table 2.

[0284] The binding kinetics of select constructs against the Omicron B.1.1.529 RBD were measured by bio-layer interferometry (BLI) on an OCTET system (Creative Biolabs, Shirley, NY). The binding kinetics values are shown in Table 3.IGM Ref. No.002-056WO2Example 3: Pseudovirus Neutralization Assay

[0285] A pseudovirus neutralization assay was used to determine how the IgM and IgG1 formats affect neutralization of pseudoviruses comprising the SARS-CoV-2 (Wuhan-Hu- 1 / D614G) spike protein. The assay was performed generally as described in Richman et al. (2003) PNAS 100(7):4144–49, in which host cells that have been infected with an HIV-based pseudovirus express luciferase. The pseudovirus was engineered to express Wuhan-Hu-1 / D614G strain spike proteins in place of HIV gp160. Various concentrations of ACE2-G1, ACE2-G2, pentameric ACE2-M3, and pentameric ACE2-M4 were incubated with the pseudovirus for 1 hr. at 37 °C. HEK-293 cells expressing human ACE- 2 were then inoculated with the antibody / pseudovirus solution. The amount of luciferase activity in the infected cells was measured 72 h post-inoculation to determine the neutralizing activity. A decrease in luciferase activity indicated neutralizing activity by the binding molecule assayed. Neutralizing activity was calculated as the percent inhibition of viral replication (luciferase activity) at each dilution compared with negative control. The IC50 and fold improvement for each IgM-based molecule over its corresponding IgG1-based molecule are shown in Table 4. Table 4: Pseudovirus Neutralization Results

[0286] The monomeric ACE2-IgG1 chimera (ACE2-G1) was not able to neutralize the pseudoviruses, so it is not possible to compute a fold improvement relative to the corresponding IgM chimera. The monomeric ACE2-IgM chimera, however, showed the strongest IC50value (9 ng / mL) in this assay of any molecule tested. The dimeric ACE2- IgM chimera showed an 11.4-fold IC50 improvement in neutralization potency relative to the corresponding IgG1 chimera.IGM Ref. No.002-056WO2 Example 4: Pseudovirus Neutralization Assay

[0287] Another set of pseudovirus neutralization assays were performed as in Example 3 above. Various concentrations of ACE2-G1, ACE2-G2, pentameric ACE2-M1, pentameric ACE2-M2, pentameric ACE2-M3, pentameric ACE2-M4, pentameric ACE2- M5, pentameric ACE2-M6, pentameric ACE2-M7, pentameric ACE2-M8, and CoV-2-14 IgM antibody (an anti-SARS-CoV-2 spike protein antibody described in US application: 2022 / 0267415, which is hereby incorporated by reference herein in its entirety) were tested against pseudoviruses expressing the S-protein from Wuhan-Hu-1 / D614G and from Omicron BA.2. A representative selection of neutralization curves against the Wuhan-Hu-1 / D614G strain are shown in FIG.5A, and against the Omicron BA.2 strain in FIG.5B. The IC50 of each molecule and the fold improvement of the ACE2-M molecule relative to its corresponding ACE2-G molecule are shown in Table 5. Table 5: Pseudovirus Neutralization Results

[0288] While all ACE-M chimeras show broadly similar neutralizing potencies against both the pseudovirus bearing the Wuhan-Hu-1 / D614G strain S-protein and the pseudovirus bearing the Omicron BA.2 strain S-protein, the CoV-2-14 IgM antibody was strongly neutralizing against the Wuhan-Hu-1 / D614G pseudovirus but was not active against the Omicron BA.2 pseudovirus. The monomeric ACE2-IgM chimeras were generally more potent neutralizers than the dimeric ACE2-IgM chimera, although there is variability observed among both sets of molecules. In every instance, the monomeric ACE2-IgM chimera was more potent than its dimeric match (compare, e.g., ACE2-M1 vs. ACE2-M2, ACE-M3 vs. ACE2-M4, and ACE2-M5 vs. ACE2-M6). The monomericIGM Ref. No.002-056WO2 ACE2-IgM chimeras also showed reproducibly higher fold improvement against their corresponding ACE2-IgG chimera than did the dimeric ACE2-IgM chimeras compared to their corresponding ACE2-IgG chimera.

[0289] In order to assess the effects of the different linker domains from Table 1 and the effects of forming the ACE2-IgM chimeras as hexamers instead of pentamers, a similar test was run against the Wuhan-Hu-1 / D614G and Omicron BA.2 pseudoviruses as above, with various dilutions of pentameric ACE2-M4, hexameric ACE2-M4, pentameric ACE2-M5, pentameric ACE2-M7, and pentameric ACE2-M9. The neutralization curves against Wuhan-Hu-1 / D614G pseudovirus are shown in FIG.6. The IC50of each molecule relative to the Wuhan-Hu-1 / D614G and Omicron BA.2 pseudoviruses are shown in Table 6. Table 6: Pseudovirus Neutralization Results (IC50, ng / mL)

[0290] While the hexameric ACE2-M4 was more potent than the pentameric ACE2-M4 against the Wuhan-Hu-1 / D614G pseudovirus, the difference between pentamer and hexamer was less clear in the Omicron BA.2 pseudovirus assay. The choice of linker did not make a notable difference for the potency against either pseudovirus.

[0291] A third pseudovirus assay was conducted according to the protocols above, using a reporter virus particles from Integral Molecular (Philadelphia, Pennsylvania). Pseudoviruses expressing the S-protein from Wuhan-Hu-1 / D614G, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4 / 5 were tested. The results are shown in Table 7. Table 7: Pseudovirus Neutralization Results (IC50, ng / mL)IGM Ref. No.002-056WO2

[0292] The hexameric ACE2-M4 was more potent than the pentameric ACE2-M4 against all strains of pseudovirus tested. The ACE2-IgM chimeras were also reproducibly more potent than were the ACE2-IgG1 chimeras against all pseudovirus strains tested.

[0293] Finally a fourth pseudovirus neutralization trial was conducted using the same protocol as described immediately above, except with a SARS-CoV-1 strain (Urbani) and more SARS-CoV-2 strains: WT D614G, Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron BA.2.75, and Omicron BA.4 / 5. The results from this experiment were consistent with thefirst pseudovirus neutralization trial. These results are shown in FIG.7 and summarized in Table 8. Table 8. IC50(ng / mL) of pseudovirus neutralization against SARS-CoV-2 and SARS-CoV-1 variantsExample 5: Live Virus Neutralization Assay

[0294] All microneutralization assays using SARS-CoV-2 were conducted at the University of Vermont BSL-3 facility under an approved Institutional Biosafety protocol. SARS-CoV-2 strain 2019-nCoV / USA_USA WA1 / 2020 (WA1), strain hCoV- 19 / USA / PHC658 / 2021 (B.1.617, Delta Variant), and strain hCoV-19 / USA / MD- HP20874 / 2021 (B.1.1.529, subvariant BA.1, Omicron Variant) were propagated in VeroIGM Ref. No.002-056WO2 E6 cells. The microneutralization assay used low passage Vero E6 cells. Vero E6 cells were maintained in complete Dulbecco’s Modified Eagle Medium (cDMEM) containing 10% fetal bovine serum (FBS), 1% HEPES Buffer Solution, and 1% penicillin- streptomycin, all purchased from Thermo Fisher Scientific (Carlsbad, CA). Cells were grown in a humidified incubator at 37ºC with 5% CO2.

[0295] Each binding molecule sample was diluted in 50 µL of cDMEM. Binding molecule concentrations tested ranged from 50µg / mL to 0.00075ng / mL, in serial 1:4 dilutions. Diluted samples were mixed with 50μL of cDMEM containing 175 focus forming units of WA1, Delta or Omicron and then incubated for 45 minutes at 37°C.

[0296] To inoculate, the media from confluent Vero E6 cell monolayers in 96-well white polystyrene microplates (Thermo Fisher Scientific) were removed and 50 µL of each antibody-virus mixture was inoculated onto the cells in duplicate technical replicates. The inoculated cells were incubated at 37°C in 5% CO2 for 30 minutes, after which the wells were overlaid with 150μL of warmed 1.2% methylcellulose in cDMEM. The methylcellulose covered cells were incubated at 37°C in 5% CO2for 24 hr.

[0297] After 24hr, infected cells were fixed in 4% formaldehyde in 3× phosphate buffered saline (PBS). Cells were permeabilized with 0.1% 100× Triton in 1× PBS for 15 min. at room temperature and then incubated with a primary, cross-reactive rabbit anti-SARS- CoV N monoclonal antibody (40143-R001, Sinobiological) (1:20,000) for 45 min. at 37°C. Following a wash, the cells were incubated with peroxidase-labeled goat anti-rabbit antibody (5220-0336, SeraCare) (1:2,000) for 30 minutes at 37°C. Following a wash, peroxidase substrate (5510-0030, SeraCare) was added to develop blue viral foci. Images of the wells were captured using the CTL Immunospot S6 Universal Analyzer. Viral foci were then quantified, with foci counts normalized to virus only control wells on each plate. IC50determinations were made using a non-linear regression curve fit ([inhibitor] vs. normalized response – variable slope) in GraphPad Prism.

[0298] The IC50 for each molecule and the fold improvement of the ACE2-IgM chimera relative to its corresponding ACE2-IgG1 chimera are shown in Table 9.IGM Ref. No.002-056WO2 Table 9: Neutralization Results (IC50, ng / mL)

[0299] All ACE2-IgM chimeras tested neutralized both strains of SARS-CoV-2 at ng / mL levels. The monomeric ACE2-IgM chimeras show greater fold improvement than the dimeric ACE2-IgM chimeras, each relative to its corresponding ACE2-IgG1 chimera. Nevertheless, the monomeric and dimeric ACE2-IgM chimeras were broadly similar in their IC50 values for each pair tested (see, e.g., ACE2-M1 vs. ACE2-M2 or ACE2-M3 vs. ACE2-M4). Just as with the Omicron pseudoviruses in Example 3 above, the CoV-2-14 IgM antibody showed little potency against the Omicron BA.1 virus.

[0300] A second neutralization trial was conducted using the same protocol as described immediately above, except with a smaller set of ACE2 constructs and with two Omicron strains (BA.1 and BA.2) but without Delta. The results from this experiment were consistent with thefirst neutralization trial. These results are shown in FIG.8 and summarized in Table 10.IGM Ref. No.002-056WO2 Example 6: In vivo prophylactic potency against Omicron

[0301] K18-hACE2 transgenic mice express human ACE2 under the control of the human keratin 18 promoter, which directs expression to epithelia. K18-hACE2 mice are susceptible to SARS-CoV-2 and SARS-CoV-1 viruses.

[0302] Four cohorts of four K18-hACE2 mice each were dosed intranasally with ACE2- M3: one at 0.625 mg / kg, one at 1.25 mg / kg, one at 2.5 mg / kg, and one at 5 mg / kg. Another four-mouse cohort was dosed with control IgM and another cohort with phosphate buffered saline (PBS). All cohorts were dosed 6 hours before intranasal delivery of 7.0×103pfu SARS-CoV-2 virus (Omicron BA.2). The mice were weighed daily for the next two weeks. On day-4 post virus, the lungs were lavaged to collect virus. Virus samples were titered on Vero-TMPRSS2 cells.

[0303] By day-7 post challenge, the average body weight of the control mice had declined ~13% (FIG.9A) relative to the day of viral dosing, while the average body weight of the ACE2-M3 mice had grown slightly. By day-14 post virus, none of the ACE2-M3 mice had died, while ~40% of the control mice had died (FIG.9B).

[0304] Viral titers recovered from the lungs of the control mice were significantly higher than those recovered from the lungs of the ACE2-M3 mice (FIG.11A). Titers from mice treated with different doses of ACE2-M3 were about 100-fold lower in most of the dose groups except 1.25 mg / kg with p=0.115, p=0.009, and p=0.017 for 5.0 mg / kg, 2.5 mg / kg, and 0.625 mg / kg dose group, respectively. The relatively high p value for 5.0 mg / kg group was reflected by the variation of the lung titer. The 1.25 mg / kg dose group showed less lung viral titer reduction comparing with the control group and was not statistically different (p=0.100) (FIG.11A). Example 7: In vivo prophylactic potency against Wuhan

[0305] Two cohorts of four K18-hACE2 mice each were dosed intranasally with ACE2- M3: one at 0.5 mg / kg and another at 5 mg / kg. Another four-mouse cohort was dosed with control IgM (5 mg / kg). All cohorts were dosed 6 hours before intranasal delivery of 103pfu SARS-CoV-2 virus (Wuhan-Hu-1 / D614G). The mice were weighed daily for the next two weeks. On day-4 post virus, the lungs were lavaged to collect virus. Virus samples were titered on Vero-TMPRSS2 cells.IGM Ref. No.002-056WO2

[0306] By day-7 post challenge, the average body weight of the control D614G-infected mice declined ~20% (FIG.9C) relative to the day of viral dosing. In contrast, the average body weight of the ACE2-M3-treated mice had declined less than ~5% on average.

[0307] Viral titers recovered from the lungs of the control mice were a million-fold higher (p<0.0001) than those recovered from the lungs of their counterparts that had been pre- treated with ACE2-M3 (FIG.11C). The titers from both cohorts of ACE2-M3-treated mice were at the lower limit of detection, so it is not possible to know whether there were meaningful differences between the 0.5 mg / kg and 5.0 mg / kg cohorts. Example 8: In vivo therapeutic potency

[0308] Six cohorts of four K18-hACE2 mice each were dosed intranasally with 7.0×103pfu SARS-CoV-2 virus (Omicron BA.2) each. Six hours later, four of those cohorts were dosed with ACE2-M3: one at 0.625 mg / kg, one at 1.25 mg / kg, one at 2.5 mg / kg, and one at 5 mg / kg. Another four-mouse cohort was dosed with both control IgM 6 hours post- infection, and another cohort with PBS 6 hours post-infection. The mice were weighed daily for the next two weeks. On day-4 post virus, the lungs were lavaged to collect virus. Virus samples were titered on Vero-TMPRSS2 cells.

[0309] By day-7 post challenge, the average body weight of the control mice had declined 20% (FIG.10A) relative to the day of viral dosing, while the average body weight of the ACE2-M3 mice remained steady. By day-14 post virus, none of the ACE2-M3 mice had died, while ~70% of the control mice had died (FIG.10B).

[0310] The lung viral titer was significantly lower in mice treated with various doses of ACE2-M3 than the mice receiving the IgM control with a p=0.041 for multiple group comparison (FIG.11B), and p=0.039, p=0.019, p=0.050, and p=0.007 for 5.0 mg / kg, 2.5 mg / kg, 1.25 mg / kg, and 0.625 mg / kg dose group, respectively, when compared individually with the IgM control group (FIG.11B). The lung titers from PBS and IgM controls did not differ (p=0.822). Example 9: Nebulization of ACE2-M3

[0311] A solution of ACE2-M3 at 1.98 mg / mL and pH 6.5 in 20 mM Histidine, 300 mM NaCl, 2%(w / v) sucrose was buffer exchanged using PD-10 columns (CYTIVA®) into the following solutions: a) - 10 mM sodium acetate, 5%(w / v) sorbitol, 0.02%(w / v) PS20, pH 4.0; b) 20 mM Histidine, 300 mM NaCl, 2%(w / v) sucrose, 0.01%(w / v) PS20, pH 6.5; andIGM Ref. No.002-056WO2 c) in 20mM Histidine, 300 mM NaCl, 2%(w / v) sucrose, 0.02%(w / v) PS20, pH 6.5. These three solutions were assayed as follows: 1) concentration & turbidity (OD350) determined by THERMO SCIENTIFIC NANODROP®; 2) osmolality determined by ADVANCED INSTRUMENTS OMSOTECH XT®; 3) viscosity determined in an ANTON PAAR® rolling ball viscometer; 4) aggregation & degradation determined by AGILENT 1260 INFINITY II SEC purity assay; and 5) hydrodynamic size and aggregation determined by WYATT NANOSTAR® II DLS. Samples of the solutions were then diluted to a 1mg / mL concentration with respective buffers and were atomized with a TELEFLEX® MAD Nasal™ syringe atomizer and nebulized using AEROGEN SOLO® vibrating-mesh nebulizer. The atomized and nebulized material was collected and characterized by all of assays # 1–5.

[0312] The results of assays #1–3 are shown in Table 11. The results of assay #4 are shown in Table 12. The results of assay #5 are shown in FIGs.12–14. Nebulization resulted in loss of protein concentration of all tested formulations relative to atomization. Turbidity, osmolality, and viscosity did not differ appreciably between the nebulized and atomized samples. Nebulization also increased the aggregate (HMW) and degradation (LMW) content of the pH 4.0 solution (Table 12), and changed the hydrodynamic size distributions of the three buffer-exchanged formulations (FIGs.12A, 13A, & 14A) into much more heterogenous distributions and generated aggregates (FIGs.12C, 13C, & 14C). By contrast, atomization had little effect on hydrodynamic size distribution compared to the control.

[0313] In summary, none of solutions (a), (b), or (c) were acceptable for nebulized delivery of the ACE2-M3 molecule. It took 10 min. & 40 sec. to nebulize solution (a) completely, 10 min. & 42 sec. to nebulize solution (b) completely, and 14 min. & 59 sec. to nebulize solution (c) completely.IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2

[0314] Two additional solutions of ACE2-M3 were then prepared in 20 mM histidine, 300 mM NaCl, 2%(w / v) sucrose, 0.2%(w / v) PS20, pH 6.5 at d) 1 mg / mL and e) 5.0 mg / mL. These were prepared by spiking PS20 solution in the drug substance solution to achieve 0.2% w / v in the formulations. These two were also nebulized, and the nebulized material collected for analysis by assays #1, 4, & 5 above. The results of #1 & #4 are shown in Table 13. The results of #5 are shown in FIG.15.IGM Ref. No.002-056WO2

[0315] Nebulization had almost no discernible effect on solutions d) or e) by any of the assays. The solutions with 0.2%(w / v) PS20 surfactant content were tolerant of nebulization. Notably, the hydrodynamic size distribution of the pre- and post- nebulization solutions were nearly identical to each other. Solution d) required only 9 min. & 4 sec. to nebulize completely, while solution e) required only 7 min.

[0316] When these pre- and post-nebulization samples were assayed for their abilities to bind to Omicron BA.5 RBD (see Example 2, above), the pre-nebulization sample showed an EC50of 45.94 ng / mL, while the post-nebulization sample EC50value was 45.48 ng / mL. Similarly, when these samples were assayed for their abilities to neutralize HIV pseudotyped with Omicron BA.5 spike protein (see Example 3, above), the pre- nebulization sample showed an IC50of 0.3093 ng / mL, while the post-nebulization sample IC50 value was 0.2416 ng / mL.

[0317] In order to define more precisely the limits of this protective effect and minimum effective concentration from surfactant, a series of 5 mg / mL ACE2-M3 formulations were made that were identical to the 5 mg / mL formulation of Table 13, except with a series of concentrations between 0.02% and 0.2%(w / v). These were prepared by spiking PS20 solution in the drug substance solution to achieve 0.05% w / v, 0.10% w / v and 0.15%w / v PS20 in the formulations. These different formulations were then nebulized as above and subjected to the same assays for concentration, clarity, aggregation, and degradation. The details of these formulations and the results of the assays are shown below in Table 14. FIG.16 shows the results of the DLS analysis. While higher order aggregates appear at peaks of 160.91 nm and 346.52 nm in the nebulized plot of FIG.16A (0.05%w / v PS20),IGM Ref. No.002-056WO2 no higher order aggregates are visible in FIGs.16B (0.10%w / v PS20) and 16C (0.15%w / v PS20).

[0318] In order to determine whether the other surfactants can protect ACE2-M3 stability as well as PS20 does, 1 mg / mL ACE2-M3 were prepared with either polysorbate 80 (PS80) at 0.04%w / v PS80 and 0.1%w / v PS80 or poloxamer 188 (P188) at 0.1%w / v PS80. These different formulations were then nebulized as above and subjected to the same assays for concentration, clarity, aggregation, and degradation. The details of these formulations and the results of the assays are shown below in Table 15 and FIG.17. While higher order aggregates appear at peaks of 169.92 nm and 896.32 nm in the nebulized plot of FIG.17A (0.04% w / v PS80), no higher order aggregates are visible in FIGs.17B (0.1%w / v PS80) and 17C (0.1%w / v P188). These data suggest that the concentration of the surfactant plays a more important role for nebulization than the surfactant type. All the surfactants mentioned above were able to successfully nebulize ACE2-M3 at 0.1%w / v in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, pH 6.5.IGM Ref. No.002-056WO2Example 10: Generation of Additional Binding Molecules

[0319] Additional binding molecules were generated as described in Example 1 each comprising a point mutation in the ACE2 domain that is involved in substrate and Zinc binding. The different mutant constructs generated are described in Table 16. Table 16. Constructs ExemplifiedIGM Ref. No.002-056WO2 Example 11: Enzymatically inactive ACE2

[0320] In order to test whether the multimeric binding molecule would retain antiviral potency absent the ACE2 enzymatic activity, the mutant multimeric binding molecules described in Table 16 were tested compared to ACE2-M3, according to the procedure described in Example 3 above, for their ability to neutralize an HIV pseudovirus engineered to express an Omicron XBB.1 strain S protein. The results of this neutralization assay are shown in Table 17 below.

[0321] The ACE2 enzymatic activity of ACE2-M12, ACE2-M11, and ACE2-M10 were all confirmed to be ablated for ACE2 physiological substrate, angiotensin II (data not shown). Example 12: Stability of Mutant ACE2 Binding Molecules

[0322] In order to determine how well ACE2-M12, ACE2-M11, and ACE2-M10 could tolerate storage under temperature stress, samples of each of these three as well as a control sample of ACE2-M3 were stored in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.2%(w / v) PS20, pH 6.5 for 2 weeks at 4°C, 1 week at 25°C, 2 weeks at 25°C, 1 week at 37°C, and 2 weeks at 37°C. At the end of the specified storage, the samples were assayed for aggregation and degradation by SEC as in Example 8 above. The results are shown in Table 18 below.IGM Ref. No.002-056WO2

[0323] After the indicated periods of storage, these formulations were also tested for their abilities to bind to the Omicron BA.4 / 5 strain RBD (see Example 2, above) and to neutralize HIV pseudotyped with the Omicron BA.4 / 5 spike protein (see Example 3, above). The results of these assays are shown in Table 19 below.

[0324] Finally, samples of ACE2-M10, ACE2-M11, ACE2-M12, and a control sample of ACE2-M3—each in 20 mM His, 300 mM NaCl, 2%(w / v) sucrose, 0.2%(w / v) PS20, pH 6.5—were nebulized. Pre- and post-nebulization samples were assayed for protein concentration, clarity, aggregation, and degradation as in Example 8 above. DLS results are shown in FIG.18, with the upper row representing pre-nebulization samples, and theIGM Ref. No.002-056WO2 post-nebulization sample for each mutant immediately below. Concentration and clarity results are shown in Table 20.

[0325] In order to assess the role of pH in promoting or inhibiting aggregation, two separate solutions of 1 mg / mL ACE2-M3 were prepared. Thefirst solution comprised 20 mM histidine, 300 mM NaCl, 2%(w / v) sucrose, and 0.02%(w / v) PS20 at pH 6.5. The second solution comprised 10 mM sodium acetate, 5%(w / v) sorbitol, and 0.02%(w / v) PS20 at pH 4.0. Twenty microliters of each solution were loaded into cuvettes for measurement in a ZETASIZER PRO (Malvern Panalytical, United Kingdom).

[0326] The experiment was started at 25°C and ramped at 5C° increments up to a maximum temperature of 75°C, and then dropped back to 25°C. Each temperature step was held for an equilibration time of 60 seconds. The hydrodynamic size data were collected at each temperature.

[0327] The hydrodynamic size data were plotted against the temperature and analyzed for the increase in hydrodynamic size—i.e., the onset of aggregation (Tonset). The results are shown in FIG.19. The ACE2-M3 aggregated into aggregates with average diameters in excess of 2500 nm in the low pH buffer (FIG.19A), as compared to average diameters no greater than 250 nm in neutral pH buffer (FIG.19B), which shows the stability imparted by the formulation at neutral pH.IGM Ref. No.002-056WO2 Table 21: Sequences of the DisclosureIGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2IGM Ref. No.002-056WO2

Claims

IGM Ref. No.002-056WO2 CLAIMS WHAT IS CLAIMED IS:

1. A multimeric binding molecule comprising five or six bivalent binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof associated with a variant of human angiotensin-converting enzyme 2 (ACE2) ectodomain consisting of amino acids 18 to 615 of SEQ ID NO: 14 or amino acids 19 to 615 of SEQ ID NO: 14, wherein the variant ACE2 ectodomain comprises a substitution of the amino acid corresponding to R237, H345, H378, or E375 of SEQ ID NO:

14.

2. The multimeric binding molecule of claim 1, wherein the ACE2 ectodomain consists of amino acids 19 to 615 of SEQ ID NO: 14 except for the substitution.

3. The multimeric binding molecule of claim 1, wherein the ACE2 ectodomain consists of amino acids 18 to 615 of SEQ ID NO: 14 except for the substitution.

4. The multimeric binding molecule of any one of claims 1 to 3, wherein the ACE2 ectodomain comprises: (a) a glutamine (Q), a leucine (L), a lysine (K), or an alanine (A) substitution of the amino acid corresponding to R237 of SEQ ID NO: 14, (b) an L, glycine (G), or proline (P) substitution of the amino acid corresponding to H345 of SEQ ID NO: 14, (c) an L, G, or asparagine (N) substitution of the amino acid corresponding to H378 of SEQ ID NO: 14, or (d) an L, A, or Q substitution of the amino acid corresponding to E375 of SEQ ID NO:

14.

5. The multimeric binding molecule of claim 4, wherein the ACE2 ectodomain comprises an L substitution of the amino acid corresponding to E375 of SEQ ID NO:

14.

6. The multimeric binding molecule of any one of claims 1 to 5, wherein each IgM heavy chain constant region or multimerizing fragment or variant thereof comprises an IgM Cµ4 domain and an IgM tailpiece domain.

7. The multimeric binding molecule of claim 6, wherein each IgM heavy chain constant region or multimerizing fragment or variant thereof further comprises an IgM Cµ3 domain situated N-terminal to the IgM Cµ4 and IgM tailpiece domains.

8. The multimeric binding molecule of claim 7, wherein each IgM heavy chain constant region or multimerizing fragment or variant thereof further comprises an IgM Cµ2 domain situated N-terminal to the IgM Cµ3 domain.IGM Ref. No.002-056WO2 9. The multimeric binding molecule of any one of claims 6 to 8, wherein each IgM heavy chain constant region is a human IgM constant region comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a multimerizing fragment or variant thereof.

10. The multimeric binding molecule of claim 9, wherein each IgM heavy chain constant region is a variant multimerizing fragment of a human IgM heavy constant region that includes the Cµ3, Cµ4, and tailpiece domains, wherein each variant multimerizing fragment consists essentially of amino acids 217 to 453 of SEQ ID NO:

35.

11. The multimeric binding molecule of claim 10, wherein each heavy chain further comprises a variant human IgG1 hinge region fused to the N-terminus of the variant multimerizing fragment of the human IgM constant region, wherein the variant human IgG1 hinge region consists of SEQ ID NO:

5.

12. The multimeric binding molecule of claim 11, comprising the amino acids 18 to 870 of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO:

32.

13. The multimeric binding molecule of claim 12, which comprises 18 to 870 of SEQ ID NO:

30.

14. The multimeric binding molecule of any one of claims 1 to 13 which comprises five bivalent binding units, and further comprises a J chain or functional fragment or variant thereof.

15. The multimeric binding molecule of claim 14, wherein the J chain is a human J chain comprising the amino acid sequence SEQ ID NO:

7.

16. The multimeric binding molecule of claim 14, wherein the J chain is a variant human J chain comprising the amino acid sequence SEQ ID NO:

8.

17. The multimeric binding molecule of claim 14, wherein the J chain or functional fragment or variant thereof is a modified J chain, and further comprises a heterologous polypeptide, wherein the heterologous polypeptide is directly or indirectly fused to the J chain or functional fragment or variant thereof.

18. The multimeric binding molecule of claim 17, wherein the heterologous polypeptide is fused to the J chain or fragment thereof via a peptide linker.

19. The multimeric binding molecule of claim 17 or claim 18, wherein the heterologous polypeptide is fused to the N-terminus of the J chain or fragment or variant thereof, the C-terminus of the J chain or fragment or variant thereof, or to both the N- terminus and C-terminus of the J chain or fragment or variant thereof.IGM Ref. No.002-056WO2 20. The multimeric binding molecule of any one of claims 1 to 19, which can neutralize infectivity of a human sarbecovirus at a lower 50% effective concentration (EC50) than a reference binding molecule, wherein the reference binding molecule is a single bivalent binding unit comprising two heavy chains, each comprising an IgG heavy chain constant region or fragment or variant thereof fused to a human angiotensin-converting enzyme 2 (ACE2) ectodomain.

21. The multimeric binding molecule of claim 20, wherein each heavy chain comprises amino acids 18 to 973 of SEQ ID NO: 23 or amino acids 18 to 848 of SEQ ID NO:

24.

22. The multimeric binding molecule of claim 20 or claim 21, wherein the EC50 is at least two-fold, at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500-fold, or at least 1000-fold lower than the EC50 of the reference binding molecule.

23. The multimeric binding molecule of any one of claims 20 to 22, which can inhibit binding of the human sarbecovirus to its receptor at a lower 50% inhibitory concentration (IC50) than the reference binding molecule.

24. The multimeric binding molecule of claim 23, wherein the IC50 is at least two-fold, at least five-fold, at least ten-fold, at least fifty-fold, at least 100-fold, at least 500- fold, or at least 1000-fold lower than the IC50 of the reference binding molecule.

25. The multimeric binding molecule of any one of claims 20 to 24, wherein the human sarbecovirus is SARS-CoV or SARS-CoV-2.

26. A composition comprising the multimeric binding molecule of any one of claims 1 to 25.

27. A polynucleotide comprising a nucleic acid sequence that encodes the multimeric binding molecule of any one of claims 1 to 25, or a subunit thereof.

28. A vector comprising the polynucleotide of claim 27.

29. A host cell comprising the polynucleotide of claim 27 or the vector of claim 28, wherein the host cell can express the multimeric binding molecule of any one of claims 1 to 25.

30. A method for treating or preventing a human sarbecovirus disease in a subject in need of treatment comprising administering to the subject an effective amount of the multimeric binding molecule of any one of claims 1 to 25, wherein the multimeric binding molecule exhibits greater potency than reference binding molecule.IGM Ref. No.002-056WO2 31. The method of claim 30, wherein the human sarbecovirus disease is severe acute respiratory syndrome (SARS) or coronavirus disease 2019 (COVID-19).

32. The method of claim 30 or claim 31, wherein the subject is human.

33. The method of any one of claims 30 to 32, wherein administering comprises intravenous, subcutaneous, intramuscular, intranasal, and / or inhalation administration.