Multivalent protein complexes und uses thereof

Abstract

Provided herein are immunoglobulin M (IgM) scaffolds as well as protein complexes comprising both an IgM scaffold and one or more single-domain antigen-binding fragment (sdAb) conjugates. Also provided are methods of using the protein complexes disclosed herein for the treat of disease, including for the treatment of infection or cancer.

Description

MULTIVALENT PROTEIN COMPLEXES UND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is an International Patent Application, which claims priority to U.S. Provisional Application No. 63 / 556,942, filed on February 23, 2024, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R35 GM137905, R01 AI163011, R01 HL169500, and R24 GM154185, awarded by the National Institutes of Health, under UL1TR004419, awarded by the National Center for Advancing Translational Sciences, and under 75N93021C00014 awarded by the Center for Research on Influenza Pathogenesis and Transmission. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on February 18, 2025, is named SeqList-084284-00310.xml and is 63,880 bytes in size. FIELD

[0004] The present disclosure relates generally to the field of molecular biology and medicine. More specifically, the disclosure relates to protein complexes that bind antigens. BACKGROUND

[0005] Antibodies are used as treatments for various diseases, including cancers and infectious diseases. These therapeutic agents can target specific cells or proteins, helping to modulate the immune response or directly attack diseased cells.

[0006] With respect to treating infectious diseases, pathogens such as viruses and bacteria often have high genetic diversity and share the ability to develop escaping mutations that undermine the effectiveness of host immunity and therapeutics. As such, the development of durable countermeasures can benefit from targeting multiple neutralizing and ideally 168506328.1evolutionarily conserved epitopes. Few mutations are observed on these sites as they may compromise pathogen fitness. While monoclonal antibodies can provide high specificity against their targets, these antibodies require frequent updates against evolving variants. A such, broad spectrum and long-lasting therapeutics for the treatment of infectious disease are urgently needed.

[0007] With respect to cancer therapies, monoclonal antibodies can be designed to target specific antigens found on the surface of cancer cells. By binding to these antigens, antibodies may directly inhibit cancer cell growth, mark cancer cells for destruction, or deliver cytotoxic agents to cancer cells. However, many cancers (including metastatic tumors, solid tumors, cancers that employ immune evasion strategies, or resistant tumors), are often difficult to treat with monoclonal antibodies. As such, new approaches to cancer treatment are urgently needed. SUMMARY

[0008] Provided herein is a protein complex including: (a) an immunoglobulin M (IgM) scaffold including IgM Cµ2, Cµ3, and Cµ4 domains and a J chain; and (b) one or more single- domain antigen-binding fragment (sdAb) conjugates, wherein: the N-termini of one or more of the IgM Cµ2 domains are each fused to a copy of a first hybridization domain; each of the one or more sdAb conjugates comprises a second hybridization domain and one or more sdAbs; and the first hybridization domain forms a covalent bond with the second hybridization domain.

[0009] In one embodiment, the N-termini of each of the IgM Cµ2 domains are each fused to a copy of the first hybridization domain.

[0010] In one embodiment, the protein complex comprises 10 sdAb conjugates.

[0011] Provided herein is a protein complex, wherein: (a) the first hybridization domain comprises a SpyCatcher domain or variant thereof and the second hybridization domain comprises a SpyTag domain or variant thereof; or (b) the first hybridization domain comprises a SpyTag domain or variant thereof and the second hybridization domain comprises a SpyCatcher domain or variant thereof.

[0012] Provided herein is a protein complex, wherein: (a) the first hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:10, 11, 61, or 62 or a portion thereof and the second hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:1, 2, or 54-60 or a portion thereof; or (b) the first hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:1, 2, or 54-60 or a portion thereof and the second hybridization domain comprises a 2 168506328.1sequence that is at least 80% identical to any one of SEQ ID NOs:10, 11, 61 or 62 or a portion thereof.

[0013] In some embodiments, the first hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:10 or SEQ ID NO:11 and the second hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:1 or SEQ ID NO:2, optionally, wherein the first hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:10 and the second hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:2.

[0014] Provided herein is a protein complex comprising a first and a second hybridization domain, wherein: (a) the first hybridization domain comprises SEQ ID NO:10 or SEQ ID NO:11 and the second hybridization domain comprises SEQ ID NO:1 or SEQ ID NO:2; or (b) the first hybridization domain comprises SEQ ID NO:1 or SEQ ID NO:2 and the second hybridization domain comprises SEQ ID NO:10 or SEQ ID NO:11.

[0015] In some embodiments, the first hybridization domain comprises SEQ ID NO:10 or SEQ ID NO:11 and the second hybridization domain comprises SEQ ID NO:1 or SEQ ID NO:2, optionally wherein the first hybridization domain comprises SEQ ID NO:10 and the second hybridization domain comprises SEQ ID NO:2.

[0016] In some embodiments, each sdAb conjugate comprises at least two sdAbs.

[0017] In some embodiments, the at least two sdAbs are connected to each other with a first linker, optionally, wherein the first linker is a polypeptide linker.

[0018] In some embodiments, the IgM Cµ2 domains are each fused to the copy of a first hybridization domain via a second linker, optionally, wherein the second linker is a polypeptide linker.

[0019] In some embodiments, the second hybridization domain is fused to one of the one or more sdAbs via a third linker, optionally, wherein the third linker is a polypeptide linker.

[0020] In some embodiments, the first linker comprises a sequence that is at least 80% identical to SEQ ID NO:52.

[0021] In one embodiment, the first linker comprises SEQ ID NO:52.

[0022] In some embodiments, at least some of the one or more sdAb conjugates include two or more sdAbs that bind to the same epitope.

[0023] In some embodiments, at least some of the one or more sdAb conjugates include two or more sdAbs that bind to different epitopes.

[0024] In some embodiments, the one or more sdAb conjugates are identical to each other. 3 168506328.1

[0025] In some embodiments, the one or more sdAbs bind to an epitope derived from a virus, a bacterium, a fungus, or a protozoan.

[0026] In some embodiments, the one or more sdAbs bind to epitopes derived from human immunodeficiency virus (HIV), influenza virus, SARS-CoV-2, or hepatitis virus.

[0027] In some embodiments, at least some of the one or more sdAb conjugates each include a sequence that is at least 80% identical to any one of SEQ ID NO:13-37.

[0028] In some embodiments, at least some of the one or more sdAb conjugates each include any one of SEQ ID NO:13-37.

[0029] In some embodiments, least some of the one or more sdAb conjugates each comprises a sequence that is at least 80% identical to any one of SEQ ID NO:38-51.

[0030] In some embodiments, at least some of the one or more sdAb conjugates each comprises any one of SEQ ID NO:38-51.

[0031] In some embodiments, at least some of the one or more sdAbs bind to a cancer antigen.

[0032] In some embodiments, at least some of the one or more sdAbs promote T-cell activation.

[0033] In some embodiments, at least some of the one or more sdAbs are inhibitors of immune checkpoint proteins. In some embodiments, the immune checkpoint proteins are selected from the group consisting of PD-1, PD-L1, PLD-2 or CTLA-4.

[0034] In one embodiment, the protein complex is further conjugated to a payload. In one embodiment, the payload is an anti-viral agent. In one embodiment, the payload is an anti- cancer agent.

[0035] In some embodiments, the J chain comprises a sequence that is at least 80% identical to SEQ ID NO:12 or SEQ ID NO:53, optionally wherein the J chain comprises SEQ ID NO:12 or SEQ ID NO:53.

[0036] Provided herein is a pharmaceutical composition comprising a protein complex disclosed herein and a pharmaceutically acceptable excipient.

[0037] Provided is a method of treating an infection with a virus, bacterium, fungus, or protozoan in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein or (ii) a pharmaceutical composition comprising a protein complex disclosed herein and a pharmaceutically acceptable excipient.

[0038] Provided is a method of treating an infection with a virus in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein or 4 168506328.1(ii) a pharmaceutical composition comprising a protein complex disclosed herein and a pharmaceutically acceptable excipient.

[0039] The method may further comprise administering to the subject an additional anti- viral agent.

[0040] Provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject (i) a protein complex of disclosed herein or (ii) a pharmaceutical composition comprising a protein complex disclosed herein.

[0041] In some embodiments, the subject is a human.

[0042] Provided herein is an IgM scaffold including IgM Cµ2, Cµ3, and Cµ4 domains and a J chain, wherein the N-termini of one or more of the IgM Cµ2 domains are each fused to a copy of a first hybridization domain, wherein the first hybridization domain comprises (a) a SpyCatcher domain or variant thereof or (b) a SpyTag domain or variant thereof.

[0043] In some embodiments, the N-termini of each of the IgM Cµ2 domains are each fused to a copy of the first hybridization domain.

[0044] In some embodiments, the first hybridization domain comprises a sequence that is (a) at least 80% identical to any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof or (b) at least 80% identical to any one of SEQ ID NOs:1, 2, or 54-60, optionally, wherein the first hybridization domain comprises a sequence that is (a) at least 80% identical to SEQ ID NO:10 or (b) at least 80% identical to SEQ ID NO:1.

[0045] In some embodiments, the first hybridization domain comprises (a) SEQ ID NOs:10, 11, 61 or 62 or a portion thereof or (b) any one of SEQ ID NOs:1, 2, or 54-60 or a portion thereof, optionally, wherein the first hybridization domain comprises (a) SEQ ID NO:10 or (b) SEQ ID NO:2.

[0046] In some embodiments, the first hybridization domain comprises SEQ ID NO:10. BRIEF DESCRIPTION OF THE FIGURES

[0047] Figs. 1A, 1B, 1C, 1D, 1E, and 1F illustrate the generation of a modular system to produce multivalent nanobodies with enhanced avidity. Fig. 1A shows schematics of conjugation and purification of Nb-IgM-Fc constructs. Nbs and recombinant IgM-Fc were incubated at a 30:1 molar ratio at 4 ˚C overnight. Excessive Nbs were removed by centrifugation using a 100 kDa molecular weight cutoff filter. Nb-IgM-Fc conjugates were efficiently recovered from the filter. Fig. 1B shows a summary of five major epitope classes targeted by the receptor-binding domain (RBD) Nbs, based on high-resolution cryo-EM structures. The PDB IDs for class I–V Nbs are PDB: 8CYA, 8CWV, 8CYJ, 8CYD, and 8CYJ, 5 168506328.1respectively. Fig.1C shows a correlation analysis of the neutralization potencies (EC50 values) of Nb-IgM-Fc conjugates and their corresponding Nb monomers against pseudotyped SARSCoV-2 Wuhan-Hu-1 (D614G) strain. Fig.1D shows a summary of neutralization EC50 values of the Nb-IgM-Fc conjugates and their corresponding Nb monomers against the pseudotyped SARS-CoV-2 variants or SARS-CoV. Each circle / dot represents the neutralization potency of a specific Nb / Nb-IgM-Fc against SARS-CoV-2 variant or SARS- CoV (details in Tables 4 and 5). Fig. 1E shows a summary of neutralization EC50s of Nb- IgM-Fc conjugates against pseudotyped SARS-CoV-2, variants, or SARS-CoV. Each bar plot is presented as mean ± STD. Welch’s t tests were used for comparison of EC50s of Nb-IgM- Fc targeting different epitopes. * p < 0.05, *** p < 0.001, **** p < 0.0001. Fig. 1F shows a plot of average solvent-accessible surface areas (SASAs) of four major RBD epitopes during the transition of an RBD from the ‘‘closed’’ to fully open state. The transition was modeled by molecular dynamics (MD) simulation. Exposed areas were presented as a function of the distance between the centroids of subdomain 1 (SD1, amino acids 531–592) and the RBD (amino acids 336–518) across different protomers of the spike protein. Accessible surface area calculations were conducted using a 7-Å probe. Traces from top to bottom at 90 Å: Epitope I, III, II, and IV.

[0048] Figs. 2A, 2B, and 2C illustrate the engineering and production of AMETA constructs. Fig. 2A shows the structure-guided design of Nb dimers to enhance cooperative binding and neutralization activities. Sequence conservation was calculated based on 19 sarbecovirus RBD sequences. The fold improvement of a dimer over its corresponding monomers was based on their average neutralization EC50s against the pseudotyped SARS- CoV-2, variants, or SARS-CoV (details in Tables 6 and 7). Fig. 2B shows a schematic representation of a representative AMETA construct. In this example, each of the five arms of the IgM scaffold comprises four different Nbs. Fig. 2C shows the design of four AMETA constructs and their potential coverage on the conserved RBD surface residues.

[0049] Figs. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H illustrate the vitro pseudovirus neutralization activities of the AMETA constructs. Figs.3A, 3B, 3C, and 3D show radar plots comparing the neutralization EC50s of four AMETA constructs and their corresponding single- epitope targeting Nb-IgM-Fc conjugates, and the highest tested concentration was 50 and 100 nM, respectively. Fig. 3A. Traces from outside to inside (for Wuhan-Hu-1): 182-IgM; AMETA1; S36-IgM; 60-IgM; 113-IgM. Fig. 3B. Traces from outside to inside (for Wuhan- Hu-1): 182-IgM; AMETA2; S36-IgM; 118-IgM; 113-IgM. Fig. 3C. Traces from outside to inside (for Wuhan-Hu-1): 182-IgM; AMETA3; S36-IgM; 118-IgM; 132-IgM. Fig.3D. Traces 6 168506328.1from outside to inside (for Wuhan-Hu-1): 182-IgM; AMETA4; 118-IgM; 132-IgM. Figs. 3E, 3F, 3G, and 3H show radar plots summaries comparing the neutralization potencies (EC50s) of four AMETA constructs and their corresponding Nb monomer against the pseudotyped SARS-CoV-2 WT (Wuhan-Hu-1 D614G), Omicron variants, and SARS-CoV. Fig.3E. Traces from outside to inside (for Wuhan-Hu-1 D614G): AMETA1; 182; 60; S36; 113. Fig.3F. Traces from outside to inside (for Wuhan-Hu-1 D614G): AMETA2; 182; 118; S36; 113. Fig. 3G. Traces from outside to inside (for Wuhan-Hu-1 D614G): AMETA3; 182; 118; S36; 132. Fig. 3H. Traces from outside to inside (for Wuhan-Hu-1 D614G): AMETA4; 182; 118; 132.

[0050] Figs. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H show the biodistribution and in vivo efficacy of AMETA4 in mouse models. Fig. 4A shows an experiment in which intranasal administration of89Zr-labeled AMETA4 (275.5 ± 43.5 mCi) in C57BL / 6 mice (n=5) was followed by a 20-minute PET scan to monitor AMETA4 distribution. Representative 3D reconstruction images were shown. The89Zr signal decreases over time. Fig. 4B shows body weight changes over time, expressed as a percentage. Experimental design summary: mouse- adapted SARS-CoV-2 (13104 PFU) was intranasally administered to three groups of 129 / S mice (n = 8). AMETA4 (2.2 nmol / kg or 2 mg / kg) was delivered intranasally either 6 h before infection for prophylactic treatment (triangles) or 6 h after infection for therapeutic treatment (squares). A control group received isotype IgM-Fc (circles, 2.2 nmol / kg), and a group of uninfected animals (n = 2) served as additional controls. Daily monitoring of animal weight changes was conducted, and animals were euthanized for lung tissue viral titer analysis at 3 days post-infection (d.p.i.). Traces from top to bottom by endpoint: Uninfected; prophylaxis; therapy; isotype. Fig.4C shows viral titers in lung tissues at 3 d.p.i. in the 129 / S mice. Dashed line indicates the detection limit. See Fig. 4B for experimental setup. Fig. 4D shows lung pathology scores at 3 d.p.i. in the 129 / S mice. Maximum score = 8. See Fig.4B for experimental setup. Fig.4E shows the weight change of the animals. Experimental design summary: mouse- adapted SARS-CoV-2 (13104 PFU) was intranasally administered to three groups of 129 / S mice (n = 8). AMETA4 (2.2 nmol / kg or 2 mg / kg) was delivered intranasally 6 h after infection (squares, bottom trace). A control group received a benchmark (humVHH72S56A)2-Fc (empty circles, middle trace, 18 nmol / kg or 2 mg / kg). Daily monitoring of animal weight changes was conducted, and animals were euthanized for lung tissue viral titer analysis at 3 d.p.i. Top trace: uninfected. Fig. 4F shows lung viral titers. See Fig. 4E for experimental setup. Left bar: Benchmark (humVHH72S56A)2-Fc. Right bar: AMETA4. Fig. 4G shows viral titers in lung tissues at 3 d.p.i. in the K18-hACE2 mice. Dashed line indicating the detection limit. Experimental design summary: SARS-CoV-2 Omicron XBB.1.5 (2.5 × 104PFU) was 7 168506328.1intranasally administered to three groups of K18-hACE2 transgenic mice (n=8). AMETA4 (1.1nmole / kg or 1 mg / kg, triangles) or (humVHH72S56A)2-Fc (9 nmole / kg or 1mg / kg, dots) was delivered intranasally 6 hours before infection for prophylaxis. A control group received isotype IgM-Fc (circles, 1mg / kg). Daily monitoring of animal weight changes was conducted, and animals were euthanized for lung tissue viral titer analysis on 3 d.p.i. Fig. 4H shows the lung pathology scores at 3 d.p.i. in the K18-hACE2 mice. Maximum score = 16. See Fig. 4G for experimental setup.

[0051] Figs. 5A, 5B, 5C, and 5D illustrate the architecture of an AMETA construct. Fig. 5A shows a structure model based on single-particle cryo-EM illustrating the full architecture of AMETA3. The SpyCatcher003-SpyTag (PDB: 4MLI, Nbs, and the IgM-Fc core were structurally determined by X-ray and cryoEM. Fusion proteins of dimeric Nb and Ig∆NSpyCatcher003were computationally modeled using structure templates and grafted to the Cryo-EM determined IgM-Fc core. Fig.5B shows root-mean-square-fluctuation (RMSF) plot of Cα atoms over 50 ns simulations of one clamp of the computationally modeled AMETA3 structure. A larger RMSF value indicates greater structural flexibility. Fig .5C shows the quantification of the number of total spikes per virion under each condition (n = 25, 35, 30, and 26 for IgM-Fc, AMETA40.1, 1, and 10 nM, respectively). Only well-defined pre- and post- fusion spikes were included in the count. Due to technical limitations, the heterogeneous and noisy densities observed around the virions in the 1–10 nM AMETA4-treated samples could not be differentiated and were excluded for quantification. Each bar plot is presented as mean ± STD. Fig.5D shows the quantification of the percentage of post-fusion spike per virion under each condition. Each bar plot is presented as mean ± STD. DETAILED DESCRIPTION

[0052] Provided herein are multivalent protein complexes comprising an immunoglobulin M (IgM) scaffold and one or more single-domain antigen-binding fragment (sdAb) conjugates. These protein complexes constitute a highly versatile and modular system that combines the strong avidity of the IgM scaffold with the high specificity and exceptional bioengineering potential of sdAbs. The miniature size and small footprints of sdAbs enable avidity binding of the protein complexes constructs to an extensive range of epitopes, including epitopes located in small nooks, crevices and conserved sites of the target proteins. The IgM / sdAB complexes are also referred to herein as “multi-epitope targeting with enhanced avidity” (AMETA)).

[0053] The versatility and modularity of the protein complexes disclosed herein represent a significant advancement over existing antibody technologies. For example, the protein 8 168506328.1complexes disclosed herein address the production challenges and bioengineering constraints associated with IgM therapy.

[0054] Further, the adaptability of the protein complexes disclosed herein to various modalities – such as nucleotides, chemical compounds, antibody fragments, and in silico- designed proteins – positions it as a highly promising platform for combating highly mutable pathogens like HIV and influenza, thereby broadening the scope of therapeutic strategies for infectious diseases and drug-resistant systems.

[0055] Beyond infectious diseases, the multispecific design of the protein complexes disclosed herein provides numerous applications in oncology. The ability of the protein complexes disclosed herein to simultaneously target multiple cancer-associated pathways or antigens directly addresses two of the most formidable challenges in cancer therapy: tumor heterogeneity and therapy resistance. By engaging diverse oncogenic drivers and evasion mechanisms within the same tumor microenvironment, the protein complexes disclosed herein can provide more comprehensive and durable therapeutic responses than traditional monoclonal antibodies.

[0056] IgM

[0057] IgM is a pentameric antibody that circulates in the mucosal membrane and blood. IgM can recognize pathogens using multivalency, which overcomes the lack of affinity maturation that other antibodies undergo. IgM is effective in agglutination and complement activation, making it crucial in early defense against pathogens. IgM’s low abundance and low binding affinity to its target have until now posed obstacles to the use of IgM-based biologics in therapy.

[0058] The predominant form of naturally occurring IgM in the blood is the pentamer. Each of the five arms of IgM comprises two heavy chains (μ chains) and two light chains (either κ or λ chains). The IgM pentamer is stabilized by disulfide bonds between the μ chains and a joining (J) chain, which is a small polypeptide that helps in the polymerization of the IgM monomers. This structure gives IgM a high molecular weight of around 970 kDa and a valency of 10, allowing it to bind multiple antigens simultaneously. In naturally occurring IgM, the heavy (µ) chains spanning Cμ2-Cμ4 are linked to the Fab region, which comprises (a) a heavy variable chain and the Cμ1 domain paired with (b) light variable chain and a light constant domain paired. In some embodiments, in the protein complexes disclosed herein, the Fab domain of the IgM molecule has been replaced with one of the two proteins in a SpyTag / SpyCatcher pair. In some embodiments, certain portions of the Fab region may be 9 168506328.1retained. The other protein of the pair is used to a sdAb conjugate, allowing the conjugation of the sdAb conjugate to the IgM scaffold. See Fig.1A for an illustration.

[0059] Single domain antibodies

[0060] As used herein, the term “single domain antibody” or “sdAb,” refers to an antibody fragment consisting of a single monomeric variable antibody domain. sdAbs can be readily produced and engineered against a variety of targets (antigens).

[0061] In one embodiment, the sdAb is derived from the antigen-binding portion of a camelid heavy-chain-only antibody. Such an sdAb is also referred to as a VHH fragment or a nanobody (Nb). Nanobodies (Nbs) are single-domain antigen-binding fragments that are fairly small (often around 15 kDa). Despite their small size, Nbs bind antigens with high affinity and specificity.

[0062] In one embodiment, the sdAb is derived from the antigen-binding portion of a cartilaginous fish heavy-chain-only antibodies (IgNAR, 'immunoglobulin new antigen receptor'). Such an sdAb is called a VNAR fragment. Alternatively, an sdAb can be generated from conventional IgGs by obtaining or engineering monomeric, stable VH or VL domains.

[0063] An sdAb generally comprises a variable region primarily responsible for antigen recognition and binding and a framework region. The “variable region” comprises the “complementarity determining region” (CDR), which comprises loops which differ extensively in size and sequence based on antigen recognition. CDRs are generally responsible for the binding specificity of the single-domain antibody. Distinct from the CDRs is the framework region. The framework region is relatively conserved and assists in overall protein structure. The framework region may comprise a large solvent-exposed surface consisting of a β-sheet and loop structure.

[0064] The sdAb may target any antigen of interest to the skilled person in the art, depending on the desired application. Non-limiting examples of sdAb antigens are disclosed throughout this disclosure.

[0065] The SpyTag / SpyCatcher System

[0066] The SpyTag / SpyCatcher conjugation technique was originally developed based on the split protein CnaB2 from Streptococcus pyogenes. See Zakeri et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin, Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7, incorporated herein by reference in its entirety. The SpyTag / SpyCatcher system comprises two fragments or domains: one named SpyTag, and the other named SpyCatcher. Once combined under nearly any common conditions, SpyTag and SpyCatcher can rapidly and efficiently covalently conjugate to each other through an 10 168506328.1intermolecular isopeptide bond. Bond formation is enabled by a catalytic triad formed between a reactive aspartic acid residue in the SpyTag and a reactive lysine residue and a glutamic acid residue in the SpyCatcher.

[0067] Multiple variants of SpyTag / SpyCatcher pairs have been developed that are suitable for use with the compositions and methods disclosed herein. See Keeble et al., Evolving Accelerated Amidation by SpyTag / SpyCatcher to Analyze Membrane Dynamics, Angew Chem Int Ed Engl. 2017 Dec 22;56(52):16521-16525, incorporated herein by reference in its entirety, for the development of SpyTag002 / SpyCatcher002. As another example, SpyCatcher003 has improved reaction rate with its reaction partner SpyTag003 and a higher structural stability than previous SpyCatcher generations. See Keeble et al., Approaching infinite affinity through engineering of peptide-protein interaction, Proc Natl Acad Sci U S A. 2019 Dec 26;116(52):26523-26533, incorporated herein by reference in its entirety.

[0068] In some embodiments, a SpyTag domain, variant, or derivative thereof is used for the compositions and methods disclosed herein. As used herein, a “SpyTag domain variant” or “SpyTag domain derivative” comprises one or more alterations when compared to a parental SpyTag domain sequence, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the variant or derivative retains at least 10% of the biological activity of the parental SpyTag peptide. As used herein, the biological activity of the SpyTag peptide is to react with a SpyCatcher domain, variant, or derivative thereof, to form a bond, preferably, a covalent bond. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The SpyTag domain variant or derivative thereof may be a portion of a parental SpyTag domain that comprises a biologically active portion of the parental SpyTag domain. In some embodiments, the SpyTag domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to its parental counterpart. In some embodiments, the SpyTag domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to its parental counterpart. The terms “amino acid mutation” and “amino acid substitution” are used interchangeably herein. In some embodiments, the SpyTag domain, variant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence disclosed in Table 1. In some embodiments, the SpyTag variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to a sequence disclosed in Table 1. In some embodiments, the SpyTag domain, variant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 11 168506328.195%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, or 54-60. In some embodiments, the SpyTag variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to any one of SEQ ID NOs: 1, 2, or 54-60. In some embodiments, the SpyTag domain, variant, or derivative thereof comprises any one of SEQ ID NOs: 1, 2, or 54-60.

[0069] In some embodiments, the SpyTag domain, variant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the SpyTag variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the SpyTag variant or derivative thereof comprises SEQ ID NO: 1 or SEQ ID NO:2.

[0070] In some embodiments, a SpyCatcher domain, variant, or derivative thereof is used for the compositions and methods disclosed herein. As used herein, a “SpyCatcher domain variant” or “SpyCatcher domain derivative” comprises one or more alterations when compared to a parental SpyCatcher domain sequence, including, but not limited to amino acid additions, substitutions, insertions, deletions, or posttranslational modifications, wherein the variant or derivative retains at least 10% of the biological activity of the parental SpyCatcher peptide. As used herein, the biological activity of the SpyCatcher peptide is to react with a SpyTag domain, variant, or derivative thereof, to form a bond, preferably, a covalent bond. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The SpyCatcher domain variant or derivative thereof may be a portion of the parental SpyCatcher domain that comprises a biologically active portion of the parental SpyCatcher domain. In some embodiments, the SpyCatcher domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to its parental counterpart. In some embodiments, the SpyCatcher domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to its parental counterpart. The terms “amino acid mutation” and “amino acid substitution” are used interchangeably herein. In some embodiments, the SpyCatcher domain, variant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence or a portion of a sequence disclosed in Table 1. In some embodiments, the SpyCatcher domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to a sequence of a portion of a sequence disclosed in Table 1. In some embodiments, the SpyCatcher domain, 12 168506328.1variant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 10, 11, 61, or 62. In some embodiments, the SpyCatcher domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to any one of SEQ ID NOs: 10, 11, 61, or 62.

[0071] In some embodiments, the SpyTag domain, variant, or derivative thereof comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the SpyCatcher domain variant or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:10. In some embodiments, the SpyTag domain, variant, or derivative thereof comprises SEQ ID NO: 10.

[0072] In the original SpyTag / SpyCatcher pair, bond formation is enabled by a catalytic triad formed between a reactive aspartic acid residue in the SpyTag domain and a reactive lysine residue and a glutamic acid residue in the SpyCatcher domain. However, alternative catalytic triads used in the SpyTag domain variant or derivative / SpyCatcher domain variant or derivative pair may be compatible with the compositions and methods disclosed herein.

[0073] As used herein, the terms “conservative amino acid substitutions” and “conservative modifications” refer to amino acid modifications that do not significantly affect or alter the function and / or activity of the presently disclosed proteins comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. 13 168506328.1

[0074] A person skilled in the art is familiar with testing whether a given SpyCatcher domain variant or derivative binds to a given SpyTag domain variant or derivative. For example, one might immobilize the SpyTag domain variant or derivative on a surface, incubate with the SpyCatcher domain variant or derivative, wash away unreacted SpyCatcher domain variant or derivative, and detect the presence of the bound SpyCatcher domain variant or derivative. Other techniques are known in the art, including the techniques described in the Examples. Table 1. Illustrative SpyTag and SpyCatcher sequences. SEQ ID Name Sequence NO L L T V

[0075] It is to be understood that in the protein complexes disclosed herein, the SpyTag, variant, or derivative thereof may be linked to the IgM scaffold. Alternatively, the SpyTag, variant, or derivative thereof may be linked to the sdAb conjugate. In one embodiment, the SpyCatcher, variant, or derivative thereof is linked to the IgM scaffold. Alternatively, the SpyCatcher, variant, or derivative thereof may be linked to the sdAb conjugate.

[0076] sdAb Conjugates 14 168506328.1

[0077] As used herein, an sdAb conjugate comprises a hybridization domain and one or more sdAbs.

[0078] In one aspect, the hybridization domain is one of the two proteins used in the SpyTag / SpyCatcher conjugation technique. In one embodiment, the sdAb conjugate comprises a hybridization domain comprising a SpyTag domain, variant, or derivative thereof. In one embodiment, the sdAb conjugate comprises a hybridization domain comprising a SpyCatcher domain, variant, or derivative thereof.

[0079] In some embodiments, the sdAb conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sdAbs. In some embodiments, the sdAb conjugate comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sdAbs. In one embodiment, the sdAb conjugate comprises one sdAb. In one embodiment, the sdAb conjugate comprises two sdAbs.

[0080] In some embodiments, the sdAb conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nanobodies. In some embodiments, the sdAb conjugate comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nanobodies. In one embodiment, the sdAb conjugate comprises one nanobody. In one embodiment, the sdAb conjugate comprises two nanobodies.

[0081] In the sdAb conjugate, the hybridization domain may be directly linked to the one or more sdAbs. Alternatively, the hybridization domain may linked to an sdAb via a linker.

[0082] In sdAb conjugates comprising two or more sdAbs, the sdAbs may be linked to each other directly or via a linker. Some of the sdAbs may be linked to each other directly. Some of the sdAbs may be linked to each via a linker.

[0083] Linkers

[0084] In some embodiments, some or all of the individual components of the IgM scaffolds or the protein complexes disclosed herein linked directly to each other without intervening sequences. In some embodiments, some or all of the individual components of the IgM scaffolds or the protein complexes disclosed herein are linked to each by a linker. An IgM scaffold or a protein complex may comprise more than one type of linker.

[0085] Suitable linkers used herein can be of any of a number of suitable lengths, such as from 1 amino acid to 30 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids. The linker can be 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. 15 168506328.1

[0086] In some embodiment, the linker is a flexible linker. Non-limiting examples of linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n(where n is an integer of at least one)), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. See, e.g., Chichili et al., Linkers in the structural biology of protein-protein interactions, Protein Sci.2013 Feb;22(2):153-67, which is incorporated herein in its entirety. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. See Scheraga, Predicting Three- Dimensional Structures of Oligopeptides, In Reviews in Computational Chemistry, John Wiley & Sons, Ltd., 1992, Vol 3; pp 73−142. The linker may predominantly consist of glycines, serines, and / or alanines.

[0087] In some embodiments, the linker is a rigid linker. Rigid polypeptide linkers comprise a sequence of amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. Rigid polypeptide linkers thus may be employed where it is desired to minimize the interaction between the different domains of a fusion protein. Rigid peptide linkers include, but are not limited to, peptide linkers rich in proline, and peptide linkers having an inflexible helical structure, such as an a-helical structure.

[0088] Flexible or rigid linkers can be used, or a combination thereof.

[0089] Provided herein are also linkers that are combinations or that contain repeats of any of the linkers disclosed herein.

[0090] In some embodiments, the linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:52. In some embodiments, the linker comprises a sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to SEQ ID NO:52. The linker may comprise SEQ ID NO:52.

[0091] Protein Complexes

[0092] Provided is an IgM scaffold comprising IgM Cµ2, Cµ3, and Cµ4 domains and a J chain, wherein the N-termini of one or more of the IgM Cµ2 domains are each fused to a copy of a hybridization domain, wherein the hybridization domain comprises (a) a SpyCatcher domain, variant, or derivative thereof or (b) a SpyTag domain, variant, or derivative thereof. 16 168506328.1In one embodiment, the N-termini of each of the IgM Cµ2 domains are each fused to a copy of the hybridization domain.

[0093] In some embodiments, the hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof. In some embodiments, the hybridization domain comprises any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof. In some embodiments, the hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs:10 or 11. In some embodiments, the hybridization domain comprises SEQ ID NOs:10 or 11. In some embodiments, the hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10. In one embodiment, the hybridization domain comprises SEQ ID NO:10.

[0094] In some embodiments, the hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:1, 2, or 54-60. In some embodiments, the hybridization domain comprises any one of SEQ ID NOs:1, 2, or 54-60. In some embodiments, the hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 or 2. In some embodiments, the hybridization domain comprises SEQ ID NO:1 or 2. In some embodiments, the hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1. In one embodiment, the hybridization domain comprises SEQ ID NO:1.

[0095] In some embodiments, the J chain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12 or SEQ ID NO:53. In some embodiments, the J chain comprises SEQ ID NO:12 or SEQ ID NO:53. In one embodiment, at least one sdAb is fused to the N- terminus of the J chain. In one embodiment, at least one sdAb is fused to the C-terminus of the J chain.

[0096] Provided is a protein complex comprising (a) an IgM scaffold comprising IgM Cµ2, Cµ3, and Cµ4 domains and a J chain (but no Fab region); and (b) one or more sdAb conjugates, wherein: the N-termini of one or more of the IgM Cµ2 domains are each fused to a copy of a first hybridization domain; each of the one or more sdAb conjugates comprises a second 17 168506328.1hybridization domain and one or more sdAbs; and the first hybridization domain forms a covalent bond with the second hybridization domain.

[0097] In some embodiments, the J chain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12 or SEQ ID NO:53. In some embodiments, the J chain comprises SEQ ID NO:12 or SEQ ID NO:53.

[0098] The J chain may comprise a tag for detection and / or purification. Examples of tags include, but are not limited to, His-tag, Glutathione S-transferase-tag, FLAG-tag, hemagglutinin-tag, Myc-tag, and Avi-tag.

[0099] In one embodiment, at least one sdAb is fused to the N-terminus of the J chain. In one embodiment, the at least one sdAb is fused to the C-terminus of the J chain.

[0100] In one embodiment, the protein complex comprises (a) one or more sdAb conjugates comprising one or more one sdAbs that activate an immune cell (including, but not limited to an sdAb that binds CD3e) and (b) a J chain used to one or more one sdAbs that target a cancer antigen. In one embodiment, the protein complex comprises (a) a J chain fused to one or more one sdAbs that activate an immune cell (including, but not limited to an sdAb that binds CD3e) and (b) one or more sdAb conjugates comprising o one or more one sdAbs that target a cancer antigen. As a result, the protein complex might achieve a more desirable immune cell:cancer cell ratio.

[0101] In one embodiment, the N-termini of each of the IgM Cµ2 domains are each fused to a copy of the first hybridization domain.

[0102] In some embodiments, the (a) the first hybridization domain comprises a SpyCatcher domain or variant thereof and the second hybridization domain comprises a SpyTag domain or variant thereof; or (b) the first hybridization domain comprises a SpyTag domain or variant thereof and the second hybridization domain comprises a SpyCatcher domain or variant thereof.

[0103] In some embodiments, the first hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof. In some embodiments, the first hybridization domain comprises any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof. In some embodiments, the first hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs:10 or 11. In some embodiments, the first hybridization domain comprises SEQ ID NOs:10 or 11. In some embodiments, the first 18 168506328.1hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10. In one embodiment, the first hybridization domain comprises SEQ ID NO:10.

[0104] In some embodiments, the first hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:1, 2, or 54-60. In some embodiments, the first hybridization domain comprises any one of SEQ ID NOs:1, 2, or 54-60. In some embodiments, the first hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 or 2. In some embodiments, the first hybridization domain comprises SEQ ID NO:1 or 2. In some embodiments, the first hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1. In one embodiment, the first hybridization domain comprises SEQ ID NO:1.

[0105] In some embodiments, the second hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof. In some embodiments, the second hybridization domain comprises any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof. In some embodiments, the second hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs:10 or 11. In some embodiments, the second hybridization domain comprises SEQ ID NOs:10 or 11. In some embodiments, the second hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10. In one embodiment, the second hybridization domain comprises SEQ ID NO:10.

[0106] In some embodiments, the second hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:1, 2, or 54-60. In some embodiments, the second hybridization domain comprises any one of SEQ ID NOs:1, 2, or 54-60. In some embodiments, the second hybridization domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 or 2. In some embodiments, the second hybridization domain comprises SEQ ID NO:1 or 2. In some embodiments, the second hybridization domain 19 168506328.1comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1. In one embodiment, the second hybridization domain comprises SEQ ID NO:1.

[0107] In some embodiments, the protein complex comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sdAb conjugates. In one embodiment, the protein complex comprises 10 sdAb conjugates.

[0108] In one embodiment, each sdAb conjugate comprises at least two sdAbs. In one embodiment, each sdAb conjugate comprises at least three sdAbs. In one embodiment, each sdAb conjugate comprises at least four sdAbs. In one embodiment, each sdAb conjugate comprises at least five sdAbs.

[0109] In one embodiment, each sdAb conjugate comprises two sdAbs. In one embodiment, each sdAb conjugate comprises three sdAbs. In one embodiment, each sdAb conjugate comprises four sdAbs. In one embodiment, each sdAb conjugate comprises five sdAbs.

[0110] In one embodiment, each Nb conjugate comprises at least two Nbs. In one embodiment, each Nb conjugate comprises at least three Nbs. In one embodiment, each Nb conjugate comprises at least four Nbs. In one embodiment, each Nb conjugate comprises at least five Nbs.

[0111] In one embodiment, each Nb conjugate comprises two Nbs. In one embodiment, each Nb conjugate comprises three Nbs. In one embodiment, each Nb conjugate comprises four Nbs. In one embodiment, each Nb conjugate comprises five Nbs.

[0112] In one embodiment, the sdAbs are connected to each other with a first linker. The first linker may be a polypeptide linker. In one embodiment, the IgM Cµ2 domains are each fused to a copy of the first hybridization domain via a second linker. The second linker may be a polypeptide linker. In one embodiment, the second hybridization domain is fused to one of the sdAbs via a third linker. The third linker may be a polypeptide linker. The first, second, and / or third linker may be any linker, including the linkers disclosed herein. The first, second, and / or third linker may comprise primarily glycines, serines, and / or alanines. In some embodiments, at least one of the first, second, and / or third linker comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:52. In some embodiments, at least one of the first, second, and / or third linker comprises SEQ ID NO:52.

[0113] A person skilled in the art will appreciate the modular nature of the sdAb conjugates. In some embodiments, the protein complex comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 sdAb conjugates that are identical to each other. In some embodiments, the protein complex comprises at least 20 168506328.1two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 sdAb conjugates that are different from each other. In some embodiments, at least one of the sdAb conjugates in the protein complex comprises two or more sdAbs that bind to the same epitope. In some embodiments, at least one of the sdAb conjugates in the protein complex comprises two or more sdAbs that bind to different epitopes. The different epitopes may be on the same target protein. The different epitope may be derived from the same tumor or the same pathogen.

[0114] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to an epitope derived from a virus, a bacterium, a fungus, or a protozoan. In some embodiments, the sdAbs are designed to bind to peptides, nucleotides, or chemical compounds. In some embodiments, the sdAbs are designed to bind to polypeptides, antibody fragments, or in silico-designed proteins or fragments thereof.

[0115] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to an epitope derived from a bacterial pathogen. Non- limiting examples of bacterial proteins which may be a source of bacterial peptides, e.g., antigenic determinants include lipopolysaccharides isolated from gram-negative bacterial cell walls and staphylococcus-specific, streptococcus-specific, pneumococcus-specific (e.g., PspA; see PCT Publication No. WO 1992 / 14488, which is incorporated herein by reference in its entirety), Neisseria gonorrhea-specific, Borrelia-specific (e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdorferi, Borrelia afzelli, and Borrelia garinii (see, e.g., U.S. Pat. No. 5,523,089; PCT Publication Nos. WO 90 / 04411, WO 91 / 09870, WO 93 / 04175, WO 96 / 06165, WO93 / 08306; PCT / US92 / 08697; Jonsson et al., Heterogeneity of outer membrane proteins in Borrelia burgdorferi: comparison of osp operons of three isolates of different geographic origins. Infect Immun. 1992 May;60(5):1845-53; Johnson et al., Incomplete protection of hamsters vaccinated with unlipidated OspA from Borrelia burgdorferi infection is associated with low levels of antibody to an epitope defined by mAb LA-2. Vaccine. 1995 Aug;13(12):1086-94; Edelman, The Sixth International Conference on Lyme Borreliosis: progress on the development of Lyme disease vaccines.19-22 June 1994, Bologna, Italy. Vaccine.1995 Jan;13(1):133-5, all of which are incorporated herein by reference in their entireties), and pseudomonas-specific proteins or peptides. Additional non-limiting examples of bacterial antigens include, e.g., antigens from Neisseria gonorrhea, Mycobacterium tuberculosis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus. Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, 21 168506328.1Campylobacter fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, and Actinobaccilus seminis.

[0116] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to an epitope derived from a viral pathogen. In some embodiments, the epitope is derived from a corona virus (e.g., receptor-binding domain of SARS-CoV-2 virus); influenza virus (e.g., surface glycoproteins hemagluttinin (HA) and neuramimidase (NA)); immunodeficiency virus (e.g., a human immunodeficiency virus antigens (HIV) such as gp120, gp160, p18 antigen Gag p17 / p24, Tat, Pol, Nef, and Env); herpesvirus (e.g., a glycoprotein from herpes simplex virus (HSV), Marek's Disease Virus, cytomegalovirus (CMV), or Epstein-Barr virus); hepatitis virus (e.g., Hepatitis B surface antigen (HBsAg)); papilloma virus; rous associated virus (e.g., RAV-1 env); infectious bronchitis virus (e.g., matrix and / or preplomer); flavivirus (e.g., a Japanese encephalitis virus (JEV) antigen, a Yellow Fever antigen, or a Dengue virus antigen); Morbillivirus (e.g., a canine distemper virus antigen, a measles antigen, or rinderpest antigen such as HA or F); rabies (e.g., rabies glycoprotein G); parvovirus (e.g., a canine parvovirus antigen); poxvirus (e.g., an ectromelia antigen, a canary poxvirus antigen, or a fowl poxvirus antigen); chicken pox virus (varicella zoster antigen); infectious bursal disease virus (e.g., VP2, VP3, or VP4); Hantaan virus, and mumps virus. In some embodiments, the epitope is derived from CMV, HIV, hepatitis virus, or SARS-CoV-2 virus. The virus may be one that infects a mammal, including, but not limited to, a human.

[0117] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to an epitope derived from a pathogenic protozoa. In one embodiment, the epitope is derived from Plasmodium, such as P. falciparum, P. vivax, P. ovale or P. malariae.

[0118] In one embodiment, the epitope is derived from a malaria-specific protein, including, but not limited to, circumsporozoite (CS) protein, Thrombospondin Related Adhesion (Anonymous) protein (TRAP), also called Sporozoite Surface Protein 2 (SSP2), LSA I, hsp70, SALSA, STARP, Hep17, MSA, RAP-1, RAP-2.

[0119] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to an epitope derived from a fungal pathogen. Non- limiting examples of fungal proteins from which antigenic determinants may be isolated 22 168506328.1include those isolated from candida (e.g., MP65 from Candida albicans), trichophyton, and ptyrosporum.

[0120] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to a helminth parasite peptide or an ectoparasite peptide.

[0121] The modular structure of the protein complexes disclosed herein allows for the rational design of highly selective, multispecific binders tailored to interact with tumor- specific antigens, immune checkpoint regulators, and / or tumor stromal components.

[0122] In one embodiment, the protein complex disclosed herein inhibits an oncogenic receptor (including, but not limited to, EGFR, c-MET, TfRc, PD-1, PDL-1) while simultaneously activating immune effector pathways or promoting T cell engagement, thus enhancing both direct tumor cell killing and immune-mediated clearance.

[0123] In some embodiments, the protein complex comprises (a) at least one sdAb conjugate comprising one or more sdAbs that inhibits an oncogenic receptor and (b) at least one sdAb conjugate comprising one or more sdAbs that activates or increases effector immune cell signaling (i.e., that activates or increases molecular signaling within immune cells that trigger specific actions to eliminate pathogens or damaged cells).

[0124] In one embodiment, the protein complex comprises (a) at least one sdAb conjugate comprising one or more sdAbs that activate an immune cell (including, but not limited to an sdAb that binds CD3e) and (b) at least one sdAb conjugate comprising one or more sdAbs that target a cancer antigen. As a result, the protein complex might achieve a more desirable immune cell:cancer cell ratio.

[0125] In some embodiments, the protein complex comprises (a) at least one sdAb conjugate comprising one or more sdAbs that inhibits an immune checkpoint protein and (b) at least one sdAb conjugate comprising one or more sdAbs that bind to a tumor antigen. Examples of immune checkpoint proteins are known in the art and include the ones disclosed herein.

[0126] In some embodiments, the protein complex comprises (a) at least one sdAb conjugate comprising one or more sdAbs that inhibit or reduce angiogenesis and (b) at least one sdAb conjugate comprising one or more sdAbs that inhibit or reduce oncogenic signaling. As such a result, the protein complex targets both tumor growth and metastasis.

[0127] In some embodiments, the protein complex disclosed herein may be engineered to recruit immune effector cells (e.g., NK cells, T cells) for enhanced tumor cell killing. 23 168506328.1

[0128] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to a cancer antigen, also referred to herein as a tumor antigen. Non-limiting examples of tumor-associated proteins from which antigenic determinants may be isolated include, e.g., adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pme117, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K- MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR- fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE- A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART- 1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR- 1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or - SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG- 3, Triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A (MART1), gp 100, tyrosinase, TRP-1 / gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin (MUC- 1) (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen (PSA) (in prostate cancer); carcinoembryonic antigen (CEA) (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE- 1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO- 24 168506328.11 / LAGE-2, NA-88, GnTV, TRP2-INT2. In some embodiments, the protein is a neo-antigen. In some embodiments, the protein is a tumor specific antigen.

[0100] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that are inhibitors of immune checkpoint proteins. In some embodiments, the immune checkpoint protein is CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, CD27, CD28, CD40, OX40, GITR, CD137, or ICOS.

[0100] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs that bind to a tumor stromal component.

[0101] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs comprising sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:13-37. In some embodiments, the protein complex comprises at least one sdAb conjugate comprising any one of SEQ ID NO:13-37.

[0102] In some embodiments, the protein complex comprises at least one sdAb conjugate comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 38-51. In some embodiments, the protein complex comprises at least one sdAb conjugate comprising any one of SEQ ID NO: 38-51. In some embodiments, the protein complex comprises at least one sdAb conjugate comprising one or more sdAbs comprising a nanobody pair as shown in Table 6. A person skilled in the art appreciates that the relative orientation of the two nanobodies in the nanobody pairs in Table 6 (i.e., which nanobody is located N-terminal with respect to the other nanobody) may be reversed. Different linkers may be used as compared to the linkers shown in Table 6.

[0103] In some embodiments, the J chain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12 or SEQ ID NO:53. In some embodiments, the J chain comprises SEQ ID NO:12 or SEQ ID NO:53. In one embodiment, at least one sdAb is fused to the N- terminus of the J chain. In one embodiment, at least one sdAb is fused to the C-terminus of the J chain.

[0104] In some embodiments, the IgM scaffolds or the protein complexes disclosed herein are conjugated to a functional moiety, also referred herein as a payload. Examples of useful functional moieties include, but are not limited to, a blocking moiety, a detectable moiety, a diagnostic moiety, a targeting moiety, and a therapeutic moiety. 25 168506328.1

[0105] In one embodiment, the blocking moiety is a polyalkylene glycol moiety, for example, a PEG moiety and preferably a PEG-maleimide moiety. Preferred pegylation moieties (or related polymers) can be, for example, polyethylene glycol (“PEG”), polypropylene glycol (“PPG”), polyoxyethylated glycerol (“POG”) and other polyoxyethylated polyols, polyvinyl alcohol (“PVA”) and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, straight chain or branched, substituted or unsubstituted as long as it has at least one active sulfone moiety. The polymeric portion can be of any length or molecular weight but these characteristics can affect the biological properties. Polymer average molecular weights particularly useful for decreasing clearance rates in pharmaceutical applications are in the range of 2,000 to 35,000 Daltons. In addition, if two groups are linked to the polymer, one at each end, the length of the polymer can impact upon the effective distance, and other spatial relationships, between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or confer the desired biological activity. PEG is useful in biological applications for several reasons. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze, and is nontoxic. Pegylation can improve pharmacokinetic performance of a molecule by increasing the molecule's apparent molecular weight. The increased apparent molecular weight reduces the rate of clearance from the body following subcutaneous or systemic administration. In many cases, pegylation can decrease antigenicity and immunogenicity. In addition, pegylation can increase the solubility of a biologically-active molecule. Other useful blocking moieties are known in the art.

[0106] Examples of detectable moieties include fluorescent moieties or labels, imaging agents, radioisotopic moieties, radiopaque moieties, and the like, e.g. detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Illustrative fluorophores include fluorescent dyes (e.g. fluorescein, rhodamine, and the like) and other luminescent molecules (e.g. luminal). A fluorophore may be environmentally-sensitive such that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g. dansyl probes). Illustrative radiolabels include small molecules containing atoms with one or more low sensitivity nuclei (13C,15N,2H,125I,123I,99Tc,43K,52Fe,67Ga,68Ga,111In and the like). Other useful detectable moieties are known in the art.

[0107] Examples of diagnostic moieties include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, a diagnostic moiety allows for determining the 26 168506328.1presence, absence, or level of a molecule, for example, a target peptide, protein, or proteins, that is associated with a disease or disorder. Such diagnostics are also suitable for prognosing and / or diagnosing a disease or disorder and its progression.

[0108] In some embodiments, the protein complex disclosed herein is conjugated to a therapeutic moiety, including, but not limited to cytotoxic agents, immune modulators, or RNA therapeutics. These targeted therapeutics can, for example, deliver precise treatments to cancer cells while minimizing off-target effects, thereby improving efficacy and reducing toxicity.

[0109] Examples of therapeutic moieties include, for example, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-viral agents, anti-bacterial agents, anti- fungal agents, anti-infective agents, or generally a therapeutic agent. The functional moiety may also have one or more of the above-mentioned functions.

[0110] Illustrative therapeutic moieties include radionuclides with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, and therefore suitable for inducing cell death (e.g., of a cancer). Illustrative high-energy radionuclides include:90Y,125I,131I,123I,111In,105Rh,153Sm,67Cu,67Ga,166Ho,177Lu,186Re and188Re. These isotopes typically produce high-energy α- or β-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0111] Illustrative therapeutic moieties also include cytotoxic agents such as cytostatics (e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross-linkers, or DNA- RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenesis agents, and the like.

[0112] Illustrative therapeutic moieties also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic agent such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5′-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Illustrative DNA-intercalators or cross-linkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, 27 168506328.1cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0113] Illustrative therapeutic moieties also include transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other illustrative cytostatic agents that are compatible with the present invention include ansamycin benzoquinones, quinonoid derivatives (e.g. quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g. carmustine, lomustine, semustine).

[0114] Illustrative therapeutic moieties also include cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; tubulin binding agents such as taxoids (e.g. paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g. Dolastatin-10, -11, or - 15), colchicine and colchicinoids (e.g. ZD6126), combretastatins (e.g. Combretastatin A-4, AVE-6032), and vinca alkaloids (e.g. vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenesis compounds such as Angiostatin K1-3, DL-α-difluoromethyl- ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0115] Illustrative therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g. prednisone), progestins (e.g. hydroxyprogesterone or medroprogesterone), estrogens, (e.g. diethylstilbestrol), antiestrogens (e.g. tamoxifen), androgens (e.g. testosterone), aromatase inhibitors (e.g. aminogluthetimide), 17-(allylamino)- 17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0116] Illustrative therapeutic moieties also include enzyme inhibitors such as, S(+)- camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

[0117] Illustrative therapeutic moieties also include gene regulators such as 5-aza-2′- deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9- cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0118] Illustrative therapeutic moieties also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members 28 168506328.1of those classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine and the like.

[0119] Still other cytotoxins that are compatible with the compositions and methods disclosed herein include auristatins (e.g. auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g. maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracindione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0120] In some embodiments, the protein complex is conjugated to a therapeutic RNA molecule. In some embodiments, the RNA is messenger RNA (mRNA), small RNA (sRNA), small interfering RNA (siRNA), small activating RNAs (saRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), long non-coding RNA (IncRNA), transfer RNA (tRNA), or small hairpin RNA (shRNA). The RNA may be single or double-stranded.

[0121] Techniques for conjugating such therapeutic moieties to immunoglobulins and protein complexes are well known.

[0122] Other types of functional moieties are known in the art and can be readily used in the methods and compositions of the present disclosure based on the teachings contained herein.

[0123] Nucleic Acids

[0124] Also provided herein are nucleic acids encoding any of the polypeptide disclosed herein, including the IgM scaffolds, sdAb conjugates, sdAbs, and hybridizations domains disclosed herein. Further provided are vectors comprising such nucleic acids and host cells comprising such nucleic acids or vectors.

[0125] The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA- RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0126] Provided herein is a nucleic acid that encodes a fusion protein disclosed herein. Provided herein is a set of nucleic acids encoding for two or more fusion proteins disclosed herein. The nucleic acids disclosed herein may be DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of 29 168506328.1

[0127] “Vector” means a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle or virus (including virus derived sequences) that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. A “vector” includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. In some embodiments, the employed vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.

[0128] Also provided herein are vectors comprising nucleic acids disclosed herein.

[0129] Cells

[0130] Provided is a host cell comprising a nucleic acid molecule described herein, or a vector described herein.

[0131] The cell can be isolated. The cell may be a mammalian cell. The cell may be human.

[0132] Pharmaceutical Compositions

[0133] In one embodiment, provided herein is a pharmaceutical composition comprising (a) protein complex or an IgM scaffold disclosed herein and (b) a pharmaceutically acceptable carrier.

[0134] Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. The pharmaceutical compositions may generally be formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0135] The terms “pharmaceutically acceptable,” “physiologically tolerable,” as referred to compositions, carriers, diluents, and reagents, are used interchangeably and include materials are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. 30 168506328.1

[0136] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the compositions disclosed herein, use of the media or compound in the compositions disclosed herein is contemplated. In some embodiments, a second therapeutic agent, such as an anti-cancer or anti-tumor, can also be incorporated into pharmaceutical compositions.

[0137] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In embodiments, the compositions disclosed herein are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0138] In some embodiments, the pharmaceutical composition further includes a cryo- protectant (e.g., glycerol, DMSO, PEG).

[0139] Methods

[0140] Provided herein are methods of treating a disease in a subject in need thereof using the protein complexes disclosed herein.

[0141] In some embodiments, the subject is a mammal. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals, include, but are not limited to, a human or non-human mammal, such as a mouse, rat, rabbit, non-human primate, bovine, equine, canine, ovine, or feline, etc. Individuals and patients are also subjects herein.

[0142] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, 31 168506328.1alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of one or more symptoms of the condition, disorder or disease state; and remission (whether partial or total), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0143] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0144] The medical uses contemplated by the specification may be formulated as protein complex for use as a medicament for prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), but could equally be formulated as (i) a method of prevention or treatment of the disease(s) and / or disorder(s) defined herein (or prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), as (ii) a protein complex for use in the preparation of a medicament for the prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein), or as (iii) use of a protein complex according for the prevention or treatment of the disease(s) and / or disorder(s) defined herein (or for the prevention of treatment of symptoms associated with the disease(s) and / or disorder(s) defined herein). Such medical uses are all envisaged by the present invention.

[0145] Provided herein are methods of treating a subject infected with an infectious agent. The infectious agent may be, without limitation, a virus, bacterium, fungus, protozoa, parasite, helminth, or ectoparasite.

[0146] Provided is a method of treating or preventing an infection with a virus, bacterium, fungus, protozoa, parasite, helminth, or ectoparasite in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein the protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an epitope derived from the virus, bacterium, fungus, protozoa, parasite, helminth, or ectoparasite or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein the protein complex comprises at 32 168506328.1least one sdAb conjugate comprising at least one sdAb that binds to an epitope derived from the virus, bacterium, fungus, protozoa, parasite, helminth, or ectoparasite.

[0147] Provided is a method of treating or preventing an infection with a virus in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an epitope derived from the virus or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an epitope derived from the virus. In one embodiment, the method further comprises administering to the subject another anti-viral agent. The virus may be any virus that can infect the subject, including any virus disclosed herein.

[0148] Provided is a method of or preventing treating an infection with a bacterium in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an epitope derived from the bacterium or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an epitope derived from the bacterium. In one embodiment, the method further comprises administering to the subject another anti-bacterial agent. The bacterium may be any bacterium that can infect the subject, including any bacterium disclosed herein.

[0149] Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to a cancer antigen or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an cancer antigen.

[0150] The term “cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Accordingly, the term “cancer” as used herein refers to an uncontrolled growth of cells, which interferes with the normal functioning of the bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject that has a cancer is a subject having objectively measurable cancer 33 168506328.1cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, are able to out- compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

[0151] Cancers that may be treated by the compositions and methods contemplated by the invention include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated include, but are not limited to benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra- epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g. , small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade / follicular non- Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as 34 168506328.1well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome. A patient can have more than one type of cancer.

[0152] Provided herein is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb that binds to an antigen derived from the tumor or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein protein complex comprises at least one sdAb that binds to an antigen derived from the tumor. “Reducing” includes inhibiting and / or reversing and can refer to, for example, the symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or the size of the dormant tumor.

[0153] Provided is a method of reducing T cell tolerance, in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb promotes T cell activation or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein protein complex comprises at least one sdAb promotes T cell activation.

[0154] Provided is a method of promoting T cell activation and / or T cell expansion, in a subject in need thereof, the method comprising administering to the subject (i) a protein complex disclosed herein, wherein protein complex comprises at least one sdAb conjugate comprising at least one sdAb promotes T cell activation or (ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a protein complex disclosed herein, wherein protein complex comprises at least one sdAb promotes T cell activation. In one embodiment, the sdAb binds to CD3e.

[0155] The embodiments of the invention may be used for treating metastasis, which relates to the spreading of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life - threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site 35 168506328.1are also significant. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.

[0156] Also contemplated are methods of reducing cancer stemness comprising the administration of the protein complexes disclosed herein. Cancer stemness may refer to the ability of a cell to self-renew and to generate an additional, phenotypically distinct cell type. Cancer stem cells (CSCs) are cancer cells that exhibit stem-cell like properties. CSCs often exhibit at least one hallmark of cancer, and is capable of generating at least one additional, phenotypically distinct cell type. Furthermore, cancer stem cells are capable of both asymmetric and symmetric replication. It is appreciated that a cancer stem cell may result from differentiated cancer cells that acquire stemness traits and / or stem cells that acquire phenotypes associated with cancer cells. Alternatively, cancer stem cells can reconstitute non-stromal cell types within a tumor.

[0157] Also provided herein are methods of reducing tumor-associated fibrosis, increasing cytokine production in the tumor microenvironment, increasing anti-tumor immunity, or increasing infiltration of a tumor with immune cells, the methods comprising administering to the subject a protein complex disclosed herein.

[0158] The compositions disclosed herein (e.g., protein complexes and pharmaceutical compositions disclosed herein) can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages can be determined by clinical trials. The precise amount of the compositions disclosed herein to be administered can be determined by a physician having accounted for individual differences in age, weight, tumor size, extent of infection or metastasis, and patient's condition (subject).

[0159] The compositions can also be administered several times at these dosages. The optimal dose and treatment regimen for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0160] The administration of the present compositions can be carried out in any convenient way, including infusion or injection, or other methods known in the art. The compositions described herein may be administered to a patient subcutaneously, intradermally, 36 168506328.1intratumorally, intranodally, intramedullary, intramuscularly, intracranially, by intravenous or intralymphatic injection, or intraperitoneally.

[0161] In certain embodiments, the protein complex is administered to the subject by intravenous infusion, i.e., introduction of the protein complex into the vein of the subject over a certain period of time. In certain embodiments, the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0162] In certain embodiments, a dose of a compound or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, once every two weeks, or once a month. In other embodiments, two, three or four doses of a compound or a composition is administered to a subject every day, every couple of days, every third day, once a week, once every two weeks or once a month. In some embodiments, a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days or 28 days. In certain embodiments, a dose of a compound or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more. The frequency may be decreased during a maintenance phase of the disease or disorder.

[0163] In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the composition is administered to the subject before, after, or concurrently with the second therapeutic agent.

[0164] “Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. For example, the compositions disclosed herein can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablating agents such as CAMPATH, anti-cancer antibodies. CD3 or other antibody therapies, cytoxine, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. 37 168506328.1

[0165] In some embodiments, the method further comprises administering a therapeutically effective amount of an immune checkpoint modulator. Checkpoint proteins interact with specific ligands that send a signal into the T cell and switch off or inhibit T cell function. By expressing high levels of checkpoint proteins on their surface, cancer cells can control the function of T cells that enter the tumor microenvironment, thus suppressing the anticancer immune response. Examples of immune checkpoint modulators include PD1, PDL1, CTLA4, TIM3, LAG3, and TRAIL. The immune checkpoint protein Programmed Death-1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1 / PD-L1 interaction can promote potent antitumor activity. Examples of PD-1 inhibitors include, but are not limited to, Pembrolizumab (MK-3475), Nivolumab (MDX- 1106), Cemiplimab-rwlc (REGN2810), Pidilizumab (CT-011), Spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym021, TSR-042. Examples of PD-L1 inhibitors include, but are not limited to, Atezolizumab (MPDL3280A), Durvalumab (MEDI4736), Avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316.

[0166] Illustrative checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies, or antigen binding fragments thereof, other binding proteins, biologic therapeutics or small molecules, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM-3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049. Illustrative immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody), anti- OX40, and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor), as well as the PD-1 and 38 168506328.1PD-L1 inhibitors described above. Checkpoint protein ligands include, but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

[0167] In some embodiments, the protein complex is administered with a TIGIT, LAP, Podoplanin, Protein C receptor, ICOS, GITR, CD226 or a CD160 inhibiting agent. In some embodiments, the protein complex is administered with a CTLA-4, a PD-1, a PD-L1, or a PD-L2 inhibiting agent.

[0168] The checkpoint modulators may be administered simultaneously, separately, or concurrently with the compositions disclosed herein.

[0169] In some embodiments, the method further comprises administering a therapeutically effective amount of a “chemotherapeutic agent,” which is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, methyldopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotics chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, 39 168506328.1methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®.; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, xeloda, gemcitabine, KRAS mutation covalent inhibitors and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additional examples include irinotecan, oxaliplatinum, and other standard colon cancer regimens.

[0170] For treatment of infections, combination therapy described herein can encompass co- administering compositions and methods described herein with an antibiotic, an anti-fungal drug, an anti-viral drug, an anti-parasitic drug, an anti-protozoal drug, or a combination thereof.

[0171] Non-limiting examples of useful antibiotics include lincosamides (clindomycin); chloramphenicols; tetracyclines (such as Tetracycline, Chlortetracycline, Demeclocycline, 40 168506328.1Methacycline, Doxycycline, Minocycline); aminoglycosides (such as Gentamicin, Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin, Neomycin); beta-lactams (such as penicillins, cephalosporins, Imipenem, Aztreonam); vancomycins; bacitracins; macrolides (erythromycins), amphotericins; sulfonamides (such as Sulfanilamide, Sulfamethoxazole, Sulfacetamide, Sulfadiazine, Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide, p-Aminobenzoic Acid, Trimethoprim-Sulfamethoxazole); Methenamin; Nitrofurantoin; Phenazopyridine; trimethoprim; rifampicins; metronidazoles; cefazolins; Lincomycin; Spectinomycin; mupirocins; quinolones (such as Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin, Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin); novobiocins; polymixins; gramicidins; and antipseudomonals (such as Carbenicillin, Carbenicillin Indanyl, Ticarcillin, Azlocillin, Mezlocillin, Piperacillin) or any salts or variants thereof. The antibiotic used will depend on the type of bacterial infection.

[0172] Non-limiting examples of useful anti-fungal agents include imidazoles (such as griseofulvin, miconazole, terbinafine, fluconazole, ketoconazole, voriconazole, and itraconizole); polyenes (such as amphotericin B and nystatin); Flucytosines; and candicidin or any salts or variants thereof.

[0173] Non-limiting examples of useful anti-viral drugs include interferon alpha, beta or gamma, didanosine, lamivudine, zanamavir, lopanivir, nelfinavir, efavirenz, indinavir, valacyclovir, zidovudine, amantadine, rimantidine, ribavirin, ganciclovir, foscarnet, and acyclovir or any salts or variants thereof.

[0174] Non-limiting examples of useful anti-parasitic agents include chloroquine, mefloquine, quinine, primaquine, atovaquone, sulfasoxine, and pyrimethamine or any salts or variants thereof.

[0175] Non-limiting examples of useful anti-protozoal drugs include metronidazole, diloxanide, iodoquinol, trimethoprim, sufamethoxazole, pentamidine, clindamycin, primaquine, pyrimethamine, and sulfadiazine or any salts or variants thereof.

[0176] Provided is a method of making a protein complex, the method comprising contacting an IgM scaffold disclosed herein with one or more sdAb conjugates as disclosed herein.

[0177] Articles of Manufacture and Kits

[0178] Also provided are kits or articles of manufacture for use in the methods described herein. In aspects, the kits comprise the compositions described herein (e.g., a protein complex or a pharmaceutical composition comprising a protein complex) in suitable packaging. Suitable 41 168506328.1packaging for compositions described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed.

[0179] Also provided are kits comprising the compositions described herein. These kits may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing the administration of the composition or performing any methods described herein.

[0180] It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed herein. It is further to be understood that this disclosure includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the disclosure, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments disclosed herein.

[0181] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[0182] All other referenced patents and applications, scientific articles and book chapters, etc., are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0183] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the disclosure. 42 168506328.1EXAMPLES

[0184] Example 1: Materials and Methods for Examples 2-7

[0185] Experimental Model and Study Participant Details

[0186] The 293T-hsACE2 cell line was purchased from Integral Molecular in the freezing media (50% cell culture media, 40% FBS, 10% DMSO). Cells were cultured in a cell culture media (DMEM, 10%FBS, 10 mM HEPES, 1x Penicillin-Streptomycin and 0.5 µg / ml Puromycin) at 37 °C, 5% (v / v) CO2. Cells were split once 80-90% confluency had been reached. After at least three passages, cells were used for the pseudovirus neutralization assay at 80-90% confluency and more than 95% viability. The cell line was authenticated by Integral Molecular using flow cytometry by an ACE2-specific monoclonal antibody (R&D system, cat# MAB9332-100).

[0187] The Expi293F cells were obtained from Thermo Fisher.1 ml of the cells was thawed at 37°C water bath and added to 29 ml of pre-warmed Expi293 Expression Media in a 125 ml non-baffled, vented flask. The cells were cultured at 37°C, 8% (v / v) CO2 on an orbital shaker at 180 r.p.m. Cells were split when density reached 3 – 5 x 106cells / ml. At least three passages were performed, and more than 98% cell viability was ensured before the transfection.

[0188] All SARS-CoV-2 Victoria variant (SARS-CoV-2 / human / AUS / VIC01 / 2020) cultures and infections were performed in the containment level 3 (CL3) lab at the Oxford Particle Imaging Centre (OPIC). 15 ml 6× 106Vero E6 cells were seeded into a T7 flask one day before infection. On the day of infection, the medium was replaced with 15 ml of DMEM (1% FBS with Glutamine supplement). 100 μl virus at titer 105were seeded in the flask and incubated for 2-3 days. Cell debris was removed by centrifugation at 400 x g for 20 min at 4°C. The supernatant was aliquoted and stored in the -80°C freezer. The frozen virus was thawed, and the virus titer was tested by plaque assay using Vero E6 cells.

[0189] HeLa-ACE2 cells (BPS Bioscience, were maintained in DMEM (Corning) supplemented with 10% FBS, 0.5 µg / mL puromycin, and penicillin / streptomycin (Corning) at 37°C and 5% CO2.

[0190] All cell lines used in this study were regularly screened for mycoplasma contamination using the MycoStrip™ - Mycoplasma Detection Kit. Cells were infected with SARS-CoV-2, isolate USA-WA1 / 2020, a previously described mouse-adapted SARS-CoV-2 strain (MA-SARS-CoV-2) and representative Beta (B.1.351), Omicron (BA.5), (XBB.1.16) and (XBB.1.5) SARS-CoV-2 variants. These variants were collected from nasopharyngeal swab specimens as part of the routine SARS-CoV-2 surveillance conducted by the Mount Sinai 43 168506328.1Pathogen Surveillance program (IRB approved, HS#13-00981). Viruses were grown in Vero- TMPRSS2 cells for 4–6 d; the supernatant was clarified by centrifugation at 4,000g for 5 min and aliquots were frozen at −80°C for long-term use. Expanded viral stocks were sequence- verified to be the identified SARS-CoV-2 variant and titered on Vero-TMPRSS2 cells before use in antiviral assays. Infections with viruses were performed under biosafety level 3 (BSL3) containment in accordance with the biosafety protocols developed by the Icahn School of Medicine at Mount Sinai.

[0191] All the antiviral animal studies were performed in an animal biosafety level 3 (BSL3) facility at the Icahn School of Medicine in Mount Sinai Hospital, New York City. Animal studies with the Wuhan-like SARS-CoV-2 / WA1 were performed using 8 or 12-week- old female 129 / S mice (Jackson Laboratory strain 002448). Animal studies with the SARS- CoV-2 Omicron XBB.1.5 were performed using 8-week-old female K18-hACE2 (B6) transgenic mice (Jackson Laboratory strain 034860).

[0192] Nb and Nb-dimer DNA synthesis and cloning

[0193] The DNA encoding SpyCatcher003 was obtained from Addgene. Genes of Nbs were synthesized and cloned into the pET-21b(+) vector at EcoRI and HindIII sites from Synbio Technologies (Table 3 and Table 6). To produce Nb-Ig∆NSpyCatcher003, the DNA fragment of ∆NSpyCatcher003 was first PCR amplified from the Addgene plasmid by using primers NSC3- F and NSC3-R to introduce a linker sequence and two restriction sites of NotI and XhoI that facilitate cloning. The PCR fragment was then inserted into the pET-21b(+) vector of different Nbs at the same restriction sites to produce the fusion constructs. To produce Nb-dimer, the DNA fragment of the Nb on the C-term was PCR amplified by using primer D-F and D-R1 / D- R2 to introduce a linker and two restriction sites HindIII and NotI, and then the PCR fragment was cloned into the N-term Nb plasmid at these two sites.

[0194] Purification of Nbs and Nb-dimers

[0195] Nb or Nb-dimer DNA constructs were transformed into BL21(DE3) cells and plated on Agar with 50 μg / mL ampicillin at 37°C overnight. Cells were cultured in an LB broth to reach an O.D. of ∼0.6–0.8 before IPTG (0.5–1 mM) induction at 16°C overnight. Cells were then harvested, sonicated, and lysed on ice with a lysis buffer (1xDPBS, 150 mM NaCl, 0.2% TX-100 with protease inhibitor). After cell lysis, protein extracts were collected by centrifugation at 21,000 x g for 10 mins and the his-tagged Nbs were purified by the His-Cobalt resin and natively eluted with a buffer containing 150 mM imidazole buffer. Eluted Nbs were subsequently dialyzed in a dialysis buffer (e.g., 1x DPBS, pH 7.4 or 20mM Na-HEPES, 150mM NaCl, pH 7.45). 44 168506328.1

[0196] Production of recombinant Nb-IgG-Fc, IgM core, Nb-IgM-Fc conjugates and AMETA

[0197] The cDNA encoding human IgM-Fc and J chain (his-tagged) were obtained from Addgene. SpyTag003 was added to the N-term of IgM-Fc using primers ST3-F and ST3-R. Plasmids of the benchmarks (humVHH72S56A-Fc and (humVHH72S56A)2-Fc) and Nb-IgG- Fc were synthesized and cloned into the IgG1-Fc pcBNC vector at the BamHI and NotI sites by Synbio Technologies (Table 3 and Table 6). To express the proteins, Expi293F cells were transiently transfected with a mixture of IgM-Fc and J chain or Nb-IgG-Fc using the ExpiFectamine 293 kit at 1-1.5 µg plasmid per ml culture. After 20-24 hrs of transfection, enhancers were added to further boost protein expression. Cell culture was harvested 3-4 days after transfection and the supernatant was collected by high-speed centrifugation at 21,000×g for 30 min. The secreted pentameric recombinant IgM-Fc or Nb-IgG-Fc in the supernatant were purified using His-Cobalt resin or protein A resin and natively eluted according to the manufacturer's protocols. Eluted proteins were subsequently dialyzed in a dialysis buffer (e.g., 1x DPBS, pH 7.4, 20mM Na-HEPES, 150mM NaCl, pH 7.45 or 50 mM Tris-HCl, 150 mM NaCl, pH 8.0).

[0198] Nbs / dimers were then incubated with IgM-Fc core at a 30:1 molar ratio at 4°C overnight (16°C for 8 hours or 23°C for 2 hours can also be used). The final conjugates were further purified by 100kDa molecular weight cutter or size-exclusion chromatography using a Superose 6 Increase 10 / 300 (for AMETA) in SEC buffer (20 mM Na-HEPEs, 150 mM NaCl, pH 7.45).

[0199] Mass photometry of AMETA samples

[0200] Mass photometry experiments were carried out using a Refeyn TwoMP (Refeyn Ltd., Oxford, UK) MP system. AcquireMP and DiscoverMP software packages were used to record movies and analyze data, respectively, using standard settings. Ready-to-use sample carrier slides (Refeyn Ltd., Oxford, UK) were used for each measurement. Samples well cassettes (6 wells, Refeyn Ltd., Oxford, UK) were used to keep the sample in droplet shape. Contrast-to-mass calibration was carried out using SEC polished γ-globulins from human blood (Millipore-Sigma: G4386), giving molecular weights of 158, 316, 474, and 632 kDa. Each sample was first diluted in PBS to 100 nM at room temperature. Droplet dilution was initiated using 18 µL PBS. Upon focusing, 2 µL of diluted sample was added with quick mixing in the droplet. Movies were then recorded when all parameters were in blue.

[0201] The percentage of conserved RBD surface covered by Nbs 45 168506328.1

[0202] Relative solvent accessible surface area (rSASA) for each RBD residue is calculated by FreeSASA. Surface residue is defined using a cutoff of rSASA > 0.8. The conservation score of each RBD residue is calculated as described in Xiang et al., Superimmunity by pan- sarbecovirus nanobodies, Cell Rep. 2022 Jun 28;39(13):111004. An amino acid residue is defined as conserved if its conservation score is larger than 0.7. The percentage of conserved RBD surface covered by Nbs is calculated as the number of conserved residues on the epitope(s) of Nb(s) by the total number of conserved RBD surface residues.

[0203] Pseudotyped SARS-CoV-2 neutralization assay

[0204] The 293T-hsACE2 cell line and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain D614G, variants and SARS-CoV) particles with luciferase reporters were purchased from the Integral Molecular, Inc.. The pseudotyped viral particles are produced in HEK-293T cells using plasmids encoding the SARS-CoV-2 spike protein, a lentiviral gag polyprotein, and a reporter gene. They are capable of a single round of infection and carry a genome that expresses luciferase optical reporter gene upon infection. The neutralization assay was carried out according to the manufacturer’s protocols. In brief, 5-fold serially diluted proteins were incubated with the pseudotyped SARS-CoV-2 particles for 1-2 hr at 37°C with 5% CO2. At least seven concentrations were tested for each protein and at least two repeats were done. Pseudovirus with PBS in the culture media was used as a negative control. 100 μL of the mixtures were then incubated with 100 μL 293T-hsACE2 cells at 2.5x10e5 cells / mL in the 96- well plates. The infection took ∼68-72 hrs at 37°C with 5% CO2. The luciferase signal was measured using the Renilla-Glo luciferase assay system with the luminometer at 1 ms integration time. The obtained relative luminescence signals (RLU) from the negative control wells were normalized and used to calculate the neutralization percentage at each concentration. Data was processed by Prism 9 (GraphPad) to fit into a 4PL curve and to calculate the EC50s (half-maximal effective concentration).

[0205] Viral growth and neutralization assay

[0206] Four thousand HeLa-ACE2 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 hours at 37°C, 5% CO2. Two hours before infection, the medium was replaced with 100 μL of DMEM (2% FBS) containing the AMETA proteins at concentrations 50% greater than those indicated. Plates were then transferred into the BSL3 facility and 100 PFU (MOI = 0.025) was added in 50 μL of DMEM (2% FBS), bringing the final protein concentration to those indicated. Plates were then incubated for 24 hours at 37°C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 hours before being removed from the BSL3 facility. The cells were then immunostained for 46 168506328.1the viral N protein (an inhouse mAb 1C7) with a DAPI counterstain. Infected cells (488 nm) and total cells (DAPI) were quantified using the Cytation 1 (Biotek) imaging cytometer. Infectivity was measured by the accumulation of viral N protein (fluorescence accumulation). Percent infection was quantified as ((Infected cells / Total cells) - Background) *100 and the PBS control were then set to 100% infection for analysis. Data was fit using nonlinear regression and IC50s for each experiment were determined using GraphPad Prism version 10.0.0 (San Diego, CA).

[0207] Preclinical evaluation of AMETA4 in the SARS-CoV-2 infection experiments

[0208] Experiment 1:

[0209] Animal studies with the Wuhan-like SARS-CoV-2 / WA1 were performed using 12- week-old female 129 / S mice (Jackson Laboratory strain 002448). There were four groups in the experiment: non-infected control, isotype control, AMETA prophylaxis (-6 hrs), and AMETA therapeutic (+6 hrs) treatment. 8 mice were used per group. Mice were intranasally infected with 1 × 104PFU of SARS-CoV-2 in 50 μl of PBS. For prophylaxis evaluation, AMETA4 or IgM-Fc were administered intranasally (i.n.) at 2.2 nmole / kg (~ 2 mg / kg) 6 hours prior to infection. For therapeutic treatment evaluation, AMETA4 was administered i.n.6 hours after infection. All dosing was performed through the intranasal (i.n.) route using PBS as a vehicle at a volume of 50 μl / mouse. Mice were weighed daily throughout the study as a measure of pathology. On day 3 post-infection, animals were humanely euthanized, and lung tissues were harvested. Lungs were homogenized in 700 µl PBS with silica glass beads and frozen at −80°C for viral titration by TCID50. Briefly, infectious supernatants were collected at 48 hrs post-infection and frozen at −80°C until later use. Infectious titers were quantified by limiting dilution titration using Vero E6 cells. Briefly, Vero E6 cells were seeded in 96-well plates at 20,000 cells / well. The next day, SARS-CoV-2-containing supernatant was applied at serial 10- fold dilutions ranging from 10−1to 10−6and, after 5 days, viral cytopathic effect (CPE) was detected by staining cell monolayers with crystal violet. TCID50 / ml was calculated using the method of Reed and Muench. The Prism software (GraphPad) was used to determine differences in lung titers using an unpaired T-test on log-transformed data.

[0210] Experiment 2:

[0211] Animal studies with the Wuhan-like SARS-CoV-2 / WA1 were performed using 8- week-old female 129 / S mice (Jackson Laboratory strain 002448). There were two groups in the experiment: benchmark ((humVHH72S56A)2-Fc) and AMETA for therapeutic (+6 hrs) treatment. 8 mice were used per group. Mice were intranasally infected with 1 × 104PFU of SARS-CoV-2 in 50 μl of PBS. AMETA4 or (humVHH72S56A)2-Fc was administered i.n. 6 47 168506328.1hours after infection. All dosing was performed through the intranasal (i.n.) route using PBS as a vehicle at a volume of 50 μl / mouse. Mice were weighed daily throughout the study as a measure of pathology. On day 3 post-infection, animals were humanely euthanized, and lung tissues were harvested. Lungs were homogenized in 700 µl PBS with silica glass beads and frozen at −80°C for viral titration by TCID50 as described above.

[0212] Experiment 3:

[0213] Animal studies with the SARS-CoV-2 Omicron XBB.1.5 were performed using 8- week-old female K18-hACE2 (B6) transgenic mice (Jackson Laboratory strain 034860). There are three groups in the experiment: isotype control, benchmark control and AMETA4 for prophylaxis (-6 hrs) treatment. 8 mice were used per group. Mice were intranasally infected with 2.5 × 104PFU of SARS-CoV-2 Omicron XBB.1.5 in 50 μl of PBS. AMETA4 (1.1 nmole / kg or 1 mg / kg) or (humVHH72S56A)2-Fc (9 nmole / kg or 1mg / kg) or an isotype control (2.2 nmole / kg or 1mg / kg) were administered i.n. 6 hours prior to infection using PBS as a vehicle at a volume of 50 μl / mouse. On day 3 post-infection, animals were humanely euthanized, and lung tissues were harvested. Lungs were homogenized in 700 µl PBS with silica glass beads and frozen at −80°C for viral titration by TCID50 as described above.

[0214] Mouse lung histological analysis

[0215] Paraffin-embedded lung tissue blocks for mouse lungs were cut into 5μm sections. Sections were stained with hematoxylin and eosin (H&E) and analyzed by Histowiz (Brooklyn, NY). Digital light microscopic scans of the whole lung processed in toto were examined by an experienced veterinary pathologist. Hematoxylin Eosin stained sections of lungs were examined by implementing a semi-quantitative, 5-point grading scheme (0 - within normal limits, 1 - mild, 2 - moderate, 3 - marked, 4 - severe) that took into account four different histopathological parameters: 1) perivascular inflammation 2) bronchial or bronchiolar epithelial degeneration or necrosis 3) bronchial or bronchiolar inflammation and 4) alveolar inflammation. These changes were absent (grade 0) in the lungs of uninfected mice.

[0216] Coupling AMETA with p-Bz-NCS-DFO

[0217] To a solution of AMETA in PBS (1.18 mg / mL, 1 mg) carbonate buffer 0.1 M was added to reach pH=8.3. A solution of deferoxamine (p-Bz-NCS-DFO) in DMSO (2.8 mL, 10 eq) was added. The reaction was stirred at 37 °C for 2 h. The reaction mixture was then purified (3 washes with PBS) and concentrated using 100k MWCO Vivaspin. DFO-AMETA was used for the radiolabeling without further purification.

[0218] Radiolabeling AMETA-DFO 48 168506328.1

[0219] A solution of89Zr in oxalic acid (20 mL, 3.3 mCi) was diluted in PBS (0.100 mL) and the solution was basified using a solution of Na2CO3to pH=7.8. The solution of AMETA- DFO was added and the reaction was stirred for half an hour at 37 °C. The complete incorporation of89Zr was checked by radio TLC. The reaction was purified with 100k MWCO Vivaspin (3 washes with PBS). A radiochemical purity of >99% was reached and the radiolabeled protein was used for in vivo studies without further purification (2.6 mCi, RCY= 79%).

[0220] PET / CT imaging of AMETA biodistribution in mice

[0221] 89Zr-labeled AMETA (275.5 ± 43.5 mCi) was intranasal injected in C57BL / 6 mice. Two, fourteen, twenty-four, and forty-eight hours later, mice were anesthetized using 1.0% isoflurane in O2 at a flow rate of ~1.0 liter / min. PET / CT scans were performed using a Mediso nanoScan PET / CT (Mediso, Budapest, Hungary). A whole-body CT scan was performed (energy, 50 kVp; current, 180 mAs; isotropic voxel size, 0.25 mm) followed by a 20-min PET scan. Reconstruction was performed with attenuation correction using the TeraTomo 3D reconstruction algorithm from the Mediso Nucline software. The coincidences were filtered with an energy window between 400 and 600 keV. The voxel size was isotropic with 0.4-mm width, and the reconstruction was applied for four full iterations, six subsets per iteration.

[0222] Biodistribution studies

[0223] C57BL / 6 mice were sacrificed 72 hours after injection of89Zr-labeled AMETA and perfused with PBS (20 ml). Tissues of interest were harvested, weighed, and gamma-counted using a Wizard22480 automatic gamma counter (PerkinElmer, Waltham, MA). Values were corrected for decay and expressed as a percentage of the injected dose per gram of tissue (%ID / g). See Table 9.

[0224] Cryo-EM sample preparation and imaging

[0225] 3.5 μl of 0.6 mg / mL AMETA were applied to ultrafoild R1.2 / 1.3 grids, and grids were blotted for 2.5 s with 0 blot force in the environmental chamber of a Vitrobot Mark IV (ThermoFisher) at 4 °C and 100% humidity. Blotted grids were plunged frozen in liquid ethane. Cryo grids were screened on a Glacios microscope (FEI / Thermo) at 200 kV and data collection was performed on a Titan Krios microscope (FEI / Thermo) at 300 kV using serialEM at PNCC. Movies were recorded on a K3 camera in super-resolution mode with a total dose of 38 electrons per Å2in a moving stack of 68 frames (dose rate 1 electron / pixel / frame) with a pixel size of 1.336 Å / pixel and a defocus range between −1 to −3 μm.

[0226] Cryo-EM data processing and model-building 49 168506328.1

[0227] Image processing was performed using CryoSPARC 4.3.1. The particles were automatically picked using the blob picker with a 300 Å diameter. The first round of reference- free 2D classification was performed with 200 classes and limited maximum resolution to 18 Å. Particles resembling IgM were selected to generate the initial volume using ab initio reconstruction. 3D refinement was first carried out using non-uniform refinement using ab initio volume as the reference without mask. A box size of 720 pixels was used to re-extract the IgM-like particle to visualize the AMETA-captured spike protein.

[0228] Models of IgM (PDB entry: 6KXS) were rigidly docked into the cryo-EM map and refined using res-space refinement in Phenix. The maps and models were displayed in ChimeraX.

[0229] Integrative Structure Modeling of AMETA

[0230] The structure model of AMETA3 was constructed using Modeller (Version: 10.3) and Alphafold2. The AMETA construct is composed of a fixed core region (five protomers of IgM-Fc homodimeric SpyTag003-Cµ2-Cµ3-Cµ4, one J chain) and ten versatile warhead regions (Nb dimer-Ig∆NSpyCatcher003). The Cryo-EM structure of the IgM-Fc pentamer core, i.e Cµ3-Cµ4 and J chain (PDB:8AE3) was used as a template for AMETA. The missing residues in hinge regions between Cµ3 and Cµ4 were fixed by Modeller. Missing structures in the J chain were fixed by using Alphafold2. The structure of Cµ2 and SpyTag003 was predicted by Alphafold and grafted to the IgM core template with the restraint of one disulfide bond in the hinge region between Cµ2 and Cµ3 domain. The fusion protein of two Nb dimers: 1) S36-182- Ig∆NSpyCatcher003and 2) 132-118-Ig∆NSpyCatcher003was constructed by Modeller with the templates of Alphafold2 prediction of NbS36, RBD-182 complex structure (PDB:8CYJ), RBD-132 complex structure (PDB:8CYD), RBD-118 complex (PDB:8CWV) and SpyCatcher-SpyTag structure (PDB:4MLI). Ten Nb dimers were then loaded to the AMETA3 core guided by the SpyCatcher-SpyTag interaction.

[0231] Molecular Dynamics Simulation of one protomer of AMETA3

[0232] Amber 20.0 (Case et al., The Amber biomolecular simulation programs, J Comput Chem.2005 Dec;26(16):1668-88) was used to perform MD simulations. All input files for MD simulations of one protomer of AMETA3 and parameter files were generated using CHARMM-GUI server (Lee et al., CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM / OpenMM Simulations Using the CHARMM36 Additive Force Field, J Chem Theory Comput.2016 Jan 12;12(1):405-13). The water-boxed AMETA3 protomer (including 450674 water molecules, 1283 chloride ions, and 1307 potassium ions) were simulated with CHARMM forcefield at 310K temperature with a step size of 2 50 168506328.1femtoseconds using one A100 enabled node with 8 processors for a period of 50 ns, after minimization and equilibrations. During MD runs, the cut-off of 12 angstroms was used for Van der Waals and short-range electrostatic interactions, along with PME conditions. After simulation, water molecules and ions were removed from the trajectory. Frames were aligned to the one of the Cµ2-Cµ3-Cµ4 chain in the first frame. The root-mean-square-fluctuations (RMSF) was calculated by pytraj python package (Roe et al., PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data, J Chem Theory Comput. 2013 Jul 9;9(7):3084-95).

[0233] Analysis of Epitope Accessibility in RBD Opening Dynamics

[0234] The MD simulation of RBD opening dynamics was obtained from (https: / / covid.molssi.org / simulations / #pmf-calculations-of-sars-cov-2-spike-opening) (Pang et al., SARS-CoV-2 spike opening dynamics and energetics reveal the individual roles of glycans and their collective impact, Commun Biol. 2022 Nov 3;5(1):1170). The trajectory without glycans was used for the analysis of Nb epitope accessibility. Nb epitope is defined by RBD residues within 8 angstrom Ca-distance to the Nb. Solvent accessible surface area (SASA) for each amino acid in each frame was calculated by the tool FreeSASA (Version 2.1.2) (Mitternacht, FreeSASA: An open source C library for solvent accessible surface area calculations, F1000Res. 2016 Feb 18;5:189). The epitope SASA was then calculated by summation of SASAs of all epitope residues. To smooth the curve, data points for each Nb were fitted to the quadratic function.

[0235] Cryo-ET sample preparation and vitrification

[0236] EM grids (G300F1, R2 / 2 Quantifoil holey carbon, gold) were glow-discharged and placed in the wells of a 12-well plate. The grids were treated with bovine fibronectin (20 μg / ml) for 30 minutes then washed with PBS and UV-treated for 30 minutes.6 × 104of Vero E6 cells resuspended in 1 ml of complete DMEM medium were seeded on top of the grids in each well of the 12-well plate, incubated for 24h at 37°C / 5% CO2 to allow cell attachment to grid carbon. The medium was moved to the BL3 facility before being replaced with the SARS-CoV-2 Victoria variant diluted to MOI 0.5 in DMEM (1% FBS with Glutamine supplement). The cells were incubated at 37°C / 5% CO2for 24h in the CL3 facility.

[0237] The AMETA or IgM-Fc were warmed up at 37°C and diluted to the desired concentration in warm PBS. The 24 well plates with grids of infected cells were washed with warm PBS once. 1 ml of diluted AMETA was added to each well and returned to 37°C / 5% CO2for 2h. PBS was used as a blank control. After incubation, the grids were washed with 51 168506328.1warm PBS and fixed with 4% PFA (EM grade) PBS solution for 1h at RT before being removed from the CL3 facility.

[0238] Grids were washed with PBS. 2 μl of PBS with 6 nm Au fiducial beads (EMS) in the carbon side and 1 μl from the Au side were added to the grids before blotting. The grids were blotted from the back for 5 seconds and plunge-frozen in liquid ethane at −183°C using the Leica GP2 plunger. Grids were clipped and stored in liquid nitrogen until imaging.

[0239] Cryo-ET data collection and processing

[0240] Tilt series of SARS-CoV-2 virus with AMETA or IgM-Fc bound were collected with Titan Krios equipped with a Selectris X energy filter and Falcon 4 detector, or a Gatan BioQuantum energy filter and K3 detector. The tilt angles are from -60 degrees to 60 degrees with a step of 3 degrees with a dose-symmetric scheme. The pixel size is 1.50 A / pixel (falcon camera) or 1.34 A / pixel (K3 camera). Each image contains 10 movie frames. Periphery of the cells that contain the freshly egressed SARS-CoV-2 virus particles were targeted for cryo- electron tomography data collection under each experimental condition. Raw movie frames were motion corrected by MotionCor2. The tilt-series were aligned by eTomo based on the tracking of gold beads fiducial markers. IsoNet neural networks were trained and then used to correct the missing wedge artifact in the reconstructed tomograms. Virus morphology and spike number and conformation were analyzed. Segmentation is processed by manually docking spikes (emd-11651) and AMETA (from this study) to corresponding areas or automatically segmented by EMAN2 after training the neural network.

[0241] Quantification and statistical analysis

[0242] GraphPad Prism (version 9.3.0) was used for all statistical calculations. Two-way ANOVA was performed within > 2 groups. For statistical difference analysis, p values less than 0.05 were considered statistically significant. ns: no significant difference; ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001.

[0243] Example 2: A modular system for conjugation of sdAbs to an IgM scaffold

[0244] A modular system was developed that allows a person skilled in the art to directly and covalently conjugate sdAbs, such as nanobodies (Nbs), to a stable human IgM scaffold using a modified SpyTag003-SpyCatcher003 conjugation technique. See Keeble et al. (2019). The SpyTag / SpyCatcher conjugation technique was originally developed based on the split protein CnaB2 from Streptococcus pyogenes. See Zakeri et al. (2012). The SpyTag / SpyCatcher system comprises two fragments: one named SpyTag, and the other named SpyCatcher. Once 52 168506328.1combined under nearly any common conditions, SpyTag and SpyCatcher can rapidly and efficiently covalently conjugate to each other through an isopeptide bond.

[0245] The core IgM scaffold used for the modular conjugation system encompassed the constant regions of the heavy (µ) chain spanning Cμ2-Cμ4, and the J chain. The Fab domain of the IgM molecule was replaced with SpyTag003 (SEQ ID NO:1), resulting in an SpyTag- IgM fusion protein (SEQ ID NO:6). See Table 2.

[0246] To reduce potential immunogenicity, a small and truncated SpyCatcher003 variant (SEQ ID NO:10) was generated by deleting the immunogenic N-terminal domain (NTD). The N-terminal deletion resulted in a compact structure that retained a single immunoglobulin (Ig) fold (Ig∆NSpyCatcher003) while exhibiting full activity comparable to the full-length SpyCatcher003. Nb-Ig∆NSpyCatcher003 fusion constructs were produced with high solubility from E.coli. The SpyTag003-IgM scaffold was expressed in high yields from mammalian cell secretion. Is has to be noted that while here, SpyCatchers were fused to the nanobodies and SpyTags were fused to the IgM scaffold, alternative embodiments are possible (e.g., SpyTags fused to nanobodies and SpyCatchers fused to the IgM scaffold).

[0247] Nb-Ig∆NSpyCatcher003 were efficiently conjugated to the SpyTag003-IgM scaffold through incubation at a high molar ratio (e.g., 30:1). The resulting Nb-IgM-Fc conjugates (also referred to herein as “multi-epitope targeting with enhanced avidity” (AMETA)) were highly stable and produced abundantly. Fully conjugated proteins were rapidly purified and concentrated using a 100 kDa molecular weight cut-off centrifugal filter (Fig. 1A). Analyses through denatured SDS and native protein gels confirmed the complete covalent attachment of Nbs across all ten arms of the IgM core (data not shown). Size-exclusion chromatography (SEC) and mass photometry further corroborated these conjugates' high purity and expected masses (data not shown). Table 2. Selected sequences. SEQ Name Sequence ID E G53 168506328.1SEQ Name Sequence ID NO L R E C T A Y C d R D L S V G T P S D V F G L L T54 168506328.1SEQ Name Sequence ID NO E A R V T R V Tp g y

[0249] To assess the avidity effects of the IgM scaffold on Nbs, a diverse collection of high- affinity anti-SARS-CoV-2 Nbs was conjugated to IgM. See Table 3. Table 3. Nb monomer sequences. Science 2020 = Xiang et al., Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2, Science.2020 Dec 18;370(6523):1479-1484.; Cell Rep 2022 = Xiang et al., Superimmunity by pan-sarbecovirus nanobodies, Cell Rep.2022 Jun 28;39(13):111004. SEQ ID Nb ID Nb ID in the Protein sequence NO used in previous T G L T V N G168506328.1SEQ ID Nb ID Nb ID in the Protein sequence NO used in previous the ublication G Y P T G A T A T S S D Y Y R T Y56 168506328.1SEQ ID Nb ID Nb ID in the Protein sequence NO used in previous the ublication T F D T S F S T Q S S S S S Q W T57 168506328.1SEQ ID Nb ID Nb ID in the Protein sequence NO used in previous the ublication G M T G I I

[0050] ese bs spec ca y arge a eas ve neura zng ep opes on e receptor- binding domain (RBD) of SARS-CoV-2 (Fig.1B).

[0251] When compared to monomeric Nbs, conjugation to the IgM-Fc significantly enhanced the Nbs’ neutralization activities against SARS-CoV-2 (Wuhan-Hu-1 D614G), with an average improvement (in molar concentrations) of approximately 500-fold, ranging from 4 to 7,000-fold as compared to the Wuhan-Hu-1 strain. See Table 4. Table 4. Summary of epitope information and the pseudovirus neutralization potencies of Nb-monomers and Nb-IgM-Fc conjugates. EC50= Neutralization EC50(pM). Epitope Nb Virus strain EC50EC50Fold ID N N i t168506328.1Epitope Nb Virus strain EC50EC50Fold ID (Nb- (Nb- improvement monomer) I M)59 168506328.1Epitope Nb Virus strain EC50EC50Fold ID (Nb- (Nb- improvement monomer) I M)60 168506328.1Epitope Nb Virus strain EC50EC50Fold ID (Nb- (Nb- improvement monomer) I M)ion Neurulation Titer (PRNT50) (data not shown). The Nb-IgM-Fc conjugates also outperformed their Nb-IgG-Fc counterparts by an average of ~ 60-fold, indicating the enhanced avidity afforded by the pentameric IgM scaffold. See Table 5. Table 5. Summary of neutralization potency EC50s (pM) of Nbs and their corresponding Nb-IgG-Fc and Nb-IgM-Fc constructs against different virus strains. Strain Nb ID Nb-monomer Nb-IgG-Fc Nb-IgM-Fc S9 1017 110 1061 168506328.1

[0253] A notable positive correlation was observed between the neutralization potency (EC50s) of individual Nbs and their corresponding conjugates (Fig.1C).

[0254] Given that highly mutated virus variants might be able to evade monomeric Nbs, it was investigated whether the Nb-IgM-Fc conjugates had elevated avidity and activity against variants such as Omicron BA.2, BA.4 / 5, XBB.1.5, XBB.1.16, EG.5, BA.2.86, and SARS-CoV. For the Nbs that showed detectable activities at the highest concentration that was tested (i.e., 2.5 micromolar / µM), the avidity-enabled conjugates drastically enhanced efficacy against these highly evolved virus variants. The most pronounced improvements were observed for epitope I and III Nbs (Fig. 1D), which strongly bound to variable receptor binding sites and semi-conserved epitopes, respectively. Epitope I and III Nb-IgM-Fc conjugates generally potently neutralized the viruses in the sub-nanomolar (nM) range (Fig. 1E). In contrast, conjugates containing Nbs targeting more conserved epitopes, such as II, IV, and V, displayed moderate improvements, with median EC50 values ranging from low nM to μM concentrations (Fig. 1E, Table 4). Of note, Epitope II Nbs and their conjugates most efficiently neutralized the SARS-CoV. See Table 4. Interestingly, more substantial improvement in antiviral potency was observed in more solvent-accessible epitopes such as I and III, implying favorable cross- linking by the Nb-IgM-Fc conjugates (Fig. 1F). However, Nbs lacking initial neutralization activity against these viruses provided limited benefit from IgM-Fc conjugation, as evidenced by the absence of conferred protection (detailed in Table 4).

[0255] Example 4: Design of multi-epitope sdAb-IgM fusions

[0256] Nine highly soluble and high-affinity RBD Nbs that bound four distinct neutralizing epitopes were selected to design multi-epitope targeting dimers with improved activities. Dimers that showed improved neutralization indicated cooperative binding and could be used to further enhance avidity. These Nbs specifically targeted four neutralizing epitopes including conserved and cryptic sites. Guided by structure information, six distinct epitope combinations were generated, predominantly heterodimers and a homodimer of epitope II Nb (118). A flexible peptide linker (27 amino acids) was introduced between the Nbs to facilitate cooperative binding. Alternative flexible linkers, for example linkers comprising primary glycines, serines, and / or alanines, can be used. The resulting dimers spanned epitopes containing 11-44% of conserved surface residues on the SARS-CoV-2 RBD (Fig. 2A) and could be rapidly produced from E.coli with high expression. See Table 6. Table 6. Nb dimer sequences. 62 168506328.1SEQ Nb ID used in Protein sequence ID NO the Examples 38 35-182 HVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAAGWFR Y P R N R Q T T E Y T N Y S A Y N D N Q A Y R N L Q Q S Q168506328.1SEQ Nb ID used in Protein sequence ID NO the Examples 44 S36-113 HVQLVESGGGLVQAGGSLTLTCAASGRTFSSETMDMGW Y S R N R N D N Q Q S R M Q Q S E Q Q Q Q V G64 168506328.1SEQ Nb ID used in Protein sequence ID NO the Examples 50 118-118 QVQLVESGGGLVQAGESLRLSCAASKPTFRNFAAGWFR N D N K Y W Y S A Yy p ion potency, indicating successful designs for cooperative binding, either within a single RBD or across two RBDs on the spike glycoprotein (Fig.2A, Table 7). Table 7. Summary of neutralization potency EC50s (pM) of Nb dimers against different strains, related to Figure 2. N.D.: not detected, N / A: not tested. Nb dimer Wuhan-Hu-1 Beta Omicron BA.2 Omicron EG.5 SARS-CoV65 168506328.1

[0258] From these 14 constructs, lead dimers were selected from each combination based on yield and potency improvements, to act as a warhead in constructing Nb-IgM multimers. To bolster valency and epitope coverage further, two unique dimer warheads were conjugated (in a 1:1 molar ratio mixture) to the IgM-Fc scaffold. Using seven lead dimers, four distinct Nb-IgM multimer constructs (also referred to herein as AMETA constructs) were generated (Figs. 2B and 2C). Nb-IgM multimer constructs were efficiently conjugated and produced in high purity, with intact masses consistent with the predictions (data not shown). The resulting AMETA constructs were each armed with 20 Nbs for superior avidity binding. They targeted three or four RBD epitopes, covering a substantial portion (between 42%- 53%) of conserved, solvent-exposed residues (Fig.2C).

[0259] Example 5: Assessment of the broad-spectrum activities of sdAb-IgM multimer constructs

[0260] Next, highly mutated SARS-CoV-2 variants, including various Omicron lineages (BA.2, BA.4 / 5, XBB.1.5, XBB.1.16, EG.5, BA.2.86) and the original SARS-CoV, were used to assess the broad-spectrum activities of the AMETA constructs designed in Example 4. Specific variants, such as BA.2.86, have been shown to resist all clinical antibodies and can dramatically evade passive immunity by vaccines.

[0261] Remarkably, all AMETA constructs potently neutralized every virus that was tested. See Table 8. AMETA1 and AMETA2 were particularly effective against earlier Omicron variants and SARS-CoV, with EC50s ranging from 10 to 50 picomolar (pM). Their potencies against later Omicron variants (i.e., XBB.1.5, XBB.1.16, EG.5, BA.2.86) were only moderately decreased, ranging from 140 to 700 pM. In contrast, AMETA3 and AMETA4 showed consistently low EC50s within the 10- 100 pM range. Their in vitro potencies may be restrained only by diffusion limits. Table 8. Summary of neutralization potencies (EC50s) of AMETAs against a panel of SARS-CoV-2, its variants, and SARS-CoV. EC50 Wuhan SARS-CoV-2 (Omicron subvariants) SARS-66 168506328.1

[0262] Moreover, a side-by-side comparisons of AMETA constructs (targeting multi- epitopes) was conducted with the corresponding Nb-IgM-Fc conjugates (targeting a single- epitope) (Figs.3A-3D). Interestingly, 182-IgM-Fc, which targets non-conserved epitope I, was completely evaded by the most recent variants at the highest concentration that was tested (100 nM). This complete loss of activity was also seen in the monomer (at 2.5 µM). Other single- epitope conjugates were either partially evaded or exhibited inferior potencies, typically in the nM range. Multi-epitope targeting with high valency are particularly useful for neutralizing highly mutated variants, as shown by AMETA’s ultrapotencies and broader activities. Notably, AMETA3 and AMETA4 outperformed the corresponding Nb-IgM-Fc conjugates by a factor of 10-10,000 against later Omicron variants and SARS-CoV (Figs. 3A-3D). Such enhancements were more dramatic – exceeding one million-fold – when compared to specific Nb monomers (Figs.3E-3H).

[0263] Example 6: sdAb-IgM fusions show high preclinical efficacy

[0264] To assess AMETA’s in vivo distribution and stability, AMETA4 were conjugated with89Zr. This tracer-conjugate was delivered intranasally to C57BL / 6 mice. Intranasal administration of therapeutics can offer protection against respiratory infection in both upper and lower airways. Whole-body PET / CT scans were performed on various time points post- delivery. The construct rapidly distributed to both upper and lower respiratory tracts (Fig.4A). PET / CT scan intensities in the nasal cavity were initially abundant and dropped below detectable levels within 24 hours. However, the presence of the construct remained highly detectable in the lung throughout the entire 48-hour analysis period. Ex vivo gamma counting at 72 hours post-delivery reveals significant and specific enrichment in the lung. Minimal signals were detected in the bloodstream and most other organs, indicating a low systemic distribution dosing. See Table 9. The exception to this was observed in the gastrointestinal tract, where PET signals were nonspecifically introduced during intranasal administration.

[0265] This data underscores the sustained presence of AMETA in the lung by targeted delivery, which is particularly useful for respiratory therapies. Table 9. Biodistribution of AMETA4 at 72 hours following intranasal injection. Mice were euthanized 72 hours post-injection, and tissues were analyzed for radioactivity. Gamma- counting was used to determine the residual tracer, corrected for decay, and expressed as a percentage of the injected dose per gram of tissue (%ID / g). Data are presented as % of injected dose per gram of tissue. 67 168506328.1Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Blood 0.047 0.002 0.011 0.017 0.022 Kid L 0509 0041 0576 0802 2005, ded into four groups. Three groups were infected intranasally with 10,000 PFU of mouse-adapted SARS-CoV-2 (USA-WA1 / 2020) strain and the fourth group was used as a non-infected control. As for prophylaxis evaluation, AMETA4 or IgM-Fc core was administered (2 mg / kg) intranasally to the first two groups 6 hours before infection. As for therapeutic evaluation, AMETA4 was delivered to the third group at 6 hours post-infection. Weight changes and clinical signs were monitored for three days before sacrificing the animals. Lung viral titers were measured using the microneutralization assay.

[0267] All infected animals with IgM-Fc core treatment experienced rapid weight loss (~ 10% by day 3 post-infection, Fig. 4B) and high viral lung titers (median ~108PFU, Fig. 4C). However, AMETA4 treatment significantly protected against these effects in both prophylaxis and treatment settings, indicated by a rebound in weight loss (Fig. 4B). Viral lung titers were significantly reduced in all AMETA-treated animals (Fig. 4C). Compared to the IgM-Fc control, prophylactic and therapeutic treatment by AMETA4 drastically reduced lung viral titers by 6-logs and 3-logs, respectively. Consistently, treatments significantly alleviated lung pathology and virus-induced inflammation (Fig.4D).

[0268] Additionally, side-by-side comparisons of AMETA4 were performed with a highly engineered and potent Nb-Fc construct ((humVHH72S56A)2-Fc) which targets a conserved sarbecovirus epitope on RBD. See Schepens Bet al., An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models, Sci Transl Med.2021 Nov 24;13(621):eabi7826.

[0269] The results showed that AMETA4, despite at a low therapeutic dose (2.2 nmole / kg, or 9 x lower than the benchmark), provided comparable protections against infection by the WT virus (Figs.4E and 4F). 68 168506328.1

[0270] Next, the prophylaxis efficacy of AMETA4 was evaluated against a highly evolved Omicron (XBB.1.5) strain in an Omicron susceptible transgenic murine model (K18-hACE2). Both (humVHH72S56A)2-Fc benchmark and an isotype control (IgM-Fc) were included.

[0271] AMETA4 protected Omicron infection at an exceedingly low dose (1.1 nmole / kg, or 9 x lower than the engineered VHH72), as evidence by significantly reduced lung viral titers and mitigated lung pathology (Fig. 4G and 4H) compared to the controls, consistent with in vitro neutralization results against Omicron subvariants (data not shown).

[0272] Collectively, these data demonstrated the high in vivo efficacy of AMETA in protecting against viral challenges. Given the accelerated virus replication in this model compared to humans, the findings indicate the significant benefits of the AMETA technology in preventing severe symptoms in humans.

[0273] Example 7: Hybrid Structural Analysis Revealing AMETA’s Dynamic Architecture and Multifaceted Antiviral Mechanisms

[0274] To explore the enhanced avidity effects, single-particle cryo-electron microscopy (cryo-EM) was employed to image the structure of a representative AEMTA construct (AMETA3). The analysis resolved most IgM-Fc core components, including the Cμ3-Cμ4 pentamer and J chain, aligning with recently published structures. Other components such as Cμ2, the Ig∆NSpyCatcher003domain, Nbs, and linker peptides were unresolved due to their inherent flexibility. Utilizing the cryo-EM findings, an in silico model of AMETA3 was developed.

[0275] The overall architecture of AMETA3 is reminiscent of a carousel, with 20 Nbs (akin to wooden horses) arranged out-of-plane around the rigid IgM-Fc core with their CDR loops highly accessible (Fig. 5A). Replacing the natural IgM’s Fab region with Nb dimers and the Ig∆NSpyCatcher003 domains may result in high flexibility, facilitating both in-plane and out-of- plane pivoting. Coupled with additional flexible linkers (between Cμ2 and the Ig∆NSpyCatcher003, between the Ig∆NSpyCatcher003 and Nb, and between bi-epitope Nbs), AMETA’s arms are likely considerably more dynamic than the Fab regions. Molecular Dynamics (MD) simulations, corroborating the cryo-EM observations, have revealed significant structural flexibility in the conjugated Nbs (Fig. 5B). Such flexibility allows different Nbs to effectively target a variety of viral epitopes including both conserved and cryptic ones, thereby improving avidity binding and antiviral efficacy.

[0276] To better understand AMETA’s marked antiviral activities, the constructs were incubated with native SARS-CoV-2 (Victoria) at different concentrations. Cryo-electron tomography (cryo-ET) was performed to visualize their binding to the virus in situ. It has been 69 168506328.1shown that spike glycoproteins on isolated inactivated SARS-CoV-2 virus particles display a significant amount of post-fusion conformation, ranging from 25% to 75%. However, spikes from intracellular and egressed virus particles in the context of infected cells are all in the pre- fusion conformation. Therefore, AMETA binding experiments were carried out directly with egressed SARS-CoV-2 in tissue culture in the BSL-3 containment lab prior to fixation and vitrification for subsequent cryo-ET analysis. In addition to blocking RBD binding to the receptor, the results indicate the presence of multiple other important mechanisms by which AMETA can leverage to inhibit the virus efficiently. At a low concentration (i.e., 0.1 nM), AMETA constructs induced inter-spike cross-linking, which may impair virus-host interactions. Additional EM densities corresponding to AMETA constructs were visualized to cross-link multiple spikes on the same virus (data not shown). As the AMETA’s concentration was increased to 1 nM, virus cross-linking was evident and became a predominant phenomenon at 1-10 nM concentration of all four AMETA tested (data not shown). Intriguingly, AMETA can also lead to massive spike disarming from the virions. Distinct and heterogenous EM densities are clearly visible outside the viruses and are no longer connected to them (data not shown), likely corresponding to the dissociated spikes and / or bound AMETA. Tomography data further supported this, showing a drastic reduction in spike numbers (both pre- and post- fusion conformations) on AMETA-treated virions. For the IgM-Fc control sample, the average number of spikes per virion was 23.4. However, the number dropped drastically to 6.3 in the AMETA4-treated samples (10 nM) (Fig. 5C). The loss of spikes may be triggered, at least in part, by the presence of epitope III (and likely epitope IV) Nbs, which can uniquely destabilize the integrity of the recombinant spike trimer at high concentrations (e.g., > 100 nM). The combined effects of enhanced avidity and multiple epitopes binding likely greatly exacerbated these destabilizing impacts, leading to the emergence of unusually "bald", disarmed virions. In addition, a significant presence of post-fusion spikes was detected up to 90.9% (at 10 nM of AMETA4), in stark contrast to the isotype control (0.9%, Fig.5D). The decrease in viral spike count and the rise in the prevalence of the post-fusion state both depend on the concentration of AMETA. This marks a rare observation of essential viral structures being disarmed or deconstructed by a therapeutic molecule in the native context– strikingly, with such high efficiency.

[0277] Collectively, the data disclosed herein have uncovered a plethora of antiviral mechanisms by AMETA, underscoring their unique properties and synergy in the development of ultrapotent, broad-spectrum and durable antiviral therapeutics. 70 168506328.1

Claims

CLAIMS We claim:

1. A protein complex comprising: (a) an immunoglobulin M (IgM) scaffold comprising IgM Cµ2, Cµ3, and Cµ4 domains and a J chain; and (b) one or more single-domain antigen-binding fragment (sdAb) conjugates, wherein: the N-termini of one or more of the IgM Cµ2 domains are each fused to a copy of a first hybridization domain; each of the one or more sdAb conjugates comprises a second hybridization domain and one or more sdAbs; and the first hybridization domain forms a covalent bond with the second hybridization domain.

2. The protein complex of claim 1, wherein the N-termini of each of the IgM Cµ2 domains are each fused to a copy of the first hybridization domain.

3. The protein complex of claim 2, wherein the protein complex comprises 10 sdAb conjugates.

4. The protein complex of any one of claims 1-3, wherein: (a) the first hybridization domain comprises a SpyCatcher domain or variant thereof and the second hybridization domain comprises a SpyTag domain or variant thereof; or (b) the first hybridization domain comprises a SpyTag domain or variant thereof and the second hybridization domain comprises a SpyCatcher domain or variant thereof.

5. The protein complex of any one of claims 1-4, wherein: (a) the first hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:10, 11, 61 or 62 or a portion thereof and the second hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:1, 2, or 54-60 or a portion thereof; or (b) the first hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:1, 2, or 54-60 or a portion thereof and the second hybridization domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs:10, 11, 61 or 62 or a portion thereof. 71 168506328.

16. The protein complex of claim 5, wherein the first hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:10 or SEQ ID NO:11 and the second hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:1 or SEQ ID NO:2, optionally, wherein the first hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:10 and the second hybridization domain comprises a sequence that is at least 80% identical to SEQ ID NO:

2.

7. The protein complex of claim 5, wherein: (a) the first hybridization domain comprises SEQ ID NO:10 or SEQ ID NO:11 and the second hybridization domain comprises SEQ ID NO:1 or SEQ ID NO:2; or (b) the first hybridization domain comprises SEQ ID NO:1 or SEQ ID NO:2 and the second hybridization domain comprises SEQ ID NO:10 or SEQ ID NO:

11.

8. The protein complex of claim 7, wherein the first hybridization domain comprises SEQ ID NO:10 or SEQ ID NO:11 and the second hybridization domain comprises SEQ ID NO:1 or SEQ ID NO:2, optionally wherein the first hybridization domain comprises SEQ ID NO:10 and the second hybridization domain comprises SEQ ID NO:

2.

9. The protein complex of any one of claims 1-8, wherein each sdAb conjugate comprises at least two sdAbs.

10. The protein complex of claim 9, wherein the at least two sdAbs are connected to each other with a first linker, optionally, wherein the first linker is a polypeptide linker.

11. The protein complex of any one of claims 1-10, wherein the IgM Cµ2 domains are each fused to the copy of the first hybridization domain via a second linker, optionally, wherein the second linker is a polypeptide linker.

12. The protein complex of any one of claims 1-11, wherein the second hybridization domain is fused to one of the one or more sdAbs via a third linker, optionally, wherein the third linker is a polypeptide linker. 72 168506328.

113. The protein complex of any one of claims 10-12, wherein the first linker comprises a sequence that is at least 80% identical to SEQ ID NO:

52.

14. The protein complex of claim 13, wherein the first linker comprises SEQ ID NO:

52.

15. The protein complex of any one of claims 1-14, wherein at least some of the one or more sdAb conjugates comprise two or more sdAbs that bind to the same epitope.

16. The protein complex of any one of claims 1-15, wherein at least some of the one or more sdAb conjugates comprise two or more sdAbs that bind to different epitopes.

17. The protein complex of any one of claims 1-15, wherein the one or more sdAb conjugates are identical to each other.

18. The protein complex of any one of claims 1-17, wherein the one or more sdAbs bind to an epitope derived from a virus, a bacterium, a fungus, or a protozoan.

19. The protein complex of claim 18, wherein the one or more sdAbs bind to epitopes derived from human immunodeficiency virus (HIV), influenza virus, SARS-CoV-2, or hepatitis virus.

20. The protein complex of claim 19, wherein at least some of the one or more sdAb conjugates each comprise a sequence that is at least 80% identical to any one of SEQ ID NO:13-37.

21. The protein complex of claim 20, wherein at least some of the one or more sdAb conjugates each comprise any one of SEQ ID NO:13-37.

22. The protein complex of claim 19, wherein at least some of the one or more sdAb conjugates each comprises a sequence that is at least 80% identical to any one of SEQ ID NO:38-51.

23. The protein complex of claim 22, wherein at least some of the one or more sdAb conjugates each comprises any one of SEQ ID NO:38-51.

24. The protein complex of any one of claims 1-17, wherein at least some of the one or more sdAbs bind to a cancer antigen. 73 168506328.

125. The protein complex of any one of claims 1-17 or 24, wherein at least some of the one or more sdAbs promote T-cell activation.

26. The protein complex of any one of claims 1-17 or 24, wherein at least some of the one or more sdAbs are inhibitors of immune checkpoint proteins.

27. The protein complex of claim 26, wherein the immune checkpoint proteins are selected from the group consisting of PD-1, PD-L1, PLD-2 or CTLA-4.

28. The protein complex of any one of claims 1-27, wherein the protein complex is further conjugated to a payload.

29. The protein complex of claim 28, wherein the payload is an anti-viral agent.

30. The protein complex of claim 28, wherein the payload is an anti-cancer agent.

31. The protein complex of any one of claims 1-30, wherein the J-chain is further conjugated to a sdAb.

32. The protein complex of any one of claims 1-31, wherein the J chain comprises a sequence that is at least 80% identical to SEQ ID NO:12 or SEQ ID NO:53, optionally wherein the J chain comprises SEQ ID NO:12 or SEQ ID NO:

53.

33. A pharmaceutical composition comprising the protein complex of any one of claims 1-32 and a pharmaceutically acceptable excipient.

34. A method of treating an infection with a virus, bacterium, fungus, or protozoan in a subject in need thereof, the method comprising administering to the subject (i) the protein complex of claim 18, 28, 31, or 32 (ii) a pharmaceutical composition comprising the protein complex of claim 18, 28, 31, or 32 and a pharmaceutically acceptable excipient.

35. A method of treating an infection with a virus in a subject in need thereof, the method comprising administering to the subject (i) the protein complex of any one of claims 19-23, 28, 74 168506328.129, 31, or 32 or (ii) a pharmaceutical composition comprising the protein complex of any one of claims 19-23, 28, 29, 31, or 32 and a pharmaceutically acceptable excipient.

36. The method of claim 35, the method further comprising administering to the subject an additional anti-viral agent.

37. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject (i) the protein complex of any one of claims 24-28 or 30-32 or (ii) a pharmaceutical composition comprising the protein complex of any one of claims 24-28 or 30-32.

38. The method of any one of claims 34-37, wherein the subject is a human.

39. An IgM scaffold comprising IgM Cµ2, Cµ3, and Cµ4 domains and a J chain, wherein the N-termini of one or more of the IgM Cµ2 domains are each fused to a copy of a first hybridization domain, wherein the first hybridization domain comprises (a) a SpyCatcher domain or variant thereof or (b) a SpyTag domain or variant thereof.

40. The IgM scaffold of claim 39, wherein the N-termini of each of the IgM Cµ2 domains are each fused to a copy of the first hybridization domain.

41. The IgM scaffold of claim 39 or 40, wherein the first hybridization domain comprises a sequence that is (a) at least 80% identical to any one of SEQ ID NOs:10, 11, 61 or 62, or a portion thereof or (b) at least 80% identical to any one of SEQ ID NOs:1, 2, or 54-60, optionally, wherein the first hybridization domain comprises a sequence that is (a) at least 80% identical to SEQ ID NO:10 or (b) at least 80% identical to SEQ ID NO:

1.

42. The IgM scaffold of claim 41, wherein the first hybridization domain comprises (a) SEQ ID NOs:10, 11, 61 or 62 or a portion thereof or (b) any one of SEQ ID NOs:1, 2, or 54-60 or a portion thereof, optionally, wherein the first hybridization domain comprises (a) SEQ ID NO:10 or (b) SEQ ID NO:

2.

43. The IgM scaffold of claim 42, wherein the first hybridization domain comprises SEQ ID NO:

10. 75 168506328.1

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