Compositions and methods for improving immune response

Abstract

Disclosed herein are compositions and methods related to conjugates comprising a variable domain on a heavy chain antibody (VHH) specific for an immunoglobulin kappa light chain covalently conjugated to at least two copies of an agent that interacts with a protein expressed on the surface of a cell or virus.

Description

CEB-00225 COMPOSITIONS AND METHODS FOR IMPROVING IMMUNE RESPONSE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. Nos.63 / 666,793 filed July 2, 2024 and 63 / 772,915 filed March 17, 2025, which are incorporated by reference herein in their entirety. BACKGROUND

[0002] Immunotherapy is typically a cost-effective form of therapy and disease prevention. Vaccines, the earliest form of immunotherapy in clinical use, have pushed to the brink of extinction or even eradicated several viral pathogens. From a public health perspective, the number of lives saved by vaccines is counted in the millions. The route of vaccine administration can be a determinant of its success. In preclinical models, intraperitoneal or intravenous administration are commonly used to deliver a vaccine, while clinical deployment relies on intramuscular or subcutaneous injection, oral delivery or delivery by inhalation. Where possible, the latter two are preferred, because they avoid the use of needles.

[0003] In particular, the discovery that camelids make both conventional four-chain (HL)2-type immunoglobulins and heavy chain only (H)2type immunoglobulins enabled the recombinant expression of just the variable region of these heavy chain only immunoglobulins, products referred to as VHHs or nanobodies. The appeal of VHHs lies in their small size, ease of expression and modification, as well as in their stability. Their single domain nature also implies that the mode of antigen recognition is distinct from that of typical immunoglobulins and may provide access to epitopes not as readily accessible to the heavy chain-light chain combination of their conventional counterparts. Their ease of site-specific modification, whether enabled by the installation of an unpaired cysteine or by enzymatic modification, has found application in the design of nanoparticles that can be targeted to the site(s) recognized by the VHHs attached to such particles. The conversion of VHHs into theranostics through installation of the appropriate radio-isotopes is a promising application because the short circulatory half-life of VHHs limits systemic exposure, while the specificity of VHHs allows their significant enrichment at the sites targeted, such as tumors. Along the same lines, modification of VHHs with cytotoxic drugsAttorney Docket No.: CEB-00225 likewise creates conjugates with a short circulatory half-life and thus limited systemic exposure, while still affording the potential for therapeutic benefit through on-target efficacy.

[0004] Innovations in immunotherapy for the treatment and / or prevention of infectious diseases and cancer are still needed. For example, globally, there are nearly a billion cases of seasonal influenza each year, and about 500,000 people die each year from the infection. Accordingly, there is a need for improved prophylactic and therapeutic VHH-based immunotherapies for the treatment of infectious diseases and cancer. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (CEB-00225_SL; size: 53 KB; and date of creation: June 26, 2025) has been submitted electronically in XML format and is herein incorporated by reference in its entirety. SUMMARY

[0006] In certain aspects, provided herein are compositions and methods related to conjugates comprising a moiety that binds to an immunoglobulin kappa light chain (e.g., a variable domain on a heavy chain antibody, or VHH) linked to at least two copies of an agent (e.g., 2, 3, 4, 5, or 6 copies of an agent) that binds to a protein on the surface of a cell or pathogen (e.g., a viral protein or a tumor associated protein). In certain embodiments, the conjugates provided herein can be used to treat or prevent a pathogenic infection (e.g., a viral infection, such as an influenza infection) or cancer.

[0007] In certain aspects, provided herein is a conjugate comprising a means for binding to an immunoglobulin kappa light chain (e.g., a VHH) covalently conjugated to at least two copies of an agent (e.g., 2, 3, 4, 5, or 6 copies of an agent). In certain embodiments, the at least two copies of an agent are 4 copies of the agent. In some embodiments, means for binding to an immunoglobulin kappa light chain is conjugated to the two or more copies of an agent via one or more linkers (e.g., each copy of the agent is conjugated via its own linker, or at least two of the copies of the agent are conjugated via the same linker). In some embodiments, the agent binds to a protein on the surface of a cell or pathogen.

[0008] In certain aspects, provided herein is a conjugate comprising a VHH specific for an immunoglobulin kappa light chain covalently conjugated to at least two copies of an agentAttorney Docket No.: CEB-00225 (e.g., 2, 3, 4, 5, or 6 copies of an agent). In certain embodiments, the at least two copies of an agent are 4 copies of the agent. In some embodiments, VHH is conjugated to the two or more copies of an agent via one or more linkers (e.g., each copy of the agent is conjugated via its own linker, or at least two of the copies of the agent are conjugated via the same linker). In some embodiments, the agent binds to a protein on the surface of a cell or pathogen. In some embodiments, the VHH comprises a set of CDR1, CDR2, and CDR3 sequences selected from the sets of CDR1, CDR2, and CDR3 sequences in Table 2. In some embodiments, the VHH comprises the amino acid sequence selected from the sequences in Table 1.

[0009] In some embodiments of the conjugates provided herein, the agent comprises a small molecule, a polypeptide, a carbohydrate, a lipid, or a nucleic acid, an aptamer, a VHH, an antibody fragment, and / or an antibody. In some embodiments, the conjugate comprises a small molecule (e.g., zanamivir or a pharmaceutically acceptable salt or analog thereof). In some embodiments of the conjugates provided herein the agent binds to the surface of a pathogen or a cell infected by the pathogen.

[0010] In some embodiments, the pathogen is a virus. In some embodiments, the virus is an influenza virus, a coronavirus, an adenovirus, an enterovirus, a rotavirus, a norovirus, a herpesvirus, a lentivirus, a poxvirus, a paramyxovirus, a rhabdovirus, an arenavirus, a flavivirus, a togavirus, a hantavirus, a pneumovirus, or an ebolavirus. In certain embodiments, the virus is a Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus (optionally wherein the SARS associated coronavirus is SARS-CoV-1 or SARS-CoV-2), a human immunodeficiency virus (HIV), or a human respiratory syncytial virus (RSV).

[0011] In some embodiments of the conjugates provided herein, the agent binds to the surface of influenza virus or a cell infected by influenza virus. In some embodiments, the influenza virus is an influenza A virus or an influenza B virus. In some embodiments, the protein is an influenza virus neuraminidase, an influenza virus hemagglutinin, or an influenza matrix-2 (M2) proton channel protein. In certain embodiments, the protein is an influenza virus neuraminidase. In some embodiments, the agent comprises zanamivir or a pharmaceutically acceptable salt or analog thereof.

[0012] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a cancer cell. In some embodiments, the cancer cell is a hematologicalAttorney Docket No.: CEB-00225 cancer cell, a lung cancer cell, a breast cancer cell, a brain cancer cell, a gastrointestinal cancer cell, a liver cancer cell, a kidney cancer cell, a bladder cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a testicular cancer cell, a prostate cancer cell, an endometrial cancer cell, a muscle cancer cell, a bone cancer cell, a neuroendocrine cancer cell, a connective tissue cancer cell, a head or neck cancer cell, or a skin cancer cell. In certain embodiments, the agent binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen comprises a MHC class I polypeptide-related sequence A (MICA) protein, a MHC class I polypeptide-related sequence B (MICB) protein, a folate receptor, a fibronectin splice variant, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor receptor 2 (VEGFR-2), C-X-C chemokine receptor type 4 (CXCR4), urokinase plasminogen activator surface receptor (uPAR), follicle- stimulating hormone receptor (FSHR), epithelial cell adhesion molecule (EpCAM), epithelial cadherin (ECAD), carcinoembryonic antigen (CEA), or mesothelin (MSLN). In certain embodiments, the tumor-associated antigen comprises a carbonic anhydrase, for example, at least one of carbonic anhydrase IX (CAIX) or carbonic anhydrase XII (CAXII).

[0013] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a bacterium. In some embodiments, the bacterium is selected from a Pasteurella species, a Staphylococcus species, a Streptococcus species, a Bacillus species, a Corynebacterium species, a Diphtheroids species, a Listeria species, an Erysipelothrix species, a Clostridium species, a Neisseria species, a Branhamella species, an Escherichia species, an Enterobacter species, a Proteus species, a Pseudomonas species, a Klebsiella species, a Salmonella species, a Shigella species, a Serratia species, an Acinetobacter species, Haemophilus species, a Brucella species, a Yersinia species, a Francisella species, a Pasturella species, a Vibrio species, a Flavobacterium species, a Pseudomonas species, a Campylobacter species, a Bacteroides species, a Fusobacterium species, a Calymmatobacterium species, a Streptobacillus species, or a Legionella species.

[0014] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a parasite. In some embodiments, the parasite is selected from a Plasmodium species, a Trypanosoma species, a Toxoplasma species, a Leishmania species, or a Cryptosporidium species. In some embodiments, the parasite is from a Plasmodium species. In certain embodiments, the Plasmodium species is Plasmodium falciparum, Plasmodium malar,Attorney Docket No.: CEB-00225 Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curlisi, or Plasmodium ovale wallikeri.

[0015] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a fungus. In some embodiments, the fungus is selected from Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Aspergillus spp., Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Candida albicans, Candida spp., Nakaseomyces glabrata, Pichia kudriavzeveii, Histoplasma spp., Eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Cryptococcus gattii, Cryptococcus spp., Cryptococcus neoformans, Talaromyces marneffei, Paecilomyces lilacinus, Pneumocystis jirovecii, and Paracoccidioides spp., Fusarium spp., Fusarium solani, Scedosporium spp., Paecilomyces lilacinus, Muccorales Fungi (e.g., Rhizopus spp., Mucor spp., Lichtheimia spp., Cunninghamella spp.), Coccidioides immitis, Blastomyces spp., Histoplasma capsulatum, dimorphic fungi (e.g. Talaromyces marneffei and Sporothrix schenckii).

[0016] In some embodiments of the conjugates provided herein, the one or more linkers comprise a cleavable or a non-cleavable linker. In some embodiments, the one or more linkers comprise an azide moiety, a bicyclononyne moiety, a tetrazine moiety, a trans-cyclooctene moiety, a polypeptide, a polynucleic acid, an alkane, an alkene, an alkyne, a polyether polyol, and / or a polythioether polyol. In certain embodiments, the one or more linkers are dibenzoyclooctyne (DBCO) linkers. In some embodiments, the one or more linkers are polyethylene glycol dibenzocyclooctyne (DBCO) linkers.

[0017] In certain embodiments, the one or more linkers comprise:

[0018] In certain embodiments (e.g., embodiments wherein there are two copies of the agent), the one or more linkers comprise:Attorney Docket No.: CEB-00225

[0019] In certain embodiments (e.g., embodiments wherein there are four copies of the agent), the one or more linkers comprise:.

[0020] In some embodiments, the structure of at least one of the one or more linkers and at least one of the agents comprise:.

[0021] In certain aspects, provided herein is a pharmaceutical composition comprising the conjugate provided herein. In some embodiments, the pharmaceutical composition is formulated for intranasal, intravenous, intraperitoneal, or intramuscular administration.

[0022] In some embodiments, the composition is formulated for intranasal administration. In certain aspects, provided herein is a nasal spray comprising a pharmaceutical composition provided herein.Attorney Docket No.: CEB-00225

[0023] In certain aspects, provided herein is method of making the conjugate provided herein, comprising conjugating the two or more copies of the agent to the VHH or the means for binding an immunoglobulin kappa chain via a linker.

[0024] In some embodiments the methods provided herein comprise attaching a conjugation handle to the VHH or the means for binding an immunoglobulin kappa chain. In certain embodiments, the conjugation handle is attached via a sortase A-mediated transpeptidation reaction. In some embodiments, the conjugation handle is attached to a glycine on the VHH or the means for binding an immunoglobulin kappa chain. In some embodiments, the conjugation handle is attached via a maleimide reaction to a cysteine on the VHH or the means for binding an immunoglobulin kappa chain. In certain embodiments, the conjugation handle is attached via a NHS ester to an N-terminal amino acid modification on the VHH or the means for binding an immunoglobulin kappa chain. In some embodiments, the VHH or the means for binding an immunoglobulin comprises a non-natural amino acid, and the conjugation handle is attached to the non-natural amino acid. In certain embodiments, the non-natural amino acid contains an azide. In some embodiments, the conjugation handle is attached via an enzymatic reaction. In some embodiments, the conjugation handle is attached via a transglutaminase, a butelase, or an OaAEP1.

[0025] In certain aspects, provided herein is method for enhancing an immune response to a pathogen or cancer in a subject, comprising administering to the subject a conjugate or pharmaceutical composition provided herein. In certain aspects, provided herein is a method of treating or preventing infection from a pathogen in a subject, comprising administering to the subject a conjugate or pharmaceutical composition provided herein.

[0026] In certain embodiments, the conjugates provided herein are for use in enhancing an immune response to a pathogen or cancer in a subject. In some embodiments, the conjugates provided herein are for use in treating or preventing infection from a pathogen in a subject. In certain aspects, provided herein is the use of a conjugate provided herein in the manufacture of a medicament for enhancing an immune response to a pathogen in a subject. In certain aspects, provided herein is the use of a conjugate provided herein in the manufacture of a medicament for treating or preventing infection from a pathogen in a subject.

[0027] In some embodiments of the methods, uses, and compositions for use provided herein, the pathogen is a virus. In some embodiments, the virus is an influenza virus, aAttorney Docket No.: CEB-00225 coronavirus, an adenovirus, an enterovirus, a rotavirus, a norovirus, a herpesvirus, a lentivirus, a poxvirus, a paramyxovirus, a rhabdovirus, an arenavirus, a flavivirus, a togavirus, a hantavirus, a pneumovirus, or an ebolavirus. In certain embodiments, the virus is a Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus (optionally wherein the SARS associated coronavirus is SARS-CoV-1 or SARS- CoV-2), a human immunodeficiency virus (HIV), or a human respiratory syncytial virus (RSV). In some embodiments, the virus is HIV. In some embodiments, the virus is an influenza A virus or an influenza B virus. In some embodiments, the pharmaceutical composition is administered intranasally.

[0028] In some embodiments of the methods, uses and compositions for use provided herein, the method is for the treatment of a cancer. In some embodiments, the cancer is a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer. In certain embodiments, the cancer expresses a tumor-associated antigen to which the agent binds. In some embodiments, the tumor-associated antigen comprises a MHC class I polypeptide- related sequence A (MICA) protein, a MHC class I polypeptide-related sequence B (MICB) protein, a folate receptor, a fibronectin splice variant, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor receptor 2 (VEGFR-2), C-X-C chemokine receptor type 4 (CXCR4), urokinase plasminogen activator surface receptor (uPAR), follicle- stimulating hormone receptor (FSHR), epithelial cell adhesion molecule (EpCAM), epithelial cadherin (ECAD), carcinoembryonic antigen (CEA), or mesothelin (MSLN).

[0029] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a bacterium. In some embodiments, the bacterium is selected from a Pasteurella species, a Staphylococcus species, a Streptococcus species, a Bacillus species, a Corynebacterium species, a Diphtheroids species, a Listeria species, an Erysipelothrix species, a Clostridium species, a Neisseria species, a Branhamella species, an Escherichia species, an Enterobacter species, a Proteus species, a Pseudomonas species, a Klebsiella species, a Salmonella species, a Shigella species, a Serratia species, an Acinetobacter species,Attorney Docket No.: CEB-00225 Haemophilus species, a Brucella species, a Yersinia species, a Francisella species, a Pasturella species, a Vibrio species, a Flavobacterium species, a Pseudomonas species, a Campylobacter species, a Bacteroides species, a Fusobacterium species, a Calymmatobacterium species, a Streptobacillus species, or a Legionella species.

[0030] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a parasite. In some embodiments, the parasite is selected from a Plasmodium species, a Trypanosoma species, a Toxoplasma species, a Leishmania species, or a Cryptosporidium species. In some embodiments, the parasite is from a Plasmodium species. In certain embodiments, the Plasmodium species is Plasmodium falciparum, Plasmodium malar, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curlisi, or Plasmodium ovale wallikeri.

[0031] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a fungus. In some embodiments, the fungus is selected from Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Aspergillus spp., Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Candida albicans, Candida spp., Nakaseomyces glabrata, Pichia kudriavzeveii, Histoplasma spp., Eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Cryptococcus gattii, Cryptococcus spp., Cryptococcus neoformans, Talaromyces marneffei, Paecilomyces lilacinus, Pneumocystis jirovecii, and Paracoccidioides spp., Fusarium spp., Fusarium solani, Scedosporium spp., Paecilomyces lilacinus, Muccorales Fungi (e.g., Rhizopus spp., Mucor spp., Lichtheimia spp., Cunninghamella spp.), Coccidioides immitis, Blastomyces spp., Histoplasma capsulatum, dimorphic fungi (e.g. Talaromyces marneffei and Sporothrix schenckii). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG.1 shows an exemplary structure of a VHHkappa linked to one zanamivir molecule. Such conjugates are called “mono zanamivir”, “VHHkappa-(zanamivir)”, “VHH- (zanamivir)” , or “VHHkappa-Zan”. FIG. 1 discloses “LPETGGG” as SEQ ID NO: 61.

[0033] FIG.2 shows an exemplary structure of a VHHkappalinked to two zanamivir molecules. Such conjugates are called “dual zanamivir”, “VHHkappa-(zanamivir)2”, “VHH- (zanamivir)2” , or “VHHkappa-Zan2”. FIG.2 discloses “LPETGGG” as SEQ ID NO: 61.Attorney Docket No.: CEB-00225

[0034] FIG.3 shows an exemplary structure of a VHHkappalinked to four zanamivir molecules. Such conjugates are called “tetra zanamivir” , “VHHkappa-(zanamivir)4”, “VHH- (zanamivir)4”, or “VHHkappa-Zan4”. FIG.3 discloses “LPETGGG” as SEQ ID NO: 61.

[0035] FIG.4A shows that VHHkappa-Zan, VHHkappa-Zan2, and VHHkappa-Zan4inhibit neuraminidase activity of influenza PR8 in vitro. The zanamivir conjugates have higher IC50s than equal amounts of free zanamivir.

[0036] FIG.4B shows that VHHkappa-Zan, VHHkappa-Zan2, and VHHkappa-Zan4inhibit neuraminidase activity of influenza Cal09 in vitro. The zanamivir conjugates have higher IC50s than equal amounts of free zanamivir.

[0037] FIG.5 shows real-time binding Biolayer interferometry (BLI) sensorgrams of VHHkappa-Zan and VHHkappa-Zan4against NA of influenza A / PR / 8 / 1934 on GatorBio Plus BLI system.

[0038] FIG.6 shows that mono, dual, and tetra zanamivir conjugates protect from influenza-induced mortality when administered intranasally (IN) in a PR / 8; H1N1 influenza infection mouse model. Survival curves for mice infected with PR / 8 and treated with either VHHkappa-Zan, VHHkappa-Zan2, or VHHkappa-Zan4.

[0039] FIG.7 shows the change in weight of the mice during the study of FIG. 6.

[0040] FIG.8 shows that mono, dual, and tetra zanamivir protect from influenza-induced mortality when administered intraperitoneally (IP) in a PR / 8; H1N1 influenza infection mouse model.

[0041] FIG.9 shows the change in weight of the mice during the study of FIG. 8.

[0042] FIG.10A shows that treatment with VHHkappa-Zan and VHHkappa-Zan2reduces the viral titer in lungs during the studies of FIGs.6-9. Viral titer was measured in the lungs 5 days post-infection. The 1 mg / kg IN and 0.3 mg / kg doses of VHHkappa-Zan2have a reduced viral titer compared to the control (p = 0.0046 and 0.0060 respectively).

[0043] FIG.10B shows that treatment with VHHkappa-Zan4 reduces the viral titer in lungs during the studies of FIGs.6-9. Viral titer was measured in the lungs 5 days post-infection. The 1 mg / kg IN and 0.3 mg / kg doses of VHHkappa-Zan2have a reduced viral titer compared to the control (p = 0.0046 and 0.0060 respectively).Attorney Docket No.: CEB-00225

[0044] FIG.11A shows that treatment with VHHkappa-Zan and VHHkappa-Zan2does not increase the titer of circulating whole influenza-specific antibodies. Viral titer was measured in serum 28 days post-infection.

[0045] FIG.11B shows serum IgG antibody titers against intact IAV PR / 8 virions for serum collected from mice that received VHHkappa-Zan4.

[0046] FIG.12A shows that treatment with VHHkappa-Zan and VHHkappa-Zan2increases the titer of circulating influenza hemagglutinin (HA)-protein-specific antibodies. Viral titer was measured in serum 28 days post-infection.

[0047] FIG.12B shows serum IgG antibody titers against PR / 8 hemagglutinin (HA) for serum collected from mice that received VHHkappa-Zan4.

[0048] FIG.13A shows that treatment with VHHkappa-Zan and VHHkappa-Zan2increases the titer of circulating influenza nucleoprotein (NP)-specific antibodies. Viral titer was measured in serum 28 days post-infection.

[0049] FIG.13B shows serum IgG antibody titers against PR / 8 nucleoprotein (NP) for serum collected from mice that received VHHkappa-Zan4.

[0050] FIG.14 shows lung homogenate IgA antibody titers against intact IAV PR / 8 virions, PR / 8 hemagglutinin (HA), and PR / 8 nucleoprotein (NP) in mice that received VHHkappa- Zan4.

[0051] FIG.15A shows that circulating antibodies exhibit enhanced virus-neutralizing activity following treatment with VHHkappa-Zan and VHHkappa-Zan2. Neutralizing titers are expressed as the sample dilution that results in 50% inhibition of IAV plaques.

[0052] FIG.15B shows that mice treated with VHHkappa-Zan and VHHkappa-Zan2exhibit greater neutralizing antibody titer in the lungs than untreated mice. Neutralizing titers are expressed as the sample dilution that results in 50% inhibition of IAV plaques.

[0053] FIG.15C shows neutralizing antibody titers against PR / 8 virus in lung homogenates and serum in mice that received VHHkappa-Zan4. Neutralizing titers are expressed as the sample dilution that results in 50% inhibition of IAV plaques.

[0054] FIG.15D shows nasal wash IgA antibody titers against PR / 8 NP and PR / 8 HA proteins in mice that received VHHkappa-Zan4.

[0055] FIG.15E shows nasal-associated lymphoid tissue (NALT) IgA antibody titers against PR / 8 NP and PR / 8 HA proteins in mice that received VHHkappa-Zan4.Attorney Docket No.: CEB-00225

[0056] FIG.16 shows that mice treated with VHHkappa-Zan and infected with PR / 8; H1N1 exhibited higher levels of inhibitor antibodies against the HK / 68 (H3N2) and influenza B strains. Serum was collected 28 days after influenza infection.

[0057] FIG.17 shows the results of prophylactic intranasal administration of VHHkappa- Zan. The conjugates were administered 31 or 14 days before infection with PR / 8; H1N1 at a 1 mg / kg dose.

[0058] FIG.18 shows the results of prophylactic intraperiotoneal administration of VHHkappa-Zan. The conjugates were administered 31 or 14 days before infection with PR / 8; H1N1 at a 1 mg / kg dose.

[0059] FIG.19 shows that long-term protection against influenza A virus-induced mortality is provided by prophylactic intranasal administration of VHHkappa-Zan2. The conjugates were administered 31 or 14 days before infection with PR / 8; H1N1 at a 1 mg / kg dose.

[0060] FIG.20 shows that long-term protection against influenza A virus-induced mortality is provided by prophylactic intraperitoneal administration of VHHkappa-Zan2. The conjugates were administered 31 or 14 days before infection with PR / 8; H1N1 at a 1 mg / kg dose.

[0061] FIG.21 shows that prophylactic intranasal treatment with VHHkappa-Zan2enhances systemic titers of antibodies specific to influenza virus. Titers are from serum IgG samples taken 28 days after infection.

[0062] FIG.22 shows that prophylactic intranasal treatment with VHHkappa-Zan2and VHHkappa-Zan4enhances systemic titers of antibodies specific to influenza virus. Titers are from serum IgG samples taken 28 days after infection.

[0063] FIG.23 shows that prophylactic intranasal treatment with VHHkappa-Zan2enhances pulmonary titers of antibodies specific to influenza virus. Titers are from lung IgA samples taken 28 days after infection.

[0064] FIG.24 shows that prophylactic intranasal treatment with VHHkappa-Zan2and VHHkappa-Zan4 enhances pulmonary titers of antibodies specific to influenza virus. Titers are from lung IgA samples taken 28 days after infection.

[0065] FIG.25 shows that prophylactic intraperitoneal treatment with VHHkappa-Zan2enhances systemic titers of antibodies specific to influenza virus. Titers are from serum IgG samples taken 28 days after infection.Attorney Docket No.: CEB-00225

[0066] FIG.26 shows that prophylactic treatment (intranasal and intraperitoneal) with VHHkappa-Zan2enhances the neutralizing capacity of systemic influenza-specific antibodies. Titers are from serum samples taken 28 days after infection.

[0067] FIG.27 shows that intranasal treatment with VHHkappa-Zan2and VHHkappa-Zan4increases systemic titers of influenza-neutralizing antibodies. Titers are from serum and lung samples taken 28 days after infection.

[0068] FIG.28 shows influenza-specific T cell responses following treatment with VHHkappa-Zan4conjugates. Measurements are from lung samples take 8 days post infection.

[0069] FIG.29 shows influenza-specific T cell responses following treatment with VHHkappa-Zan4. Measurements are from lung samples take 8 days post infection.

[0070] FIG.30 shows that late administration of VHHkappa-Zan4protects against a lethal IAV challenge. (Top) Experimental scheme and viral titers in the lung on day 5 post-infection. (Bottom) Weight loss and survival curves for mice infected with Cal / 09 and treated with VHHkappa-Zan4on day 3 or 4 post-infection.

[0071] FIG.31 shows that prophylactic administration of VHHkappa-Zan4provides extended protection against IAV. (Top row) Experimental scheme and survival curves for VHHkappa-Zan4treated mice. (Second row) Serum IgG antibody titers against intact IAV PR / 8 virions, PR / 8 NP, and PR / 8 HA. (Third row) Lung homogenate IgA antibody titers against intact IAV PR / 8 virions, PR / 8 NP, and PR / 8 HA. (Fourth row) Neutralizing antibody titers against PR / 8 virus in lung homogenates and serum. Neutralizing titers are expressed as the sample dilution that results in 50% inhibition of IAV plaques (IC50).

[0072] FIG.32 shows that prophylactic administration of VHHkappa-Zan4promotes enhanced mucosal IgA antibody titers. (Top) Nasal wash IgA antibody titers against PR / 8 NP and PR / 8 HA proteins. (Bottom) Nasal-associated lymphoid tissue (NALT) IgA antibody titers against PR / 8 NP and PR / 8 HA proteins. DETAILED DESCRIPTION

[0073] In certain aspects, provided herein are compositions and methods related to conjugates comprising a moiety that binds to an immunoglobulin kappa light chain (e.g., a variable domain on a heavy chain antibody, or VHH) linked to at least two copies of an agent (e.g., 2, 3, 4, 5, or 6 copies of an agent) that binds to a protein on the surface of a cell or pathogen (e.g., a viral protein or a tumor associated protein). In certain embodiments, the conjugatesAttorney Docket No.: CEB-00225 provided herein can be used to treat or prevent a pathogenic infection (e.g., a viral infection, such as an influenza infection) or cancer. Definitions

[0074] As used herein, the term “administration” typically refers to delivery of a composition to a subject or system to achieve presence and / or activity of an agent that is, or is included in, the composition. For example, therapeutic compositions can be administered by a variety of means known in the art, including without limitation parenteral delivery (e.g., intravenous, subcutaneous, intramuscular, or intrathecal administration), enteral (e.g., oral or rectal administration), topical, intranasal, bronchial, or ocular. Administration can be to a mammalian subject, e.g., a human subject.

[0075] As used herein, “affinity” refers to the strength of the sum total of non-covalent interactions between a particular binding agent (e.g., a viral vector), and / or a binding moiety thereof, with a binding target (e.g., a cell). Unless indicated otherwise, as used herein, “binding affinity” refers to a 1:1 interaction between a binding agent and a binding target thereof (e.g., an antibody with an antigen target of the antibody). Those of skill in the art appreciate that a change in affinity can be described by comparison to a reference (e.g., increased or decreased relative to a reference), or can be described numerically. Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and / or equilibrium association constant (KA). KDis the quotient of koff / kon, whereas KAis the quotient of kon / koff, whererefers to the association rate constant of, e.g., viral vector with target cell, and koffrefers to the dissociation of, e.g., viral vector from target cell. The konand koffcan be determined by techniques known to those of skill in the art.

[0076] As used herein, the term “agent” or “moiety” may refer to any chemical or biological entity, including without limitation any of one or more of an atom, molecule, compound, amino acid, polypeptide, nucleotide, nucleic acid, protein, protein complex, liquid, solution, saccharide, polysaccharide, lipid, or combination or complex thereof.

[0077] In its broadest sense the term “amino acid”, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-Attorney Docket No.: CEB-00225 amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino- terminal amino acid in a polypeptide, can contain a structural modification as compared with a typical or canonical amino acid structure. For example, in some embodiments, an amino acid can be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, sulfation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification can, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” can be used to refer to a free amino acid; in some embodiments it can be used to refer to an amino acid residue of a polypeptide.

[0078] As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0079] As used herein, the term “antibody” refers to a polypeptide that includes one or more immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents including theAttorney Docket No.: CEB-00225 same. Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0080] As is well known in the art, typical human immunoglobulins are approximately 150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CH1, CH2 and CH3. A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of a heavy and / or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells (e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. When natural immunoglobulins fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0081] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at leastAttorney Docket No.: CEB-00225 one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (“SMIPsTM”), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies,, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®s, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0082] In various embodiments, an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0083] An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE andIgM, based on heavy chain constant domain amino acid sequence (e.g., alpha ( ), delta ( ),epsilon ( ), gamma ( ) and mu (μ)). IgG subclasses are also well known to those in the art andinclude but are not limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. Asused herein, a “light chain” can be of a distinct type, e.g., kappa ( ) or lambda ( ), based on theamino acid sequence of the light chain constant domain. In some embodiments, an antibody hasAttorney Docket No.: CEB-00225 constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally-produced immunoglobulins are glycosylated, typically on the CH2 domain. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0084] As used herein, an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen- binding portion or variable region thereof. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0085] As used herein, the term “VHH” refers to a variable domain from a heavy chain antibody, and are also referred to as nanobodies or single-domain antibodies (sd-Abs). VHHs are single-domain antibody fragments that contain heavy chain CDRs. VHHs can be generated, for example, by immunization of species that produce heavy-chain only antibodies (e.g., dromedaries, camels, llamas, alpacas, sharks), can be selected using screening technologies, such as phage display or ribosome display, and / or can be engineered from multi-chain antibodies (e.g., murine, rabbit, or human IgG antibodies).

[0086] As used herein, the term “binding” refers to a non-covalent association between or among two or more agents. In some embodiments, the binding is direct. Direct binding involves physical contact between agents. In some embodiments, the binding is indirect. Indirect binding involves physical interaction by way of physical contact with one or more intermediate agents. Binding between two or more agents can occur and / or be assessed in any of a variety ofAttorney Docket No.: CEB-00225 contexts, including where interacting agents are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier agents and / or in a biological system or cell).

[0087] As used herein, the term “cancer” refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they display an abnormally elevated proliferation rate and / or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer can include one or more tumors. In some embodiments, a cancer can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a cancer can be or include a solid tumor. In some embodiments, a cancer can be or include a hematologic tumor.

[0088] As used herein, “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, or the like.

[0089] As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. TheAttorney Docket No.: CEB-00225 comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool).

[0090] As used herein, the term “inhibitory agent” refers to an entity, condition, or event whose presence, level, or degree correlates with decreased level, expression, or activity of a target). In some embodiments, an inhibitory agent may be act directly (in which case it exerts its influence directly upon its target, for example by binding to the target); in some embodiments, an inhibitory agent may act indirectly (in which case it exerts its influence by interacting with and / or otherwise altering a regulator of the target, so that level and / or activity of the target is reduced). In some embodiments, an inhibitory agent is one whose presence or level correlates with a target level or activity that is reduced relative to a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known inhibitory agent, or absence of the inhibitory agent in question, etc.).

[0091] As used herein, “linker” is used to refer to that portion of a multi-element conjugate that connects different elements to one another.

[0092] As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, the term nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside), and in some embodiments refers to a polynucleotide chain including a plurality of individual nucleic acid residues. A nucleic acid can be or include DNA, RNA, or a combination thereof. A nucleic acid can include natural nucleic acid residues, nucleic acid analogs, and / or synthetic residues. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with optional substitutions, e.g., 2’ methoxy or 2’ halide substitutions. In some embodiments, a nucleic acid includes natural nucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a nucleic acid is or includes of one or more nucleotide analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-Attorney Docket No.: CEB-00225 cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, 5-methoxyuridine, pseudouridine, N1-methylpseudouridine, derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid has a nucleotide sequence that is or encodes a functional gene product such as an RNA (e.g., an mRNA or gRNA) or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, a nucleic acid includes one or more genes. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid can include one or more peptide nucleic acids, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate, methylphosphonate, and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, a nucleic acid is partly or wholly single stranded, or partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence including at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0093] As used herein, the term “pharmaceutically acceptable,” as applied to one or more, or all, component(s) for formulation of a composition as disclosed herein, means that eachAttorney Docket No.: CEB-00225 component must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0094] As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates formulation of an agent (e.g., a pharmaceutical agent), modifies bioavailability of an agent, or facilitates transport of an agent from one organ or portion of a subject to another. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non- toxic compatible substances employed in pharmaceutical formulations.

[0095] As used herein, the term “pharmaceutical composition” or “formulation” refers to a composition in which a therapeutic agent is formulated together with one or more pharmaceutically acceptable carriers. The therapeutic agent can be formulated for a particular route of administration, e.g., as set forth herein, to facilitate delivery of a therapeutically effective dosage, and / or in a particular form, e.g., in a solution, suspension, tablet, capsule, pessary, suppository, bolus, powder, granule, paste, cream, foam, ointment, patch, or spray.

[0096] As used herein, “polypeptide” refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through human intervention. In some embodiments, a polypeptide may be or include of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may be or include only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide can include D-amino acids, L-amino acids, or both. In someAttorney Docket No.: CEB-00225 embodiments, a polypeptide may include only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., one or more amino acid side chains, e.g., at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, at non-terminal amino acids, or at any combination thereof. In some embodiments, such pendant groups or modifications may be selected from acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may include a cyclic portion.

[0097] In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure to indicate a class of polypeptides that share a relevant activity or structure. For such classes, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class. For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that can in some embodiments be or include a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and, in some instances, up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide can be or include a fragment of a parent polypeptide. In some embodiments, a useful polypeptide may be or include a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in aAttorney Docket No.: CEB-00225 different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0098] As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins can include moieties other than amino acids (e.g., can be glycoproteins, proteoglycans, etc.) and / or can be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D-amino acids, or both and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, proteins are antibodies, antibody

[0099] As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is a therapeutic agent. Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form ofAttorney Docket No.: CEB-00225 a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance with the present disclosure in a form different from that in which it exists or is found in nature.

[0100] As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

[0101] As used herein, the term “therapeutic agent” refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition. In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agencyAttorney Docket No.: CEB-00225 before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0102] As used herein, “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that a therapeutically effective amount does not necessarily achieve successful treatment in every particular treated individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0103] As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject who exhibits only early signs of the disease, disorder, or condition. Alternatively, or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors thatAttorney Docket No.: CEB-00225 are statistically correlated with increased risk of development of the relevant disease, disorder, or condition. In some embodiments, treatment comprises reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease e.g., a disease caused by a virus, a disease caused by a bacterium, a disease caused by a parasite, a disease caused by a fungus, a cancer) described herein. In some embodiments, treating a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In some embodiments, “treating” can also mean prolonging survival as compared to expected survival if not receiving treatment. The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, an infection (e.g. caused by a virus or pathogen), or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of infection includes, for example, reducing the number of detectable viral molecules or pathogenic cells in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of symptoms in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0104] As used herein, the term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence, absence, or level of one or more chemical moieties as compared with the reference entity. In some embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. A variant can be a molecule comparable, but not identical to, a reference. For example, a variant nucleic acid can differ from a reference nucleic acid at one or more differences in nucleotide sequence. In some embodiments, a variant nucleic acid shows an overall sequence identity with a reference nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, orAttorney Docket No.: CEB-00225 99%. In many embodiments, a nucleic acid of interest is considered to be a “variant” of a reference nucleic acid if the nucleic acid of interest has a sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residues as compared with a reference. In some embodiments, a variant has not more than 5, 4, 3, 2, or 1 residue additions, substitutions, or deletions as compared with the reference. In various embodiments, the number of additions, substitutions, or deletions is fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. Conjugates

[0105] In certain aspects, provided herein is a conjugate comprising a means for binding to an immunoglobulin kappa light chain (e.g., a VHH) covalently conjugated to at least two copies of an agent (e.g., 2, 3, 4, 5, or 6 copies of an agent). In some embodiments, the at least two copies of an agent are 2 copies of the agent. In some embodiments, the at least two copies of an agent are 3 copies of the agent. In certain embodiments, the at least two copies of an agent are 4 copies of the agent. In some embodiments, the at least two copies of an agent are 5 copies of the agent. In some embodiments, the at least two copies of an agent are 6 copies of the agent. In some embodiments, the means for binding to an immunoglobulin kappa light chain is present in a 1:2, 1:3, 1:4, 1:5, or 1:6 ratio with the agent. In some embodiments, the means for binding to an immunoglobulin kappa light chain is conjugated to the two or more copies of an agent via one or more linkers. In some embodiments each copy of the agent is conjugated via its own linker. In other embodiments, at least two of the copies of the agent are conjugated via the same linker. In some embodiments, the agent binds to a protein on the surface of a cell or pathogen.

[0106] In certain aspects, provided herein is a conjugate comprising a VHH specific for an immunoglobulin kappa light chain covalently conjugated to at least two copies of an agent (e.g., 2, 3, 4, 5, or 6 copies of an agent). In some embodiments, the at least two copies of an agent are 2 copies of the agent. In some embodiments, the at least two copies of an agent are 3 copies of the agent. In certain embodiments, the at least two copies of an agent are 4 copies of the agent. In some embodiments, the at least two copies of an agent are 5 copies of the agent. In some embodiments, the at least two copies of an agent are 6 copies of the agent. In some embodiments, the means for binding to an immunoglobulin kappa light chain is present in a 1:2,Attorney Docket No.: CEB-00225 1:3, 1:4, 1:5, or 1:6 ratio with the agent. In some embodiments, VHH is conjugated to the two or more copies of an agent via one or more linkers (e.g., each copy of the agent is conjugated via its own linker, or at least two of the copies of the agent are conjugated via the same linker). In some embodiments each copy of the agent is conjugated via its own linker. In other embodiments, at least two of the copies of the agent are conjugated via the same linker. In some embodiments, the agent binds to a protein on the surface of a cell or pathogen. In some embodiments, the VHH comprises a set of CDR1, CDR2, and CDR3 sequences selected from the sets of CDR1, CDR2, and CDR3 sequences in Table 2. In some embodiments, the VHH comprises the amino acid sequence selected from the sequences in Table 1. In some embodiments, the conjugate is the conjugate represented in FIG.2 or FIG.3. Kappa Light Chain Binders

[0107] In certain aspects, the conjugates provided herein comprise a moiety that specifically binds specifically to a kappa light chain (e.g., a kappa light chain of a human antibody). In certain embodiments, the binder binds to an epitope within the constant region of the kappa light chain. In some embodiments, the kappa light chain binder is an antibody. In certain embodiments the kappa light chain binder is a VHH.

[0108] In certain aspects, provided herein are conjugates comprising a VHH specific for a kappa light chain (e.g., a human kappa light chain). In some embodiments, the VHH comprises CDR1, CDR2, and CDR3 sequences selected from the CDR1, CDR2, and CDR3 sequences in Table 2. In some embodiments, the VHH comprises the amino acid sequence selected from the sequences in Table 1. In some embodiments, the VHH comprises a set of CDR1, CDR2, and CDR3 sequences or comprises an amino acid sequence disclosed in PCT Application Number PCT / NL2005 / 000829, published as WO 2006 / 059904 A1, or PCT Application Number PCT / US2023 / 060893, published as WO 2023 / 141500 A1, each of which is hereby incorporated by reference in its entirety.

[0109] In some embodiments, the VHH comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a VHHkappa sequences in Table 1. In some embodiments, the VHH comprises SEQ ID NO: 56.

[0110] In some embodiments, the VHH comprises a sequence in Table 1 with at least one additional amino acid on its C-terminus. In some embodiments, the VHH comprises two glycine amino acids on its C-terminus. In some embodiments, the VHH comprises three glycine aminoAttorney Docket No.: CEB-00225 acids on its C-terminus. In some embodiments, additional amino acids are added to the C- terminus during the linking or conjugation process. In other embodiments, additional amino acids are present on the VHH before the linking or conjugation process. In some embodiments, the C-terminus of the VHH comprises a sortase recognition sequence, such as LPETGGHHHHHH (SEQ ID NO: 57), prior to being linked to the agent. In some embodiments, the C-terminus of the VHH comprises two or three glycine residues and a sortase recognition sequence. In some embodiments, the VHH comprises a sequence in Table 1 with two glycine residues and a sortase recognition sequence added to its C-terminus. In some embodiments, the VHH comprises a sequence in Table 1 with three glycine residues and a sortase recognition sequence added to its C-terminus. Table 1. Sequences of exemplary VHHkappaproteinsAttorney Docket No.: CEB-00225Table 2. Sequences of exemplary VHH CDR domainsAttorney Docket No.: CEB-00225Agents

[0111] In certain aspects, the conjugates provided herein comprise at least two copies (e.g., 2, 3, 4, 5, or 6 copies) of an agent that binds to a protein on the surface of a cell or pathogen. In some embodiments, the at least two copies of an agent are 4 copies of the agent.

[0112] In some embodiments, the agent comprises a small molecule, a peptide, a protein, a carbohydrate, a lipid, a nucleotide, a nucleic acid, an oligonucleotide, an aptamer, a variable domain on a heavy chain antibody, an antibody, or an antibody fragment. In some embodiments, the agent comprises a small molecule. In some embodiments, the agent is an antibody or an antibody fragment thereof. In some embodiments, the agent is a VHH.

[0113] In some embodiments, the agent binds specifically to a target on the surface of a particular type of cell or pathogen (e.g., a target on the surface of a particular virus, bacterium, parasite, fungus, or human cell, such as a cancer cell), or of a range of related types (e.g., where the types express substantially similar versions of the target protein on their surface). In some embodiments, the agent is an inhibitory agent.

[0114] In some embodiments, the agent binds specifically to a target protein present on the surface of a pathogen, such as a pathogenic virus, bacterium, parasite, or fungus. In some embodiments, the agent binds specifically to a target protein expressed on the surface of a cell infected by the pathogen (e.g., a cell infected by a viral pathogen).

[0115] In some embodiments, the pathogen is a virus. In some embodiments, the virus is selected from an influenza virus, a coronavirus, an adenovirus, an enterovirus, a rotavirus, a norovirus, a herpesvirus, a lentivirus, a poxvirus, a paramyxovirus, a rhabdovirus, an arenavirus, a flavivirus, a togavirus, a hantavirus, a pneumovirus, or an ebolavirus.

[0116] In some embodiments, the virus is an influenza virus. In some embodiments, the influenza virus is an influenza A virus or an influenza B virus. In some embodiments, the protein on the surface of a cell, a pathogen, or cells infected by the pathogen is an influenza virus neuraminidase, an influenza virus hemagglutinin, or an influenza matrix-2 (M2) proton channel protein. In some embodiments, the target to which the agent binds is an influenza virusAttorney Docket No.: CEB-00225 neuraminidase or an influenza virus hemagglutinin, such as an influenza virus neuraminidase or an influenza virus hemagglutinin expressed on the surface of an influenza A virus or an influenza B virus. In some embodiments, the protein is an influenza virus neuraminidase. In some embodiments, the agent is a small molecule that binds to an influenza virus neuraminidase. In some embodiments, the agent comprises sialic acid or neuraminic acid or a pharmaceutically acceptable salt or an analog thereof. In some embodiments, the agent comprises zanamivir, oseltamivir, peramivir, or a pharmaceutically acceptable salt or an analog thereof. In some embodiments, the agent comprises zanamivir or a pharmaceutically acceptable salt or analog thereof. In some embodiments, the agent is an antibody or an antibody fragment that binds to an influenza virus neuraminidase or an influenza virus hemagglutinin. In some embodiments, the agent is a VHH that binds to an influenza virus hemagglutinin. An example of a VHH that binds to influenza virus hemagglutinin is as follows: Anti -HA VHH (SD36): EVQLVESGGGLVQAGGSLKLSCAASGRTYAMGWFRQAPGKEREFVAHINALGTRTYY SDSVKGRFTISRDNAKNTEYLEMNNLKPEDTAVYYCTAQGQWRAAPVAVAAEYEFWG QGTQVTVSSGG (SEQ ID NO: 58).

[0117] In some embodiments, the virus is a Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus (optionally wherein the SARS associated coronavirus is SARS-CoV-1 or SARS- CoV-2), a human immunodeficiency virus (HIV), or a human respiratory syncytial virus (RSV). In some embodiments, the target to which the agent binds is a MERS-CoV spike protein, a SARS-CoV-1 spike protein, or a SARS-CoV-2 spike protein. In some embodiments, the target to which the agent binds is a MERS-CoV spike protein receptor binding domain (RBD), a SARS- CoV-1 spike protein RBD, or a SARS-CoV-2 spike protein RBD. In some embodiments, the virus is a lentivirus. In some embodiments, the lentivirus is a human immunodeficiency virus (HIV). In some embodiments, the target to which the second agent binds is a HIV envelope glycoprotein gpl20. In some embodiments, the second agent specifically binds to a target expressed on the surface of HIV. In some embodiments, the virus is a pneumovirus. In some embodiments, the pneumovirus is a human respiratory syncytial virus (RSV). In some embodiments, the target to which the second agent binds is a RSV fusion (F) protein.

[0118] In some embodiments, the agent binds specifically to a target present on the surface of a cancer cell. In some embodiments, the cell is a cancer cell, a transformed cell, aAttorney Docket No.: CEB-00225 healthy cell, or a cell that is undergoing or has undergone a phenotypic change in response to cellular stress. In some embodiments, the cancer cell is a hematological cancer cell, a lung cancer cell, a breast cancer cell, a brain cancer cell, a gastrointestinal cancer cell, a liver cancer cell, a kidney cancer cell, a bladder cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a testicular cancer cell, a prostate cancer cell, an endometrial cancer cell, a muscle cancer cell, a bone cancer cell, a neuroendocrine cancer cell, a connective tissue cancer cell, a head or neck cancer cell, or a skin cancer cell.

[0119] In some embodiments, the agent binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen comprises a MHC class I polypeptide-related sequence A (MICA) protein, a MHC class I polypeptide-related sequence B (MICB) protein, a folate receptor, a fibronectin splice variant, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor receptor 2 (VEGFR-2), C-X-C chemokine receptor type 4 (CXCR4), urokinase plasminogen activator surface receptor (uPAR), follicle- stimulating hormone receptor (FSHR), epithelial cell adhesion molecule (EpCAM), epithelial cadherin (ECAD), carcinoembryonic antigen (CEA), or mesothelin (MSLN).

[0120] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a bacterium. In some embodiments, the bacterium is selected from a Pasteurella species, a Staphylococcus species, a Streptococcus species, a Bacillus species, a Corynebacterium species, a Diphtheroids species, a Listeria species, an Erysipelothrix species, a Clostridium species, a Neisseria species, a Branhamella species, an Escherichia species, an Enterobacter species, a Proteus species, a Pseudomonas species, a Klebsiella species, a Salmonella species, a Shigella species, a Serratia species, an Acinetobacter species, Haemophilus species, a Brucella species, a Yersinia species, a Francisella species, a Pasturella species, a Vibrio species, a Flavobacterium species, a Pseudomonas species, a Campylobacter species, a Bacteroides species, a Fusobacterium species, a Calymmatobacterium species, a Streptobacillus species, or a Legionella species.

[0121] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a parasite. In some embodiments, the parasite is selected from a Plasmodium species, a Trypanosoma species, a Toxoplasma species, a Leishmania species, or a Cryptosporidium species. In some embodiments, the parasite is from a Plasmodium species. InAttorney Docket No.: CEB-00225 certain embodiments, the Plasmodium species is Plasmodium falciparum, Plasmodium malar, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curlisi, or Plasmodium ovale wallikeri.

[0122] In some embodiments of the conjugates provided herein, the agent binds to a protein on the surface of a fungus. In some embodiments, the fungus is selected from Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Aspergillus spp., Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Candida albicans, Candida spp., Nakaseomyces glabrata, Pichia kudriavzeveii, Histoplasma spp., Eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Cryptococcus gattii, Cryptococcus spp., Cryptococcus neoformans, Talaromyces marneffei, Paecilomyces lilacinus, Pneumocystis jirovecii, and Paracoccidioides spp., Fusarium spp., Fusarium solani, Scedosporium spp., Paecilomyces lilacinus, Muccorales Fungi (e.g., Rhizopus spp., Mucor spp., Lichtheimia spp., Cunninghamella spp.), Coccidioides immitis, Blastomyces spp., Histoplasma capsulatum, dimorphic fungi (e.g. Talaromyces marneffei and Sporothrix schenckii). Linkers

[0123] In some embodiments, the two or more copies of the agent are covalently conjugated to a kappa light chain binder, such as a VHH, via one or more linkers.

[0124] In some embodiments, the linker comprises a cleavable or a non-cleavable linker. A “cleavable linker” refers to a linker within which one or more covalent bonds are cleaved (broken) under certain conditions, such as those occurring in a cell or subject. A “non-cleavable” linker refers to a linker which for all intents and purposes cannot be efficiently or reliably cleaved under certain conditions, such as those occurring in a cell or subject. Examples of cleavable linkers familiar to those of skill in the relevant art include peptide linkers (e.g., Val- Cit), disulfide linkers, and hydrazone linkers, which are cleaved by proteolysis, reduction, and low pH, respectively. Examples of non-cleavable linkers include, for example, N-succinimidyl- 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) and polyethylene glycol (PEG). Additional examples of linkers known in the art include, for example, those of Lu, et al. “Linkers Having a Crucial Role in Antibody -Drug Conjugates” IntJMol Sci, 17(4), 561., the contents of which are herein incorporated by reference in their entirety.Attorney Docket No.: CEB-00225

[0125] In some embodiments, the linker comprises an azide moiety, a bicyclononyne moiety, a tetrazine moiety, a trans-cyclooctene moiety, a polypeptide, a polynucleic acid, an alkane, an alkene, an alkyne, a polyether polyol, and / or a polythioether polyol. In some embodiments, the linker comprises a polyether polyol. In certain embodiments, the linker comprises a dibenzoyclooctyne (DBCO) linker. In certain embodiments, the linker comprises a polyethylene glycol dibenzocyclooctyne (DBCO) linker. In certain embodiments, the linker comprises.

[0126] In certain embodiments, the linker is a triglycine dibenzylcyclooctyne linker.

[0127] In some embodiments the linker comprises a dibenzylcyclooctyne (DBCO) linker and the agent is zanamivir. In some embodiments, the structure of the linker and agent comprises:.

[0128] In certain embodiments (e.g., embodiments wherein there are two copies of the agent), the one or more linkers comprise:Attorney Docket No.: CEB-00225some embodiments (e.g., embodiments wherein there are two copies of the agent), the one or more linkers comprise:. In certain embodiments (e.g., embodiments wherein there are four copies of the agent), the one or more linkers comprise:. In some embodiments (e.g., embodiments wherein there are four copies of the agent), the one or more linkers comprise:.

[0129] In some embodiments, the linker comprises a polypeptide. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more GAttorney Docket No.: CEB-00225 some embodiments, the linker comprises one or more amino acids linking the VHH or means for binding to an immunoglobulin kappa light chain and the agent, such as, for example, a repeating linker comprising glycine and / or serine, or another amino acid linker that is generally known in the art. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three- dimensional structure, but rather provides flexibility to the polypeptide. A variety of different polypeptide linkers are known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

[0130] Additional examples of suitable linkers are known in the art, such as in WO2022 / 032188A1, which is hereby incorporated by reference in its entirety. Methods of Making

[0131] In another aspect, the present disclosure provides a method of making the conjugates provided herein. In some embodiments, method comprises conjugating the copies of the agent to the VHH via a linker. In another aspect, the present disclosure provides a method of making the conjugates provided herein, comprising conjugating the copies of the agent to the means for binding an immunoglobulin kappa chain via a linker.

[0132] In some embodiments, a conjugation handle is attached to the VHH or the means for binding an immunoglobulin kappa chain. In some embodiments, the conjugation handle is attached via an enzymatic reaction. In some embodiments, the conjugation handle is attached via a sortase A-mediated transpeptidation reaction. In some embodiments, the conjugation handle is attached to a glycine on the VHH or the means for binding an immunoglobulin kappa chain. In some embodiments, the conjugation handle is attached via a maleimide reaction to a cysteine on the VHH or the means for binding an immunoglobulin kappa chain. In some embodiments, the conjugation handle is attached via a NHS ester to an N-terminal amino acid modification on the VHH or the means for binding an immunoglobulin kappa chain. In some embodiments, the VHH or the means for binding an immunoglobulin comprises a non-natural amino acid, and the conjugation handle is attached to the non-natural amino acid. As used herein, the phrase “non- natural amino acid” refers to an entity having the chemical structure of an amino acid (i.e.,:therefore being capable of participating in at least two peptide bonds, but having an R group that differs from those found in nature. In some embodiments, non-naturalAttorney Docket No.: CEB-00225 amino acids can also have a second R group rather than a hydrogen, and / or can have one or more other substitutions on the amino or carboxylic acid moieties. In some embodiments, the non- natural amino acid contains an azide. In some embodiments, the conjugation handle is attached via a transglutaminase, a butelase, or an OaAEP1.

[0133] In some embodiments, the agent is linked to the VHH or means for binding to an immunoglobulin kappa light chain by means of a sortase enzyme (e.g., sortase A). Sortases are a class of enzyme that specifically targets the amino acid motif LPXTG, where X is any amino acid (SEQ ID NO: 60), by cleaving C-terminal to the threonine residue, generating a peptide bond between the threonine and sortase that is subsequently transferred to the N-terminus of another protein. In some embodiments, the C-terminus of the VHH or means for binding to an immunoglobulin kappa light chain is labeled with a sortase recognition sequence, such as LPETGGHHHHHH (SEQ ID NO: 57), prior to being linked to the agent. In some embodiments, the agent is a protein or peptide (e.g., an antibody or antibody fragment that is labeled at its C- terminus with a sortase recognition sequence, prior to being linked to the VHH or means for binding to an immunoglobulin kappa light chain.

[0134] In some embodiments, the agent is a protein or polypeptide (e.g., an antibody or antibody fragment) and the linker is a peptide bond, an isopeptide bond, or a disulfide bond. In some embodiments, the linker comprises one or more amino acids linking the VHH or means for binding to an immunoglobulin kappa light chain and the agent. In some embodiments, the VHH or means for binding to an immunoglobulin kappa light chain and the agent are translated separately (e.g., in an in vitro translation system or recombinantly expressed in a cell) and post- translationally linked. In some embodiments, the VHH or means for binding to an immunoglobulin kappa light chain and agent are translated as a fusion protein (e.g., in an in vitro translation system or recombinantly expressed in a cell), wherein the VHH or means for binding to an immunoglobulin kappa light chain and the agent are linked by one or more peptide bonds. In some embodiments, the conjugate is produced recombinantly, by inserting one or more nucleic acids (e.g., DNA or RNA) encoding the VHH or means for binding to an immunoglobulin kappa light chain and the agent to a prokaryotic (e.g., bacterial) or eukaryotic (e.g., fungal or mammalian) cell for expression, and subsequently isolating the conjugate using one or more techniques that are generally known in the art, such as, for example, affinity chromatography or size and / or size exclusion chromatography. In some embodiments, the VHHAttorney Docket No.: CEB-00225 or means for binding to an immunoglobulin kappa light chain and the agent are encoded by different nucleic acids (e.g., DNA or RNA). In some embodiments, the VHH or means for binding to an immunoglobulin kappa light chain and the agent are encoded by the same nucleic acid (e.g., DNA or RNA). In some embodiments, the VHH or means for binding to an immunoglobulin kappa light chain and agent are encoded by one or more plasmids or mRNAs and inserted (transfected) into cells by any means known in the art (e.g., electroporation). In some embodiments, nucleic acids encoding the VHH or means for binding to an immunoglobulin kappa light chain and agent are inserted (transfected) into the cell using a viral vector (e.g., an adenoviral vector, a lentiviral vector), by any means known in the art. In some embodiments, nucleic acids encoding the VHH or means for binding to an immunoglobulin kappa light chain and agent are chromosomally inserted into cell by any means known in the art. Pharmaceutical Compositions

[0135] In some embodiments, compositions (e.g., pharmaceutical compositions) of the present disclosure comprise a conjugate described herein. In some embodiments, compositions (e.g., pharmaceutical compositions) of the present disclosure comprise two or more conjugates described herein. In some embodiments, a composition comprising two or more conjugates comprises two or more conjugates specific for the same antigen or target. In some embodiments, a composition comprising two or more conjugates comprises only one conjugate specific for each antigen. As contemplated herein, the terms “composition” and “formulation” may be used interchangeably.

[0136] In some embodiments, a composition may comprise one or more conjugates described herein and one or more pharmacologically acceptable excipients. A pharmacologically acceptable excipient may enhance stability of a conjugate described herein, enhance delivery of the conjugate to cells (e.g., immune cells) of a subject to which the composition is administered, permit sustained or delayed release of the conjugate upon administration, alter the biodistribution of the conjugate (e.g., target the conjugate to specific tissues or cell types), or reduce host immunity against the conjugate. Examples of pharmacologically acceptable excipients includes any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and preservatives, as are known in the art. In some embodiments, a pharmacologically acceptable excipient comprises an aqueous solution or buffer. In some embodiments, the composition is isotonic,Attorney Docket No.: CEB-00225 relative to a biological fluid of a subject (i.e., blood) to which the composition is to be administered. In some embodiments, the composition has a pH between 7 and 8, or optimally a pH of about 7.4. In some embodiments, the composition comprises phosphate-buffered saline (PBS). Pharmaceutically acceptable excipients are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a conjugate such as a conjugate disclosed herein. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0137] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the conjugate which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.Attorney Docket No.: CEB-00225

[0138] Methods of preparing these formulations or compositions include the step of bringing into association an active conjugate, such as a conjugate disclosed herein, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a conjugate disclosed herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0139] Formulations disclosed herein suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a conjugate disclosed herein as an active ingredient. Compositions or conjugates may also be administered as a bolus, electuary or paste.

[0140] Liquid dosage forms useful for intranasal administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions, in addition to the active conjugates, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0141] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition,Attorney Docket No.: CEB-00225 prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. Kits and Nasal Sprays

[0142] Also encompassed by the present disclosure are kits (e.g., pharmaceutical packs). In some embodiments, the kits provided are nasal sprays. The kits provided may comprise a pharmaceutical composition or conjugate described herein and a container (e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other container suitable for storage and / or administration. In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or conjugate described herein. In some embodiments, the pharmaceutical composition or conjugate described herein is provided in the first container and is combined with the second container to form one dosage unit.

[0143] Thus, in one aspect, provided herein are kits including a first container comprising a conjugate or pharmaceutical composition described herein. In certain embodiments, the kits are useful for enhancing or eliciting an immune response toward a particular cell or pathogen in a subject (e.g., a pathogenic cell or a cell of the subject). In certain embodiments, the kits are useful for treating a disease (e.g., a disease caused by a virus, a disease caused by a bacterium, a disease caused by a parasite, a disease caused by a fungus, a cancer) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., a disease caused by a virus, a disease caused by a bacterium, a disease caused by a parasite, a disease caused by a fungus, a cancer) in a subject in need thereof.

[0144] In certain embodiments, a kit described herein further includes instructions for using the pharmaceutical composition or conjugate included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for enhancing or eliciting an immune response toward a cell or pathogen in a subject (e.g., a pathogenic cell or a cell of the subject). In certain embodiments, the kits and instructions provide for treating a disease (e.g., a disease caused by a virus, a disease caused by a bacterium, a disease caused by a parasite, a disease caused by a fungus, a cancer) in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., a disease caused byAttorney Docket No.: CEB-00225 a virus, a disease caused by a bacterium, a disease caused by a parasite, a disease caused by a fungus, a cancer) in a subject in need thereof. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

[0145] Compositions may be formulated in nasal sprays or inhalation solutions or suspensions using approaches known and acceptable in the art and in the medical field and clinical practice. The FDA provides guideline and guidance with regard to such sprays, solutions and suspensions and spray drug products, including in Guidance for Industry documents available at fda.gov. An exemplary July 2002 Guidance for Industry document entitled Nasal Spray and Inhalation Solution, Suspension and Spray Drug Products Chemistry, Manufacturing and Controls Documentation includes details regarding formulation components and compositions, specifications therefore, manufacturing, and closed container systems.

[0146] In certain embodiments, nasal sprays comprise the conjugates or compositions discussed herein dissolved or suspended in a formulation, typically aqueous-based, which can contain other excipients and are intended for use by nasal inhalation. Container closure systems for nasal sprays include the container and all components that are responsible for metering, atomization, and delivery of the formulation to the patient. The container closure system can be a nonpressurized dispenser that delivers a spray containing a metered dose of the active ingredient. The dose can be metered by the spray pump or could have been premetered during manufacture. In some embodiments, the nasal spray is designed for unit dosing. In some embodiments, the nasal spray can discharge numerous metered sprays of formulation containing the drug substance. In some embodiments, the nasal sprays are for application to the nasal cavity to induce local and / or systemic effects. In some embodiments, the nasal sprays are for administration to the lungs by nasal inhalation for local and / or systemic effects and are to be used with a specified nebulizer.

[0147] Current container closure system designs for inhalation sprays include both premetered and device-metered presentations using mechanical or power assistance and / or energy from patient inspiration for production of the spray plume. Premetered presentations contain previously measured doses or a dose fraction in some type of units (e.g., single or multiple blisters or other cavities) that are subsequently inserted into the device during manufacture or by the patient before use. Typical device-metered units have a reservoir containing formulation sufficient for multiple doses that are delivered as metered sprays by theAttorney Docket No.: CEB-00225 device itself when activated by the patient. Devices for administration or delivery to the nasal cavity, respiratory tract, and / or airway(s) are known and recognized in the skilled art and in clinical or medical practice and are applicable in the methods, protocols and compositions disclosed herein. Devices include the metered dose inhaler, metered spray pumps, hand-bulb atomizer, small or large volume nebulizers, ultrasonic nebulizer and dry powder inhaler.

[0148] A prolonged residence time in the nasal cavity may also be achieved by using bioadhesive polymers or microspheres (e.g., as made from chitosan, starch, albumin, dextran, agarose, alginate, and / or gelatin) or by increasing the viscosity of the formulation. In some embodiments, the nasal spray comprises a humectant (e.g., glycerin, sorbitol and mannitol).

[0149] The selection of delivery system depends upon the drug being used, proposed indication, patient population and last but not least, marketing preferences. Some of these delivery systems include nasal drops, nasal sprays, nasal gels, and nasal powders. Methods of Treatment

[0150] In some embodiments, a conjugate provided herein provides a therapeutic effect when administered to a subject. In some embodiments, a conjugate provided herein enhances the association (proximity) between one or more immune cells (e.g., one or more immune cell types) and a cell or pathogen when the conjugate is administered to a subject. In some embodiments, administration of a conjugate provided herein to a subject results in the killing of a cell or pathogen in the subject. In some embodiments, administration of a conjugate provided herein to a subject results in the inactivation of a cell or pathogen in the subject. In some embodiments, the subject to which a conjugate provided herein provides a therapeutic effect is a mammal. In some embodiments, the subject to which a conjugate provided herein provides a therapeutic effect is a human.

[0151] In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed in a subject. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms e.g., in light of a history of symptoms for the disease, in light of a risk of relapse or reoccurrence of the disease, and / or in light of exposure to a pathogen that is causative for the disease or the likelihood for future exposure to a pathogen that is causative for the disease). Treatment may also be continued after symptoms have resolved, for example, to delay or preventAttorney Docket No.: CEB-00225 relapse or recurrence. Prophylactic treatment refers to the treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease and is at risk of relapse or regression of the disease. In some embodiments, the subject is at a higher risk of developing the disease or at a higher risk of relapse or regression of the disease than an average healthy member of a population.

[0152] In some embodiments, treatment may be administered before infection. In some embodiments, treatment may be administered 6, 5, 4, 3, 2, or 1 week before infection, preferably about 1 month, about 4 weeks, or about 2 weeks before infection. In some embodiments, treatment may be administered about 31 days or about 14 days before infection. In some embodiments, treatment may be administered after infection. In certain embodiments, treatment may be administered 1, 2, 3, 4, 5, 6, or 7 days after infection, preferably about 3 or 4 days after infection. In some embodiments, treatment may be administered after symptoms arise, preferably on the first day that symptoms of infection arise. In some embodiments, treatment may be administered less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, or less than 7 days after symptoms of infection arise.

[0153] An effective amount of a composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of a conjugate described herein, the condition being treated, the mode of administration, and the age and health of the subject. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount sufficient for prophylactic treatment. In some embodiments, an effective amount is the amount of a conjugate described herein administered in a single dose. In some embodiments, an effective amount is the combined amount (sum) of a conjugate described herein administered in multiple doses. Where an effective amount of a composition is referred to herein, the amount that is therapeutically and / or prophylactically effective is signified, depending on the subject and / or the disease to be treated.

[0154] A composition or conjugate described herein may be administered systemically (e.g., via intravenous injection) or locally (e.g., via local injection). In some embodiments, the composition or conjugate described herein is administered orally, intravenously, topically, intranasally, or sublingually. Parenteral administrating is also contemplated. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, intradermally, andAttorney Docket No.: CEB-00225 intracranial injection or infusion techniques. In some embodiments, the administering is done intramuscularly, intradermally, orally, intravenously, topically, intranasally, intravaginally, or sublingually. In some embodiments, the composition or conjugate described herein is administered prophylactically. In some embodiments, the conjugates and compositions are for intranasal administration. The term intranasal as used herein includes, but is not limited to, administering, administration or occurring within or via the nose or nasal structures. The term intranasal as used herein and as exemplified as an embodiment in the examples in not intended to be limited to or to imply limitation to administration directly or specifically or solely via the nose or nasal cavity, particularly in serving to exclude other means of administration whereby the conjugate or composition is delivered or otherwise provided to, deposited in or at or otherwise distributed to the respiratory tract.

[0155] In certain aspects, provided herein is method for enhancing an immune response to a pathogen or cancer in a subject, comprising administering to the subject a conjugate or pharmaceutical composition provided herein. In certain aspects, provided herein is a method of treating or preventing infection from a pathogen in a subject, comprising administering to the subject a conjugate or pharmaceutical composition provided herein.

[0156] In certain embodiments, the conjugates provided herein are for use in enhancing an immune response to a pathogen or cancer in a subject. In some embodiments, the conjugates provided herein are for use in treating or preventing infection from a pathogen in a subject.

[0157] In certain aspects, provided herein is the use of a conjugate provided herein in the manufacture of a medicament for enhancing an immune response to a pathogen in a subject. In certain aspects, provided herein is the use of a conjugate provided herein in the manufacture of a medicament for treating or preventing infection from a pathogen in a subject.

[0158] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a virus. In some embodiments, the virus is an influenza virus, a coronavirus, an adenovirus, an enterovirus, a rotavirus, a norovirus, a herpesvirus, a lentivirus, a poxvirus, a paramyxovirus, a rhabdovirus, an arenavirus, a flavivirus, a togavirus, a hantavirus, a pneumovirus, or an ebolavirus. In certain embodiments, the virus is a Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus (optionally wherein the SARS associated coronavirus is SARS-CoV-1 or SARS- CoV-2), a human immunodeficiency virus (HIV), or a human respiratory syncytial virus (RSV).Attorney Docket No.: CEB-00225 In some embodiments, the virus is HIV. In some embodiments, the virus is an influenza A virus or an influenza B virus. In some embodiments, the pharmaceutical composition is administered intranasally.

[0159] In some embodiments of the methods, uses and compositions for use provided herein, the method is for the treatment of a cancer. In some embodiments, the cancer is a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer. In certain embodiments, the cancer expresses a tumor-associated antigen to which the agent binds. In some embodiments, the tumor-associated antigen comprises a MHC class I polypeptide- related sequence A (MICA) protein, a MHC class I polypeptide-related sequence B (MICB) protein, a folate receptor, a fibronectin splice variant, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor receptor 2 (VEGFR-2), C-X-C chemokine receptor type 4 (CXCR4), urokinase plasminogen activator surface receptor (uPAR), follicle- stimulating hormone receptor (FSHR), epithelial cell adhesion molecule (EpCAM), epithelial cadherin (ECAD), carcinoembryonic antigen (CEA), or mesothelin (MSLN).

[0160] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a bacterium. In some embodiments, the bacterium is selected from a Pasteurella species, a Staphylococcus species, a Streptococcus species, a Bacillus species, a Corynebacterium species, a Diphtheroids species, a Listeria species, an Erysipelothrix species, a Clostridium species, a Neisseria species, a Branhamella species, an Escherichia species, an Enterobacter species, a Proteus species, a Pseudomonas species, a Klebsiella species, a Salmonella species, a Shigella species, a Serratia species, an Acinetobacter species, Haemophilus species, a Brucella species, a Yersinia species, a Francisella species, a Pasturella species, a Vibrio species, a Flavobacterium species, a Pseudomonas species, a Campylobacter species, a Bacteroides species, a Fusobacterium species, a Calymmatobacterium species, a Streptobacillus species, or a Legionella species.

[0161] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a parasite. In some embodiments, the parasite is selected from aAttorney Docket No.: CEB-00225 Plasmodium species, a Trypanosoma species, a Toxoplasma species, a Leishmania species, or a Cryptosporidium species. In some embodiments, the parasite is from a Plasmodium species. In certain embodiments, the Plasmodium species is Plasmodium falciparum, Plasmodium malar, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curlisi, or Plasmodium ovale wallikeri.

[0162] In some embodiments of the methods, uses and compositions for use provided herein, the pathogen is a fungus. In some embodiments, the fungus is selected from Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces glabrata, Pichia kudriavzeveii, Histoplasma spp., Eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, Candida parapsilosis, Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp..

[0163] In some embodiments, a composition or conjugate described herein is administered once or is administered repeatedly (e.g., 2, 3, 4, 5, or more times). For multiple administrations, the administrations may be done over a period of time (e.g., 1 day, 1 week, 1 month, 6 months, 1 year, 2 years, 5 years, 10 years, or longer). For repeating administrations, the administrations may be done over a fixed period of time (e.g., 1 day, 1 week, 1 month, 6 months, 1 year, 2 years, 5 years, 10 years, or longer), or a variable period of time. In some embodiments, the composition or conjugate described herein is administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later). In some embodiments, the composition or conjugate described herein is administered more than twice, is administered until a subject is free of symptoms of a disease (e.g., a disease caused by a virus, a disease caused by a bacterium, a disease caused by a parasite, a disease caused by a fungus, a cancer), or is administered until the risk of developing the disease subsides. In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of enhancing or eliciting an immune response toward a cell or pathogen in a subject (e.g., a pathogenic cell or a cell of the subject). In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of treating or preventingAttorney Docket No.: CEB-00225 an infection by a pathogen. In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of treating or preventing a viral infection. In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of treating or preventing a bacterial infection. In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of treating or preventing a parasitic infection. In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of treating or preventing a fungal infection. In some embodiments, a composition or conjugate described herein is administered to a subject for the purpose of treating or preventing a cancer.

[0164] In some embodiments, administration of a composition or conjugate described herein to a subject enhances or elicits an innate (cell-mediated) immune response in the subject. In some embodiments, following administration of a composition or conjugate described herein to a subject, the conjugate binds to a cell or pathogen in the subject and to an immunoglobulin of the subject, wherein the immunoglobulin further binds to a subject’s immune cell. In some embodiments, the subject’s immunoglobulin comprises an immunoglobulin kappa light chain or an immunoglobulin lambda light chain. In some embodiments, the subject’s immunoglobulin comprises an immunoglobulin kappa light chain. In some embodiments, the immune cell is a macrophage, a dendritic cell, a natural killer cell, a neutrophil, a basophil, an eosinophil, or a mast cell. In some embodiments, administration of a composition or conjugate described herein induces the production of one or more cytokines or chemokines by the immune cell. In some embodiments, administration of a composition or conjugate described herein induces the production of one or more proinflammatory cytokines or proinflammatory chemokines by the immune cell. In some embodiments, administration of a composition or conjugate described herein induces phagocytosis of a cell or pathogen in the subject by the immune cell of the subject. In some embodiments, administration of a composition or conjugate described herein results in killing of a cell or pathogen in the subject. In some embodiments, administration of a composition or conjugate described herein results in inactivation of a cell or pathogen in the subject (i.e., the cell or pathogen is no longer able to replicate or reproduce). In some embodiments, administration of a composition or conjugate described herein increases the prevalence or activity of CD4 or CD8 T cells in the subject.Attorney Docket No.: CEB-00225

[0165] In some embodiments, administration of a composition or conjugate described herein to a subject protects from pathogen-induced morality. In some embodiments, the pathogen is influenza.

[0166] In some embodiments, administration of a composition or conjugation described herein to a subject reduces viral titer in the lungs. In some embodiments, administration of a composition or a conjugate described herein to a subject increases the titer of target-specific antibodies (e.g., antibodies that specifically bind a target, wherein the target is the binding target of the agent) in the subject. In some embodiments, the target-specific antibodies are circulating (e.g., in the blood or serum). In some embodiments, the target-specific antibodies are in the subject’s lungs. In some embodiments, the target-specific antibodies exhibit enhanced virus- neutralizing activity.

[0167] In some embodiments, the subject is a subject that has or is at risk for developing an infection by a pathogen (e.g., a viral infection, a bacterial infection, a parasitic infection, a fungal infection). In some embodiments, the subject has or is at risk for developing a cancer, such as, but not limited to, a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer. In some embodiments, the cancer is a metastatic cancer.

[0168] In certain embodiments of the methods, uses, and compositions for use provided herein, the subject has a weak or compromised immune system. In certain embodiments, the subject has received immunosuppressants. In some embodiments, the immunosuppressants are at least one of corticosteroids (e.g., prednisone, cortisone, hydrocortisone, or methylprednisolone), biologics or antibodies (e.g., adalimumab, infliximab, or basiliximab), calcineurin inhibitors (e.g., tacrolimus or cyclosporine), inosine monophosphate dehydrogenase inhibitors (e.g., mycophenolate mofetil), Janus kinase inhibitors (e.g., tofacitinib), or mTOR inhibitors (e.g., sirolimus). In some embodiments, the subject has a human immunodeficiency virus (HIV) infection. In some embodiments, the HIV infection is advanced. In some embodiments, the subject has received a transplant (e.g., an organ or bone marrow transplant). In some embodiments, the subject is pregnant. In other embodiments, the subject is a child under 10 years of age.Attorney Docket No.: CEB-00225

[0169] In some embodiments, a subject is a mammal. In some embodiments, the subject is a non-human animal [e.g., primate (e.g., cynomolgus monkey or rhesus monkey), a commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or a bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)]. In some embodiments the subject is a domesticated animal (e.g., cattle, pig, horse, sheep, goat) or a companion animal (i.e., a pet or service animal, e.g., cat or dog). In some embodiments, the subject is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal.

[0170] In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the human infant is a neonate that is less than 28 days of age. In some embodiments, the human infant is less than 1, 2, 3, 4, 5 ,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days of age at the time of administration.

[0171] In some embodiments, the human subject is more than 28 days of age (e.g., more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, or 17 years of age). In some embodiments, the human subject is an adult (e.g., more than 18 years of age). In some embodiments, the human subject is an elderly subject (e.g., more than 60 years of age). In some embodiments, the human subject is more than 60 years, more than 65 years, more than 70 years, more than 75 years, more than 80 years, more than 85 years, more than 90 years, more than 95 years, more than 100 years, or more than 100 years of age.

[0172] In some embodiments, the human subject is part of one or more immunologically vulnerable populations. In some embodiments, the human subject is frail (e.g., a subject having frailty syndrome, a malnourished subject, or a subject with a chronic disease-causing frailty). In some embodiments, the human subject has a weak immune system, such as an undeveloped (e.g., an infant or a neonate subject), immunosenescent (e.g., an elderly subject), or compromised immune system. Immunosenescent subjects include, without limitation, subjects exhibiting a decline in immune function associated with advanced age. Immunocompromised subjects include, without limitation, subjects with primary immunodeficiency or acquired immunodeficiency such as those suffering from sepsis, HIV infection, and cancers, includingAttorney Docket No.: CEB-00225 those undergoing chemotherapy and / or radiotherapy, as well as subjects to which immunosuppressants are administered, as for organ or tissue transplantation. In some embodiments, the human subject has or is suspected of having one or more disorders or diseases that reduce immune system function and / or increase the risk of infection in the subject by one or more pathogens (e.g., a virus, a bacterium, a parasite, a fungus). In some embodiments, the human subject is, for example, a subject that has or is suspected of having chronic lung disease, asthma, cardiovascular disease, cancer, a metabolic disorder (e.g., obesity or diabetes mellitus), chronic kidney disease, or liver disease.

[0173] Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure. EXAMPLES Example – VHH conjugates as building blocks for intranasally delivered immune that protect against influenza virus infections.

[0174] Herein is described the production and use of VHH conjugates, comprising a VHHkappathat recognizes mouse immunoglobulin kappa light chains and the small molecule influenza virus neuraminidase inhibitor zanamivir. Such conjugates achieve half-life extension of zanamivir, while recruiting polyclonal immunoglobulins of all isotypes, regardless of specificity to infected cells for antibody-dependent cell mediated cytotoxicity (ADCC) and complement- dependent cellular cytotoxicity (CDC). Animals infected with a 10 x LD50and protected by administration of VHHkappa-zanamivir given two days later mount a strong serum response against the hemagglutinin, neuraminidase and nucleoprotein. Notwithstanding the large dose of live virus to which the animals were exposed, it was found that the serum response is qualitatively and quantitatively similar to that observed in animals that received a sublethal dose of IAV and made a full recovery without further treatment. Increasing the number of zanamivir molecules per VHH increases their antiviral potency and allows intranasal delivery to achieve protection. A VHH conjugate that contains 4 zanamivir molecules is the most potent produced to date. It affords protection when given intranasally 14 days prior to infection with influenza AAttorney Docket No.: CEB-00225 virus (IAV). This adduct can be applied prophylactically and therapeutically and does not require prior immunization for protection against a lethal challenge with IAV. This approach may find application in the generation of VHH conjugates that target other cells in need of elimination, including pathogen-infected or even cancerous cells.

[0175] The following example describes the production and use of adducts that comprise VHHkappa, a single-domain antibody that recognizes mouse immunoglobulin kappa light chains, and one or more copies of the small molecule influenza virus neuraminidase (NA) inhibitor, zanamivir. Such compounds achieve half-life extension of zanamivir, while recruiting polyclonal immunoglobulins of all isotypes regardless of specificity to infected cells for antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC). Since the influenza A virus (IAV) NA is a tetramer, VHHkappaadducts with 1, 2, or 4 zanamivir molecules attached in site-specific manner were produced to allow multivalent engagement of NA. Administration of a VHHkappaadduct modified with 4 zanamivir molecules (“VHHkappa- Zan4” or “VHHkappa-(zanamivir)4) was ~10-fold more potent in protection against infection with IAV than VHHkappa-Zan carrying only a single zanamivir molecule. VHHkappa-Zan4can be given intranasally to confer full protection against a lethal IAV challenge. The neutralizing antibody titers in the respiratory mucosa and in the circulation, as well as the serum IgG antibody response against the hemagglutinin and nucleoprotein, are higher in VHHkappa-Zan4treated mice that survived the lethal challenge than in controls infected with a sublethal dose of virus. VHHkappa- Zan4affords protection even when administered intranasally weeks prior to a challenge with a lethal dose of IAV (both A and B strains of the virus). This type of adduct can therefore be applied prophylactically and therapeutically and does not require prior immunization for protection against a lethal dose of IAV.

[0176] Influenza virus critically relies on the activity of its NA to ensure the release of newly formed virions. Because zanamivir is a strict sialic acid analog, emergence of resistance to it is rare. The NAs of both influenza A (IAV) and influenza B (IBV) strains are sensitive to zanamivir. The drug is administered within 48 hours of diagnosis by inhalation, or it is given by intravenous injection to critically ill patients. As a small molecule, zanamivir is rapidly eliminated from the circulation and thus requires repeated dosing. In contrast, anti-light chain nanobodies (VHHkappa) equipped with a small molecule influenza virus NA inhibitor, zanamivir, have delayed elimination. The conjugation of zanamivir to the C-terminus of VHHkappaachievesAttorney Docket No.: CEB-00225 half-life extension of the drug, as the adduct will bind to immunoglobulins of all isotypes in the circulation, regardless of their specificity. Based on the tetrameric structure of neuraminidase (NA), a conjugate in which four molecules of zanamivir are flexibly linked to the C-terminus of the VHHkappawas designed. This construct shows remarkable efficacy. Even at very low concentrations, it can rescue mice infected with a lethal dose of influenza A, significantly reduce viral load in the lungs, enhance the quality of neutralizing antibodies in infected mice, and provide protection for mice up to two weeks prior to infection. Of note, the half-life of mouse immunoglobulins is ~7 days, while that of human immunoglobulins is ~30 days. Furthermore, because of the inherent stability characteristics of VHHs, this conjugate can be stored at 4 ºC for months without losing efficacy and can be nebulized without the risk of denaturation. These unique properties make it possible to administer this drug by inhalation. This avoids the use of needles, which can significantly improve ease of administration and adoption without compromising the efficacy of the drug.

[0177] A significant benefit of the conjugates are that they can be used for both therapy and prophylaxis. VHHkappabinds the kappa light chains of all immunoglobulins (all IgG subtypes, IgE, IgA, IgM). VHHkappais conjugated to a molecule that binds to, and inhibits, a pathogen of interest, in this case neuraminidase in flu. This conjugate, once in the circulation of an infected animal / individual, brings a large variety of antibodies into close proximity of an infected cell or pathogen. The diverse Fc portions of the various antibody classes then attract a broad range of FcR+ effector cells, leading to the killing of the infected cell and elimination of the pathogen. This involvement of a broad population of effector cells, including T cells, neutrophils, NK cells, and macrophages, is expected to prime the immune system more effectively, leading to a stronger and more diverse immunological memory than natural recovery.

[0178] The present disclosure describes new single-domain antibody-based antivirals that can be given intranasally to achieve full protection, even when administered weeks prior to infection. Context of design and selection of VHH as means for binding immunoglobulin

[0179] For a successful vaccine to elicit a desired response, the relevant antigen must be delivered under inflammatory conditions, either through the deployment of attenuated strains of a pathogen, or by inclusion of an adjuvant in the case of subunit vaccines. The route of vaccine administration can be a determinant of its success. In preclinical models, intraperitoneal orAttorney Docket No.: CEB-00225 intravenous administration are commonly used to deliver a vaccine, while clinical deployment relies on intramuscular or subcutaneous injection, oral delivery or delivery by inhalation. Where possible, the latter two are preferred, because they avoid the use of needles.

[0180] While the correlates of protection are not always easy to identify, the adaptive immune response plays an obvious and important role. The production of microbicidal or neutralizing antibodies is often used as a proxy. The effector functions of immunoglobulins, carried in their Fc portion, allow the recruitment of Fc receptor-positive cells with cytotoxic and cytokine producing activity (ADCC), as well as the activation of complement for direct lytic activity or through recruitment and activation of cytotoxic cells (complement-dependent cell mediated cytotoxicity; CDC). Antibody production against proteinaceous antigens requires T cell help, provided by CD4 T cells that are guided in their response by the Class II products of the Major Histocompatibility Complex. Because carbohydrates are not usually presented by Class II MHC products, induction of antibodies against polysaccharide or other carbohydrate antigens requires their conjugation to a potential source of peptides, included in vaccine preparations as a carrier protein. Where cytotoxic CD8 T cells are required for protection, professional antigen presenting cells (APCs), capable of cross-presentation of acquired antigens by Class I MHC products, are involved. The most efficient APCs capable of cross-presentation are dendritic cells (DCs) , which may require licensing by CD4 T cells to display their full spectrum of co- stimulatory molecules, to release cytokines, and to acquire the necessary migratory characteristics that ensure interaction with T cells.

[0181] There is a counterpoint to the notion that Class II MHC-positive APCs are required for the induction of an adaptive immune response: if antigen is delivered to APCs under non-inflammatory conditions, the outcome may be the imposition of tolerance, a state of immunological non-responsiveness. The protective anti-pathogen responses evoked by vaccines and imposition of tolerance are both restricted and guided by MHC products, because they are both T cell-dependent.

[0182] For vaccine design, the mere presence of an antigen is not sufficient. A combination of antigen with an appropriate inflammatory stimulus is required. Thus, the engineering goal for novel immunotherapies is to deliver the right combination of antigen and immune stimulant to the right anatomical location for optimal effect.Attorney Docket No.: CEB-00225

[0183] The discovery that camelids make both conventional four-chain (HL)2-type immunoglobulins and heavy chain only (H)2type immunoglobulins enabled the recombinant expression of just the variable region of these heavy chain only immunoglobulins, products referred to as VHHs. The appeal of VHHs lies in their small size, ease of expression and modification, as well as in their stability. Their single domain nature also implies that the mode of antigen recognition is distinct from that of typical immunoglobulins, and may provide access to epitopes not as readily accessible to the heavy chain-light chain combination of their conventional counterparts. Their ease of site-specific modification, whether enabled by the installation of an unpaired cysteine or by enzymatic modification, has found application in the design of nanoparticles that can be targeted to the site(s) recognized by the VHHs attached to such particles. The conversion of VHHs into theranostics through installation of the appropriate radio-isotopes is a promising application because the short circulatory half-life of VHHs limits systemic exposure, while the specificity of VHHs allows their significant enrichment at the sites targeted, such as tumors. Along the same lines, modification of VHHs with cytotoxic drugs likewise creates conjugates with a short circulatory half-life and thus limited systemic exposure, while still affording a therapeutic benefit through on-target efficacy. Finally, VHHs are finding application in adoptive cell therapy. The construction of chimeric antigen receptors (CARs) using VHHs as building blocks has moved from preclinical studies to clinical application. The BCMA-specific CAR T cells, used to treat multiple myeloma, comprises two BCMA-specific VHHs in tandem. In a proof-of-concept study, CAR T cells engineered to secrete a VHH specific for CD47 into the tumor micro-environment improved elimination of tumor cells by blocking the ‘don’t eat me’ signal. This indicates that VHHs can be used as building blocks for immune interceptors, capable of eliminating pathogens. Targeted delivery of antigens to antigen presenting cells improves antibody production.

[0184] Adaptive immunity involves the action of professional APCs to get things started. APCs fulfill the essential task of capturing antigen, processing it and then present fragments (peptides) of these processed antigens via surface-displayed products encoded by the major histocompatibility complex. Class II MHC molecules are positioned throughout the secretory and endocytic pathway of APCs whereas Class I MHC products, essential for the action of CD8 T cells, are confined primarily to the secretory pathway and the cell surface. All professional APCs (dendritic cells, macrophages, B cells) express Class II MHC products, which can be used forAttorney Docket No.: CEB-00225 targeted delivery of payloads to APCs. Upon receipt of the proper inflammatory stimuli, peptide loaded Class II molecules are delivered to the surface of the APC where they are available for inspection by and activation of antigen-specific T cells. These T cells, in turn, control the production of high affinity immunoglobulins by B cells, and regulate numerous aspects of T cell biology and their interactions with APCs.

[0185] Dendritic cells, notably DEC205, are important in triggering an adaptive immune response. By creating covalent adducts of antibodies that recognize DEC205 with an antigenic payload of interest, strong responses against the attached antigen can be elicited. Not all APCs express DEC205, which prompted focus on Class II MHC molecules instead. VHHs that recognize mouse H-2 I-A products in a seemingly monomorphic pattern of recognition have been generated: all I-A allelic products tested were recognized by VHH7. The amino acid sequence of VHH7 prior to conjugation is as follows: QVQLQESGGGLVQAGDSLRLSCAASGRTFSRGVMGWFRRAPGKEREFVAIFSGSSWSG RSTYYSDSVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYCAAGYPEAYSAYGRESTY DYWGQGTQVTVSSGG (SEQ ID NO: 59).

[0186] Upon conjugation of VHH7 to a variety of different antigens, attached in a sortase-catalyzed transpeptidation reaction, and delivery under inflammatory conditions, a string antibody response against the attached antigenic payload was observed. Certain antigens such as ubiquitin or cyclotides, small cystine-knotted cyclical peptides, administered under inflammatory conditions but lacking the targeting VHH, were poorly immunogenic or failed to yield an antibody response at all. However, when conjugated to VHH7 and given under inflammatory conditions (poly-dI-dC; anti CD40), a strong response against ubiquitin or the cyclotide MCoTI-I was obtained. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. By attaching a synthetic peptide corresponding to the conserved stem region of influenza HA to VHH7, under similar conditions a strong anti HA response capable of protecting mice from a challenge with flu was obtained. The anti-Class II MHC VHH was equipped with the receptor binding domain of the SARS-CoV-2 spike protein. Administration of this adduct together with poly dI-dC and anti CD40 elicited a protective immune response against the virus A signature advantage ofAttorney Docket No.: CEB-00225 sortase-catalyzed installation of antigens onto the Class II MHC VHH, VHH7, is the option of including molecular structures that cannot be encoded by a recombinant DNA or RNA construct. For example, by attaching a modified O-linked sugar to VHH7 as part of a tumor-associated MUC1(Tn) epitope, a MUC1-specific T cell response was obtained in vitro that included both a CD4 and a CD8 component. Using VHHs to recruit immunoglobulin effector functions

[0187] VHHs that recognize immunoglobulin light chains of mouse or human origin can be used to recruit immunoglobulin. Anti-light chain VHHs were equipped with a small molecule influenza virus neuraminidase inhibitor, zanamivir. Influenza virus critically relies on the activity of its neuraminidase to ensure the release of newly formed virions. Because zanamivir is a strict sialic acid analog, emergence of resistance to it is rare. The neuraminidases of both influenza A (IAV) and influenza B (IBV) strains are sensitive to zanamivir. The drug is administered within 48 hours of diagnosis by inhalation or it is given by intravenous injection to critically ill patients. As a small molecule, zanamivir is rapidly eliminated from the circulation and thus requires repeated dosing. By covalently attaching zanamivir to the C-terminus of VHHkappa, a VHH that recognizes immunoglobulin kappa light chains, a half-life extension of the drug was achieved, as the adduct will bind to immunoglobulins of all isotypes in the circulation, regardless of their specificity. Because VHHkapparecognizes kappa light chains monovalently, no crosslinking of the targeted immunoglobulins is possible. When the circulatory half-life of VHHkappa-zanamivir was measured in mice, a value of xx hours was obtained. For comparison, the circulatory half-lives of immunoglobulins in mice are as follows. IgM:~2days; IgG1 and IgG3: 6-8 days; IgG2b:4-6 days; IgA:<4days;IgE:12 hours.

[0188] Upon challenge of mice with a 10 x LD50 of IAV, some weight loss was observed in animals that received 0.3 mg / kg of VHHkappa-zanamivir, but they were nonetheless fully protected as measured by overall survival. The covalent complex is required: giving a mixture of zanamivir and VHHkappadoes not afford protection. Mere half-life extension of zanamivir also did not account for this protective effect, because a similar adduct comprised of an anti-serum albumin VHH and zanamivir showed a similar circulatory half-life, but required a far higher dose for protection. This suggests that extension of circulatory half-life renders zanamivir more effective than the drug alone, but it does account for the level of protection seen with VHHkappa-zanamivir. The mechanism responsible for protection requires the presence ofAttorney Docket No.: CEB-00225 immunoglobulins. RAG-deficient mice, which have no B or T cells and therefore no immunoglobulins in the circulation, are killed by IAV. Administration of VHHkappa-zanamivir fails to protect RAG-deficient animals, unless they also receive a dose of polyclonal Ig. Administration of polyclonal Ig in the absence of the VHHkappa-zanamivir adduct does not afford protection. Only the combination of polyclonal Ig and VHHkappa-zanamivir protects mice against IAV. An in vitro antibody dependent cytotoxicity (ADCC) and complement-dependent cell mediated cytotoxicity (CDC) assay was performed to corroborate the involvement of immunoglobulin effector functions and found that both mechanisms likely participate.

[0189] The serum response to influenza virus proteins on the assumption that exposure to a 10 x LD50 of live virus would far exceed what a susceptible host would encounter. Mice that received such a dose could be kept alive only by giving them VHHkappa-zanamivir at xx mg / kg. Mice were infected with a 10 x LD50 of IAV and then treated mice 2 days later with xx mg / kg of VHHkappa-zanamivir. In view of the large dose of live IAV administered, it was expected to see a robust antibody response. The serum response was measured by ELISA and by immunoblot using a pool of serum obtained from 5 mice. The response was examined against viral proteins by SDS-PAGE and immunoblot. To distinguish between the virus-encoded glycoproteins and non-glycosylated proteins such as M and NP, egg-grown IAV H1N1 extracts were digested with PNGase F to remove N-linked glycans. Polypeptides susceptible to digestion with PNGase F are proteins that carry N-linked glycans, IAV HA and N. Mice that had received 10 x LD50 of IAV and that had been treated with a protective dose of VHHkappa-zanamivir were then challenged with a second dose of virus 20 days later and found this cohort to be completely resistant to re- infection as determined by weight loss. In view of the dose of VHHkappa-zanamivir given for protection, even after 20 days a sufficient amount of the adduct should have persisted to afford protection. The obvious presence of anti IAV antibodies in the treated mice also contributes to protection. The serum response of the ‘rescued’ 10 x LD50 IAV cohort was then compared with that of mice that received a sublethal dose of the virus. The serum response as measured by immunoblot was very similar, if not indistinguishable, from that of the ‘rescued’ 10 x LD50 IAV cohort. If transposed to the clinic, it is concluded that VHHkappa-zanamivir is a potent antiviral that might be efficacious in patients seriously ill with IAV or IBV, even when given several days after diagnosis. Increasing the number of zanamivir molecules on VHHkappa.Attorney Docket No.: CEB-00225

[0190] Influenza virus NA (also referred to as Flu NANAse) is a homotetramer. With each monomer having a binding pocket for a single sialic acid (or zanamivir) molecule. VHHkappa-zanamivir carries only a single molecule of drug per monomer (FIG.1). To assess whether increasing the number of zanamivir molecules per VHHkappamonomer would increase anti-viral efficacy, constructs with multiple zanamivir moieties were made. A multivalent version would be expected to show enhanced avidity, with two possible, not mutually exclusive outcomes: stronger inhibition of neuraminidase activity (resulting in a greater reduction in release of viral particles) and / or more effective recruitment of polyclonal Ig and its attendant improvement in ADCC and CDC.

[0191] A synthetic route to obtain VHHkappa-(zanamivir)2was devised (FIG.2), and its activity was compared against IAV with VHHkappa-zanamivir. VHHkappa-(zanamivir)2was more effective than VHHkappa-zanamivir in preventing weight loss and improving survival. Because VHHs have been used not only for intravenous or intraperitoneal delivery, but also for delivery by inhalation, the protective effect of VHHkappa-zanamivir and VHHkappa-(zanamivir)2by intranasal delivery was also tested. If these drugs could be given in a manner that avoids parenteral administration, it would facilitate clinical deployment. Indeed, intranasal delivery resulted in decreased viral titers in the lung, and an increase in neutralizing antibodies in the lung. It was effective, but at a dose that would be difficult to attain clinically.

[0192] This prompted the design of a synthetic route to further increase the number of zanamivir molecules on VHHkappa. By creating VHHkappa-(zanamivir)4(FIG. 3) a more complete occupancy of the active sites on neuraminidase, and hence greater anti-viral activity, was predicted. The intranasal dose required for full protection was then compared to the previous conjugates, and it was found that 0.3 mg / kg of VHHkappa-(zanamivir)4was indeed sufficient. When VHHkappa-(zanamivir)4was given intranasally at 0.3 mg / kg, mice were still protected when challenged with live IAV 14 days later. Therefore, increasing the number of zanamivir molecules per VHHkappa monomer achieves improved protection and allows administration via the intranasal route.

[0193] The two and four zanamivir molecules are conjugated in a site-specific and flexible manner. This flexible arrangement can enable simultaneous engagement of several active sites per NA tetramer. For VHHkappa-Zan4, more complete occupancy of the active sites on NA, resulting in enhanced NA-inhibitory and antiviral activity, was anticipated. To assess thisAttorney Docket No.: CEB-00225 potential improvement, NA activity in the presence of the different VHHkappa-zanamivir conjugates was measured (FIG.4A-B). As expected, the IC50of the conjugates shows a linear positive correlation with the number of zanamivir molecules conjugated to VHHkappa. Specifically, the IC50 of VHHkappa-Zan4is approximately 2-fold lower than that of VHHkappa- Zan2and 4-fold lower than that of VHHkappa-Zan. It is worth noting that the VHHkappa-bound inhibitors exhibited reduced activity compared to ‘free’ zanamivir, likely due to the chemical modifications at C7 employed during the conjugation process. The enhanced binding avidity VHHkappa-Zan4for NA was confirmed by measuring the binding kinetics of VHHkappa-Zan and VHHkappa-Zan4using biolayer interferometry (FIG.5). By immobilizing the VHH conjugates on a biosensor and exposing them to varying concentrations of NA protein from the PR / 8 strain, we found the dissociation constant (Kd) for VHHkappa-Zan to be 11 nM and for VHHkappa-Zan4to be 3.5 nM. The multivalent VHHkappa-Zan4 conjugate thus shows significantly higher avidity for NA compared to the monovalent VHHkappa-Zan. The improvement in antiviral activity for VHHkappa- Zan4exceeds its ~4-fold increase in avidity for NA, as shown below. VHHkappa-Zan4provides complete protection against IAV at doses 10x lower than VHHkappa-Zan

[0194] Since VHHs have been delivered not only intravenously or intraperitoneally but also by inhalation, the protective effect of VHHkappa-Zan, VHHkappa-Zan2, and VHHkappa-Zan4given intranasally (IN) was tested, as intranasal administration is an appealing delivery route to combat a virus that targets the respiratory tract. The efficacy of the VHHkappa-Zan, VHHkappa- Zan2, and VHHkappa-Zan4in an influenza A virus (IAV) challenge experiment were comparted. Upon infection of mice with a 15x LD50 dose of IAV, VHHkappa-Zan4was more effective than either VHHkappa-Zan2or VHHkappa-Zan in preventing weight loss (FIG. 7) and improving survival when administered at equimolar doses one day after infection (FIG. 6).

[0195] VHHkappa-Zan4protected 100% of mice when given at 0.3 mg / kg, while VHHkappa-Zan protected only 40% and VHHkappa-Zan2protected 70% of the mice. ‘Free’ zanamivir provided protection only when given at ~200 g per day (equivalent to ~20 mg / kg) over the course of infection. Increasing the number of zanamivir molecules per VHHkappamonomer thus improves protection and survival outcomes following IN administration. Intraperitoneal (IP) delivery requires higher doses to achieve the same level of protection as IN administration (See supporting information). These results demonstrate that the increased inhibitory properties and avidity against NA observed for VHHkappa-Zan4translate to increasedAttorney Docket No.: CEB-00225 protection from lethal IAV challenge in vivo. Similar results were observed with intraperitoneal (IP) administration, as shown in FIG.8 and FIG.9.

[0196] Next, the viral load in the lungs on day 5 post-infection was measured. Intranasal delivery of VHHkappa-Zan2and VHHkappa-Zan4conjugates reduced viral titers in the lungs compared to the control group (FIGs. 10A-B). Significant reductions were observed in groups treated with either 0.3mg / kg or 1.0mg / kg of the VHHkappa-Zan adduct. While ‘free’ zanamivir improved survival, even twice-daily treatments failed to promote viral clearance from the lungs. This suggests that VHHkappa-Zan2and VHHkappa-Zan4facilitate early clearance of IAV.

[0197] The mechanism of action of VHHkappa-Zan(n) conjugates involves engagement of the kappa light chain of a highly diverse repertoire of immunoglobulins and may improve engagement of the immune system by ADCC and / or CDC. This might lead to higher titers of IAV-specific antibodies in the treated mice and to what is referred to as a “vaccinal effect”. To test this hypothesis, antibody titers against intact IAV and two immunogenic IAV proteins, the hemagglutinin (HA) and nucleoprotein (NP), were measured in mice treated with 1 mg / kg of VHHkappa-Zan4. Interestingly, no significant differences in IAV-specific serum IgG titers were observed between VHHkappa-Zan4treated mice and mice that received twice-daily doses of zanamivir or the control group that received a sublethal IAV infection (FIGs. 11A, 11B, 12A, 12B, 13A, and 13B). As the adduct is administered intranasally, it is possible that any enhancement in IAV-specific immunity occurs at the site of administration. Thus, the IgA antibody titers within the lung tissue were assessed. IgA plays a crucial role in mucosal immunity and provides a critical first response against respiratory pathogens. A significant increase in IgA titers in the lung homogenates of mice treated with VHHkappa-Zan4compared to controls, including animals treated with ‘free’ zanamivir, was observed (FIG.14). Next, when the IAV-neutralizing capacity of antibodies in mice treated with VHHkappa-Zan4were assessed, both systemic IgG and lung-localized IgA showed improved neutralizing activity against live IAV compared to the controls (FIGs. 15A-C). Finally, the IgA antibody titers in the upper respiratory tract were evaluated, in both nasal washes collected by flushing saline through the nasal passages and in the nasal-associated lymphoid tissue (NALT). The titers of IgA were higher in mice treated with VHHkappa-Zan4compared to control groups, suggesting that IN administration of VHHkappa-Zan4promotes enhanced antibody responses in the upper respiratoryAttorney Docket No.: CEB-00225 mucosa (FIGs.15D-E). Early administration of VHHkappa-Zan4thus provides a vaccinal effect by enhancing the generation of the IAV-specific humoral immune response. VHHkappa-Zan4does not enhance the IAV-specific T cell response

[0198] An influenza virus specific T cell response precedes the development of the virus specific antibody response and plays a role in viral clearance. To test whether there is a T cell- mediated vaccinal effect of the VHHkappa-Zan4, mice were infected with a lethal dose of IAV and treated them with VHHkappa-Zan4one day later. Seven days post-infection, the lungs were harvested and analyzed for the presence of IAV-specific T cells. Using the following influenza- specific tetramers: H-2Db-restricted NP366-374, H-2Dbrestricted Polymerase Acidic Protein PA224-233, and I-Ab-restricted NP311-325, the IAV-specific CD4+ and CD8+ T cell populations were enumerated. No significant differences were observed in these populations when comparing VHHkappa-Zan4treated mice with those that survived owing to treatment with free zanamivir or with mice infected with a sublethal dose of IAV. VHHkappa-Zan4treatment provides complete protection against IAV 3 days post infection

[0199] Conventional antiviral treatments including zanamivir demonstrate the highest efficacy when given early after infection.

[0200] Mice treated with VHHkappa-Zan and infected with PR / 8; H1N1 exhibited higher levels of inhibitor antibodies against the HK / 68 (H3N2) and influenza B strains. Serum was collected 28 days after influenza infection. (FIG. 16). Long-term protection against influenza A virus-induced mortality was provided by prophylactic administration of VHHkappa-Zan2. (FIGs. 17-20). Prophylactic treatment with VHHkappa-Zan2enhanced systemic and pulmonary titers of antibodies specific to influenza virus (FIGs.21-25, 27). Prophylactic treatment (intranasal and intraperitoneal) with VHHkappa-Zan2enhances the neutralizing capacity of systemic influenza- specific antibodies (FIG. 26). FIGs.28-29 show influenza-specific T cell responses following treatment with VHHkappa-Zan4conjugates.

[0201] Experiments were performed to probe the extent to which treatment could be delayed and protection against a lethal IAV challenge could be maintained. VHHkappa-Zan4given on either day 3 or 4 of infection with IAV promoted early viral clearance in the lungs (FIG.30, top). This is all the more striking given that there was only a 24-hour window between the treatment on day 4 and the assessment of viral titers on day 5. Treatment with either 2mg / kg or 1mg / kg VHHkappa-Zan4on day 3 rescued mice from further weight loss and protected 100% ofAttorney Docket No.: CEB-00225 mice from IAV-induced death (FIG.30, bottom). Finally, a 2mg / kg dose of VHHkappa-Zan4given on day 4 post-infection protected 25% of mice from IAV-induced death. Thus, treatment can be delayed until day 3 post-infection while maintaining a robust efficacy against a lethal IAV challenge. Prophylactic administration of VHHkappa-Zan4provides extended protection against a lethal IAV challenge

[0202] The circulatory half-life of immunoglobulins is a key determinant of how long protection afforded by VHHkappa-Zan(n)conjugates lasts. The VHHkappa-Zan adduct given intraperitoneally protects mice from a lethal dose of IAV when given up to 7 days prior to infection. Experiments were performed to determine the allowable delay between intranasal (IN) delivery of VHHkappa-Zan4and an IAV challenge. Animals received a single IN dose of VHHkappa-Zan4at 1 or 2 mg / kg, either 4 or 2 weeks prior to a challenge with 15xLD50 of IAV. Mice that received VHHkappa-Zan4at 2 weeks before infection, regardless of the dose, afforded full protection (FIG.31). While mice that received a single 1mg / kg dose 4 weeks before infection did not survive the challenge, 80% of those that received 2mg / kg VHHkappa-Zan4survived. Similarly, to mice that were treated 24 hours post-infection, no improvement was observed in serum IgG antibody titers of those treated 2 or 4 weeks prior to infection (FIG. 31). Animals treated with VHHkappa-Zan42 weeks previously did show increased NP-specific IgA in lung samples, as well as increased neutralizing activity in both serum and lung samples (FIG. 31). The average half-life of mouse immunoglobulins is only ~7 days, much shorter than the ~30-day half-life typical in humans. These results suggest the potential for development of an adduct that could protect human patients for a period of months.

[0203] Finally, the IgA antibody titers in the upper respiratory tract in mice that were prophylactically treated with 2mg / kg of VHHkappa-Zan4two or four weeks prior to an IAV infection were evaluated. Mice that received VHHkappa-Zan4at 2 weeks before infection demonstrated enhanced IgA titers specific for the NP and HA proteins in both the NALT and in fluid collected from the nasal passages (FIG.32). Animals treated 4 weeks prior to infection showed more modestly increased IgA titers compared to controls. These data suggest that IN administration of VHHkappa-Zan4promotes enhanced antibody responses in the upper respiratory tract. Overall, these data demonstrate that intranasal treatment with VHHkappa-Zan4 can beAttorney Docket No.: CEB-00225 utilized both prophylactically or therapeutically to provide complete protection against a lethal IAV infection.

[0204] The approaches described herein for the various modifications of VHHkappamerit further comment.

[0205] First, no immunization is required for VHHkappa-Zan(n)to reap the benefits of all effector functions associated with the various Ig isotypes and subclasses, regardless of antibody specificity.

[0206] Second, the small molecule zanamivir, used here as a recognition module, can be swapped out for other entities, including VHHs. Any small molecule (or VHH / antibody fragment) capable of interacting with the surface of a pathogen or pathogen-infected cell deserves exploration as a modifier of VHHkappa to achieve recruitment of Ig. However, increasing the size of these types of adduct, as would be the case upon installation of multiple nanobodies, may compromise the possibility of intranasal delivery.

[0207] Third, the recognition unit, be it a small molecule or a VHH, need not be neutralizing or otherwise have antiviral or anti-bacterial properties on its own, as long as it enables the recruitment of immunoglobulins to the pathogen or infected cell.

[0208] Fourth, the efficacy of these VHHkappaadducts, to a first approximation, lasts as long as their complex with circulating immunoglobulins persists. This means that in mice, where the circulatory half-life of immunoglobulins is several days, a dose can be chosen that retains activity for several half-lives, thus extending protection. If transposed to the human setting -with VHHs that recognize human kappa light chains- protection is expected to last much longer, based on the circulatory half-life of human Ig, which is measured in weeks. VHHkappa-Zan(n)adducts are covalent conjugates via stable amide and carbamate linkages, without relying on maleimide-type linkers. They are expected to survive as long as the adduct is present.

[0209] Fifth, protection against the targeted pathogen is achieved very soon after delivery of the administered modified VHHkappa. Both prophylactic and therapeutic applications are therefore possible, as no induction of an immune response is required.

[0210] Sixth, the modest size of VHHkappa-(zanamivir)nis most likely responsible for its ability to exert its effect when administered intranasally.Attorney Docket No.: CEB-00225 Finally, in preliminary experiments it was shown that this approach is not limited to the elimination of virus and virus-infected cells. Fusions of VHHkappawith anti-Class II MHC VHHs efficiently reduce the number of Class II MHC+ cells in vivo, while fusions of VHHkappawith an anti-CTLA4 VHH result in starkly reduced tumor growth through a reduction in the number of intratumoral regulatory T cells. Example 2 – Materials and Methods of Example 1 Influenza viruses

[0211] Influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (NR-348) and A / California / 07 / 2009 (H1N1) (NR-13663) were obtained from BEI resources and propagated in MDCK cells (American Type Culture Collection [ATCC], Manassas, VA). Neuraminidase inhibition assay

[0212] Inhibition of influenza neuraminidase activity was assayed using the NA-Fluor Influenza Neuraminidase Assay Kit (Thermo Fisher). Briefly, influenza H1N1 viruses A / Puerto Rico / 8 / 1934 and A / California / 07 / 2009 were assayed at serial 2-fold dilutions of 1:1-1:1024 to determine an appropriate working concentration in the linear portion of the neuraminidase activity curve. Using these viruses at a 1:4 dilution (after dilution: 6.5×105PFU / mL of A / Puerto Rico / 8 / 1934 and 1.5×103PFU / mL of A / California / 07 / 2009), the various VHH-zanamivir conjugates were tested against the respective concentration of zanamivir to assess neuraminidase activity inhibition. For each drug, a 50% neuraminidase inhibitory concentration (IC50) value was calculated by fitting a sigmoidal dose-response (four-point logistic) curve using GraphPad Prism 10 software. Mouse Experiments

[0213] 8–10-week-old female C57Bl / 6 mice were purchased from the Jackson Laboratory and housed in the animal facility at Mispro Biotech Services. All experimental procedures using mice were approved and carried out in accordance with Institutional Animal Care and Use Committee (IACUC) under protocol #2023-CRB-03. Mice were infected IN with either A / Puerto Rico / 8 / 1934 (PR / 8) or A / California / 07 / 2009 (Cal / 09) virus while anesthetized with isoflurane. Infected mice were monitored daily for weight loss and mortality, and humanely euthanized at a threshold of 80% weight loss. When indicated, mice received one intranasal dose of VHHkappaadduct at the indicated concentration in 75ul volume while anesthetized withAttorney Docket No.: CEB-00225 isoflurane. Intraperitoneal administration of the VHHkappaadduct was given in 100ul total volume diluted in sterile PBS. Zanamivir treated groups received either one IN dose at 10 mg / kg administered 24 hours post-infection or received a 5 mg / kg IN dose twice per day during the first 5 days of infection. Plaque assay for viral titers

[0214] Whole lungs were homogenized, and supernatant was flash frozen and stored at - 80°C until further analysis. Samples were diluted 4-fold starting at 1:10 then added to MDCK cells in six-well plates and incubated at 37°C for 1 hour. Wells were washed with PBS and overlaid with 1:1 mixture 2X Eagle MEM (Quality Biological) and 1.6% SeaKem ME agarose (Lonza) supplemented with 2ug / mL TPCK-trypsin. Plates were incubated for 3 days at 37°C with 5% CO2. Following incubation, the agar plug was removed from wells and cells were fixed with 70% Ethanol. Cells were stained with a 1% crystal violet solution and viral plaques were counted after 24 hours. Antibody ELISA

[0215] Serum, lung homogenates, and nasal-associated lymphoid tissue were collected on day 28 post-infection and stored at -80°C until further analysis. Nasal wash fluid was collected by cannulation of the trachea, and 300ul of sterile PBS was washed through the nasal cavity, collected out the nose, and stored at -80°C. Polystyrene high-binding plates (Corning) were coated overnight at 4°C with either live A / Puerto Rico / 8 / 34 virus (1x104PFU / well), A / Puerto Rico / 8 / 34 Nucleoprotein (1ug / mL) (Sino Biological), or A / Puerto Rico / 8 / 34 Hemagglutinin (1ug / mL) (Sino Biological). Plates were blocked with 5% nonfat dry milk in PBS for 1 hour at 37°C. Samples were serially diluted 5-fold starting at 1:50 (serum) or 1:4 (lung), and plates were incubated overnight at 4°C. Goat anti-mouse HRP-conjugated antibody specific for IgG or IgA (Southern Biotech) was added at 1:4000 dilution and incubated for 1 hour at 37°C. Plates were developed in 3,3’,5,5’-tetramethylbenzidine solution (Sigma-Aldrich), and the reaction was stopped with ELISA stop solution (Thermo Fisher). Absorbance values were measured at 450nm. Neutralizing antibody titers

[0216] Serum or lung homogenates were collected on day 28 post-infection and stored at -80°C until further analysis. Samples were diluted 5-fold starting at 1:50 (serum) or 1:4 (lung)Attorney Docket No.: CEB-00225 and mixed with 500 PFU IAV PR / 8 in a 96-well round bottom plate prior to incubation at 37°C for 1 hour. 100ul of virus / sample mixture was added to confluent MDCK cells in 6-well plates and further incubated at 37°C for 1 hour. Plates were washed, overlaid, and stained as described above for the viral titer plaque assay. A four-parameter fit curve analysis was used to determine the serum dilution that resulted in 50% inhibition of IAV viral plaques. Antibody staining and flow cytometry

[0217] Whole lungs were digested in 4 mL RPMI supplemented with 0.5mg / mL DNase I (Sigma-Aldrich) and 2.5mg / mL collagenase I (Sigma-Aldrich) for 30 min at 37°C. Digested lungs were homogenized with an AutoMACS (Miltenyi) and passed through a 70um filter to generate a single-cell suspension. Cells were stained with live / dead Fixable Aqua (Thermo Fisher) for 30 min at 4°C. For tetramer staining, cells were stained with either IAV-specific NP311-325(4ug / mL) for 3 hours at 37°C, or IAV-specific NP366-374(2ug / mL) and IAV-specific PA224-233(2ug / mL) for 30 min at 4°C. Extracellular staining was then performed using antibodies for CD45.2 (104; Thermo Fisher), CD90.2 (53-2.1; Thermo Fisher), CD11a (M17 / 4; Thermo Fisher), CD44 (IM7; Thermo Fisher), CD4 (RM4-5; Thermo Fisher), and CD8 (53-6.7; Thermo Fisher) for 30 min at 4°C. For intracellular cytokine staining, cells were stimulated with eBioscience cell stimulation cocktail (Thermo Fisher) for 5 hours followed by staining for live / dead Fixable Aqua and extracellular markers as described above. Cells were fixed using eBioscience Intracellular Fixation and Permeabilization Buffet set (Thermo Fisher) followed bystaining with antibodies for IFN- (XMG1.2; Thermo Fisher) and TNF (MP6-XT22; ThermoFisher) for 30 min at 4°C. Samples were run on an Attune CytPix (Thermo Fisher) and analyzed using FlowJo software (BD Biosciences). Synthesis of VHHkappa-(zanamivir)2and VHHkappa-(zanamivir)4

[0218] VHHkappa-(zanamivir) (“mono zanamivir conjugate”) is prepared by a sortase- mediated conjugation of triglycine modified zanamivir to VHHkappa. This process is described in PCT Application No. PCT / US2023 / 060893, published as WO 2023 / 141500 A2, as well as Liu et al. “An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections.” Sci. Immunol. (2023), each of which is hereby incorporated by reference in its entirety. VHHkappa-(zanamivir)2and VHHkappa-(zanamivir)4are prepared by the same method.

[0219] Briefly, to obtain VHHkappa-Zan2and VHHkappa-Zan4, a Sortase A-based approach for VHH bioconjugation was applied. Through solid-phase peptide synthesis severalAttorney Docket No.: CEB-00225 peptide / PEG-based linkers, each modified with 1, 2, or 4 azido moieties, were generated. These flexible linkers were then sortagged onto VHHkappato generate three distinct modified VHHkappa- based adducts, with 1, 2, or 4 "clickable" reactive azide moieties. Zanamivir was equipped with a DBCO moiety separated by a PEG linker. In a copper-free click reaction, zanamivir could thus be installed on the branched nucleophiles. Synthesis mono, dual and tetra azido-linkers

[0220] Peptide-based linkers were synthesized manually using standard Fmoc-SPPS chemistry protocol. The following protected-amino acids with side-chain protection groups were used: Fmoc-Lys(N3)-OH, Fmoc-Lys(mtt)-OH, Boc-Gly-Gly-Gly-OH, Fmoc-Lys(Fmoc)-OH as well as the azide-carrying PEG linker: N,N-Bis(PEG1-azide)-N-PEG2-acid.SPPS was performed on Rink-amide polystyrene resin. Manual loading of the first amino acid residue on the resin and subsequent Fmoc-SPPS, followed established standard protocols.

[0221] In brief: Fmoc-deprotections were performed with 20% piperidine in DMF. 2 x 8 min. Couplings were performed with Fmoc-amino acid (4.0 equiv relative to resin substitution), HATU (3.9 equiv) and DIPEA (8.0 equiv) in DMF for 45 min. For coupling of the azido-PEG linker, the reaction time was extended to 90 min.

[0222] The completed peptide was cleaved from the Rink Amide polystyrene resin using a cleavage cocktail of 95:2.5:2.5 TFA:TiPS:H2O and shaken for 2 h. The resin was removed by filtration and washed with TFA (5 mL / g resin), the filtrate was placed in a plastic centrifugal tube (40 mL) and volatiles removed under reduced pressure. The residue was triturated with Et2O (ca.30 mL / g resin), centrifuged (3500 g, 3 min) and the supernatant was removed by decantation. The crude material was dried using N2 flow and dissolved in a suitable solvent (1:1 CH3CN:H2O + 0.1% TFA) for RP-HPLC purification.

[0223] The linkers were purified by reverse phase high performance liquid chromatography (RP-HPLC) The mobile phase for RP-HPLC were Milipore-H2O containing 0.1% TFA and HPLC-grade CH3CN containing 0.1% TFA. Preparative HPLC was performed on a C18 column (5 m, 100 Å pore size, 20 mm I.D. x 250 mm), at the flow rate of 20 mL / min.

[0224] The purified fractions containing the desired product were combined and lyophilized to yield the desired product as a white powder. Synthesis of zanamivir-PEG6-DBCOAttorney Docket No.: CEB-00225

[0225] Zanamivir-PEG6-DBCO was prepared following the previously described Procedure. To a THF solution containing the protected and carbonyldiimidazole-activated zanamivir compound, DBCO-PEG6-amine and diisopropylethylamine were added and stirred overnight at room temperature. The final step is identical to the previously described work (ref). LC-MS [M + H]+ = 970.04. VHHkappaproduction

[0226] VHHkappawas cloned into a pHEN6 and recombinantly expressed in WK6 E. coli. Cells were then grown at 37°C in Terrific Broth containing ampicillin (100 mg / L) until the optical density at 600 nm (OD600) reached 0.6 to 0.8. To induce the VHH expression, 1 mMisopropyl- -D-thiogalactopyranoside was added, and incubation was continued overnight at30°C. Cells were harvested by centrifugation. VHHs were released by osmotic shock using tris / EDTA / sucrose (TES) buffer [200 mM tris, 0.65 mM EDTA, and 0.5 M sucrose (pH 8)].

[0227] VHHs were isolated on Ni-NTA beads and further purified by size exclusion chromatography using a Superdex 7510 / 600 column. To deplete lipopolysaccharide (LPS), purified VHHs were reloaded on Ni-NTA beads, which was then washed with PBS solution (40 column volumes) containing 0.1% (v / v) Triton X-114. Elution was performed with endotoxin- free PBS containing 500 mM imidazole. Imidazole was removed by desalting on a PD10 column using LPS-free PBS as the elution buffer. VHHkappa-Zanamivir

[0228] VHHkappa-LPETGGHHHHHH (SEQ ID NO: 57) was first modified with the desired linker (mono, dual and tetra azido-linker) using sortase A mediated ligation. The general procedure is as follow: to 0.5 mL of VHHkappa-LPETGGHHHHHH (SEQ ID NO: 57) in PBS (250 uM) was added the 50 uL of the desired linker (1.0 mM) and 40 uL of sortase A 7M (0.5 mM). The resulting mixture was stirred overnight at 15 ºC. The completion of the reaction was monitored by LCMS and when completion reached over 80%, the reaction mixture was filtered through Ni-NTA resin to remove the sortase, the unreacted VHHkappa and the cleaved histidine tag. The remaining excess of the linker was removed via PD10 column to afford the clean VHHkappa-linker-(N3)n.

[0229] The final conjugation was done by mixing into a PBS solution of VHHkappa- linker-(N3)n (100 uM), 1.5 equivalent of zanamivir-PEG6-DBCO compared to n. The reaction was a stirred at 15 ºC and. the completion was monitored by LCMS. The remaining excess ofAttorney Docket No.: CEB-00225 zanamivir-PEG6-DBCO was removed by PD10 column and the resulting conjugates were used without further purification.

[0230] For example, to prepare VHHkappa-Zan4, 200 uL of VHHkappa-linker-(N3)4(100 uM) in PBS was mixed with 40 uL of DBCO-PEG-zanamivir (2.4 mM) and stirred at 15 ºC. Completion was reached after 20 hours and the reaction mixture was passed through PD10 column to remove the excess of DBCO-PEG-zanamivir. OTHER EMBODIMENTS

[0231] It will be appreciated that the scope of the present disclosure is to be defined by that which may be understood from the disclosure and claims rather than by the specific embodiments that have been presented by way of example. Elements described with respect to one aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. Moreover, recitation of claim elements in connection with a particular independent claim support recitation of such elements in connection with other independent claims. Throughout the disclosure and claims, where compositions or methods are described as having, including, or comprising specific elements, compositions that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed. While specific embodiments disclosed herein have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the subject matter disclosed herein should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. All references, including publications and patents, mentioned or cited herein are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Claims

Attorney Docket No.: CEB-00225 CLAIMS We claim:

1. A conjugate comprising a means for binding to an immunoglobulin kappa light chain covalently conjugated to at least two copies of an agent via one or more linkers, wherein the agent binds to a protein on the surface of a cell or pathogen.

2. A conjugate comprising a variable domain on a heavy chain antibody (VHH) specific for an immunoglobulin kappa light chain covalently conjugated to at least two copies of an agent via one or more linkers, wherein the agent binds to a protein on the surface of a cell or pathogen.

3. The conjugate of claim 2, wherein the VHH comprises a set of CDR1, CDR2, and CDR3 sequences selected from the sets of CDR1, CDR2, and CDR3 sequences in Table 2.

4. The conjugate of claim 2, wherein the VHH comprises the amino acid sequence selected from the sequences in Table 1.

5. The conjugate of any one of claims 1-4, wherein the at least two copies of an agent are 2, 3, 4, 5, or 6 copies of the agent.

6. The conjugate of claim 5, wherein the at least two copies of an agent are 4 copies of the agent.

7. The conjugate of any one of claims 1-6, wherein the agent comprises a small molecule, a polypeptide, a carbohydrate, a lipid, or a nucleic acid, an aptamer, a VHH, an antibody, or an antibody fragment.

8. The conjugate of claim 7, wherein the agent comprises a small molecule.

9. The conjugate of any one of claims 1-8, wherein the agent binds to the surface of a pathogen or a cell infected by the pathogen.

10. The conjugate of claim 9, wherein the pathogen is a virus.Attorney Docket No.: CEB-00225 11. The conjugate of claim 10, wherein the virus is an influenza virus, a coronavirus, an adenovirus, an enterovirus, a rotavirus, a norovirus, a herpesvirus, a lentivirus, a poxvirus, a paramyxovirus, a rhabdovirus, an arenavirus, a flavivirus, a togavirus, a hantavirus, a pneumovirus, or an ebolavirus.

12. The conjugate of claim 11, wherein the virus is an influenza virus.

13. The conjugate of claim 12, wherein the influenza virus is an influenza A virus or an influenza B virus.

14. The conjugate of claim 12 or 13, wherein the protein is an influenza virus neuraminidase, an influenza virus hemagglutinin, or an influenza matrix-2 (M2) proton channel protein.

15. The conjugate of claim 14, wherein the protein is an influenza virus neuraminidase.

16. The conjugate of claim 15, wherein the agent is a small molecule that binds to an influenza virus neuraminidase.

17. The conjugate of claim 16, wherein the agent comprises zanamivir or a pharmaceutically acceptable salt or analog thereof.

18. The conjugate of claim 10, wherein the virus is a Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus (optionally wherein the SARS associated coronavirus is SARS-CoV-1 or SARS- CoV-2), a human immunodeficiency virus (HIV), or a human respiratory syncytial virus (RSV).

19. The conjugate of any one of claims 1-8, wherein the agent binds to a protein on the surface of a cancer cell.

20. The conjugate of claim 19, wherein the cancer cell is a hematological cancer cell, a lung cancer cell, a breast cancer cell, a brain cancer cell, a gastrointestinal cancer cell, a liver cancer cell, a kidney cancer cell, a bladder cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a testicular cancer cell, a prostate cancer cell, an endometrial cancer cell, a muscle cancer cell, a bone cancer cell, a neuroendocrine cancer cell, a connective tissue cancer cell, a head or neck cancer cell, or a skin cancer cell.Attorney Docket No.: CEB-00225 21. The conjugate of claim 19 or 20, wherein the agent binds to a tumor-associated antigen.

22. The conjugate of claim 21, wherein the tumor-associated antigen comprises a MHC class I polypeptide-related sequence A (MICA) protein, a MHC class I polypeptide-related sequence B (MICB) protein, a folate receptor, a fibronectin splice variant, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor receptor 2 (VEGFR-2), C-X-C chemokine receptor type 4 (CXCR4), urokinase plasminogen activator surface receptor (uPAR), follicle- stimulating hormone receptor (FSHR), epithelial cell adhesion molecule (EpCAM), epithelial cadherin (ECAD), carcinoembryonic antigen (CEA), or mesothelin (MSLN).

23. The conjugate of claim 9, wherein the pathogen is a bacterium.

24. The conjugate of claim 23, wherein the bacterium is selected from a Pasteurella species, a Staphylococcus species, a Streptococcus species, a Bacillus species, a Corynebacterium species, a Diphtheroids species, a Listeria species, an Erysipelothrix species, a Clostridium species, a Neisseria species, a Branhamella species, an Escherichia species, an Enterobacter species, a Proteus species, a Pseudomonas species, a Klebsiella species, a Salmonella species, a Shigella species, a Serratia species, an Acinetobacter species, Haemophilus species, a Brucella species, a Yersinia species, a Francisella species, a Pasturella species, a Vibrio species, a Flavobacterium species, a Pseudomonas species, a Campylobacter species, a Bacteroides species, a Fusobacterium species, a Calymmatobacterium species, a Streptobacillus species, or a Legionella species.

25. The conjugate of claim 9, wherein the pathogen is a parasite.

26. The conjugate of claim 25, wherein the parasite is selected from a Plasmodium species, a Trypanosoma species, a Toxoplasma species, a Leishmania species, or a Cryptosporidium species.

27. The conjugate of claim 26, wherein the parasite is from a Plasmodium species.Attorney Docket No.: CEB-00225 28. The conjugate of claim 27, wherein the Plasmodium species is Plasmodium falciparum, Plasmodium malar, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curlisi, or Plasmodium ovale wallikeri.

29. The conjugate of any one of claims 1-28, wherein each agent is covalently conjugated via a separate linker.

30. The conjugate of any one of claims 1-28, wherein at least two of the agents are covalently conjugated via the same linker.

31. The conjugate of any one of claims 1-20, wherein the one or more linkers comprise a cleavable or a non-cleavable linker.

32. The conjugate of claim 31, wherein the one or more linkers comprise an azide moiety, a bicyclononyne moiety, a tetrazine moiety, a trans-cyclooctene moiety, a polypeptide, a polynucleic acid, an alkane, an alkene, an alkyne, a polyether polyol, and / or a polythioether polyol.

33. The conjugate of any one of claims 1-32, wherein the one or more linkers are dibenzoyclooctyne (DBCO) linkers.

34. The conjugate of any one of claims 1-33, wherein the one or more linkers are polyethylene glycol dibenzocyclooctyne (DBCO) linkers.

35. The conjugate of any one of claims 1-31, wherein the one or more linkers comprise:Attorney Docket No.: CEB-00225.

36. The conjugate of any one of claims 1-35, wherein the structure of at least one of the one or more linkers and at least one of the agents comprise:.

37. A pharmaceutical composition comprising the conjugate of any one of claims 1-36.

38. The pharmaceutical composition of claim 37, wherein the composition is formulated for intranasal, intravenous, intraperitoneal, or intramuscular administration.

39. The pharmaceutical composition of claim 38, wherein the composition is formulated for intranasal administration.

40. A nasal spray comprising the pharmaceutical composition of any one of claims 37-39.Attorney Docket No.: CEB-00225 41. A method of making the conjugate of claim 1-36, comprising conjugating the copies of the agent to the VHH or the means for binding an immunoglobulin kappa chain via a linker.

42. The method of claim 41, comprising attaching a conjugation handle to the VHH or the means for binding an immunoglobulin kappa chain.

43. The method of claim 42, wherein the conjugation handle is attached via a sortase A- mediated transpeptidation reaction.

44. The method of claim 42 or 43, wherein the conjugation handle is attached to a glycine on the VHH or the means for binding an immunoglobulin kappa chain.

45. The method of claim 44, wherein the conjugation handle is attached via a maleimide reaction to a cysteine on the VHH or the means for binding an immunoglobulin kappa chain.

46. The method of claim 45, wherein the conjugation handle is attached via a NHS ester to an N-terminal amino acid modification on the VHH or the means for binding an immunoglobulin kappa chain.

47. The method of claim 42, wherein the VHH or the means for binding an immunoglobulin comprises a non-natural amino acid, and the conjugation handle is attached to the non-natural amino acid.

48. The method of claim 47, wherein the non-natural amino acid contains an azide.

49. The method of claim 42, wherein the conjugation handle is attached via an enzymatic reaction.

50. The method of claim 49, wherein the conjugation handle is attached via a transglutaminase, a butelase, or an OaAEP1.

51. A method for enhancing an immune response to a pathogen or cancer in a subject, comprising administering to the subject the pharmaceutical composition of any one of claims 37- 39.Attorney Docket No.: CEB-00225 52. A method of treating or preventing infection from a pathogen in a subject, comprising administering to the subject the pharmaceutical composition of any one of claims 37-39.

53. The method of claim 51 or 52, wherein the pathogen is a virus.

54. The method of claim 53, wherein the virus is an influenza A virus or an influenza B virus.

55. The method of any one of claims 51-55, wherein the pharmaceutical composition is administered intranasally.

56. A conjugate of any one of claims 1-36 for use in enhancing an immune response to a pathogen or cancer in a subject.

57. A conjugate of any one of claims 1-36 for use in treating or preventing infection from a pathogen in a subject.

58. The conjugate for use of claim 56 or 57, wherein the pathogen is a virus.

59. The conjugate for use of claim 58, wherein the virus is an influenza A virus or an influenza B virus.

60. Use of a conjugate of any one of claims 1-36 in the manufacture of a medicament for enhancing an immune response to a pathogen in a subject.

61. Use of a conjugate of any one of claims 1-36 in the manufacture of a medicament for treating or preventing infection from a pathogen in a subject.

62. The use of claim 60 or 61, wherein the pathogen is a virus.

63. The use of claim 62, wherein the virus is an influenza A virus or an influenza B virus.

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