Nanobody-drug adducts and uses thereof

Abstract

Provided herein is a conjugate molecule comprising an antibody or antibody fragment capable of binding to polyclonal immunoglobulin in a subject. Such conjugates are useful for recruiting immune cells of a subject to one or more cell types targeted by the conjugate. Also provided herein is a composition comprising the conjugate, including a pharmaceutical composition that may be administered to a subject for purposes such as treating or preventing disease.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 300,995, filed January 19, 2022; and U.S. Provisional Patent Application No. 63 / 423,667, filed November 8, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Federally funded research This invention was made with Government support under Grant No. AI150593 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] background Fragment crystallizable (Fc) receptors are immunoglobulin receptors located on the cell membrane of many immune cell types, including macrophages, dendritic cells, natural killer cells, neutrophils, basophils, eosinophils, and mast cells. These cells typically have limited ability to directly target antigens, such as those located on the surface of viruses, microbial pathogens, and cancer cells. However, these immune cells can bind to polyclonal antibodies via their surface Fc receptors. These polyclonal antibodies can then bind to antigens located on the surface of cells (including bacteria, parasites, and fungi) or viruses, thereby allowing the immune cells to act on these targets. Summary of the Invention

[0004] overview The immune system encompasses a variety of specialized cell types, many of which are responsible for actively targeting and eliminating viruses, foreign cells, especially pathogenic microorganisms (e.g., bacteria, parasites, and fungi), and cells that are not foreign but have undergone certain harmful phenotypic changes (e.g., damaged cells, virus-infected cells, and transformed cells such as cancer cells). Although immune cells protect the host from this wide range of pathogenic or otherwise dangerous cell types, these immune cells are generally unable to bind directly to their targets. Instead, many specialized immune cells bind to immunoglobulins produced by their host via Fc receptors on their surface, which are then specific for specific antigens located on the surface of target cells or pathogens. This system allows for effective immunization against a wide range of possible targets, but is limited by the amount of immunoglobulin specific for a particular antigen present in the host at any given time. The level of immunoglobulin specific for a given target antigen requires prior exposure to these antigens, for example, by natural infection or by deliberate immunization with that particular antigen.

[0005] Molecules that enhance the interaction between immune cells and potential targets, regardless of the specificity of the host immunoglobulin, would be particularly useful for the treatment and prevention of various diseases. Described herein is one strategy for designing and producing such molecules, which involves conjugating a first agent specific for an immunoglobulin produced by the host to a second agent specific for an antigen on the surface of a cell or pathogen. By selecting a first agent that specifically binds to a structural feature shared by a wide range of host immunoglobulins, such as kappa light chains or lambda light chains, such conjugates can simultaneously bind to a virus or cell surface antigen and any one of a wide range of host immunoglobulins, and then bind to Fc receptor-positive immune cells, such as natural killer (NK) cells, macrophages, or other cells of the myeloid lineage. In this way, these conjugates can be used to recruit Fc receptor-positive immune cells to target cells or pathogens, without relying on the clonality or specificity of the immunoglobulin that links the immune cell to the target. These conjugates may be tailored to target immune cells to any possible antigen and are useful for enhancing or eliciting an immune response against a particular cell or pathogen in a subject.

[0006] Some aspects of the disclosure provide conjugates comprising a first agent that binds to an immunoglobulin and a second agent that binds to a target on the surface of a cell or pathogen, wherein the first agent and the second agent are covalently conjugated via a linker in a chemical reaction.

[0007] In some embodiments, the first agent is an antibody fragment that comprises a variable region capable of binding to an antigen. In some embodiments, the antibody fragment comprises a heavy chain variable region. In some embodiments, the first agent is a single domain antibody fragment. In some embodiments, the immunoglobulin recruited by the conjugate comprises an immunoglobulin kappa light chain or an immunoglobulin lambda light chain. In some embodiments, the immunoglobulin kappa light chain is a human immunoglobulin kappa light chain, and the immunoglobulin lambda light chain is a human immunoglobulin lambda light chain. In some embodiments, the first agent binds to a human immunoglobulin kappa light chain.

[0008] In some embodiments, the second agent comprises a small molecule, a peptide, a protein, a carbohydrate, a lipid, a nucleotide, a nucleic acid, an oligonucleotide, an aptamer, or an antibody.In some embodiments, the second agent is an antibody that is a single domain antibody.In some embodiments, the second agent has a therapeutic effect when administered to a subject.

[0009] In some embodiments, the linker comprises a cleavable or non-cleavable linker. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the cleavable linker is a peptide, disulfide, or hydrazone linker.

[0010] In some embodiments, the cell is a cell infected by a pathogen, a cancer cell, a transformed cell, a healthy cell, a cell that is undergoing or has undergone a phenotypic change in response to a cellular stress, in some embodiments, the pathogen is a virus, a bacterium, a parasite, or a fungus.

[0011] In some embodiments, the pathogen is a virus selected from influenza virus, coronavirus, adenovirus, enterovirus, rotavirus, norovirus, herpesvirus, lentivirus, poxvirus, paramyxovirus, rhabdovirus, arenavirus, flavivirus, togavirus, hantavirus, pneumovirus, or Ebola virus.

[0012] In some embodiments, the influenza virus is an influenza A virus or an influenza B virus. In some embodiments, the second agent binds to influenza virus neuraminidase or influenza virus hemagglutinin. In some embodiments, the second agent comprises a small molecule that binds to influenza virus neuraminidase. In some embodiments, the second agent comprises zanamivir or an analog thereof. In some embodiments, the linker is a triglycine dibenzylcyclooctyne (DBCO) linker. In some embodiments, the second agent comprises an antibody or antibody fragment that binds to influenza virus neuraminidase.

[0013] In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the betacoronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-associated coronavirus (SARS-CoV)-1, or SARS-CoV-2. In some embodiments, the second agent binds to MERS-CoV spike protein, SARS-CoV-1 spike protein, or SARS-CoV-2 spike protein. In some embodiments, the second agent binds to MERS-CoV spike protein receptor binding domain (RBD), SARS-CoV-1 spike protein RBD, or SARS-CoV-2 spike protein RBD.

[0014] In some embodiments, the lentivirus is a human immunodeficiency virus (HIV). In some embodiments, the second agent binds to the HIV envelope glycoprotein gp120.

[0015] In some embodiments, the pneumovirus is a human respiratory syncytial virus (RSV). In some embodiments, the second agent binds to the RSV fusion (F) protein.

[0016] In some embodiments, the pathogen is a bacterium selected from Pasteurella species, Staphylococcus species, Streptococcus species, Bacillus species, Corynebacterium species, Diphtheroids species, Listeria species, Erysipelothrix species, Clostridium species, Neisseria species, Branhamella species, Escherichia species, Enterobacter species, Proteus species, Pseudomonas species, Klebsiella species, Salmonella species, Shigella species, Serratia species, Acinetobacter species, Haemophilus species, Brucella species, Yersinia species, Francisella species, Pasturella species, Vibrio cholera species, Flavobacterium species, Pseudomonas species, Campylobacter species, Bacteroides species, Fusobacterium species, Calymmatobacterium species, Streptobacillus species, or Legionella species.

[0017] In some embodiments, the pathogen is a parasite selected from Plasmodium species, Trypanosoma species, Toxoplasma species, Leishmania species, or Cryptosporidium species. In some embodiments, the Plasmodium is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curtisi, or Plasmodium ovale wallikeri. In some embodiments, the second agent binds to a Plasmodium surface protein. In some embodiments, the Plasmodium surface protein is merozoite surface protein 1 (MSP-1). In some embodiments, the second agent is an antibody that binds to MSP-1, and optionally, the antibody is a nanobody. In some embodiments, the antibody comprises the CDR-H1, CDR-H2, and CDR-H3 of any one of the antibodies listed in Table 1. In some embodiments, the antibody comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%%) identical to any one of SEQ ID NOs: 6-17. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 6-17.

[0018] In some embodiments, the cancer cell is a blood 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 and neck cancer cell, or a skin cancer cell. In some embodiments, the second agent binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen comprises MHC class I polypeptide-related sequence A (MICA) protein, MHC class I polypeptide-related sequence B (MICB) protein, folate receptor, 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), CXC 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 some embodiments, the second agent is an antibody that binds to MICA, and optionally, the antibody is a nanobody. In some embodiments, the antibody comprises the CDR-H1, CDR-H2, and CDR-H3 of any one of the antibodies listed in Table 2. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs:19-27.

[0019] In some embodiments, the cells are cancerous or healthy bone marrow cells. In some embodiments, the myeloid-associated antigen is cluster of differentiation 45 (CD45).

[0020] In some embodiments, the cell is a cancerous or healthy immune cell. In some embodiments, the cell is a cancerous or healthy T cell or B cell. In some embodiments, the second agent binds to an immune cell-associated antigen. In some embodiments, the immune cell-associated antigen is cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), T cell receptor (TCR), or B cell receptor (BCR).

[0021] In some embodiments, the conjugate provides a therapeutic effect when administered to a subject. In some embodiments, the conjugate enhances the association between one or more immune cells expressing a fragment crystallizable (Fc) receptor and a cell or pathogen when administered to a subject. In some embodiments, the conjugate provides killing of a cell or pathogen when administered to a subject. In some embodiments, the conjugate provides inactivation of a cell or pathogen when administered to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0022] In another aspect, the present disclosure provides a composition comprising any one of the conjugates described herein. In some embodiments, such a composition further comprises a pharmacologic acceptable excipient.

[0023] In another aspect, the present disclosure provides a method for enhancing immune response against a cell or pathogen in a subject, comprising administering to the subject an effective amount of any one of the conjugates or compositions described herein.In some embodiments, the cell is a pathogen-infected cell, a cancer cell, a transformed cell, a healthy cell, a cell that is undergoing or has undergone phenotypic change in response to cellular stress.In some embodiments, the pathogen is a virus, a bacterium, a parasite, or a fungus.In some embodiments, the cell is a subject's cell.

[0024] In some embodiments, the pathogen is a virus selected from influenza virus, coronavirus, adenovirus, enterovirus, rotavirus, norovirus, herpes virus, lentivirus, poxvirus, paramyxovirus, rhabdovirus, arenavirus, flavivirus, togavirus, hantavirus, pneumovirus, or Ebola virus. In some embodiments, the virus is influenza A virus or influenza B virus. In some embodiments, the virus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-related Coronavirus (SARS-CoV)-1, or SARS-CoV-2. In some embodiments, the virus is human immunodeficiency virus (HIV). In some embodiments, the virus is human respiratory syncytial virus (RSV).

[0025] In some embodiments, the pathogen is a bacterium selected from Pasteurella species, Staphylococcus species, Streptococcus species, Bacillus species, Corynebacterium species, Diphtheroids species, Listeria species, Erysipelothrix species, Clostridium species, Neisseria species, Branhamella species, Escherichia species, Enterobacter species, Proteus species, Pseudomonas species, Klebsiella species, Salmonella species, Shigella species, Serratia species, Acinetobacter species, Haemophilus species, Brucella species, Yersinia species, Francisella species, Pasturella species, Vibrio cholera species, Flavobacterium species, Pseudomonas species, Campylobacter species, Bacteroides species, Fusobacterium species, Calymmatobacterium species, Streptobacillus species, or Legionella species.

[0026] In some embodiments, the pathogen is a parasite selected from Plasmodium species, Trypanosoma species, Toxoplasma species, Leishmania species, or Cryptosporidium species. In some embodiments, the parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curtisi, or Plasmodium ovale wallikeri.

[0027] In some embodiments, the cancer cell is a blood cancer cell, a lung cancer cell, a breast cancer cell, a brain cancer cell, a gastrointestinal cancer cell, a liver cancer cell, a renal 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 and neck cancer cell, or a skin cancer cell.

[0028] In some embodiments, the cells are cancerous or healthy bone marrow cells.

[0029] In some embodiments, the cell is a cancerous or healthy immune cell. In some embodiments, the cell is a cancerous or healthy T cell or B cell.

[0030] In some embodiments, the immune response comprises an innate immune response. In some embodiments, the conjugate binds to the cell or pathogen and the subject's immunoglobulin, which further binds to an immune cell of the subject expressing a fragment crystallizable (Fc) receptor on its surface. 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, the administration induces production of one or more cytokines or chemokines by the immune cell. In some embodiments, the one or more cytokines or chemokines are pro-inflammatory cytokines or chemokines. In some embodiments, the administration induces phagocytosis of the cell or pathogen by the immune cell. In some embodiments, the administration results in killing of the cell or pathogen. In some embodiments, the administration results in inactivation of the cell or pathogen.

[0031] In some embodiments, the subject has a viral infection or is at risk of developing a viral infection. In some embodiments, the subject has cancer or is at risk of developing cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human neonate, a human infant, a human adult, or an elderly human. In some embodiments, the subject is a companion animal, a research animal, or a livestock animal.

[0032] In some embodiments, administration is intravenous, intramuscular, intradermal, subcutaneous, or by inhalation. In some embodiments, administration is performed more than once. In some embodiments, administration is prophylactic.

[0033] In another aspect, the present disclosure provides a method for treating or reducing the risk of disease in a subject in need thereof, comprising administering to the subject an effective amount of any one of the conjugates or compositions described herein. In some embodiments, the disease is a disease caused by a virus, a bacterium, a parasite, a fungus, or cancer.

[0034] In some embodiments, the virus is influenza virus, coronavirus, adenovirus, enterovirus, rotavirus, norovirus, herpes virus, lentivirus, poxvirus, paramyxovirus, rhabdovirus, arenavirus, flavivirus, togavirus, hantavirus, pneumovirus, or Ebola virus. In some embodiments, the virus is influenza A virus or influenza B virus. In some embodiments, the virus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-related Coronavirus (SARS-CoV)-1, or SARS-CoV-2. In some embodiments, the virus is human immunodeficiency virus (HIV). In some embodiments, the virus is human respiratory syncytial virus (RSV).

[0035] In some embodiments, the bacteria are Pasteurella sp., Staphylococcus sp., Streptococcus sp., Bacillus sp., Corynebacterium sp., Diphtheroids sp., Listeria sp., Erysipelothrix sp., Clostridium sp., Neisseria sp., Branhamella sp., Esch erichia species, Enterobacter species, Proteus species, Pseudomonas species, Klebsiella species, Salmonella species, Shigella species, Serratia species, Acinetobacter species, Haemophilus species, Brucella species, Yersinia species, Francisella species, Pasturella species, Vibrio cholera species, Flavobacterium species, Pseudomonas species, Campylobacter species, Bacteroides species, Fusobacterium species, Calymmatobacterium species, Streptobacillus species, or Legionella species.

[0036] In some embodiments, the parasite is a Plasmodium species, Trypanosoma species, Toxoplasma species, Leishmania species, or Cryptosporidium species. In some embodiments, the parasite is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curtisi, or Plasmodium ovale wallikeri.

[0037] In some embodiments, the cancer is blood cancer, lung cancer, breast cancer, brain cancer, gastrointestinal cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, ovarian cancer, testicular cancer, prostate cancer, endometrial cancer, muscle cancer, bone cancer, neuroendocrine cancer, connective tissue cancer, head and neck cancer, or skin cancer. In some embodiments, the cancer is metastatic cancer.

[0038] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human neonate, a human infant, a human adult, or an elderly human. In some embodiments, the subject is a companion animal, a research animal, or a livestock animal.

[0039] In some embodiments, administration is intravenous, intramuscular, intradermal, subcutaneous, or by inhalation. In some embodiments, administration is performed more than once. In some embodiments, administration is prophylactic.

[0040] Another aspect of the disclosure provides an antibody comprising the CDR-H1, CDR-H2 and CDR-H3 of any one of the antibodies listed in Table 1. In some embodiments, the antibody comprises an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%%) identical to any one of SEQ ID NOs: 6-17. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 6-17.

[0041] Another aspect of the disclosure provides an antibody comprising the CDR-H1, CDR-H2 and CDR-H3 of any one of the antibodies listed in Table 2. In some embodiments, the antibody comprises an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%%) identical to any one of SEQ ID NOs: 19-27. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 19-27.

[0042] The above summary is meant to describe, in a non-limiting manner, some of the aspects, advantages, features, and uses of the technology disclosed herein. Other aspects, advantages, features, and uses of the technology disclosed herein will become apparent from the detailed description, drawings, examples, and claims. [Brief description of the drawings]

[0043] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, every component may not be labeled in every drawing. In the drawings: [Figure 1A-1C] Figure 1A: Schematic diagram showing the mechanism by which nanobody-drug adducts enhance immunity against cells and / or pathogens in a subject. The nanobody-drug adducts contain an antibody fragment (e.g., VHH kappa) with sufficient affinity to bind to the kappa light chain of a host immunoglobulin, which then interacts with the Fc rQVQeceptor on the host immune cells. At the same time, the nanobody-drug adducts contain an agent (e.g., zanamivir) that is sufficient to bind to the surface of a target cell and / or pathogen (e.g., a cell infected by a pathogen, e.g., a cell infected by influenza virus), resulting in the inactivation and / or killing of the cell and / or pathogen by the host immune cells. Figure 1B: Schematic diagram of the mode of action of the viral neuraminidase-targeting VHH kappa-zanamivir adduct and the viral hemagglutinin-targeting VHH kappa-SD36 adduct. Conjugation with VHH kappa extends the circulating half-life of zanamivir and SD36, allowing them to kill virus-infected cells by attracting immune effectors. Figure 1C: Structures of VHH kappa-zanamivir and VHH kappa-SD36. VHH kappa-zanamivir is prepared by sortase-mediated conjugation of triglycine-modified zanamivir to VHH kappa. VHH kappa-SD36 is expressed as a genetically fused heterobivalent nanobody with a C-terminal sortase recognition motif (LPETG). [Diagram 2]Figure 2: VHH kappa binds to mouse IgG with nanomolar affinity. The affinity of the VHH kappa-biotin constructs was assessed using mouse IgG2b coated ELISA plates. HRP-conjugated streptavidin was used as secondary reagent for detection. SD36 is a nanobody that recognizes influenza hemagglutinin (HA). Affinities are reported as dissociation constants (Kd). Binding for each construct is shown according to the color code. [Figure 3A-3C] Figures 3A-3C: Schematic diagram showing steps in synthetic nanobody-drug adducts including Gly-Gly-Gly-zanamivir. Figure 3A: Schematic diagram showing synthesis of zanamivir targeting ligand from zanamivir. Figure 3B: Schematic diagram showing synthesis of Gly-Gly-Gly-zanamivir from Gly-Gly-Gly-DBCO and the zanamivir targeting ligand shown in Figure 3A. Figure 3C: Schematic diagram showing tethering of VHH kappa to Gly-Gly-Gly-zanamivir by a sortase reaction (e.g., SrtA) to generate VHH kappa-zanamivir nanobody-drug adduct.

[0044] [Figure 4A-4B] Figures 4A and 4B: Evaluation of VHH kappa-zanamivir nanobody-drug adducts synthesized as shown in Figures 3A-C. Figure 4A: 15% reducing SDS-PAGE shows that VHH kappa-zanamivir is synthesized predominantly as a single species with a mass of approximately 14 kDa. Figure 4B. Mass spectrum of VHH kappa-zanamivir shows the purity of VHH kappa-zanamivir synthesized as shown in Figures 3A-C. [Figure 5A]Figure 5A-D: VHH kappa-zanamivir binds to influenza neuraminidase with nanomolar affinity. Figure 5A: Madin-Darby Canine Kidney (MDCK) cells were infected with influenza A virus A / Wisconsin / 629-D00015 / 2009 (H1N1) to express influenza A neuraminidase within 24 hours post-infection, at which point infected cells were treated with VHH kappa-zanamivir nanobody-drug adducts. The affinity of VHH kappa-zanamivir for cell surface-localized influenza A neuraminidase was assessed by saturation binding assay. Mouse IgG-phycoerythrin (PE) was used as secondary antibody to assess VHH kappa-zanamivir affinity. The nanomolar affinity of VHH kappa-zanamivir for influenza A neuraminidase was determined from the logarithmic regression of the observed binding and is reported as the dissociation constant (Kd). [Figure 5B-5C] Figure 5B: The affinity of VHH kappa-zanamivir for influenza A neuraminidase was assessed as in Figure 5A, but instead MDCK cells were infected with influenza A virus A / Hong Kong / 8 / 1968 (H3N2). The nanomolar affinity of VHH kappa-zanamivir for influenza A neuraminidase was determined from the logarithmic regression of the observed binding and is reported as the dissociation constant (Kd). Figure 5C: Madin-Darby Canine Kidney (MDCK) cells were infected with influenza B virus--B / Florida / 4 / 2006 to express influenza B neuraminidase within 24 h post-infection, at which point infected cells were treated with VHH kappa-zanamivir nanobody-drug adducts. The affinity of VHH kappa-zanamivir for cell surface-localized influenza B neuraminidase was assessed by saturation binding assays. Mouse IgG-phycoerythrin (PE) was used as a secondary antibody to assess VHH kappa-zanamivir affinity. The nanomolar affinity of VHH kappa-zanamivir for influenza B neuraminidase was determined from the logarithmic regression of the observed binding and is reported as the dissociation constant (Kd). [Figure 5D]Figure 5D: The affinity of VHH kappa-zanamivir for influenza B neuraminidase was assessed as in Figure 5A, but instead MDCK cells were infected with influenza B virus-B / Brisbane / 60 / 2008. The nanomolar affinity of VHH kappa-zanamivir for influenza B neuraminidase was determined from the logarithmic regression of the observed binding and is reported as the dissociation constant (Kd).

[0045] [Figure 6A] Figure 6A-6E: A single intraperitoneal injection of VHH kappa-zanamivir protects mice against lethal influenza infection. Figure 6A: Mice were infected with 50 mL of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (10 LD50) at day 0. Mice received intraperitoneal PBS control, a single dose of VHH kappa-zanamivir or its components intraperitoneally at day 0 post-infection, or a dose of VHH kappa-zanamivir intraperitoneally at days 0, 2 and 4 post-infection. The percent change in body weight of infected mice was monitored daily for up to 14 days post-infection. Mice were treated with VHH kappa-zanamivir or its components as indicated according to the color code. [Figure 6B-6C] Figure 6B: Infected mice were treated as in Figure 6A and survival was monitored daily for up to 14 days post-infection. Mice treated with ≥ 1 mg / kg VHH kappa-zanamivir on day 0 post-infection did not show influenza lethality within 14 days post-infection. Mice were treated with VHH kappa-zanamivir or its components as indicated according to the color code. Figure 6C: Infected mice were treated as in Figure 6A and survival was monitored daily for up to 14 days post-infection. Efficacy of VHH kappa-zanamivir against different strains of influenza virus as indicated. [Figure 6D-6E] Figure 6D: Delayed addition of VHH kappa-zanamivir on days 1, 2 or 3 post-infection. Figure 6E: Infection of mice with influenza A / Puerto Rico / 8 / 1934 (H1N1) 7 days after a single dose of VHH kappa-zanamivir. [Figure 7A-7B]Figure 7A-7E: Synthesis of SD36-VH kappa adducts by copper-free click reactions. Figure 7A: Synthesis of SD36-azide, sortase A catalyzes the addition of a triglycine azido-lysine peptide to the C-terminus of SD36. Figure 7B: Synthesis of VHH kappa-DBCO, sortase A catalyzes the addition of a triglycine DBCO functionalized cysteine ​​peptide to the C-terminus of anti-mouse VHH kappa. [Figure 7C-7D] Figure 7C: SD36-azide is conjugated to VHH kappa-DBCO by a copper-free click reaction. Figure 7D: Schematic of gene fusion anti-mouse VHH kappa-SD36. [Figure 7E] Figure 7E: 15% reducing SDS-PAGE shows that VHH kappa-SD36 is mainly synthesized as a single species with a mass of approximately 30 kDa. The mass spectrum of VHH kappa-SD36 shows the purity.

[0046] [Figure 8A-8B] Figures 8A-8C: Synthesis of VHH kappa-biotin, VHH kappa-SD36-biotin and SD36-biotin. Figure 8A: Sortase A catalyzes the addition of a triglycine biotin functionalized cysteine ​​peptide to the C-terminus of an anti-mouse VHH kappa. Figure 8B: Sortase A catalyzes the addition of a triglycine biotin functionalized cysteine ​​peptide to the C-terminus of a genetically fused anti-mouse VHH kappa-SD36 conjugate. [Figure 8C] FIG. 8C: Sortase A catalyzes the addition of a triglycine biotin-functionalized cysteine ​​peptide to the C-terminus of SD36. [Figure 9A-9C]9A-9C: Binding affinity of SD36 and VH-kappa-SD36 to various influenza virus hemagglutinins. FIG. 9A: Saturation binding curve of SD36-biotin to hemagglutinin (HA) expressed on influenza virus-infected MDCK cells. Streptavidin-phycoerythrin (PE) was used to quantify the amount of SD36-biotin bound to HA. FIG. 9B: Saturation binding curve of anti-mouse VHH kappa-SD36-biotin (genetic fusion) to hemagglutinin (HA) expressed on influenza virus-infected MDCK cells. Streptavidin-phycoerythrin (PE) was used to quantify the amount of anti-mouse VHH kappa-SD36-biotin bound to HA. FIG. 9C: Saturation binding curve of anti-mouse VHH kappa-SD36 (conjugation of C-body C by click reaction) to hemagglutinin (HA) expressed on influenza virus-infected MDCK cells. Mouse IgG-phycoerythrin (PE) was used to quantitate the amount of anti-mouse VHH kappa-SD36 bound to HA. Data are presented as mean ± SD (n=3). [Figure 10A-10C] Figure 10A-C: A single intraperitoneal injection of VHH kappa-SD36 protects against lethal influenza virus infection. Mice were infected intranasally with 50 μL of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (=10 LD50). Mice received a single dose of anti-mouse VHH kappa-SD36 gene fusion, i.e., Figure 10A, or anti-mouse VHH kappa-SD36 (C to C conjugation by click reaction, Figure 10B), i.e., Figure 10B, intraperitoneally on the day of infection. Figure 10C, delayed addition of VHH kappa-SD36 at 1, 2 or 3 days post-infection. Mice that lost more than 25% of their initial body weight or became moribund were considered dead for survival curves. Body weight change curves (left) and survival curves (right) are shown for each treatment. Body weight change values ​​(%) are shown as mean ± SD. [Figure 11A-11B]Figure 11A and Figure 11B: Preparation of SD36-DFO (chelated 89Zr) and VHH kappa-SD36-DFO (chelated 89Zr). Figure 11A: Sortase A catalyzes the addition of triglycine desferrioxamine (DFO) peptide to the C-terminus of SD36. Zirconium-89 (89Zr) chelates with DFO at room temperature under pH 7. Figure 11B: Sortase A catalyzes the addition of triglycine desferrioxamine (DFO) peptide to the C-terminus of anti-mouse VHH kappa-SD36 conjugate. Zirconium-89 (89Zr) chelates with DFO at room temperature under pH 7.

[0047] [Figure 12] Figure 12: ImmunoPET imaging of influenza virus infection using VHH kappa-SD36-DFO (89Zr chelated) and SD36-DFO (89Zr chelated). Mice were infected intranasally with 50 μL of influenza virus A / Hong Kong / 8 / 1968 (H3N2) (=10 LD50). Four days after infection, mice were injected retro-orbitally with a single dose of 60 μCi of SD36-DFO (89Zr chelated) or anti-mouse VHH kappa-SD36-DFO (89Zr chelated). Mice were scanned for 10 min with a PET scanner. Images were processed by Vivoquant using the same intensity settings. Images were taken at different time points after injection of the contrast agent. Images taken 48 hours after contrast agent injection were enlarged to confirm detailed virus infection in the mouse chest. [Figure 13A-13D]Figures 13A-D: VHH nanobodies specifically bind to Plasmodium falciparum merozoite surface protein 1 (MSP-1). Figure 13A: Schematic of PfMSP-1 propeptide and four inclusive subunits: p83, p30, p38 and p42. Figure 13B: Purified anti-p84 B4, anti-p38 B8, anti-p42 A6 and anti-p42 G11 VHHs were biotinylated and these VHHs were incubated with plate-bound subunit proteins as indicated. Binding ELISA was detected by using streptavidin-HRP and tetramethylbenzidine (TMB). Data are expressed as optical density (OD). Error bars indicate SEM. Figure 13C: Western blot of VHHs against three PfMSP-1 subunits, full-length p190 propeptide and several 3D7 lysates from 3D7 schizonts at approximately 38-44 hours. FIG. 13D: Flow cytometry of synchronized 3D7 schizonts approximately 38-44 hours old using fluorescently (Cy5)-labeled VHHs. [Figures 14A-14C] Figures 14A-C: Synthesis of VHH kappa-VHH7 adducts by copper-free click reaction. Figure 14A: Synthesis of VHH7-azide, sortase A catalyzes the addition of a triglycine azide-lysine peptide to the C-terminus of VHH7. Figure 14B: Synthesis of VHH kappa-DBCO, sortase A catalyzes the addition of a triglycine DBCO functionalized cysteine ​​peptide to the C-terminus of anti-mouse VHH kappa. Figure 14C: VHH7-azide is conjugated to VHH kappa-DBCO by copper-free click reaction. [Figure 14D]Figure 14D: 6-9 week old female BALB / c mice were infected with 10 LD50 of influenza virus. Mice were treated by intraperitoneal injection with the indicated doses of VHH kappa-SD36, a mixture of VHH kappa and SD36, or an equal volume of PBS. Mice were euthanized when they lost 25% of their body weight or became moribund. Weight loss curves (left) and survival curves (right) are shown. For weight loss curves, % body weight change values ​​represent the mean ± standard deviation. Mean values ​​of % body weight change over 14 days between any two groups were compared using one-way ANOVA analysis with Tukey's multiple comparison test, and statistical differences between the indicated groups and the PBS-treated group are shown (*P<0.05, **P<0.01, ***P<0.001, see all comparisons and P values ​​in Table S1). For survival curves, statistical differences between the indicated groups and the PBS-treated group were calculated by the Log-rank (Mantel-Cox) test (*P<0.05, **P<0.01).

[0048] [Figure 15A-15B] Figures 15A and 15B: Complement dependent cytotoxicity (CDC) of A20 cells induced by VHH kappa-VHH7 adducts. Figure 15A: Schematic showing the experimental procedure of the complement dependent cytotoxicity assay. Figure 15B: Bar graph showing the percentage of cytotoxicity induced by VHHs. Error bars represent standard deviation (n=4). [Figure 16A-16B] Figures 16A and 16B: Antibody-dependent cellular cytotoxicity (ADCC) of A20 cells induced by VHH kappa-VHH7 adducts. Figure 16A: Schematic showing the experimental procedure of the antibody-dependent cellular cytotoxicity assay. Figure 16B: Bar graph showing the percentage of total cell lysis induced by VHHs. Error bars represent standard deviation (n=3). [Figures 17A-17C]Figures 17A-E: Development of MHC class I polypeptide-related sequence A (MICA) specific nanobodies. Figure 17A: Comparison of amino acid sequences of nine MICA specific nanobodies identified and cloned into pHen6 expression vector. Figure 17B: Immunoblot showing specific binding of biotinylated A1 and H3 anti-MICA nanobody clones to purified MICA*009 antigen in whole cell lysates. Figure 17C: Quantification of ELISA cross-competition assay to determine binding epitopes of anti-MICA nanobodies. Reduction of intensity measured at 450 nm compared to single nanobody alone indicates binding to the same epitope. Cross-competition is shown for A1 (left) and H3 (right) anti-MICA nanobody clones. [Fig. 17D-17E] Figure 17D: Quantification of anti-MICA nanobody binding to MICA allele products as determined by ELISA. A significant increase in intensity measured at 450 nm compared to uncoated ELISA control indicates binding. Figure 17E: Flow cytometry of B16F10 cells transfected with empty vector, MICA, or MHC class I polypeptide-related sequence B (MICB) using A1 and H3 anti-MICA nanobody clones.

[0049] [Figure 18A-18B] Figures 18A-C: Affinity of VHH kappa-biotin and VHH kappa-SD36-biotin to mouse immunoglobulins. Figure 18A: Saturation binding curves of VHH kappa-biotin and VHH kappa-SD36-biotin to mouse polyclonal IgG. Figure 18B: Saturation binding curves of VHH kappa-biotin and VHH kappa-SD36-biotin to monoclonal mouse IgM. [Figure 18C] Figure 18C: Saturation binding curves of VHH kappa-biotin and VHH kappa-SD36-biotin to mouse polyclonal IgA coated on ELISA plates. Streptavidin-phycoerythrin (PE) was used to quantitate the amount of VHH bound to mouse immunoglobulin. [Figure 19A-19B]Figures 19A-B: Neuraminidase inhibition assay. The neuraminidase inhibitory activity of VHH kappa-zanamivir, ALB1-zanamivir, zanamivir, and VHH kappa was measured by NA-Star™ Influenza Neuraminidase Inhibitor Resistance Detection Kit. Different influenza strains were used as neuraminidase sources. Figure 19A: Summary of half-maximal inhibitory concentration (IC50) values ​​of tested molecules. Figure 19B: Dose inhibition curves of tested molecules. [Figure 20A-20B] Figures 20A-B: Saturation binding curves of VHH kappa-SD36 to hemagglutinin expressed on influenza virus-infected MDCK cells. Mouse IgG-phycoerythrin (PE) was used to quantitate the amount of VHH kappa-SD36 bound to hemagglutinin. Data represent the mean ± standard deviation (n=3). [Figure 21] Figure 21: Comparison of therapeutic efficacy between VHH kappa-zanamivir, MEDI8852, and VHH kappa-E11. 6-9 week old female BALB / c mice were infected with 10 LD50 of influenza virus. Mice were treated by intraperitoneal injection with the indicated doses of VHH kappa-zanamivir, MEDI8852, or VHH kappa-E11 (SARS CoV-2 spike-specific nanobody). Mice were euthanized when they lost 25% of their body weight or became moribund. Weight loss curves (left) and survival curves (right) are shown. For weight loss curves, % body weight change represents the mean ± standard deviation. Mean values ​​of % body weight change over 14 days between any two groups were compared using one-way ANOVA analysis with Tukey's multiple comparison test. Statistical differences between the indicated groups and the PBS-treated group are indicated (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). For survival curves, statistical differences between the indicated groups and the PBS-treated group were calculated by the Log-rank (Mantel-Cox) test (*P<0.05, **P<0.01).

[0050] [Fig. 22A-22B]Figures 22A-B: Preparation of SD36-DFO and VHH kappa-SD36-DFO for PET imaging. Figure 22A: Nanobody-DFO adducts were prepared by sortase-mediated conjugation of triglycine-modified DFO to the nanobody. Figure 22B: The final products of SD36-DFO (left) and VHH kappa-SD36-DFO (right) were analyzed by SDS-PAGE. [Figures 23A-23C] Figures 23A-E: VHH kappa-zanamivir induces CDC and ADCC. Figure 23A: Influenza virus-infected MDCK cells were killed by VHH kappa-zanamivir in the presence of rabbit complement and mouse polyclonal mouse IgG. The difference in % cytotoxicity between VHH kappa-zanamivir-treated groups and groups treated with individual components as a mixture was analyzed by t-test (data represent the mean ± standard deviation, n=5, *P<0.05, **P<0.01, ***P<0.001). Figure 23B: Virus-infected MDCK cells induced luciferase expression in luciferase-expressing reporter cells upon engagement of mouse FcγRIV receptors in the presence of VHH kappa-zanamivir and mouse polyclonal mouse IgG. Induction of ADCC was calculated by dividing the luminescence intensity of the indicated samples by the mean value of control samples containing virus-infected cells and reporter cells without VHH. The difference in fold induction between the VHH kappa-zanamivir treated group and the group treated with the mixture of the individual components was analyzed by t-test (data represent the mean ± standard deviation, n=5, *P<0.05, **P<0.01, ***P<0.001). Results represent at least two independent experiments for CDC and ADCC assays. Figure 23C: Weight loss curves (left) and survival curves (right) for the comparison of efficacy between VHH kappa-zanamivir and ALB1-zanamivir. [Fig. 23D-23E]FIG. 23D: Measurement of the clearance rate of nanobodies after post-orbital injection of 89Zr-labeled constructs. Each individual measurement of 89Zr decay rate as counts per minute (CPM) from 10 μL of whole blood (y-axis) is shown as blue squares (VHH kappa-DFO-89Zr, n=3), red circles (ALB1-DFO-89Zr, n=4) or black triangles (SD36-DFO-89Zr, n=3) for each blood collection time point after the first injection of the construct (x-axis: 10 min, 1 h, 24 h, 48 h, 72 h, 96 h, and 144 h). All mice received an initial dose of 250 μCi of 89Zr-labeled VHH (equivalent to 1 mg / kg VHH). Each data point represents the mean ± standard deviation. The half-life (fast and slow phases) of each VHH was estimated using a biphasic decay model. Total VHH exposure over the first 144 hours post-injection was calculated by integrating the VHH concentration in the blood over time. This is expressed as the "area under the curve" (AUC). Figure 23E: Weight loss curves (left) and survival curves (right) of virus-infected Rag1 knockout mice receiving anti-mouse VHH kappa-zanamivir + mouse polyclonal IgG. For panels (C) and (E), the % body weight change represents the mean ± standard deviation in panel (C), while the weight change curves of individual mice are shown in panel (E). The mean values ​​of % body weight change over 14 days between any two groups were compared using one-way ANOVA analysis with Tukey's multiple comparison test. Statistical differences between the indicated groups and the PBS-treated group are indicated (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). For survival curves, statistical differences between the indicated groups and the PBS-treated group were calculated by the Log-rank (Mantel-Cox) test (*P<0.05, **P<0.01).

[0051] [Figure 24A]Figure 24A and Figure 24B: VHH kappa-SD36 induces ADCC but not CDC. Figure 24A: Virus-infected MDCK cells induced luciferase expression in luciferase-expressing reporter cells upon engagement of mouse FcγRIV receptors in the presence of VHH kappa-SD36 and mouse polyclonal mouse IgG. Fold induction was calculated by dividing the luminescence intensity of the indicated samples by the mean value of control samples containing virus-infected cells and reporter cells without VHH. The difference in fold induction between VHH kappa-SD36-treated groups and groups treated with mixtures of individual components was analyzed by t-test (data represent the mean ± standard deviation, n=5, *P<0.05, **P<0.01, ***P<0.001). [Figure 24B] Figure 24B: Influenza virus-infected MDCK cells were not significantly killed by VHH kappa-SD36 in the presence of rabbit complement and polyclonal mouse IgG. The difference in % cytotoxicity between VHH kappa-SD36-treated groups and groups treated with the individual components as a mixture was analyzed by t-test (data represent the mean ± standard deviation, n=5, *P<0.05, **P<0.01, ***P<0.001). Results represent at least two independent experiments for ADCC and CDC assays. [Fig. 25A-25B] Figure 25A and Figure 25B: Preparation of ALB1-zanamivir. Figure 25A: Amino acid sequence of anti-serum albumin nanobody (ALB1). A sortase recognition motif (LPETG) is attached to the C-terminus of the nanobody. Figure 25B: ALB1-zanamivir was prepared by sortase-mediated conjugation of triglycine-modified zanamivir to ALB1. The identity of the final product, ALB1-zanamivir, was confirmed by SDS-PAGE (left) and mass spectrometry (right). [Figure 26]Figure 26: Preparation of VHH kappa-DFO, ALB1-DFO, and SD36-DFO for PET imaging. Nanobody-DFO adducts were prepared by sortase-mediated conjugation of triglycine-modified DFO to nanobodies. Nanobody-DFO adducts were analyzed by SDS-PAGE (for each gel, from left to right: 1: sortase, 2: unconjugated nanobody, 3: reaction mixture, 4-9: different fractions obtained after PD-10 column elution, the nanobody-DFO adduct shown as #6 on the gel was used for PET imaging). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Some aspects of the present disclosure are based at least in part on the discovery that conjugate molecules that specifically bind both antigens located on the surface of a cell or pathogen and structural features shared by many distinct immunoglobulins produced in a host, such as kappa or lambda light chain sequences, may be used to recruit immune cells with cell surface Fc receptors to cells or pathogens expressing the antigen, regardless of the clonality or specificity of the host immunoglobulin. As described herein, these conjugates may be used to enhance or induce an immune response to a unique virus or cell in a subject. This approach is useful for treating or reducing the risk of a disease associated with a target virus or cell in a subject. In some embodiments, the conjugates described herein are useful for treating (both prophylactically and therapeutically) one or more diseases in a subject, including infections and cancers caused by pathogens (e.g., viruses, bacteria, parasites, fungi). As further described, the disclosed conjugates are also useful for eliminating a unique type of cell in a subject, regardless of whether the cell is associated with a disease.

[0053] Conjugates Without wishing to be bound by theory, during an innate (cell-mediated) immune response, various immune cell types directly target potential pathogens or other harmful cells and kill or inactivate these cells (i.e., prevent reproduction). In this way, immune cells such as macrophages, dendritic cells, natural killer cells, neutrophils, basophils, eosinophils, and mast cells contain and destroy pathogenic infections and cancers occurring in their hosts. However, these immune cells are generally unable to directly interact with their targets because they lack receptors on their surface to do so. Instead, many immune cells express receptor proteins on their surface called fragment crystallizable (Fc) receptors. Fc receptors can bind to the subject's host immunoglobulins, each specific for a particular antigen. When an immunoglobulin bound to an Fc receptor binds to its target antigen, such as an antigen present on the surface of a pathogen or other target cell, it brings the target into sufficient proximity to the immune cell to result in the killing or inactivation of the pathogen or other target cell.

[0054] Although this system allows protection against a wide range of possible targets, the effectiveness of many immune cells is largely based on the amount of immunoglobulin in a subject that is specific for a particular antigen. In other words, if the amount of immunoglobulin specific for an antigen on the surface of a pathogen or cancer cell is insufficient, certain immune cell types are limited in their ability to mount an effective response. Fortunately, immune responses may be enhanced or induced in a subject by providing a molecule that can enhance the proximity between Fc receptor-positive immune cells and their targets. Provided herein are such molecules that are conjugates between a first agent that is specific for immunoglobulin produced by a host and a second agent that is specific for an antigen on the surface of a cell or pathogen. By selecting a first agent that specifically binds to a structural feature shared by many host immunoglobulins, such as kappa light chain or lambda light chain, these conjugates can simultaneously bind to the target and the immunoglobulin bound by Fc receptor-positive immune cells. By effectively cross-linking immune cells with their targets, these conjugates allow immune enhancement against virtually any potential target, without relying on the specificity of the host immunoglobulin, simply by customizing the targeting specificity of the second agent.

[0055] Thus, some aspects of the present disclosure describe a conjugate comprising a first agent that binds to immunoglobulin and a second agent that binds to a target on the surface of a cell or pathogen, wherein the first agent and the second agent are covalently conjugated via a linker. In some embodiments, the first agent specifically binds to immunoglobulin. In some embodiments, the second agent specifically binds to a target on the surface of a cell or pathogen.

[0056] In some embodiments, the first agent is an antibody or an antibody fragment thereof (for example, but not limited to, a recombinant antibody fragment such as a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a single domain antibody (sdAb)). In some embodiments, the first agent is a single domain antibody, alternatively referred to in the art as a "nanobody". In some embodiments, the first agent is a single domain antibody that is a heavy chain antibody, i.e., a single domain antibody fragment derived from an immunoglobulin containing only a heavy chain, as typically found in mammalian species belonging to the family Camelidae. Such fragments are referred to in the art as "VHH" and are recombinantly expressed as "nanobodies". In some embodiments, the first agent is a recombinant single domain antibody, such as a recombinant VHH or nanobody.

[0057] The first agent of the conjugate described herein binds to an immunoglobulin. "Immunoglobulin" refers to an antibody protein complex produced and secreted by lymphocytes or plasma cells. Immunoglobulins typically include one or more heavy chains and one or more light chains covalently linked to each other by disulfide bonds. The heavy and light chains of an immunoglobulin may each include a variable region (e.g., heavy chain variable region and light chain variable region) that varies between immunoglobulin clones and includes amino acid sequences that determine which antigen the immunoglobulin binds to, and a constant region (e.g., heavy chain constant region and light chain constant region) that is constant across antibody clones. An immunoglobulin may belong to any one of several structural classes (isotypes) according to its overall mass and number of antigen-binding sites. For example, an immunoglobulin may be immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), or immunoglobulin M (IgM). Immunoglobulins may further belong to specific structural subclasses, such as, for example, IgG subclass 1 (IgG1), IgG subclass 2 (IgG2), IgG subclass 3 (IgG3), and IgG subclass 4 (IgG4). The immunoglobulin classes and subclasses determine many of the functional properties of an immunoglobulin, including, but not limited to, its biodistribution. In some embodiments, the immunoglobulins described herein comprise a heavy chain that includes a fragment crystallizable (Fc) region. The Fc region is contained in the heavy chain constant region and is necessary for binding to Fc receptors, such as Fc receptors on the surface of immune cells (e.g., macrophages, dendritic cells, natural killer cells, neutrophils, basophils, eosinophils, mast cells). All circulating immunoglobulins contain an Fc region.

[0058] The present disclosure particularly relates to immunoglobulins comprising a light chain that is a kappa (κ) light chain or a lambda (λ) light chain. Kappa light chains and lambda light chains are expressed from genes in different loci located on different chromosomes (e.g., in humans, chromosome 2 for IGK and chromosome 22 for IGL), called IGK (NCBI gene ID: 50802) and IGL (NCBI gene ID: 3535), respectively. In some embodiments, the immunoglobulins of the present disclosure comprise a kappa light chain expressed from the IGK locus. In some embodiments, the immunoglobulins of the present disclosure comprise a lambda light chain expressed from the IGL locus.

[0059] In some embodiments, the first agent binds to an immunoglobulin kappa light chain or an immunoglobulin lambda light chain. In some embodiments, the first agent is a nanobody or single domain antibody (e.g., a VHH) that binds to an immunoglobulin kappa light chain or an immunoglobulin lambda light chain. In some embodiments, the first agent is a nanobody or single domain antibody (e.g., a VHH) that binds to an immunoglobulin kappa light chain or an immunoglobulin lambda light chain of a particular species, such as human. In some embodiments, the first agent is a nanobody or single domain antibody (e.g., a VHH) that specifically binds to an immunoglobulin kappa light chain, such as a human immunoglobulin kappa light chain. As used herein, a nanobody or single domain antibody that is a VHH and is specific for a kappa light chain is referred to as a "VHH". カッパ Exemplary VHHs are referred to as カッパ The amino acid sequence is as follows: Anti-mouse IgG kappa light chain VHH: QVQLVESGGGWVQPGGSLRLSCAASGFTFSDTAMMWVRQAPGKGREWVAAIDTGGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTARYYCAKTYSGNYYSNYTVANYGTTGRGTLVTVSSGG (SEQ ID NO: 1) Anti-human IgG kappa light chain VHH: QVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPENTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSGG (SEQ ID NO: 2)

[0060] In some embodiments, the second agent comprises a small molecule, a peptide, a protein, a carbohydrate, a lipid, a nucleotide, a nucleic acid, an oligonucleotide, an aptamer, or an antibody or an antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a nanobody or single domain antibody (sdAb)). In some embodiments, the second agent is a small molecule, a peptide, a protein, a carbohydrate, a lipid, a nucleotide, a nucleic acid, an oligonucleotide, an aptamer, or an antibody or an antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a nanobody or single domain antibody (sdAb)). In some embodiments, the second agent is a small molecule (e.g., a small molecule inhibitor). In some embodiments, the second agent is an antibody or an antibody fragment thereof. In some embodiments, the second agent is a single domain antibody. In some embodiments, the second agent has a therapeutic effect when administered to a subject (e.g., when the second agent is administered alone to a subject). In some embodiments, the second agent has no therapeutic effect when administered to a subject (e.g., when the second agent is administered alone to a subject). In some embodiments, the second agent specifically binds to a target on the surface of a particular species 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 a range of related species (e.g., where the species express a substantially similar version of the target on their surface).

[0061] In some embodiments, the first agent and the second agent are covalently linked via a linker. In some embodiments, the linker comprises a cleavable or non-cleavable linker. A "cleavable linker" refers to a linker in which one or more covalent bonds are cleaved (destroyed) under certain conditions, such as conditions occurring in a cell or a subject. A "non-cleavable" linker refers to a linker that, for all intents and purposes, cannot be efficiently or reliably cleaved under certain conditions, such as conditions occurring in a cell or a subject. Examples of cleavable linkers well known to those skilled in the 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-1-carboxylate (SMCC) and polyethylene glycol (PEG). Further examples of linkers known in the art include, for example, those described in Lu, et al. "Linkers Having a Crucial Role in Antibody-Drug Conjugates" Int J Mol Sci, 17(4), 561., the contents of which are incorporated herein by reference.

[0062] In some embodiments, the linker is a triglycine dibenzylcyclooctyne (DBCO) linker, which is as follows: [ka]

[0063] In some embodiments, the second agent is a protein or peptide (e.g., an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a nanobody or single domain antibody (sdAb)) 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 first agent and the second agent, such as a repeated linker comprising glycine and / or serine, or another amino acid linker commonly known in the art. In some embodiments, the first agent and the second agent are translated separately (e.g., in an in vitro translation system or recombinantly expressed in a cell) and linked post-translationally. In some embodiments, the first agent and the second agent are translated as a fusion protein (e.g., in In some embodiments, the conjugate is recombinantly produced by inserting and expressing one or more nucleic acids (e.g., DNA or RNA) encoding the first and second agents into a prokaryotic cell (e.g., bacterial) or eukaryotic cell (e.g., fungal or mammalian), followed by isolating the conjugate using one or more techniques commonly known in the art, such as, for example, affinity chromatography or size and / or size exclusion chromatography. In some embodiments, the first and second agents are encoded by different nucleic acids (e.g., DNA or RNA). In some embodiments, the first and second agents are encoded by the same nucleic acid (e.g., DNA or RNA). In some embodiments, the first and second agents are encoded by one or more plasmids or mRNA and inserted (transfected) into the cell by any means known in the art (e.g., electroporation).In some embodiments, the nucleic acids encoding the first and second agents are inserted (transfected) into the cell using a viral vector (e.g., adenoviral vector, lentiviral vector) by any means known in the art. In some embodiments, the nucleic acids encoding the first and second agents are chromosomally inserted into the cell by any means known in the art.

[0064] In some embodiments, the second agent is linked to the first agent by a sortase enzyme (e.g., sortase A). Sortases are a class of enzymes that specifically target the amino acid motif LPXTG (where X is any amino acid) by cleaving the C-terminus of a threonine residue and generating a peptide bond between the threonine and the sortase, which is then transferred to the N-terminus of another protein. In some embodiments, the first agent (e.g., VHH カッパ ) is labeled with a sortase recognition sequence, such as LPETGGHHHHHH (SEQ ID NO: 3), before being linked to the second agent. In some embodiments, the second agent is a protein or peptide (e.g., an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a nanobody or single domain antibody (sdAb)) that is labeled at its C-terminus with a sortase recognition sequence before being linked to the first agent.

[0065] In some embodiments, the second agent specifically binds to a target present on the surface of a particular cell, such as a cell infected by a pathogen (e.g., a virus, a bacterium, a parasite, a fungus), a cancer cell, a transformed cell, a healthy cell, or a cell that is undergoing or has undergone a phenotypic change in response to a cellular stress. A cell that is undergoing or has undergone a phenotypic change in response to a cellular stress may be a cell that is undergoing or has undergone a phenotypic change in response to a cellular stress caused by, but not limited to, oxidative stress, nutritional stress, hypoxia, heat shock, ionizing radiation, exposure to heavy metals, or exposure to mutagens, or physical injury.

[0066] In some embodiments, the second agent specifically binds to the target present on the surface of pathogens such as pathogenic viruses, bacteria, parasites, or fungi.In some embodiments, the pathogen is a virus.In some embodiments, the virus is influenza virus, coronavirus, adenovirus, enterovirus, rotavirus, norovirus, herpesvirus, lentivirus, poxvirus, paramyxovirus, rhabdovirus, arenavirus, flavivirus, togavirus, hantavirus, pneumovirus, or ebolavirus.

[0067] In some embodiments, the virus is an influenza virus. In some embodiments, the virus is an influenza A virus or an influenza B virus. In some embodiments, the target to which the second agent binds is an influenza virus neuraminidase or influenza virus hemagglutinin, such as influenza virus neuraminidase or influenza virus hemagglutinin expressed on the surface of influenza A virus or influenza B virus. In some embodiments, the second agent is a small molecule inhibitor, such as a small molecule inhibitor that binds to influenza virus neuraminidase. In some embodiments, the second agent comprises zanamivir, oseltamivir, peramivir, or an analog thereof. In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to influenza virus neuraminidase or influenza virus hemagglutinin. In some embodiments, the second agent is a VHH that binds to influenza virus hemagglutinin. Examples of VHHs that bind to influenza virus hemagglutinin are: Anti-HA VHH(SD36): EVQLVESGGGLVQAGGSLKLSCAASGRTYAMGWFRQAPGKEREFVAHINALGTRTYYSDSVKGRFTISRDNAKNTEYLEMNNLKPEDTAVYYCTAQGQWRAAPVAVAAEYEFWGQGTQVTVSSGG (SEQ ID NO: 4).

[0068] In some embodiments, the first and second agents are linked by a triglycine dibenzylcyclooctyne (DBCO) linker. In some embodiments, the first and second agents are linked by a triglycine dibenzylcyclooctyne (DBCO) linker and the second agent is zanamivir.

[0069] In some embodiments, the virus is a betacoronavirus. In some embodiments, the betacoronavirus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-associated coronavirus (SARS-CoV)-1, or SARS-CoV-2. In some embodiments, the target to which the second agent binds is MERS-CoV spike protein, SARS-CoV-1 spike protein, or SARS-CoV-2 spike protein. In some embodiments, the target to which the second agent binds is MERS-CoV spike protein receptor binding domain (RBD), SARS-CoV-1 spike protein RBD, or SARS-CoV-2 spike protein RBD. In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen binding (Fab) fragment, a single chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to a target on the surface of MERS-CoV, SARS-CoV-1, or SARS-CoV-2, such as the MERS-CoV spike protein, SARS-CoV-1 spike protein, or SARS-CoV-2 spike protein, or the MERS-CoV spike protein RBD, SARS-CoV-1 spike protein RBD, or SARS-CoV-2 spike protein RBD. In some embodiments, the second agent specifically binds to a target expressed on the surface of SARS-CoV-2. In some embodiments, the second agent specifically binds to a target expressed on the surface of SARS-CoV-2, as first discovered.In some embodiments, the second agent specifically binds to a target expressed on the surface of identified SARS-CoV-2 variants, such as variants of concern (VOCs) identified by the Centers for Disease Control and Prevention (CDC), including but not limited to existing variants B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.427 and B.1.429 (epsilon), B.1.525 (eta), B.1.526 (iota), B.1.617.1 (kappa), B.1.1.529 (omicron), B.1.621 (mu), and P.2 (zeta) variants, as well as variants that have not yet emerged for SARS-CoV-2.

[0070] 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 the HIV envelope glycoprotein gp120. In some embodiments, the second agent specifically binds to a target expressed on the surface of HIV. In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to a target on the surface of HIV, such as the HIV envelope glycoprotein gp120.

[0071] 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 the RSV fusion (F) protein. In some embodiments, the second agent specifically binds to a target expressed on the surface of RSV. In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single-chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to a target on the surface of RSV, such as the RSV F protein.

[0072] In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is a Pasteurella species, a Staphylococcus species, a Streptococcus species, a Bacillus species, a Corynebacterium species, a Diphtheroids species, a Listeria species, a Erysipelothrix species, a Clostridium species, a Neisseria species, a Branhamella species, a Escherichia species, a Enterobacter species, a Proteus species, a Pseudomonas species, a Klebsiella species, a Salmonella species, a Shigella species, a Serratia species, a Acinetobacter species, a 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. In some embodiments, the second agent specifically binds to a target expressed on the surface of the bacteria. In some embodiments, the second agent is an antibody or an antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single-chain variable fragment (scFv), or a single domain antibody (sdAb)) that binds to a target on the surface of the bacteria.

[0073] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a Plasmodium species, a Trypanosoma species, a Toxoplasma species, a Leishmania species, or a Cryptosporidium species. In some embodiments, the Plasmodium species is Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale curtisi, or Plasmodium ovale wallikeri. In some embodiments, the target to which the second agent binds is a Plasmodium surface protein, such as merozoite surface protein 1 (MSP-1). In some embodiments, the second agent specifically binds to a target expressed on the surface of the parasite. In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a nanobody or single domain antibody (sdAb)) that binds to a target on the surface of a parasite, such as a target on the surface of a Plasmodium species. In some embodiments, the target on the surface of a Plasmodium species is a Plasmodium surface protein, e.g., merozoite surface protein 1 (MSP-1). In some embodiments, the second agent is an antibody that binds to MSP-1, and optionally, the antibody is a nanobody. In some embodiments, the antibody comprises the CDR-H1, CDR-H2, and CDR-H3 of any one of the antibodies listed in Table 1. In some embodiments, the antibody comprises an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%%) identical to any one of SEQ ID NOs: 6-17. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 6-17.

[0074] In some embodiments, the second agent specifically binds to the target present on the surface of cancer cell.In some embodiments, the cancer cell is blood cancer cell, lung cancer cell, breast cancer cell, brain cancer cell, gastrointestinal cancer cell, liver cancer cell, kidney cancer cell, bladder cancer cell, pancreatic cancer cell, ovarian cancer cell, testicular cancer cell, prostate cancer cell, endometrial cancer cell, muscle cancer cell, bone cancer cell, neuroendocrine cancer cell, connective tissue cancer cell, head and neck cancer cell, or skin cancer cell.In some embodiments, the cancer cell is human cancer cell. In some embodiments, the target to which the second agent binds is a tumor-associated antigen, such as, but not limited to, MHC class I polypeptide-related sequence A (MICA) protein, MHC class I polypeptide-related sequence B (MICB) protein, folate receptor, 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), CXC 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 some embodiments, the second agent is a small molecule (e.g., a small molecule inhibitor) that binds to a target on the surface of a cancer cell, such as a tumor-associated antigen. In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to a target on the surface of a cancer cell, such as a tumor-associated antigen. In some embodiments, the second agent is an antibody that binds to MICA, and optionally, the antibody is a nanobody. In some embodiments, the antibody comprises the CDR-H1, CDR-H2, and CDR-H3 of any one of the antibodies listed in Table 2. In some embodiments, the antibody comprises an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%%) identical to any one of SEQ ID NOs: 19-27.In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs:19-27.

[0075] In some embodiments, the cells are cancerous or healthy bone marrow cells. In some embodiments, the target to which the second agent binds is a myeloid-associated antigen, such as, but not limited to, cluster of differentiation 45 (CD45). In some embodiments, the second agent is an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single-chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to a target on the surface of a myeloid cell, such as a myeloid-associated antigen.

[0076] In some embodiments, the cell is a cancerous or healthy immune cell, such as, but not limited to, a cancerous or healthy T cell or B cell. In some embodiments, the target to which the second agent binds is an immune cell-associated antigen, such as, but not limited to, cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), T cell receptor (TCR), or B cell receptor (BCR). In some embodiments, the second agent is a target on the surface of an immune cell, such as an antibody or antibody fragment thereof (e.g., a fragment antigen-binding (Fab) fragment, a single chain variable fragment (ScFv), or a single domain antibody (sdAb)) that binds to an immune cell-associated antigen.

[0077] In some embodiments, the conjugates provided herein provide a therapeutic effect when administered to a subject. In some embodiments, the conjugates provided herein enhance the association (proximity) between one or more immune cells (e.g., one or more immune cell types) expressing a fragment crystallizable (Fc) receptor and a cell or pathogen when the conjugates are administered to a subject. In some embodiments, administration of the conjugates provided herein to a subject results in the killing of a cell or pathogen in the subject. In some embodiments, administration of the conjugates provided herein to a subject results in the inactivation of a cell or pathogen in the subject. In some embodiments, the subject to which the conjugates provided herein provide a therapeutic effect is a mammal. In some embodiments, the subject to which the conjugates provided herein provide a therapeutic effect is a human.

[0078] composition In some embodiments, the composition of the present disclosure (e.g., pharmaceutical composition) comprises a conjugate described herein. In some embodiments, the composition of the present disclosure (e.g., pharmaceutical composition) comprises two or more conjugates described herein. In some embodiments, the composition comprising two or more conjugates comprises two or more conjugates specific for the same antigen. In some embodiments, the 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.

[0079] In some embodiments, the composition may comprise one or more conjugates described herein and one or more pharmacologically acceptable excipients. The pharmacologically acceptable excipients may enhance the stability of the conjugates described herein, enhance the delivery of the conjugates to cells (e.g., immune cells) of the subject to which the composition is administered, allow sustained or delayed release of the conjugates upon administration, alter the biodistribution of the conjugates (e.g., target the conjugates to a specific tissue or cell type), or reduce host immunity to the conjugates. Examples of pharmacologically acceptable excipients include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, and preservatives, as known in the art. In some embodiments, the pharmacologically acceptable excipients include aqueous solutions or buffer solutions. In some embodiments, the composition is isotonic with respect to the biological fluids (i.e., blood) of the subject 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.

[0080] kit Kits (e.g., pharmaceutical packs) are also covered by the present disclosure. The kits provided may include the pharmaceutical compositions or conjugates described herein and containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser packages, or other containers suitable for storage and / or administration). In some embodiments, the kits provided may optionally further include a second container that includes pharmaceutical excipients for diluting or suspending the pharmaceutical compositions or conjugates described herein. In some embodiments, the pharmaceutical compositions or conjugates described herein are provided in a first container and are combined with a second container to form a dosage unit.

[0081] Thus, in one aspect, provided herein is a kit comprising a first container comprising the conjugate or pharmaceutical composition described herein.In certain embodiments, the kit is useful for enhancing or inducing immune response against a specific cell or pathogen in a subject (e.g., pathogenic cell or cells of a subject).In certain embodiments, the kit is useful for treating disease in a subject in need thereof (e.g., disease caused by virus, disease caused by bacteria, disease caused by parasite, disease caused by fungus, cancer).In certain embodiments, the kit is useful for preventing disease in a subject in need thereof (e.g., disease caused by virus, disease caused by bacteria, disease caused by parasite, disease caused by fungus, cancer).

[0082] In certain embodiments, the kits described herein further include instructions for using the pharmaceutical composition or conjugate included in the kit. The kits described herein may also include information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kits and instructions are provided for enhancing or eliciting an immune response against a cell or pathogen in a subject (e.g., a pathogenic cell or cells in a subject). In certain embodiments, the kits and instructions are provided for treating a disease in a subject in need thereof (e.g., a disease caused by a virus, a disease caused by a bacteria, a disease caused by a parasite, a disease caused by a fungus, cancer). In certain embodiments, the kits and instructions are provided for preventing a disease in a subject in need thereof (e.g., a disease caused by a virus, a disease caused by a bacteria, a disease caused by a parasite, a disease caused by a fungus, cancer). The kits described herein may include one or more additional pharmaceutical agents described herein as separate compositions.

[0083] Administration of the conjugate The terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of, a disease described herein (e.g., a disease caused by a virus, a disease caused by a bacteria, a disease caused by a parasite, a disease caused by a fungus, cancer). In some embodiments, treatment may be administered after one or more signs or symptoms of a disease have developed or been observed in a subject. In other embodiments, treatment may be administered in the absence of signs or symptoms of a 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 disease symptoms, in light of a risk of relapse or recurrence of the disease, and / or in light of exposure to a disease-causing pathogen or possible future exposure to a disease-causing pathogen). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent relapse or recurrence. Prophylactic treatment refers to treatment of subjects who do not have the disease and who did not have the disease but are at risk of developing the disease, or subjects who have the disease and are at risk of relapse or regression of the disease. In some embodiments, the subject is at higher risk of developing the disease, or at higher risk of relapse or regression of the disease, than the average healthy member of the population.

[0084] An "effective amount" of a composition described herein refers to an amount sufficient to induce a desired biological response. The effective amount of a composition described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the conjugate described herein, the condition being treated, the mode of administration, and the age and health of the subject. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is an amount sufficient for prophylactic treatment. In some embodiments, the effective amount is the amount of the conjugate described herein administered in a single dose. In some embodiments, the effective amount is the total amount (total) of the conjugate described herein administered in multiple doses. When an effective amount of a composition is referred to herein, the therapeutically and / or prophylactically effective amount is indicated depending on the subject and / or disease being treated. The determination of the effective amount and / or dosage is within the capabilities of one of ordinary skill in the art.

[0085] The terms "administer", "administering" or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing a composition or conjugate described herein into or on a subject. The composition or conjugate described herein may be administered systemically (e.g., by intravenous injection) or locally (e.g., by local injection). In some embodiments, the composition or conjugate described herein is administered orally, intravenously, topically, intranasally, or sublingually. Parenteral administration is also contemplated. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intradermal, and intracranial injection or infusion techniques. In some embodiments, administering is performed intramuscularly, intradermal, orally, intravenously, topically, intranasally, intravaginally, or sublingually. In some embodiments, the composition or conjugate described herein is administered prophylactically.

[0086] In some embodiments, the compositions or conjugates described herein are administered once or repeatedly (e.g., 2, 3, 4, 5, or more times). In multiple administrations, administration may be 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). In repeated administrations, administration may be 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) or over a variable period of time. In some embodiments, the compositions or conjugates described herein are administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months later, 0 and 6 months later, 0 and 9 months later, 0 and 12 months later, 0 and 18 months later, 0 and 2 years later, 0 and 5 years later, or 0 and 10 years later). In some embodiments, the compositions or conjugates described herein are administered more than twice and are administered until the subject is free of symptoms of the disease (e.g., a disease caused by a virus, a disease caused by a bacteria, a disease caused by a parasite, a disease caused by a fungus, cancer) or is at a reduced risk of developing the disease.

[0087] In some embodiments, the compositions or conjugates described herein are administered to a subject to enhance or elicit an immune response against a cell or pathogen in the subject (e.g., a pathogenic cell or cells of the subject). In some embodiments, the compositions or conjugates described herein are administered to a subject to treat or prevent infection by a pathogen. In some embodiments, the compositions or conjugates described herein are administered to a subject to treat or prevent a viral infection. In some embodiments, the compositions or conjugates described herein are administered to a subject to treat or prevent a bacterial infection. In some embodiments, the compositions or conjugates described herein are administered to a subject to treat or prevent a parasitic infection. In some embodiments, the compositions or conjugates described herein are administered to a subject to treat or prevent a fungal infection. In some embodiments, the compositions or conjugates described herein are administered to a subject to treat or prevent a cancer.

[0088] In some embodiments, administration of a composition or conjugate described herein to a subject enhances or induces an innate (cell-mediated) immune response in the subject. In some embodiments, after administration of a composition or conjugate described herein to a subject, the conjugate binds to a cell or pathogen in the subject and to the subject's immunoglobulin, where the immunoglobulin further binds to an immune cell of the subject that expresses a fragment crystallizable (Fc) receptor on its surface. 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 production of one or more cytokines or chemokines by the immune cell. In some embodiments, administration of the compositions or conjugates described herein induces the production of one or more proinflammatory cytokines or chemokines by immune cells. In some embodiments, administration of the compositions or conjugates described herein induces phagocytosis of cells or pathogens in a subject by immune cells of the subject. In some embodiments, administration of the compositions or conjugates described herein results in the killing of cells or pathogens in a subject. In some embodiments, administration of the compositions or conjugates described herein results in the inactivation of cells or pathogens in a subject (i.e., the cells or pathogens can no longer replicate or reproduce).

[0089] In some embodiments, the subject has or is at risk of developing an infection by a pathogen (e.g., viral infection, bacterial infection, parasitic infection, fungal infection). In some embodiments, the subject has or is at risk of developing cancer, such as, but not limited to, blood cancer, lung cancer, breast cancer, brain cancer, gastrointestinal cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, ovarian cancer, testicular cancer, prostate cancer, endometrial cancer, muscle cancer, bone cancer, neuroendocrine cancer, connective tissue cancer, head and neck cancer, or skin cancer. In some embodiments, the cancer is metastatic cancer.

[0090] As defined herein, "subject" refers to an organism to which administration is intended. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human animal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird, e.g., a chicken, a duck, a goose, or a turkey)). In some embodiments, the subject is a livestock animal (e.g., a cow, a pig, a horse, a sheep, a goat) or a companion animal (i.e., a pet or a service animal, e.g., a cat or a dog). In some embodiments, the subject is a fish, a reptile, or an amphibian. The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic or genetically engineered animal.

[0091] 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 newborn less than 28 days old. In some embodiments, the human infant is less than 1 day old, 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 old at the time of administration.

[0092] In some embodiments, the human subject is over 28 days old (e.g., 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 old, 3 years old, 4 years old, 5 years old, 10 years old, 11 years old, 12 years old, 13 years old, 14 years old, 15 years old, 16 years old, 17 years old). In some embodiments, the human subject is an adult (e.g., over 18 years old). In some embodiments, the human subject is an elderly subject (e.g., over 60 years old). In some embodiments, the human subject is 60, 65, 70, 75, 80, 85, 90, 95, 100 or over 100 years old.

[0093] 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 with frailty syndrome, a malnourished subject, or a subject with a chronic disease that causes frailty). In some embodiments, the human subject has a weakened immune system, such as an underdeveloped (e.g., an infant or neonatal subject), an immunosenescent (e.g., an elderly subject), or a compromised immune system. Immunosenescent subjects include, but are not limited to, subjects that exhibit age-related decline in immune function. Immunocompromised subjects include, but are not limited to, subjects with primary or acquired immune deficiencies, such as subjects suffering from sepsis, HIV infection, and cancer (including those undergoing chemotherapy and / or radiation therapy), and subjects receiving immunosuppressants in connection with 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 with one or more pathogens (e.g., viruses, bacteria, parasites, fungi). In some embodiments, the human subject is one who has or is suspected of having, e.g., chronic lung disease, asthma, cardiovascular disease, cancer, a metabolic disorder (e.g., obesity or diabetes mellitus), chronic kidney disease, or liver disease.

[0094] Some of the aspects, advantages, features, and uses of the technology disclosed herein will be more fully understood from the following examples, which are intended to illustrate some of the benefits of the disclosure and to illustrate certain aspects, but are not intended to exemplify the entire scope of the disclosure and therefore are not intended to limit the scope of the disclosure.

[0095] example Example 1 - Nanobody-drug adducts for the treatment of influenza Influenza is an acute and potentially life-threatening respiratory infection caused by influenza viruses. Influenza in humans can be caused by influenza A and influenza B viruses, which typically spread during seasonal influenza epidemics. The human cost of annual influenza epidemics is high, resulting in an estimated 3-5 million severe influenza cases each year and 250,000-500,000 deaths per year, including approximately 12,000-79,000 deaths per year in the United States. Influenza viruses also pose a significant health risk to human society due to their high transmissibility and the potential for transmission of new influenza variants from animal reservoirs (e.g., non-human mammalian or avian species), including many migratory bird species, to humans. These factors make influenza viruses particularly likely to cause pandemics. In fact, at least four separate influenza pandemics have occurred in the last century (e.g., 1918-1920, 1957-1958, 1968-1969, and 2009-2010), collectively causing tens of millions of deaths.

[0096] Fortunately, several therapeutics have been clinically approved for use in treating influenza infections. These include several antiviral drugs that specifically target molecular features on the surface of influenza virions and infected cells, such as zanamivir, oseltamivir, and peramivir, which specifically bind to and inhibit the neuraminidase enzyme encoded by influenza A and influenza B viruses, thereby preventing the release of new virions from infected cells. Timely administration of these therapeutics may shorten the duration of influenza infection by about 1-2 days, although it is unclear whether administration actually reduces the risk of developing severe disease (e.g., illness requiring hospitalization). Furthermore, it is unclear whether these therapeutics effectively prevent infection when administered prophylactically, and therefore prophylactic use of these therapeutics is not recommended due to the risk of adverse drug reactions.

[0097] One approach to improve the efficacy of influenza treatment is to fuse a therapeutic agent specific for influenza to a separate agent capable of binding to polyclonal immunoglobulins produced by the host. The bound immunoglobulins can then recruit immune cells expressing Fc receptors on their surface, which then kill or inactivate the virions or infected cells to which they are recruited. This strategy can be used to fuse existing therapeutic agents specific for influenza, such as zanamivir, to a kappa light chain (VHH カッパ This may be achieved by fusing the VHH to an antibody or antibody fragment that binds broadly to host immunoglobulins, such as the variable region of the heavy chain of a camelid antibody specific for influenza VHH (Figure 1). Since any host immunoglobulin containing a kappa light chain and Fc region can be used and immune cells can be recruited to influenza virions or infected cells regardless of the host immunoglobulin specificity, this strategy could potentially be used to stimulate a stronger immune response to influenza than could be achieved by other methods. カッパZanamivir fused to VHH may retain its inhibitory activity, but in principle any agent specific for one or more targets on the surface of influenza virions and / or influenza-infected cells may be used, including, for example, antibodies or antibody fragments specific for viral neuraminidase or hemagglutinin. カッパ Fusion to may also extend the circulating half-life of zanamivir or another therapeutic agent specific for influenza compared to the free drug alone, thus further enhancing the efficacy of the therapeutic agent.

[0098] To initially evaluate the efficacy of this strategy, we used VHHs against immunoglobulins. カッパ The specificity of was tested in vitro using various concentrations of VHH fused to biotin. カッパ (VHH カッパ -biotin) was exposed to mouse IgG2b-coated enzyme-linked immunosorbent assay (ELISA) plates. For this assay, a commercially available anti-mouse kappa chain VHH was used (nanobody clone TP1170). Subsequently, horseradish peroxidase fused to streptavidin (HRP-streptavidin) was used as a secondary agent to bind the VHH カッパ As a control, a separate nanobody SD36 specific for influenza hemagglutinin, but not kappa light chain, was fused to biotin and tested (SD36-biotin), as was the binding between SD36-biotin and the VHH カッパ Fusion with (VHH カッパ -SD36-biotin-1 and VHH カッパ -SD36-Biotin-2) was also tested. After 2 hours of incubation at ambient temperature, the ELISA plates were washed and then incubated with streptavidin-HRP, followed by washing, treatment with TMB substrate solution (Biolegend, Cat. No. 421101), quenched with H2SO4, and measurement. From the plot of the average absorbance values ​​at 450 nm against the concentration of VHH, the dissociation constants (K d ) was calculated. カッパ-Biotin, VHH カッパ -SD36-biotin-1 and VHH カッパ -SD36-biotin-2 binds mouse IgG2b with low nanomolar affinity (dissociation constants K of 2.0–2.4 nM), respectively. d ) (Figure 2). In contrast, no binding was detected between SD36-biotin and IgG2b. These results indicate that VHHs against host immunoglobulins カッパ We confirmed the high specificity of VHHs to influenza-specific drugs. カッパ These results suggest that fusion of the IgG1A-specific ...

[0099] Next, VHH カッパ A method was devised to covalently link VHH to an influenza-specific drug, e.g., zanamivir. Although in principle any cleavable or non-cleavable linker could be used, a method was developed to modify zanamivir by attaching a triglycine dibenzylcyclooctyne (DBCO) linker to the 7-hydroxyl group of zanamivir. First, a zanamivir targeting ligand was prepared (Figure 3A), which was then reacted with Gly-Gly-Gly-DBCO to generate Gly-Gly-Gly-zanamivir (Figure 3B). The VHH was then reacted with Gly-Gly-Gly-DBCO to generate Gly-Gly-Gly-zanamivir (Figure 3B). カッパ To generate the -zanamivir adduct, a sortase (sortase A; SrtA) reaction was used to convert Gly-Gly-Gly-zanamivir to a VHH カッパ Briefly, pentamutant sortase A was used and the VHH カッパVHHs synthesized by this method were purified using a PD-10 desalting column to catalyze the addition of a sortase-ready nucleophile to the C-terminal LPETG motif of VHHs. The sortase reaction was carried out in PBS containing 20 μM sortase A and 10 μM CaCl2. After overnight incubation at 4 °C, Ni-NTA beads were added to the reaction mixture and incubated at 4 °C for 30 min to remove unreacted VHHs and His-tagged sortase A. The reaction mixture was then loaded onto a PD-10 desalting column to remove excess nucleophile. ... カッパ The -zanamivir adduct was subsequently determined to be approximately 14 kDa in size and substantially pure by SDS-PAGE and mass spectrometry (Figures 4A and 4B).

[0100] Then, VHH against influenza-infected cells カッパ The specificity of the -zanamivir adduct was evaluated in vitro using influenza-infected cell cultures. Madin-Darby Canine Kidney (MDCK) cells were infected with influenza virus and neuraminidase was expressed 24 hours after infection, at which point infected MDCK cells were treated with various concentrations of VHH カッパ -Treated with zanamivir adduct. VHH カッパ The binding between -zanamivir adducts and infected cells was measured by saturation binding assay. Briefly, medium was removed from infected cells and replaced with medium containing various concentrations of the adduct. After 1 h of incubation, infected cells were washed and treated with mouse IgG-phycoerythrin (R&D Systems #IC002P) in fresh serum-free medium. After 30 min of incubation, infected cells were washed, lysed in 1% aqueous sodium dodecyl sulfate (SDS) and cell-associated fluorescence was measured using an excitation wavelength of 560 nm and an emission wavelength of 620 nm. The dissociation constant (K d ) was calculated. カッパ Similar to the specificity of VHH (Figure 2), カッパThe -zanamivir adduct showed low nanomolar specificity against influenza A virus (Figure 5A and Figure 5B). Furthermore, the VHH カッパ The -zanamivir adduct showed low nanomolar specificity against MDCK cells infected with two separate strains of influenza A, A / Wisconsin / 629-D00015 / 2009, an H1N1 influenza subtype (Figure 5A) and A / Hong Kong / 8 / 1968, an H3N2 influenza subtype (Figure 5B). カッパ The -zanamivir adduct showed low nanomolar specificity against MDCK cells infected with two separate strains of influenza B, B / Florida / 4 / 2006 (Figure 5C) and B / Brisbane / 60 / 2008 (Figure 5D). These results support the conclusion that the VHH カッパ It is confirmed that the -zanamivir adduct is as specific for influenza neuraminidase as free zanamivir and should be effective in treating a range of influenza subtypes.

[0101] In vitro VHH カッパ After establishing the efficacy of the zanamivir adduct, VHH カッパ -Zanamivir adducts were evaluated in animal models. Mice were infected with 50 μL of influenza A virus A / Puerto Rico / 8 / 1934, subtype H1N1, on day 0. 50 μL of A / Puerto Rico / 8 / 1934 influenza A virus was shown to have a LD of A / Puerto Rico / 8 / 1934 influenza A. 50 Mice were then transfected with various amounts of VHH カッパ -zanamivir adduct (0.1, 1 mg / kg, or 3 mg / kg), 1 mg / kg of non-covalently fused VH カッパ and zanamivir, or mock treatment with phosphate-buffered saline (PBS), were administered intraperitoneally. カッパ Mice that received the -zanamivir adduct were treated with VHH either on day 0 only or on days 0, 2, and 4 post-infection. カッパ -Received zanamivir adduct. VHH カッパAfter administration of the 0.1 mg / kg VH-zanamivir adduct, the body weight (mass) and survival rate of infected mice were monitored once a day for 14 days (Figure 6A and Figure 6B). カッパ Mice treated with the -zanamivir adduct showed substantial weight loss and complete mortality by days 8 and 10 post-infection, respectively. Importantly, 1 mg / kg of VHH カッパ Mice treated with 1 or 3 mg / kg VHH and zanamivir (separately) also showed weight loss and mortality by day 9 post-infection. カッパ Mice treated with the -zanamivir adduct did not exhibit weight loss or mortality as a result of influenza infection. In fact, VHH at 3 mg / kg on days 0, 2, and 4 post-infection カッパ -Zanamivir adduct or 1mg / kg VHH カッパ -zanamivir adduct-treated mice gained a small amount of weight over the 14 days post-infection.

[0102] To establish this efficacy across multiple virus species, mice were infected with influenza AA / California / 07 / 2009 (H1N1); influenza AA / Hong Kong / 1 / 1968; or influenza BB / Florida / 4 / 2006 in the same manner; and administered a single dose of VHH on the same day. カッパ -Treated with zanamivir (3 mg / kg) (Figure 6C). カッパ -Zanamivir has been shown to result in high survival rates for all infected mice for up to 14 days.

[0103] By days 1, 2 or 3 post-infection with influenza AA / Puerto Rico / 8 / 1934 (Figure 6D), VHH カッパ Further experiments were performed to delay the addition of zanamivir (10 mg / kg). Treated mice showed high survival rates for up to 14 days.

[0104] Finally, a prevention experiment was performed. Mice were immunized with VHH カッパ-zanamivir (5 mg / kg) was administered 7 days before infection with influenza AA / Puerto Rico / 8 / 1934 (Figure 6E). These data support the conclusion that pre-infection VHH カッパ -Trusted Source showed that administration of zanamivir resulted in a higher survival rate.

[0105] These results suggest that VHH カッパ The present invention shows that administration of a fusion between an IgG4 fusion protein and an influenza-specific drug is highly effective in enhancing immunity to influenza and reducing the risk of severe disease.

[0106] These results were obtained in a mouse model, but using mouse-specific VHHs. カッパ VHH specific for human kappa light chain カッパ VHHs effective for treating influenza in humans simply by replacing カッパ -zanamivir adducts can be obtained. As mentioned above, similar results may be obtained with any adduct containing an agent specific for influenza virions and / or influenza infected cells, such as a small molecule or peptide specific for hemagglutinin, or an antibody or antibody fragment (e.g., nanobody) specific for neuraminidase, hemagglutinin, or another target (e.g., protein) on the surface of influenza virions and / or infected cells. Unless designed for specificity for influenza A or influenza B, VHHs may be used, particularly since zanamivir and other influenza therapeutics are known to be active against both influenza A and influenza B. カッパ An adduct containing is very likely to be effective for treating both types of influenza.

[0107] Example 2 - Nanobody-Nanobody Adducts for the Treatment of Influenza Alternatively, immune cell-specific nanobodies (e.g., VHHs) could be used instead of influenza-specific small molecules such as zanamivir. カッパ) and influenza virus-specific nanobodies. To this end, nanobody VHHs may be developed that are useful for the treatment of influenza. カッパ was conjugated to a previously reported single domain antibody (VHH) specific for influenza virus hemagglutinin, SD36 (see, e.g., Laursen, et al. "Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin." 2018. Science, 362(6414), 598-602). カッパ The -SD36 adduct was first prepared by combining a terminal azide (Figure 7A) and a VHH linked via Gly-Gly-Gly-Cys-DBCO (SEQ ID NO: 28) (Figure 7B), each by a sortase reaction as described in Example 1. カッパ The VHHs were then combined to produce the VHH カッパ The VHH-SD36 adduct was generated (Figure 7C). The final product was purified by size exclusion chromatography using a Superdex 75 10 / 300 column. カッパ A genetic fusion version of the -SD36 adduct is recombinantly expressed and isolated, wherein the VHH カッパ is linked at its N-terminus to SD36 by a flexible linker (GGGGS)3 (SEQ ID NO: 29) (Figure 7D). カッパ , SD36, and genetically conjugated VHHs カッパ A biotinylated version of -SD36 was also generated (Figures 8A-8C). The amino acid sequence of the gene fusion conjugate is as follows: VHH カッパ -SD36 nanobody conjugates: QVQLVESGGGWVQPGGSLRLSCAASGFTFSDTAMMWVRQAPGKGREWVAAIDTGGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTARYYCAKTYSGNYYSNYTVANYGTTGRGTLVTVSSAAAGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLKLSCAASGRTYAMGWFRQAPGKEREFVAHINALGTRTYYSDSVKGRFTISRDNAKNTEYLEMNNLKPEDTAVYYCTAQGQWRAAPVAVAAEYEFWGQGTQVTVSSGGLPETGGHHHHHH (sequence number 5).

[0108] We then assayed the binding of biotinylated SD36, VHHs using a saturation binding assay (Figure 5A and Figure 5B) that was identical to that previously described, except that 10 μg / mL streptavidin-PE (Biolegend #405204) was used to treat cells incubated with SD36-biotin. カッパ -SD36, and genetically conjugated VHHs カッパ The binding between VHH and VHH conjugates was evaluated. As expected, each VHH and VHH conjugate bound to influenza A hemagglutinin with low nanomolar affinity (Figures 9A-C). カッパ -VHH genetically conjugated to SD36 カッパ Both VHHs synthesized by click chemistry and VHH-SD36 bound to influenza A virus-infected MDCK cells. カッパ -SD36 bound with higher affinity (Fig. 9C).

[0109] In vitro VHH カッパ After establishing the efficacy of the -SD36 conjugate, each conjugate was then evaluated in an animal model as before (Figure 6A and Figure 6B). カッパ -SD36 (Figure 6A) or VHH conjugated by click chemistry カッパMice treated with either -SD36 (Figure 6B) were fully protected from a lethal dose of influenza A virus, whereas mice treated with 10 mg / kg of either conjugate showed no further weight loss as a result of infection (Figures 10A and 10B).

[0110] By days 1, 2 or 3 post-infection with influenza AA / Hong Kong / 1 / 1968 (Figure 10C), VHH カッパ Further experiments were performed to delay the addition of -SD36 (10 mg / kg). Treated mice showed high survival rates for up to 14 days.

[0111] From these results, VHH in Example 1 カッパ -VHH targeting influenza antigens, similar to zanamivir adducts カッパ Administration of fusions between and nanobodies has been shown to be highly effective in boosting immunity against influenza and reducing the risk of severe disease.

[0112] Finally, VHH カッパ The in vivo half-life of the VHH-SD36 conjugate was evaluated. カッパ -SD36 Zirconium-89( 89 Zr) radiolabeled version (generated by click chemistry) of the VHH カッパ -SD36-DFO or SD36-DFO control was incubated in 2.0 M Na2CO3 at pH 6.8–7.5. 89 Zr 4+ The radiolabeled conjugates were synthesized by treatment with a stock solution for 1 h (Figures 11A and 11B). The location and half-life of the radiolabeled conjugates in mice were then assessed by immunopositron emission tomography (Immuno-PET). Briefly, lethal doses (10 LD 50 Mice infected with influenza virus A / Hong Kong / 8 / 1968 (H3N2) from カッパ - injected with SD36-DFO and scanned every 24 hours, and administered either SD36-DFO or VHHカッパ The location and total levels of -SD36-DFO were determined (Figure 12). カッパ -SD36-DFO was observed to have an extended half-life in vivo compared to SD36 alone and accumulated to a greater extent at sites of infection, particularly in the lungs.

[0113] Together, these results suggest that conjugates involving different modalities of binding to influenza antigens (e.g., small molecules to bind neuraminidase compared to nanobodies to bind hemagglutinin) are effective in treating influenza and preventing severe disease, and therefore, VHHs. カッパ The flexibility of the conjugate is demonstrated.

[0114] Example 3 - Development of Malaria-specific Nanobodies Next, we investigated the specificity of VHH against another pathogen, Plasmodium falciparum. カッパ Adjuncts containing P. falciparum have been developed. P. falciparum is a single-celled protozoan species that is one of five parasites known to cause malaria in humans when transmitted by infected females. The World Health Organization estimates that there were approximately 241 million malaria cases worldwide in 2020, resulting in approximately 627,000 deaths. Cases caused by P. falciparum carry the highest risk of death. Malaria is treatable and curable, especially with the use of artemisinin-based combination therapy (ACT), but drug-resistant malaria has emerged as a global health threat. Additionally, while generally effective, ACTs are somewhat toxic and can cause fatigue, headache, dizziness, nausea, vomiting and abdominal pain.

[0115] Example 1 demonstrates the use of the small molecule zanamivir for influenza virions and influenza-infected cells, where a set of VHH nanobodies specific for different regions of P. falciparum merozoite surface protein 1 (MSP-1) was developed. MSP-1 is a propeptide (designated p190) that contains four subunits, p83, p30, p38 and p42, and is expressed in Plasmodium parasites at the beginning of their asexual reproduction stage (Figure 13A). Upon cleavage, these subunits assemble to form the mature MSP-1 complex on the surface of Plasmodium cells, where it is used to bind and infect red blood cells. The isolated VHH sequences that bind to MSP-1 are shown in Table 1 below.

[0116] [Table 1]

[0117] The purified VHH nanobodies were biotinylated and tested for specificity against isolated MSP-1 subunits. Anti-p83 B4, anti-p38 B8, anti-p42 A6, and anti-p42 G11 VHHs were incubated with plate-bound p83, p38, p42, and p42, respectively, and binding to the plate was detected by enzyme-linked immunoassay (ELISA) using streptavidin-horseradish peroxidase (HRP) and tetramethylbenzidine (TMB). Each VHH was observed to specifically bind to one subunit of the MSP-1 complex, whereas the control VHH did not (Figure 13B). This binding was further confirmed by subsequent gel electrophoresis, which showed that each antibody bound to one subunit of MSP-1 and to the MSP-1 propeptide (p190), but not to any other subunits (Figure 13C). VHH clones B4 and B8 were also tested against lysates from 3D7 schizonts aged approximately 38-44 hours and observed to bind. Finally, purified VHHs were tested against 3D7 schizonts viable for approximately 38-44 hours in a flow cytometry assay. Cy5 fluorescently labeled VHHs were observed to bind specifically to live Plasmodium schizonts, with higher fluorescence intensity than that observed with the control VHH, anti-major histocompatibility complex II (MHC-II) (Figure 13D).

[0118] Together, these data demonstrate the specificity of VHH nanobodies targeting Plasmodium parasites and, as demonstrated in Example 1, in a similar manner as demonstrated for influenza. カッパ and provides proof of concept that adducts comprising any one of these VHHs can be used to effectively treat malaria.

[0119] Example 4 – Nanobody-drug adducts to enhance immunity against cancer cells The efficacy of pathogen-targeting adducts has been demonstrated above (Examples 1 and 2), as well as the development of nanobodies for use in nanobody-nanobody adducts to treat diseases caused by pathogens (Examples 2 and 3). However, VHH カッパ may further be useful for enhancing or eliciting an immune response against cancer. カッパ Adducts were developed containing VHH7, a previously reported VHH targeting mouse major histocompatibility complex II (MHC-II) (see Fang, et al., “Structurally Defined αMHC-II Nanobody-Drug Conjugates: A Therapeutic and Imaging System for B-Cell Lymphoma.” 2016. Angewandte Chemie, 55(7), 2416-2420). MHC-II is typically located on the surface of professional antigen-presenting cells (e.g., dendritic cells), but is also expressed on the surface of lymphoid cells, including certain lymphomas. Thus, adducts containing nanobodies targeting MHC-II could be used to recruit immune cells to lymphoma cells. Similar to those previously prepared to target influenza hemagglutinin (Figures 7A-C), click chemistry was used to conjugate the VHH カッパ -VHH7 adduct was synthesized (Figure 14A-C). The amino acid sequence of VHH7 before conjugation is as follows: VHH7 nanobodies: QVQLQESGGGLVQAGDSLRLSCAASGRTFSRGVMGWFRRAPGKEREFVAIFSGSSWSGRSTYYSDSVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYCAAGYPEAYSAYGRESTYDYWGQGTQVTVSSGG (SEQ ID NO: 18).

[0120] Subsequently, the VHHs were inhibited in a complement-dependent cytotoxicity (CDC) assay against the mouse lymphoma cell line, A20 cells. カッパThe -VHH7 adducts were tested in vitro. Briefly, A20 cells were plated in 96-well white-walled plates and incubated with anti-mouse VHH カッパ -VHH7 adduct or anti-mouse VHH カッパ and a mixture of VHH7 (final concentrations: 10 nM and 100 nM). After 30 min incubation at ambient temperature, fresh serum-free medium containing normal mouse IgG isotype control (Invitrogen #10400C) and 40% (v / v) rabbit complement serum (Sigma-Aldrich #S7764) was added to the cells. After a further 4 h incubation at 37°C, cell viability was measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7572). Maximal killing was measured by treating cells with 5% H2O2. VHH カッパ The percent cytotoxicity induced by the -VHH7 adduct was calculated as follows: Cytotoxicity % = (Luminescence VHHなし -Luminescence VHHあり ) / (Luminescence VHHなし -Luminescence 最大殺滅 ) x 100. VHH カッパ The -VHH7 adduct was found to be effective in inducing cytotoxicity in A20 cells at both 10 nM and 100 nM, whereas treatment with each component separately was ineffective (Figures 15A and 15B).

[0121] Antibody-dependent cellular cytotoxicity (ADCC) assays were used to detect VHHs カッパ The efficacy of the -VHH7 adduct was further demonstrated. Briefly, mouse natural killer (NK) cells were harvested from the spleen of BALB / c mice by EasySep™ Mouse NK Cell Isolation Kit (STEMCELL, #19855RF) for use as effector cells in vitro. A20 cells were plated in a 96-well plate and transfected with anti-mouse VHH カッパ -VHH7 adduct or anti-mouse VHH カッパand VHH7 mixture (final concentrations: 10 nM and 100 nM), followed by mouse IgG2a kappa isotype control antibody (final concentration: 20 μg / mL). After 30 min of incubation, a suspension of mouse NK cells was added to each well at 1 × 10 6 Cells / well were added and incubated for an additional 4 hours at 37° C. Cell viability was measured by CytoTox 96® Non-Radioactive Cytotoxicity Assay (LDH) (Promega, #G1780) measuring absorbance at 490 nm. Spontaneous signals generated by effector cells alone were also evaluated. カッパ The total lysis percentage induced by the -VHH7 adduct was calculated as Total Lysis% = (A490 実験 -A490 エフェクター自発性 ) / (A490 標的最大値 ) × 100. As expected, VHH カッパ Treatment with the -VHH7 adduct, at both 10 nM and 100 nM, lysed approximately 30%-40% of A20 cells within this time frame (Figures 16A and 16B). カッパ The efficacy of the -VHH7 adduct was evaluated using separate VHH カッパ and VHH7. These data support the use of VHHs to effectively target and treat cancer cells by enhancing immune cell responses, in addition to their use to treat pathogenic infections. カッパ It has been demonstrated that additions can also be used.

[0122] VHH カッパ -SD36 adduct, 10 LD 50 The study was further carried out in 6- to 9-week-old female BALB / c mice infected with influenza virus at the indicated doses. カッパ -SD36, VHH カッパMice were treated with a mixture of VHH and SD36 or an equal volume of PBS by intraperitoneal injection. Mice were euthanized when they lost 25% of their body weight or became moribund. Weight loss curves (left) and survival curves (right) are shown (Figure 14D). These data support the conclusion that VHH カッパ The results demonstrate that the -SD36 conjugate is effective in treating infected mice.

[0123] Example 5 - Nanobody-drug adducts to enhance immunity against target pathogens or other cell types The efficacy of the adduct useful for treating influenza and malaria infections is demonstrated in Examples 1-3. Additionally, the efficacy of the adduct for treating cancer is demonstrated in Example 4. However, the VHH カッパ The adducts can be modified in several ways for specificity against different pathogens and / or cell types. For example, VHHs カッパ Alternatively, the VHH may be linked to an agent specific for another target, such as a target (e.g., a protein) located on the surface of another pathogen, such as another virus, bacterium, parasite, or fungus. For example, rather than zanamivir, the VHH カッパ can be linked to a small molecule, peptide, protein, carbohydrate, lipid, nucleotide, nucleic acid, oligonucleotide, aptamer, or antibody (or antibody fragment) that specifically recognizes a target on the surface of a betacoronavirus, e.g., Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus (SARS-CoV)-1, or SARS-CoV-2. For example, the VHH カッパ are linked to an agent that specifically recognizes the MERS-CoV, SARS-CoV-1, or SARS-CoV-2 spike protein, or the spike protein receptor binding domain (RBD), to form VHHs specific for and useful for the treatment of MERS-CoV, SARS-CoV-1, or SARS-CoV-2 infection. カッパ Similarly, VHH may form adducts. カッパcan be linked to an agent specific for the human immunodeficiency virus (HIV) envelope glycoprotein gp120 to produce a VHH specific for and useful in the treatment of HIV infection. カッパ Adducts may be formed. カッパ or linked to an agent specific for the human respiratory syncytial virus (RSV) fusion (F) protein to provide a VHH specific for and useful in the treatment of RSV infection. カッパ Adducts may be formed. VHHs useful for treating parasites such as Plasmodium カッパ The adduct may be, for example, a VHH カッパ may be generated by linking to another agent specific for merozoite surface protein 1 (MSP-1) or to another surface protein of the parasite. Similar strategies may be employed to generate adducts capable of recruiting host immunoglobulins and immune cells to infectious bacteria and fungi.

[0124] Alternatively, related strategies may be used to target host immunoglobulins and immune cells to the subject's own cells. For example, VHHs specific for and useful in the treatment of cancer can be used to target host immunoglobulins and immune cells to the subject's own cells. カッパ The adducts can be VHHs to agents specific for tumor associated antigens such as, but not limited to, folate receptor, fibronectin splice variants, 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), CXC 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). カッパ When administered systemically, such adducts are useful for targeting cancer cells throughout the body and are therefore particularly useful for the treatment of metastatic cancer. Adducts can also be produced by linking a VHH to an agent specific for a target that is disproportionately expressed on the surface of target cells compared to non-target cells.カッパ By linking VHHs to other cell types, including cells that are undergoing or have undergone phenotypic changes such as in response to cellular stress, adducts may be designed to target host immunoglobulins and immune cells to other cell types, including cells that are undergoing or have undergone phenotypic changes such as in response to cellular stress. Additionally, adducts may be designed to target host immunoglobulins and immune cells to healthy cells in addition to diseased cells. For example, adducts specific for bone marrow may be designed to target host immunoglobulins and immune cells to other cell types, including cells that are undergoing or have undergone phenotypic changes such as in response to cellular stress, such as in response to cellular stress. カッパ VHHs may be generated by linking the VHHs to an agent specific for bone marrow cells or their immediate precursors. Such agents may be useful for ablating bone marrow in a subject without the need for potentially harmful radiation or chemotherapeutic agents, as are typically administered prior to bone marrow transplantation. Similarly, VHHs may be linked to agents specific for target cells, such as cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), T cell receptor (TCR), or B cell receptor (BCR). カッパ may be used to generate adducts specific for healthy and / or diseased immune cells. Such adducts may be used, for example, to eliminate immune cells specific for autoantigens (i.e., antigens associated with autoimmunity) or specific for particular allergens.

[0125] Alternatively, or in addition to any of the examples mentioned above, the adducts may also be modified to include alternative antibodies or antibody fragments known in the art (e.g., whole antibodies, Fab fragments or single chain fragment variable (ScFv), etc.). Adducts that are specific for and useful for enhancing immune responses to target pathogens and / or cells also include those that bind agents specific for such pathogens and / or cells, such as VHHs. カッパ Instead of lambda light chain (VHH ラムダ ) to the heavy chain of a camelid antibody specific for VHH ラムダ The adducts are VHHs only in their specificity for host immunoglobulins that contain lambda light chains but not kappa light chains. カッパ Different from the adduct. VHH ラムダThe adducts can be attached to any host immunoglobulin that contains a lambda light chain and an Fc region to recruit immune cells to target pathogens and / or cells, regardless of the specificity of the host immunoglobulin.

[0126] As mentioned above, in principle any linker known in the art can be used to link the VHH カッパ or VHH ラムダ The linker may be a cleavable linker, such as a peptide, disulfide or hydrazone linker, or a VHH, such as one that links the target-specific agent to the C-terminus. カッパ Or VHH ラムダ Formation of the conjugate can be by chemical reaction between the individual components of the conjugate, or by isolating the desired conjugate (e.g., VHH) by specifying the amino acid sequence of the desired conjugate when expressed in bacterial or eukaryotic cells. カッパ or VHH ラムダ This may be achieved by the generation of nucleic acid constructs, RNA or DNA based, resulting in a target-specific antibody or antibody fragment (e.g., a target-specific nanobody or single domain antibody) conjugated to the

[0127] Example 6 - Development of MICA-specific nanobodies MHC class I polypeptide-related sequence A (MICA), a class I MHC-like molecule, is a cell stress-inducible glycoprotein frequently found enriched on the surface of malignantly transformed cells. MICA is recognized by natural killer group 2D (NKG2D), also known as Killer Cell Lectin Like Receptor K1 (KLRK1), an activating receptor on the surface of natural killer (NK) cells that enables immunity against MICA-positive targets such as tumor cells. High levels of MICA expression positively correlate with improved prognosis, for example, in cholangiocarcinoma (see, for example, Oliviero B, et al. Oncoimmunology. 2022; 11(1): 2035919). Downregulation of MICA can occur by shedding catalyzed by ADAM family matrix metalloproteases (see, for example, Waldhauer I, et al. Cancer Res. 2008; 68(15): 6368-76). Loss of MICA surface expression renders tumor cells less susceptible to NKG2D-positive NK cells, whereas soluble MICA itself can occupy the NKG2D receptor on NK cells and impair NK cell-to-NK cell interactions with MICA-positive targets. VHH nanobodies specific for MICA can be used to selectively induce immune responses against MICA-positive tumor cells or to deliver cytotoxic or cytostatic agents to such cells for therapy as part of a VHH nanobody adduct.

[0128] The generation of VHH nanobodies that specifically recognize MICA is described herein. These nanobodies are expected to have a short circulating half-life and excellent tissue penetration compared to conventional two-chain immunoglobulins, properties that are desirable for both in vivo imaging and immunotherapeutic agents. Because MICA is expressed on the surface of stressed and cancerous cells, the ability to non-invasively detect such abnormalities in vivo would be an important diagnostic tool for detecting precancerous and malignant lesions. MICA-specific nanobodies may also be present as part of a therapeutic nanobody adduct.

[0129] To obtain MICA-specific VHH nanobodies, alpacas were immunized with the purified extracellular domain of MICA and a phage display library was created from which MICA-specific nanobody sequences were isolated. Briefly, alpacas were immunized with 250 μg of purified MICA*009 in alum adjuvant followed by three booster injections separated by 2-week intervals. The immune response of immunized alpacas was monitored by immunoblotting serum samples collected before each booster injection. Although the signal generated in the immunoblot cannot be distinguished between conventional immunoglobulins or heavy chain-only (nanobody) immunoglobulins, a positive signal indicated successful immunization and subsequent immune response. Since it was determined that the immunization was successful after the final booster injection, a phage display library was constructed and screened using established techniques.

[0130] DNA from positive clones was sequenced and a total of nine clones were selected for further characterization. The relevant VHH sequences were subcloned into the pHEN6 expression vector with certain modifications, namely, that each nanobody sequence has a C-terminal LPETG motif and a His6 tag recognized by sortase A to facilitate recovery and purification (Figure 17A). This arrangement allows for the installation of fluorophores, biotin, or other moieties by a site-specific and efficient sortase-catalyzed transpeptidation reaction. Because the LPETG sequence is cleaved during transpeptidation, the His6 tag immediately C-terminal to the LPETG motif is lost upon combining the nanobody with another moiety. This allows for enrichment of the desired modified nanobody by depleting the His6-tagged sortase and unreacted input nanobody on a nickel-nitriloacetic acid (NiNTA) matrix, followed by recovery of the unbound fraction containing the modified nanobody. The amino acid sequences of the nine isolated anti-MICA nanobody clones are shown below in Table 2.

[0131] [Table 2]

[0132] Although nanobodies are not necessarily suitable for immunoblotting experiments, biotinylated versions of clones A1 and H3, when used at a dilution of 1ug / mL, produced surprisingly strong and specific luminescent signals on immunoblots (Figure 17B). Immunoblots were prepared by separating samples of total cell lysates spiked with purified MICA*009 antigen by SDS-PAGE, immunoblotting with isolated nanobodies, and treating the immunoblots with streptavidin-horseradish peroxidase (HRP) as the secondary detection agent.

[0133] To determine whether the isolated nanobodies recognize similar or distinct epitopes on the MICA antigen, the specificity of anti-MICA VHH nanobody binding was further evaluated by performing ELISA cross-competition experiments. Competition between unlabeled and biotinylated nanobodies for binding to MICA showed that the isolated nanobodies recognize two distinct epitopes, one exemplified by the A1 nanobody and the other by the H3 nanobody (Figure 17C). Interestingly, none of the isolated nanobodies competed for binding with the previously reported 7C6 anti-MICA monoclonal antibody (see, for example, Ferrari de Andrade, et al. Science. 2018;359(6383):1537-1542), indicating that these nanobodies bind to previously unrecognized epitopes of the MICA antigen.

[0134] Next, it was evaluated whether the isolated anti-MICA VHH nanobodies were selective for certain MICA allele products. MICA is a highly polymorphic locus in the human genome, resulting in the expression of a wide variety of allele products in the human population, including some variants associated with disease states (see, for example, Shi C, et al. Open Rheumatol J. 2015;9:60-64). It is therefore possible that the isolated anti-MICA nanobodies preferentially bind some MICA variants over others. To determine whether this is the case, the A1, B11, E9, and H3 anti-MICA nanobody clones, which were determined to bind to either of two distinct epitopes, were evaluated in ELISA assays for binding to a set of MICA variants, as well as to a stress-induced glycoprotein, MHC class I chain-related protein B (MICB), and a ferritin control. Each of the isolated nanobodies was observed to bind to the MICA*008 and MICA*009 variants, but not to MICA*002 (Figure 17D). Considering that MICA*008 and MICA*009 were observed to occur in just over half of participants in a study of 1.2 million donors of German descent, occurring in approximately 42.3% and 8.8% of participants, respectively (see, for example, Klussmeier A, et al. Front Immunol. 2020;11:314.), the isolated nanobodies will have broad utility for either imaging or therapeutic applications in subjects with MICA-expressing tumors.

[0135] To further evaluate whether the isolated anti-MICA VHH nanobodies bind to MICA on the cell surface, A1 and H3 nanobody clones were generated against soluble recombinant MICA*009 and cell binding was assessed by flow cytometry. Binding was assessed using B16F10 melanoma cells transfected to express either MICA or MICB, or empty vector (EV) as a negative control. Biotinylated A1 and H3 nanobodies were labeled with streptavidin-phycoerythrin (PE) and used for cytofluorometry of transfected B16F10 cells. Both A1 and H3 nanobody clones demonstrated robust staining of MICA-transfected cells (Figure 17E). Furthermore, neither nanobody clone stained MICB- or EV-transfected cells, confirming that the isolated nanobodies are capable of selectively binding to MICA-positive tumor cells. These results further demonstrate the utility of some of the isolated anti-MICA nanobodies, including MICA-specific nanobody adducts.

[0136] To further establish the usefulness of the isolated anti-MICA VHH nanobodies, further assays may be performed in vivo. First, the binding of the labeled nanobodies may be evaluated in a mouse xenograft model expressing MICA-positive tumors. For example, C57 / B6 mice may be inoculated with MICA-positive B16F10 cells and subsequently treated with biotinylated nanobodies. Given that the mice do not express MICA or cross-reactive species, the biotinylated nanobodies are expected to bind only to the MICA-positive B16F10 tumors. The mice are then further treated with streptavidin-conjugated fluorescent or luminescent agents and imaged. Fluorescence / luminescence signals localized at the xenograft site indicate that the labeled nanobodies are specific for MICA-positive cancer cells. Second, the isolated nanobodies may be further tested as imaging agents for positron emission tomography (Immuno-PET) due to their small size, efficient tissue penetration, and short circulatory half-life. To test the utility of isolated nanobodies for imaging by immuno-PET, C57 / B6 mice may be implanted with B16F10 control cells or MICA-positive B16F10 cells. Once B16F10 tumors are established, the mice are subsequently 89 Patients may be treated with Zr-labeled A1 or H3 nanobody clones and imaged via Immuno-PET. Given the high specificity of the isolated nanobodies (Figure 17E), these studies are expected to further demonstrate the diagnostic and clinical utility of these novel anti-MICA nanobodies.

[0137] Example 7 - Evaluation of conjugates VHH カッパ -Biotin and VHH カッパ-SD36-biotin conjugates were tested for their binding affinity to mouse immunoglobulins (Figures 18A-C). 96-well ELISA high-binding plates were coated overnight at 4°C with 100 μl of 5 μg / ml mouse Ig (mouse IgG isotype control: Invitrogen, Cat. No. 10400C; mouse IgA isotype control: Invitrogen, Cat. No. 14-4762-81; mouse IgM isotype control: BioLegend, Cat. No. 401601). Plates were washed three times with washing buffer (PBS supplemented with 0.1% (v / v) Tween-20) and incubated with blocking buffer (1% (w / v) BSA in PBS) for 1 h at room temperature. After washing four times with washing buffer, each well was treated with 100 μl of serial 4-fold dilutions of VHH kappa-biotin, VHH kappa-SD36-biotin, or SD36-biotin in blocking buffer. After 2 hours of incubation at room temperature, the plate was washed four times with washing buffer and incubated with streptavidin-HRP (1:1000 dilution, Biolegend, Cat. No. 405210) for 1 hour at room temperature. After washing four times with washing buffer, each well was incubated with 100 μl of TMB substrate solution (Biolegend, Cat. No. 421101) for 10 minutes at room temperature, after which 100 μl of 1N H2SO4 was added to stop the enzymatic reaction. The optical density was then read at OD450. Dissociation constants (Kd) were calculated from plots of mean absorbance values ​​at 450 nm versus VHH concentration using the saturation binding equation in GraphPad Prism 7 (saturation binding equation, 1 site--total). カッパ -Biotin and VHH カッパ These results demonstrate that the -SD36-biotin conjugate has high binding affinity for mouse immunoglobulins.

[0138] VHHs against neuraminidase from various influenza species カッパ -zanamivir, ALB1-zanamivir, zanamivir, and VHH カッパThe neuraminidase inhibitory activity of VHH kappa-zanamivir, ALB1-zanamivir, zanamivir, and VHH kappa was measured by NA-Star™ Influenza Neuraminidase Inhibitor Resistance Detection Kit (Invitrogen, Cat. No. 4374422). Influenza strains are shown in the figure. S12 was used as the neuraminidase source. All viruses were diluted to a signal:noise ratio of 40:1 (luminescence intensity of virus-containing wells:NA-Star assay buffer containing wells). Briefly, serial dilutions (25 μL) of tested molecules were incubated with 25 μL of virus in the NA-Star™ detection microplate for 20 min at 37°C. Then, 10 μL of NA-Star substrate was added to each well and incubated at room temperature for 30 minutes. Finally, 60 μL of NA-Star promoter solution was added to all wells and their luminescence intensity was immediately read by a plate reader (SpectraMax® iD5, Molecular Devices). The half-maximal inhibitory concentration (IC50) values ​​were calculated by GraphPad Prism 7. VHH カッパ -zanamivir and ALB1-zanamivir demonstrated half-maximal inhibitory concentration (IC50) efficacy rates (Figures 19A-B).

[0139] The amino acid sequence of ALB1 is: AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGLPETGGHHHHHH (sequence number 38).

[0140] VHH that binds to hemagglutinin expressed on influenza virus-infected MDCK cells カッパThe ability of -SD36 to infect MDCK cells was tested. MDCK cells were seeded in 24-well plates and grown to confluence overnight. Infection of MDCK cells with influenza virus (10 TCID50) was performed according to the Manual for Laboratory Diagnosis and Virological Surveillance of Influenza (World Health Organization-2011).

[0141] The affinity of VHHs for viral hemagglutinin on the surface of infected MDCK cells was determined using a saturation binding assay. Briefly, spent medium was aspirated from 24-well plates containing virus-infected MDCK cells and then replaced with 0.5 mL of fresh serum-free medium containing various concentrations of VHH kappa-SD36. After 1 h of incubation at 37°C, virus-infected cells were rinsed with fresh medium (2 x 0.5 mL) to remove unbound VHHs. To quantify the amount of VHHs bound to HA, mouse IgG-phycoerythrin (PE) (1:20 dilution, R&D Systems, catalog number IC002P) in 0.25 mL of fresh serum-free medium was added to the VHH kappa-SD36-containing wells. After 30 min of incubation at 37°C, virus-infected cells were rinsed again with fresh medium (2 x 0.5 mL) and then dissolved in 0.5 mL of 1% (w / v) sodium dodecyl sulfate (SDS). Cell-associated fluorescence was measured using an excitation wavelength of 560 nm and emission at 620 nm. Dissociation constants (Kd) were calculated from plots of cell-associated fluorescence intensity versus concentration of VHH using the saturation binding equation in GraphPad Prism 7 (saturation binding equation, 1 site--total). VHH カッパ -SD36 bound with high affinity to hemagglutinin expressed on influenza A virus strains (Figures 20A-B).

[0142] SD36-DFO and VHH カッパ -SD36-DFO was prepared for PET imaging. Nanobody-DFO adducts were prepared by sortase-mediated conjugation of triglycine-modified DFO to nanobodies. SD36-DFO (left) and VHH カッパThe final product of -SD36-DFO (right) was analyzed by SDS-PAGE.

[0143] Example 8 - Performance of conjugates in vivo VHH カッパ -Zanamivir, MEDI8852, and VHH カッパ The therapeutic efficacy during the first 6-9 week period was tested and compared. 50 Mice were infected with the indicated doses of VHH カッパ -zanamivir, MEDI8852 (a monoclonal antibody (mAb) that neutralizes both group I and group II influenza A viruses (IAV) in vitro), or VHH カッパ Mice were treated with 100-mL PBS-E11 (SARS CoV-2 spike-specific nanobody) by intraperitoneal injection. Mice were euthanized when they had lost 25% of their body weight or were moribund. Body weight loss curves (left) and survival curves (right) are shown. For body weight loss curves, % body weight change represents the mean ± standard deviation. Mean values ​​of % body weight change over 14 days between any two groups were compared using one-way ANOVA analysis with Tukey's multiple comparison test. Statistical differences between the indicated groups and the PBS-treated group are shown (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). For survival curves, statistical differences between the indicated groups and the PBS-treated group were calculated by Log-rank (Mantel-Cox) test (*P<0.05, **P<0.01). These data were compared using the VHH カッパ - Zanamivir has been shown to be associated with a statistically significant improvement in body weight and / or survival in MEDI8852 and VHH カッパ -E11 (Figure 21)

[0144] Example 9 VHHs that induce complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) カッパ -The ability of zanamivir was tested.

[0145] For the CDC assay, 10,000 cells / well of MDCK cells were seeded in a 96-well plate and incubated with 100 TCID 50 The cells were incubated with 100 μl of influenza virus A / NWS / 33 (H1N1) for 24 h. Spent medium was aspirated from the 96-well plate containing the virus-infected MDCK cells, and then 50 μl of VHH カッパ -Zanamivir (or VHH カッパ -SD36) or VHH カッパ and zanamivir (or SD36) (final concentration: 10 nM). After 30 min incubation at ambient temperature, 50 μl of fresh serum-free medium containing 40 μg / mL normal mouse IgG isotype control (Invitrogen, Cat. No. 10400C) and 40% (v / v) rabbit complement serum (Sigma-Aldrich, Cat. No. S7764) was added to the cells. The plates were then incubated at 37°C for 2.5 h. Cell viability was measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat. No. G7572). Maximum cell killing was achieved by treating the cells with 5% H2O2. VHH カッパ - The percentage of zanamivir-induced cytotoxicity was calculated as follows:

number

[0146] Influenza virus-infected MDCK cells express VHHs in the presence of rabbit complement and mouse polyclonal mouse IgG, as evidenced by high cytotoxicity in infected cells. カッパ -Killed by zanamivir (Figure 23A).

[0147] For the ADCC assay, 10,000 cells / well of MDCK cells were seeded in a 96-well plate and 100 TCID 50The cells were incubated with 100 μl of influenza virus A / NWS / 33 (H1N1) for 24 h. Spent medium was aspirated from the 96-well plate containing the virus-infected MDCK cells, and then 25 μl of VHH カッパ -Zanamivir (or VHH カッパ -SD36) or VHH カッパ After treatment with a mixture of zanamivir and SD36 (final concentration: 10 nM), 25 μl of 40 μg / mL normal mouse IgG isotype control (Invitrogen, Cat. No. 10400C) was added. After incubation at room temperature for 30 min, 25 μl of ADCC reporter cells (Promega, Cat. No. 10400C) were added at 75,000 cells / well and incubated at 37° C. for 6 h. To measure luciferase produced by ADCC reporter cells, 75 μl of Bio-Glo™ reagent (Promega, Cat. No. 10400C) was added to each well and the luminescence intensity was measured by a plate reader (SpectraMax® iD5, Molecular Devices).

[0148] Virus-infected MDCK cells were transfected with VHH カッパ Upon engagement of the mouse FcγRIV receptor in the presence of -zanamivir and mouse polyclonal mouse IgG, luciferase expression was induced in luciferase-expressing reporter cells. The induction of ADCC was calculated by dividing the luminescence intensity of the indicated samples by the average value of the control samples containing virus-infected cells and reporter cells without VHH. カッパ -zanamivir conjugates bind to VHHs in infected cells. カッパ and zanamivir mixture (Figure 23B).

[0149] VHH カッパ -zanamivir and ALB1-zanamivir were found to have similar clearance rates (Figure 23D). 89Each individual measurement of Zr decay rate is represented by a blue square (VHH カッパ -DFO- 89 Zr, n = 3), red circle (ALB1-DFO- 89 Zr, n=4) or black triangle (SD36-DFO- 89 All mice received an initial dose of 250 μCi. 89 Each subject received Zr-labeled VHH (equivalent to 1 mg / kg VHH). Each data point represents the mean ± standard deviation. The half-life (fast and slow phases) of each VHH was estimated using a biphasic decay model. Total VHH exposure over the first 144 h after injection was calculated by integrating the VHH concentration in the blood over time. This is expressed as the "area under the curve" (AUC).

[0150] VHH カッパ - Using experiments similar to those performed with zanamivir, VHH カッパ It was found that -SD36 induced ADCC but did not induce CDC (Figures 24A-B).

[0151] Example 10 ALB1-zanamivir conjugate was prepared. ALB1 is an anti-serum albumin nanobody (ALB1) with the amino acid sequence shown in Figure 25A. A sortase recognition motif (LPETG) was attached to the C-terminus of the nanobody. ALB1-zanamivir was prepared by sortase-mediated conjugation of triglycine-modified zanamivir to ALB1. The identity of the final product, ALB1-zanamivir, was confirmed by SDS-PAGE and mass spectrometry (Figure 25B).

[0152] VHH for PET imaging カッパ-DFO, ALB1-DFO, and SD36-DFO were prepared. Nanobody-DFO adducts were prepared by sortase-mediated conjugation of triglycine-modified DFO to nanobodies. Nanobody-DFO adducts were analyzed by SDS-PAGE (for each gel, from left to right: 1: sortase, 2: unconjugated nanobody, 3: reaction mixture, 4-9: different fractions obtained after PD-10 column elution; the nanobody-DFO adduct shown as #6 on the gel was used for PET imaging).

[0153] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as defined by the appended claims.

[0154] Unless indicated to the contrary or otherwise clear from the context, articles such as "a," "an," and "the" may mean one or more than one. Unless indicated to the contrary or otherwise clear from the context, a claim or description that includes "or" between two or more members of a group is considered to be satisfied when one, more than two, or all of the members of the group are present. The disclosure of a group that includes "or" between two or more members of the group provides embodiments in which exactly one member of the group is present, embodiments in which two or more members of the group are present, and embodiments in which all of the members of the group are present. For the sake of brevity, these embodiments are not individually described herein, but it will be understood that each of these embodiments may be provided herein, specifically claimed, or disclaimed.

[0155] It should be understood that the present disclosure covers all variations, combinations, and substitutions in which one or more limitations, elements, clauses, or descriptive terms from one or more of the claims or from one or more relevant parts of the specification are introduced into another claim. For example, a claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, when a claim describes a composition, it should be understood that the claim includes a method of making or using the composition according to any of the methods of making or using disclosed herein, or according to any method known in the art, unless otherwise indicated or unless it is obvious to a person skilled in the art that a contradiction or inconsistency would occur.

[0156] When elements are presented as a list in Markush group format as an example, it is understood that all possible subgroups of elements are also disclosed, and that any element or subgroup of elements may be removed from the group. It is also noted that the term "comprising" is intended to be open, allowing for the inclusion of additional elements or steps. In general, when an embodiment, product, or method is referred to as comprising a particular element, feature, or step, it is understood that an embodiment, product, or method consisting of or consisting essentially of such element, feature, or step is also provided. For the sake of brevity, these embodiments are not individually described herein, but it will be understood that each of these embodiments may be provided herein, specifically claimed, or disclaimed.

[0157] When a range is given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and / or the understanding of a person skilled in the art, unless the context clearly states otherwise, it should be understood that values ​​expressed as ranges may in some embodiments take any particular value within the stated range to one tenth of the unit of the lower limit of the range. For brevity, each range value is not individually recited herein, but it will be understood that each of these values ​​may be provided herein and specifically claimed or disclaimed. It should also be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of a person skilled in the art, values ​​expressed as ranges may take any subrange within the given range, with the endpoints of the subranges being expressed with the same precision as one tenth of the unit of the lower limit of the range.

[0158] Where websites are provided, URL addresses are provided as non-browser executable code with brackets around the periods in each web address. The actual web addresses do not contain the brackets.

[0159] In addition, it should be understood that any particular embodiment of the present disclosure may be expressly excluded from any one or more of the claims. Where ranges are given, any value within the range may be expressly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more of the claims. For the sake of brevity, not all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are expressly specified herein.

Claims

[Claim 1] A conjugate comprising a first agent that binds to an immunoglobulin and a second agent that binds to a target on the surface of a cell or pathogen, wherein the first agent and the second agent are covalently conjugated via a linker.

Images