WO2007150020A1 - Targeted immune conjugates - Google Patents

Targeted immune conjugates Download PDF

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Publication number
WO2007150020A1
WO2007150020A1 PCT/US2007/071875 US2007071875W WO2007150020A1 WO 2007150020 A1 WO2007150020 A1 WO 2007150020A1 US 2007071875 W US2007071875 W US 2007071875W WO 2007150020 A1 WO2007150020 A1 WO 2007150020A1
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WIPO (PCT)
Prior art keywords
immune conjugate
cell
immune
antibody
immunogen
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PCT/US2007/071875
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French (fr)
Inventor
Paul M. Simon
Original Assignee
Simon Paul M
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Simon Paul M filed Critical Simon Paul M
Priority to US12/306,457 priority Critical patent/US8318912B2/en
Priority to EP07798929A priority patent/EP2041175A4/en
Priority to CA2690973A priority patent/CA2690973A1/en
Publication of WO2007150020A1 publication Critical patent/WO2007150020A1/en
Priority to US13/591,687 priority patent/US20130071413A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • This invention relates to mate ⁇ als and methods involved m immunotherapy
  • Vaccination is one of the most important medical interventions for preventing disease
  • the purpose of vaccination is to induce an optimal immune response that provides preventive or therapeutic benefit to the host
  • Vaccines typically contain one or more immunogens (IMGs) that are harmless variants or derivatives of pathogens, which act to stimulate the immune system to mount defenses against the actual pathogen
  • IMGs immunogens
  • FMGs immunogens
  • Some BVIGs are very effective inducers of the desired immune response, while others require the co-admmistration of non-specific immune stimulants, or adjuvants, or the coupling of the IMG to a carrier protein or microparticulate substances
  • Still other IMGs are inherently poor at inducing effective immune responses, despite combination with adjuvants and repeated boosts
  • Many of these inherently weak immunogens are involved m diseases e g , influenza and cancer, that remain major causes of human mor
  • immune conjugates that are useful for inducing or enhancing an immune response against an antigen
  • immune conjugates that include a ligand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, and an immunogen coupled to the ligand, wherein the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen
  • the immune conjugate includes a
  • ligand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, wherein the molecule is selected from the group consisting of CR2, glycophorin A, band 3, Ter-119, blood group antigen H, blood group antigen A, blood group antigen B, CD41a, CD14, CD56, CD66d, CD83, CMKLRl, and BDCA-4, and an immunogen coupled to the ligand, where the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen
  • a circulating non- lymphoid cell can be any cell that circulates through the body of a mammal in the blood and/or lymph system, and is not a B lymphocyte or a T lymphocyte
  • the circulating non-lymphoid cell can be, for example, a red blood cell, a platelet, a natural killer cell, a monocyte, a granulocyte or a plasmacytoid dendritic cell
  • the circulating non-lymphoid cell is a red blood cell
  • the circulating non-lymphoid cell is a platelet
  • the circulating non- lymphoid cell can be derived from any mammal, e g , humans, a non-human primates, cattle, horses, pigs, sheep, goats, deer, elk, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, or mice
  • the cell-surface molecule can be any molecule that is differentially expressed on circulating non-lymphoid cells relative to the levels of the same molecule on a cell type that is not a circulating non-lymphoid cell
  • the cell surface molecule can be a polypeptide, a carbohydrate, a phospholipid, or a glycohpid
  • the cell surface molecule can be a molecule on the surface of a red blood cell, including, for example, complement receptor 2 (CR2), glycophonnA (CD235A), band 3 (CD233), TER-119, the ABO blood group antigens, e g , blood group antigen A, blood group antigen B, blood group antigen H, and phosphatidyl se ⁇ ne
  • the cell surface molecule can be a molecule on the surface of a platelet, including, for example, gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectm) and CDl 51
  • the cell surface molecule can be a molecule on the surface of a natural killer (NK) cell, including, for example, CD56 (NCAM, Leu-19, NKHl)
  • the cell surface molecule can be a molecule on the surface of a monocyte, including, for example, CD14 (LPS-recept
  • the ligand can be any molecule capable of binding a specific cell surface molecule on a circulating non-lymphoid cell
  • a ligand can include, for example, a polypeptide, a carbohydrate, glycolipid or biomimetic of a polypeptide, carbohydrate or glycohpid, as long as the ligand binds specifically to a cell surface molecule that is differentially expressed on a circulating non-lymphoid cell
  • a ligand can be a polypeptide, provided that it is not C3d, a heat shock protein, muramyl dipeptide, or muramyl t ⁇ peptide
  • a polypeptide ligand can be an antibody
  • the antibody can be a monoclonal antibody, a polyclonal antibody, a holoantibody, a single chain variable fragment immunoglobulin, or a chimeric molecule that contains the constant region of an immunoglobulin and cell-binding sequences from a different source grafted in place of the immunoglobulin variable regions
  • the antibody can be, for example, an antibody that recognizes targets on red blood cells eg , complement receptor 2 (CR2), glycopho ⁇ n A (CD235A), band 3 (CD233), TER- 119, blood group antigen A, blood group antigen B, and blood group antigen H
  • the antibody can be, for example, an antibody that recognizes targets on platelets, e g , gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectm) and CD151
  • the antibody can be, for example, an antibody that recognizes targets on monocytes, e
  • a ligand can also be a polypeptide that is not an immunoglobulin, including for example, the erythrocyte-bmdmg antigen 175 (EBA- 175) of Plasmodium falciparum or a fragment of EBA- 175 that binds to a red blood cell, for example, SEQ ID NO 7, a complement fragment that binds to CR2, for example, C4b, C3b, iC3b, CIq, C3d or a peptide derived from these complement fragments, e g , C3 residues 1201-1214, or a
  • EBA- 175 erythrocyte-bmdmg antigen 175
  • a fragment of EBA- 175 that binds to a red blood cell for example, SEQ ID NO 7, a complement fragment that binds to CR2, for example, C4b, C3b, iC3b, CIq, C3d or a peptide derived from these complement fragments,
  • lectin e g
  • glycoprotein that recognizes blood group antigen A, blood group antigen B or blood group antigen H.
  • the polypeptide hgand can include post-translational modifications, e g , biotinylation, glycosylation, acetylation, alkylation, isoprenylation, hpoylation, phosphorylation
  • a hgand can also be a carbohydrate or glycolipid, including, for example, bacte ⁇ al lipopolysacchande or a fragment of it, microbial products bound by Toll-like receptors, bacte ⁇ al diacyl and t ⁇ acyl hpopeptides, lipoteichoic acid or zymosan
  • a hgand can be a nucleic acid, including, for example, single- and double-stranded viral RNA and CpG DNA
  • the immunogen can be any molecule capable of eliciting a functional immune response in a mature T or a B lymphocyte or a precursor of a T or a B lymphocyte
  • Irnrnunogens can include polypeptides, carbohydrates, glycolipids, haptens or biomimetics thereof
  • the immunogen can be a molecule expressed or released by an infectious agent, including, for example, viruses, viroids, bactena, fungi, p ⁇ ons or parasites
  • An infectious agent can include for example, Orthomyxovmdae, e g , influenza viruses, including the strain A(H5N1), Rhadbovi ⁇ dae, Hepadnavmdae, e g , hepatitis B, Picornavindae, e g , hepatitis A, Flavivindae, e g , hepatitis C, Retrovi ⁇ dae, e g , human immunodeficiency viruses
  • Immunogens derived from pathogenic organisms can include, for example, influenza A M2 protein, hepatitis B surface antigen, HBV preSl protein, HIV tat, HIV gpl20, anthrax protective antigen, botuhnum toxm, and Streptococcus pneumoniae pneumococcal polysaccharides
  • An influenza M2 protein antigen can be the ectodomam peptide M2e, for example SEQ ID NO 1, or a variant of the ectodomam
  • HBV preSl protein can include the preSl protein peptide 35-49, for example SEQ ID NO 2
  • the immunogen can also be a molecule expressed by a mammal
  • an immunogen can be a molecule whose expression is correlated with a particular disease for example, cancer or neurodegenerative disease
  • the immunogen can be a tumor-associated antigen (TAA), including for example, MART-I, Muc-1, MAGE, RAGE, or CEA
  • TAA tumor-associated antigen
  • the immunogen can be an antigen that is involved in the initiation or progression of neurodegenerative diseases, e g Alzheimer's disease and Transmissible Spongiform Encephalopathies (TSEs), including for example, beta-amyloid, tau protein, alpha synuclem, or a prion-related protem
  • the immunogen can be a germ cell antigen, including for example, sperm adhesion molecule 1 (SPAM-I), and human lntra-acrosomal protein
  • the immunogen can be a non-toxic variant of a toxic substance, including for example, ricm, botulmum
  • linker can be any reagent, molecule or macromolecule that connects the ligand and the immunogen such that a) the immune complex is stable under physiological conditions, b) the connection between the linker and the ligand does not alter the ability of the ligand to bmd to its target on the surface of a circulating non- lymphoid cell, and c) the connection between the linker and the immunogen does not abolish the capacity of the immunogen to induce an effective immune response in a host against an infectious agent, cell or molecule on which the immunogen is naturally found
  • a linker can be a peptide bond
  • the ligand and the immunogen can be a fusion polypeptide comprising one or more ammo acid segments from the ligand and one or more ammo acid segments from the immunogen
  • the ammo acid segments of the ligand can be contiguous with the ammo acid segments of the immunogen or they can be separated by ammo acids inserted as a structural spacer
  • a spacer segment can be one or more ammo acids
  • the one or more ammo acids can include ammo acids that are the same or that are different
  • nucleic acids composing a nucleotide sequence that encodes the fusion proteins, a vector (e g , s. vector that includes a transcriptional regulatory element (TRE) operably linked to the nucleotide sequence) containing the nucleic acid, and a cell (e g , a prokaryotic cell or a eukaryotic cell) containing the vector
  • the hgand and immunogen can be obtained separately, either through chemical synthesis or synthesis m vivo, purified and then linked non-covalently or covalently
  • the non-covalent linkage can be a for example, a biotm-avidm (or streptavidm) linkage
  • the covalent linkage can be through a chemical cross-linking agent, for example, a homobifunctional cross-lmkmg reagent or a heterobifunctional cross-lmkmg reagent
  • the hgand and the immunogen can be connected through a linking polymer, including, for example, linear or branched polymers or co-polymers (e g , polyalkylene, poly(ethylene-lysme), polymethacrylate, polyammo acids, poly- or oligosaccharides, or dend ⁇ mers)
  • the hgand and the immunogen can be connected through a microparticle, including, for example, micelles, liposomes, fullerenes, nanotubes, or other colloidal complexes such as lipoproteins
  • the hgand and the immunogen can be attached to the linking molecule or microparticle through a non- covalent high affinity linkage, e g , avidm-biotin high affinity binding, adsorbed or incorporated into a hydrophobic microparticle by hydrophobic affinity, or covalent chemical cross-lmkmg techniques
  • the immune conjugates provided herein can include one or more of the same hgands or any combination of different hgands
  • the immune conjugates can also include one or more of the same immunogens or any combination of different immunogens
  • Also provided are methods and mate ⁇ als for inducing or enhancing an immune response m a mammal comprising administering to the mammal an effective amount of a composition comprising an immune conjugate that includes a hgand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, and an immunogen coupled to the hgand, wherem the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen
  • the immune conjugate includes a hgand which bmds specifically to a cell surface molecule on a circulating non-lymphoid cell, wherem the molecule is selected from the group consisting of CR2, glycophorm A, band 3, Ter-119, blood group antigen H, blood group antigen A, blood group antigen B, CD41a, CD14, CD56, CD66d, CD83, CMKLRl, and BDCA-4, and an immunogen coupled to the hgand,
  • the mammals can be, for example, humans, non-human p ⁇ mates, horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, and mice
  • the mammal can have, be likely to have, or be at risk for having, an infectious disease, e g , a viral disease, a bacte ⁇ al disease, a protozooal disease, or a fungal disease
  • infectious diseases can include, for example, influenza, HTV-AIDS, hepatitis, botulism, smallpox, viral hemorrhagic fevers, gastrointestinal disease induced by pathogenic forms of E coh, Salmonella and Shigella, Staphylococcal infection, trypanosomiasis, and malaria
  • the mammal can have, be likely to have, or be at nsk for having, a proliferative cell disease, e g , a
  • methods and mate ⁇ als for inducing or enhancing an immune response in a mammal against a germ cell antigen are provided.
  • methods and mate ⁇ als for inducing or enhancing an immune response in a mammal against toxic substances, e g , ricm, botulinum toxm, nicotine and drugs of abuse are provided.
  • compositions featuring immune conjugates that include a hgand which binds specifically to a cell surface molecule on a circulating non- lymphoid cell, and an immunogen coupled to the hgand, wherem the immune conjugate, when administered to an individual, induces or enhances an immune response agamst the immunogen m a pharmaceutically acceptable earner or excipient hi another embodiment, the composition can include an adjuvant [0026] Also provided are articles of manufacture that can include immune conjugates as desc ⁇ bed herein An article of manufacture can include, for example, one or more immune conjugates In addition, an article of manufacture further may include, for example, packaging mate ⁇ als, instructions for use, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required
  • Figure 1 depicts the electrophoretic mobility of tripartite immune conjugates containing biotmylated antibody, streptavidm, and varying amounts of biotmylated M2e peptide (SEQ ID NO 1)
  • Figure 2 depicts the electrophoretic mobility of tripartite immune conjugates containing biotmylated antibody and a constant ratio of streptavidm, and biotmylated M2e peptide (SEQ ID NO 1)
  • the mammalian immune system mounts two different types of responses to immunogens (IMGs), humoral and cellular
  • IMGs immunogens
  • B lymphocytes defends primarily against extracellular pathogens through the production of circulating antibodies that mark foreign cells and molecules for destruction by other specialized cells and proteins
  • T lymphocytes defends predominantly against intracellular pathogens and cancers by directly binding to and destroying the infected or cancerous cells
  • Both responses depend upon specialized cells that internalize through endocytosis, pinocytosis or phagocytosis, and process IMGs, fragments of the IMGs are then presented to T lymphocytes, which m turn, help to trigger B-lymphoctye responses against the immunogens and/or the T cells to attack the target directly
  • the phagocytic cells that function as antigen-presentmg cells are part of the reticuloendothelial system (RES)
  • the RES is a diffuse system comprised of circulating and tissue-fixed cells including monocytes, macrophages, dendritic cells, Kupffer cells in the liver, Langerhans cells in the skin and microglial cells in the brain
  • tissues and organs by virtue of their wealth of phagocytic and specialized endothelial cells, comprise critical locations for the clearance and antigen presentation functions of the RES These include the liver, spleen, bone marrow and lymphatic tissues
  • the RES plays an important role in clearing potentially harmful materials from the blood including micro-organisms, bacte ⁇ al endotoxins, immune complexes, tumor cells and senescent and damaged cells of the blood and lymph systems
  • the cells that cycle through the RES e g red blood cells (or erythrocytes), platelets, natural killer cells, monocytes,
  • targeted immune conjugates are provided that specifically bind to circulating non-lymphoid cells, e g , red blood cells, platelets, natural killer cells, monocytes, granulocytes, and plasmacytoid dend ⁇ tic cells, that are cleared through the RES
  • the circulating non-lymphoid cells act as vehicles for the specific delivery of IMGs to the RES and thereby to the immune system
  • the term "targeting immune conjugate” includes a hgand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell coupled to an IMG, where the targeted immune conjugate, when administered to an individual, induces or enhances an immune response against the IMG
  • Targeted immune conjugates provide injectable vaccines for efficient imm
  • the targeting immune conjugates provided herein have the general formula
  • L is a ligand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell
  • X is a linker
  • BVIG is an immunogen
  • y and z are integers having a value of one or greater than one
  • a circulating non-lymphoid cell can be any cell that a) circulates through the body of a mammal m the blood and/or lymph system, and b) is not a B lymphocyte or a T lymphocyte
  • circulating non-lymphoid cells include erythrocytes, i e , red blood cells, platelets, natural killer cells, monocytes, granulocytes, i e , neutrophils, eosinophils, and basophils, and plasmcytoid dendritic cells
  • B and T lymphocytes are ultimately denved from hematopoietic stem cells and perform the p ⁇ ncipal functions of the immune system
  • T lymphocytes mature through the thymus and are generally identified by their expression of CD3 (which is associated with the T cell receptor) and either CD4 or CD8 CD8-expressing (or CD8+) T cells are principally involved with direct cell killing, or cytotoxicity CD4+ T cells are primarily regulatory cells which stimulate and suppress immune responses as needed
  • B lymphocytes are characterized by their expression of CD19 or CD20, among other
  • B cells are also effective antigen presenting cells
  • Erythrocytes also known as red blood cells, are the most abundant cell type in mammalian blood They are small disc-shaped, anucleated, biconcave cells whose primary function is to carry oxygen and carbon dioxide to and from the tissues Red blood cells express a distinctive complement of cell surface markers, including the human blood group antigens, glycopho ⁇ n, band 3 and the Lewis antigens
  • Platelets are denved from megakaryocytes, they are centrally involved in blood clotting, and can be identified by their surface expression of CD41a (or gpIIb/IIIa)
  • Natural killer cells also referred to as large granular lymphocytes, are denved from the bone marrow and do not express T-cell antigen receptors (TCR), the pan-T marker CD3 or surface immunoglobulins (Ig) B cell receptor, but typically express the surface markers CD 16 (Fc ⁇ RIII) and CD56
  • Monocytes are denved from myeloid stem cells and are found pnmanly in the circulation They are competent phagocytes Upon their binding of pathogens and/or stimulation by various cytokines, monocytes mature mto macrophages, which are even more avid phagocytes and producers of many cytokines, degradative enzymes and other molecules that mediate inflammatory reactions Macrophages are generally found bound to vascular endothelium or within vanous tissues Monocytes (and
  • Granulocytes are also denved from myeloid stem cells and are charactenzed by the presence of abundant granules in their cytoplasm, different classes of granulocytes, e g , eosinophils, basophils and neutrophils, are distinguished by their ability stain with eosin, basophilic dyes or neither, respectively Eosinophils are involved m defense against parasitic pathogens and allergens, basophils are also involved m allergic reactions Neutrophils are early and aggressive phagocytes at the site of infections and release products that induce inflammatory reactions
  • Plasmacytoid dendntic cells are distmct from myeloid dendntic cells Both are found m the circulation and in tissues pDC are an important link between the innate and adaptive immune responses, in particular in mounting anti-viral
  • the circulating non-lymphoid cells can be de ⁇ ved from any mammal, e g , humans, a non-human primates, cattle, horses, pigs, sheep, goats, deer, elk, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, or mice
  • any cell surface molecule that is differentially expressed on circulating non lymphoid cells relative to the levels of the same molecule on a cell type that is not a circulating non-lymphoid cell is a suitable target for the hgand
  • the cell-surface molecule can be a polypeptide, a carbohydrate, or a glycohpid Full-length molecules, epitopes, analogs, mutants, and functional fragments thereof are encompassed by this definition
  • a "functional fragment" of a molecule is a fragment of the molecule that is smaller (shorter where the molecule is a polypeptide) than the molecule per se but has at least 10% ⁇ e g , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99 5%, 100% or even more) of the hgand-bindmg activity of the molecule per se
  • a cell-surface molecule on a circulating non-lymphoid cell can be classified as being differentially expressed if the molecule is present at a level that is greater than the average level observed in cells that are not circulating non-lymphoid cells if the expression levels differ by at least 50% ⁇ eg , 50, 100, 200, 300% or more) Any method can be used to determine whether or not a specific gene product is expressed at a level that is greater or less than the average level of expression observed in control cells
  • the level of expression of a cell surface polypeptide can be measured using any method such as immuno-based assays (e g , immunofluorescence, flow cytometry, ELISA), western blotting, or polyacrylamide gel electrophoresis with silver staining
  • Levels of particular carbohydrates or lipids can be measured by immunodetection e g , ELISA, flow cytometry, or immunostammg using fluorochrome- or radioisotope-labeled antibodies or
  • ligand refers to a molecule capable of binding a specific cell surface molecule on a circulating non lymphoid cell
  • a ligand can be a polypeptide, a carbohydrate, glycolipid or biomimetic of a polypeptide, carbohydrate or glycolipid, as long as the ligand binds specifically to a cell surface molecule that is differentially expressed on a circulating non-lymphoid cell
  • ligands are defined to be “specifically binding” if 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross react with related target molecules
  • the binding affinity of a ligand can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann NY Acad Sci 51 660-672, 1949)
  • a ligand disclosed herein can bind to its target with at least 1 5-fold, 2-fold, 5-fold, 10-fbld, 100-fold, 103- fold, 104-fold, 105-fold, 106-foldor greater affinity for the target than for a closely related or unrelated polypeptide
  • a ligand can bind its target with high affinity (10 "4 M or less, 10 7 M or less, 10 9 M or less, or with subnanomolar affinity (0 9, 0 8, 0 7, 0 6, 0 5, 04, 0 3, 02, 0 1 nM or even less)
  • Ligands can also be descn
  • the hgands disclosed herein do not bind to known related molecules
  • the hgands disclosed herein can bmd to orthologs, homologs, paralogs or variants, or combinations and subcombmations thereof, of their targets
  • a hgand that specifically binds to circulating non- lymphoid cells is a hgand the binds to circulating non-lymphoid cells and does not significantly bind to lymphoid cells
  • an immune conjugate that includes a hgand that specifically binds to red blood cells will bind to red blood cells and not significantly bind to other circulating cells, e g , lymphocytes, platelets, natural killer cells, monocytes, granulocytes or dendritic cells
  • an immune conjugate that includes a hgand that specifically binds to platelets will bind to platelets and not significantly bind to other circulating cells, e g , lymphoid cells, red blood cells, natural killer cells, monocytes, granulocytes or dendritic cells
  • Ligands may be screened against known related target polypeptides to isolate a hgand that specifically binds the target
  • a hgand specific to a target will flow through an affinity chromatography column comprising other closely related target molecules adhered to insoluble matrix under appropriate buffer conditions
  • Screening and isolation of specific antibodies is well known m the art (see, Fundamental Immunology, W Paul (ed ), Raven Press, 1993, Getzoff et al , Adv in Immunol 43 1-98, 1988, Monoclonal Antibodies Principles and Practice, Goding, J W (ed ), Academic Press Ltd , 1996, Benjamin et al , Ann Rev Immunol 2 67
  • a hgand can be a polypeptide, provided that it is not C3d, a heat shock protein, muramyl dipeptide, or muramyl t ⁇ peptide
  • polypeptide refers to a compound of two or more subunit ammo acids, ammo acid analogs, or other peptidomimetics, regardless of post-translational modification, e g , phosphorylation or glycosylation The subumts may be linked by peptide bonds or other bonds such as, for example, dicysteme, ester or ether bonds
  • amino acid refers to natural and/or unnatural or synthetic ammo acids, including D/L optical isomers Full-length proteins, analogs, mutants, and fragments thereof are encompassed by this definition
  • ammo acid sequence of the hgands disclosed herem can be identical to the wild-type sequences of approp ⁇ ate components Alternatively, any of the components can contain mutations such as deletions, additions, or substitutions All that is required is that the variant hgand have at least 5% (e g , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the hgand containing only wild-type sequences to specifically bind the target on the circulating non- lymphoid cell Substitutions will preferably be conservative substitutions Conservative substitutions typically include substitutions within the following groups glycine and alanine, valine, lsoleucme, and leucine, aspartic acid and glutamic acid, asparagme, glutamme, se ⁇ ne and threonine, lysine, mstidine and argmme, and phenylalanine and tyrosine
  • Any method can be used to make a polypeptide including, for example, expression by prokaryotic systems, expression by eukaryotic systems, and chemical synthesis techniques Any method can be used to purify a polypeptide including, without limitation, fractionation, cent ⁇ fugation, and chromatography, e g , gel filtration, ion exchange chromatography, reverse-phase HPLC and immunoaffmity purification
  • a suitable polypeptide ligand can be an antibody
  • the antibody can be a monoclonal antibody, ; e , homogeneous antibodies of identical antigenic specificity produced by a smgle clone of antibody-producing cells
  • the antibody can be a polyclonal antibody, i e , heterogenous antibodies that can recognize different epitopes on the same antigen and that are produced by more than one clone of antibody producing cells
  • the antibody can include any class of immunoglobulin, e g , IgG, IgA, IgM, and can be derived from any species e g , humans, mice, rats, or can be a humanized version of a non-human antibody
  • An antibody can include, without limitations, a holoantibody, i e , an antibody that includes one or more of an immunoglobulin monomer units of two heavy and two light chains, a single cham variable fragment immunoglobulin, or a chime ⁇ c molecule that contains the constant region of an immunoglobulin and cell-binding sequences from a different source grafted in place of the immunoglobulin variable regions
  • the cell-binding regions can be from a different antibody, a lectin, a cytokine, a microbial protein fragment or any other molecule that binds the target cell receptor molecule with specificity
  • Antibodies can be purified by chromatographic methods known to those of skill m the art, including ion exchange and gel filtration chromatography (for example, Came et al , Protein Expr Punf (1996) 8(2) 159-166) Alternatively or in addition, antibodies can be purchased from commercial sources, for example, Invitrogen (Carlsbad, CA), MP Biomedicals (Solon, OH), Nventa Biopharmaceuticals (San Diego, CA) (formerly Stressgen), Serologicals Corp (Norcross, GA)
  • the antibody can be, for example, an antibody that recognizes targets on red blood cells, including for example, without limitation, glycopho ⁇ n A (CD235A), band 3 (CD233), TER-119, blood group antigen A, blood group antigen B, and blood group antigen H
  • One useful antibody is anti-TER-119, which specifically binds to TER- 119, a region corresponding to the extracelllular domain of glycopho ⁇ n A
  • Other suitable antibodies include, without limitation, antibodies that recognize targets on platelets, eg , gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectin) and CD151, monocytes, e g , CD14, NK cells, e g , CD56, granulocytes, e g , CD66d, and plasmacytoid dendritic cells, e g , CMKLRl,BDCA-2 (CD303) and BDCA-4 (CD304)
  • a ligand can also be a polypeptide that is not an immunoglobulin
  • One non-immunoglobulm type ligand can be the erythrocyte-bindmg antigen 175 (EBA-175) of Plasmodium falciparum, which specifically hmds the red blood cell surface protein band 3, or a fragment of EBA-175 that binds to a red blood cell, for example EBA-175 peptide 1085-96, SEQ ED NO 7
  • Another polypeptide ligand can also be a complement fragment that binds to CR2, for example, C4b, C3b, iC3b, CIq or a peptide derived from the complement fragments, e g , C3 residues 1201-1214 (Tsokos et al , Journal of Immunol 144 1640-45, 1990) or residues 727 768 (Becherer, Biochemistry 31(6) 1787- 94 1992)
  • a polypeptide ligand can also be
  • a ligand can also be a peptidomimetic, a small protem-hke chain containing non-peptidic structural elements that is capable of mimicking or antagonizing the biological action(s) of a natural parent peptide
  • Peptidomimetic compounds are synthetic, non-peptide compounds having a three-dimensional conformation (; e , a "peptide motif) that is substantially the same as the three dimensional conformation of a selected peptide
  • the peptide motif provides the peptidomimetic compound with the ability to bind the ligand in a manner qualitatively identical to that of the parent peptide from which the peptiomimetic was de ⁇ ved Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased prolonged biological half-life
  • the peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based
  • side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based
  • Several types of chemical bonds eg , ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known m the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics
  • any peptidomimetic that binds specifically and selectively to a cell surface molecule that is differentially expressed on a circulating non-lymphoid cell can be used.
  • useful peptidomimetics include those that mimic the ability of antibodies that recognize, for example, complement receptor 1 (CRl), complement receptor 2 (CR2), glycophorm A (CD235A), band 3 (CD233), TER-119, blood group antigen A, blood group antigen B, and blood group antigen H, gpIIb/IJIa (CD41a), CD42d, CD61, CD62P (P-selectm), CD151, CD14, CD56, CD66d, CMKLRl and BDCA-2
  • the polypeptide can include post-translational modifications, i e , chemical modification of the polypeptide after its synthesis
  • Chemical modifications can be naturally occurring modifications made m vivo following translation of the mRNA encoding the polypeptide or synthetic modifications made in vitro
  • a polypeptide can include one or more post-translational modifications, in any combination of naturally occurring, i e , in vivo, and synthetic modifications made in vitro
  • post- translational modifications include, but are not limited to, biotinylation, e g , acylation of lysine or other reactive ammo acid residues with a biotm molecule, glycosylation, e g , addition of a glycosyl group to either asparagmes, hydroxylysme, se ⁇ ne or threonine residues to generate a glycoprotein or glycopeptide, acetylation, e g , the addition of an acetyl group, typically at the N-termmus of a polypeptide
  • a highly suitable post-translational modification can be biotmylation Biotm, also known as vitamin H or B7 is a water-soluble B-complex vitamin that binds avidm or streptavidm with very high affinity (10 15 M) Both egg avidm and bacte ⁇ al streptavidm have 4 biotm-bmdmg sites and thus can serve to couple several biotinylated ligands or to couple biotinylated hgand(s) to other biotinylated molecules (e g , IMGs)
  • Polypeptides can be convalently linked to one or more biotm molecules through primary amines, e g lysine and N-termmus), carboxyl groups found on aspartic- and glutamic- acid residues and at the C-termmus, sulfhydryl groups, or carbohydrate modifications on glycoproteins
  • Methods for denvatizmg polypeptides with biotm are well known in the art and there are many commercial sources for such
  • a ligand can also be a carbohydrate or glycolipid
  • carbohydrate and glycolipid ligands include, without limitation, bacte ⁇ al lipopolysacchande (LPS) or a fragment of it, microbial products bound by Toll-like receptors (TLRs), bacterial diacyl and tnacyl hpopeptides and lipoteichoic acid from bacte ⁇ a, and zymosan from yeast cell walls
  • a ligand can also be a nucleic acid
  • nucleic acids include, without limitation, single- and double-stranded RNA from viruses, and CpG DNA from bacte ⁇ a or viruses
  • an immunogen is any molecule capable of eliciting a functional immune response (e g , a cytotoxic or helper T cell response or an antibody producing response) m a T or a B cell
  • an "effector T lymphocyte” is a T lymphocyte having immunological activity Such immunological activity can be, without limitation, cytotoxic activity, helper activity, suppressive activity, immune-deviating activity, inflammatory activity, or pro-inflammatory activity
  • an "effector T lymphocyte precursor cell” is a T lymphocyte that, subsequent to activation, has any of the above immunological activities Activation can occur, without limitation, by recognition of a complex of the relevant immunogenic peptide epitope and the major histocompatibility complex (MHC) molecule by the T cell receptor (TCR) on the effector T lymphocyte or by a non-specific stimulus, e g , a T cell mitogen such as concanavalm A
  • an effector T lymphocyte cell can be a "virgin” T lymphocyte that has never previously been activated or a "memory” T lymphocyte that has previously been activated or the progeny of such a memory T lymphocyte
  • a "cytotoxic activity cytotoxic activity
  • helper activity suppressive activity
  • lymphocyte is a T lymphocyte that can kill a target cell expressing on its surface a peptide epitope-MHC molecular complex for which the TCR of the CTL is specific
  • a "CTL cell” is a T lymphocyte that can, subsequent to activation, kill a target cell expressing on its surface a peptide epitope-MHC molecular complex for which the TCR of the CTL is specific Activation can be, without limitation, by recognition of the relevant peptide epitope- MHC molecular complex by a TCR on the CTL or by a non-specific stimulus, e g , a T cell mitogen such as concanavalm A
  • a CTL cell can be a "virgin” T lymphocyte that has never previously been activated or a "memory" T lymphocyte that has previously been activated or the progeny of such a memory T lymphocyte
  • a "helper T lymphocyte” is a T lymphocyte that provides helper or regulatory activity in an immune response
  • Such an immune response can be, for example, an antibody-producing response of a B lymphocyte, a response of a CTL precursor cell, or an inflammatory or pro-inflammatory response of a variety of leukocyte types
  • a "Th cell” is a T lymphocyte that, subsequent to activation, provides helper activity m an immune response such as those listed above for Th Activation can be as indicated above for CTL cells
  • a Th cell can be a "virgin" T lymphocyte that has never previously been activated or a "memory" T lymphocyte that has previously been activated or the progeny of such a memory T lymphocyte
  • a B cell is a B lymphocyte that, subsequent to activation, can produce antibody molecules Activation of a B cell can be, without limitation, by recognition of an antigen by an antigen specific immunoglobulin receptor on the B cell surface or by a non-specific stimulus, e g , a B cell mitogen such as hpopolysaccha ⁇ de or pokeweed mitogen
  • a B cell can be a "virgin" B lymphocyte that has never previously been activated or a "memory" B lymphocyte that has previously been activated or the progeny of such a B lymphocyte
  • antigenic means capable of being recognized by an effector lymphocyte or an antibody molecule
  • a substance is antigenic if it is recognized by an antigen specific receptor on, for example, a CTL, a Th, or a B
  • lymphocyte producing antibody molecules or by an antibody molecule physically unassociated with a B lymphocyte
  • An immunogen can be a polypeptide, carbohydrate, glycohpid, hapten or biomimetic thereof
  • a polypeptide immunogen, as defined above, can include without limitation, a compound of two or more subumt ammo acids, ammo acid analogs, or other peptidomimetics, regardless of post-translational modification, e g , phosphorylation or glycosylation
  • the subumts may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds
  • the term "ammo acid” refers to natural and/or unnatural or synthetic ammo acids, including D/L optical isomers Full-length proteins, analogs, mutants, and fragments thereof are encompassed by this definition
  • ammo acid sequence of the immunogens disclosed herein can be identical to the wild-type sequences of approp ⁇ ate components Alternatively, any of the components can contain mutations such as deletions, additions, or substitutions All that is required is that the variant hgand have at least 5% (e g , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the immunogen contaimng only wild-type sequences to induce an immune response against the naturally ocur ⁇ ng wild-type immunogen Substitutions will preferably be conservative substitutions Conservative substitutions typically include substitutions withm the following groups glycine and alanine, valine, isoleucme, and leucine, aspartic acid and glutamic acid, asparagme, glutamme, serine and threonine, lysine, histidme and argmme, and phenylalanine and tyrosine
  • a polypeptide immunogen can include any peptide epitopes of a variety of lengths, for example, 7 - 50 (eg , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, or 50) ammo acid residues long
  • a polypeptide immunogen can include one or more epitopes, for example, 1, 2, 4, 6, 10, 20, 30, 50 or more
  • Polypeptide immunogens can include one or more post-transc ⁇ ptional modifications as desc ⁇ bed above in the Ligands section
  • An immunogen can be, without limitation, biotmylated, glycosylated, acetylated, alkylated, isoprenylated, hpoylated, or phosphorylated
  • An immunogen can also be a molecule that is not a protein, e g , a carbohydrate or glycolipid
  • carbohydrate and glycolipid lmmunogens include, without limitation, bacte ⁇ al hpopolysacchande (LPS) or a fragement of it, microbial products bound by various Toll like receptors (TLRs), bacterial diacyl and triacyl lopopeptides and lipoteichoic acid from bacte ⁇ a, and zymosan from yeast cell walls
  • the immunogen can be present m a killed or attenuated organism, m a crude cellular extract, a cell lysate or partially or substantially pure
  • substantially pure with respect to a naturally-occur ⁇ ng immunogen refers to an immunogen that has been separated from cellular components by which it is naturally accompanied, such that it is at least 60% (e g , 70%, 80%, 90%, 95%, or 99%), by weight, free from naturally-occur ⁇ ng organic molecules with which it is naturally associated
  • Methods for purifying lmmunogens are known to those in the art For example, in general, a substantially pure polypeptide will yield a single major band on a non-reducmg polyacrylamide gel
  • the immunogen can be a molecule expressed or released by any of a wide range of infectious agents, including, without limitation, viruses, viroids, bacte ⁇ a, fungi, p ⁇ ons or parasites
  • viral pathogens can include, without limitation, influenza viruses, including the strain A(H1N5), hepatitis viruses (e g, Hepatitis A, B, C and D), Arenaviruses, Bunyaviruses, Flavivimses, Filoviruses, Alphaviruses, (e g , Venezuelan equme encephalitis, eastern equme encephalitis, western equine encephalitis), Hantaviruses, human immunodeficiency viruses HIVl and HIV2, felme immunodeficiency virus, simian immunodeficiency virus, measles virus, rabies virus, rotaviruses, papilloma virus, respiratory syncytial virus, Variola, and viral encephalitides, (e g , West Nile Virus, LaCrosse, California encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, Kyasanur Forest Virus) Bacte ⁇
  • influenza viruses including the
  • Fungal pathogens can include, without limitation, members of the genera Aspergilllus, Penecillium, Stachybotrys, Tnchoderma, mycoplasma, Histoplasma capsulatum, Cryptococcus neoformans, Chlamydia trachomatis, and Candida albicans
  • Pathogenic protozoa can include, for example, members of the genera Cryptosporidium, e g , Cryptosporidium parvum , Giardia lambha, Microsporidia and Toxoplasma e g, Toxoplasma brucei, Toxoplasma gondii, Entamoeba histolytica, Plasmodium falciparum, Leishmama major and Cyclospora cayatanensis
  • influenza M2 protein antigen can be the ectodomam peptide M2e, for example, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO 1), or a variant of the ectodomam peptide M2e, for example, SEQ ID NO 3 or SEQ ID 4
  • An HBV preSl protein can include the preSl protein peptide 35-49, e g ,
  • FGANSNNPDWDFNPNKDHWPEANQVGA SEQ ID NO 2
  • useful non- peptidic immunogens include the pneumococcal polysaccha ⁇ des from Streptococcus pneumoniae
  • the immunogen can also be a molecule expressed by a mammal
  • an immunogen can be a molecule whose expression is correlated with a particular disease state, for example, cancer or neurodegenerative disease
  • the immunogen can be a tumor-associated antigen (TAA)
  • TAA tumor-associated antigen
  • a TAA is a molecule (e g , a polypeptide, carbohydrate or lipid) that is expressed by a tumor cell and either (a) differs qualitatively from its counterpart expressed m normal cells, or (b) is expressed at a higher level m tumor cells than in normal cells
  • a TAA can differ (e g , by one or more ammo acid residues where the molecule is a protein) from, or it can be identical to, its counterpart expressed m normal cells
  • it is not expressed by normal cells
  • it is expressed at a level at least two-fold higher (e g , a two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold, 100-fold, 500-fold, 1, 000-fold, 5,000-fold, or 15,000-fold higher) m a tumor cell than in the tumor cell's normal counterpart
  • relevant cancers include,
  • immunogens that can be included in the immune conjugates disclosed herein are those derived from antigens that are involved in the initiation or progression of neurodegenerative diseases, e g Alzheimer's disease and Transmissible Spongiform Encephalopathies (TSEs), e g , human p ⁇ on diseases such as Creutzfeld- Jacob disease (CJD), variant CJD ("mad cow disease'), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal familial Insomnia (FFI), animal prion diseases such as Scrapie in sheep, bovme spongiform encephalopathy (BSE) in cows, transmissible mink encephalopathy (TME) in mink, chronic wasting disease (CWD) in elk and deer
  • CJD Creutzfeld- Jacob disease
  • variant CJD ("mad cow disease')
  • GSS Gerstmann-Straussler-Scheinker syndrome
  • FFI Fatal familial Insomnia
  • a neurodegenerative antigen is a molecule (e g , a polypeptide, carbohydrate or lipid) that is expressed by a neuronal cell in an individual with a neurodegenerative disease and either (a) differs qualitatively from its counterpart expressed in cells from an individual who does not have the neurodegenerative disease, e g , the molecule appears m abnormal locations within the body or is associated with other molecules not normally found with the antigen m healthy individuals who do not have the neurodegenerative disease, or (b) is expressed at a higher level m cells from an individual who does not have the neurodegenerative disease
  • a neurodegenerative antigen can differ (e g , by one or more ammo acid residues where the molecule is a protem) from, or it can be identical to, its counterpart expressed in normal cells It is preferably not expressed by normal cells Alternatively, it is expressed at a level at least two-fold higher ⁇ e g , 3. two-fold, three-fold, five
  • Examples of neurodegenerative antigens found in Alzheimer's disease include beta-amyloid, tau protein, alpha synuclem
  • Other neurodegenerative disease antigens can be de ⁇ ved from prions
  • a prion is small protemaceous infectious particle that resists mactivation by procedures that modify nucleic acids Prions are encoded by the p ⁇ on-related protem gene (PrP) Mutant forms of the PrP protein aggregate as p ⁇ ons which can lead to fatal neurodegenerative disease
  • an immunogen can be a PrP polypeptide
  • Germ cell immunogens can be useful in the generation of immune responses that block the function of germ cells, thereby interfering with conception Germ cell antigens can include antigens on sperm cells Examples include, without limitation sperm adhesion molecule 1 (SPAM-I), and human intra-acrosomal protein
  • An immunogen can also be a non-toxic variant of a toxic substance (a "toxoid") that can be used to stimulate an immune response against the harmful form of the toxm
  • a toxoid can be, without limitation, a toxm that has been rendered less toxic or completely non-toxic through treatment with high temperature, aggregation, chemical reaction (e g , formalin fixation), couplmg to a carrier molecule, or molecular alteration (e g , deletion, augmentation or substitution)
  • a toxoid can be thus denved from a toxm such as, for example, ⁇ cm, anthrax or botuhnum toxm types A, B, C, D, E, F or G
  • An immunogen can also be a substance of abuse such as nicotine, or an opiate or opiate derivative Such an immunogen can induce antibodies capable of binding and neutralizing the corresponding substance of abuse
  • linker can be any reagent, molecule or macromolecule that connects the hgand and the immunogen such that a) the immune complex is stable under physiological conditions, b) the connection between the linker and the hgand does not alter the ability of the hgand to bind to its target on the surface of a circulating non-lymphoid cell, and c) the connection between the linker and the immunogen does not substantially affect the capacity of the immunogen to induce an effective immune response m a host against an infectious agent, cell or molecule on which the immunogen is naturally found
  • a linker can be a peptide bond That is, the ligand and the immunogen can be a fusion polypeptide comprising one or more ammo acid segments from the hgand and one or more ammo acid segments from the immunogen
  • the term "ammo acid segment" as used herein refers to a contiguous stretch of ammo acids within a polypeptide
  • the ammo acid residues 30 to 40 withm a 100 ammo acid polypeptide would be considered an ammo acid segment
  • An ammo acid segment can be a length greater than eight ammo acid residues (e g , greater than about nine, ten, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 500, 1000, or more ammo acid residues)
  • an ammo acid segment can have a length less than 1000 ammo acid residues (e g , less than 500, less than 400, less than 350, less than 300, less than 200, or less than 100 ammo acid residues)
  • ammo acid segments of the ligand can be contiguous with the ammo acid segments of the immunogen or they can be separated by ammo acids inserted as a structural spacer
  • a spacer segment can be one or more ammo acids
  • the one or more ammo acids can include amino acids that are the same or that are different
  • a spacer can be a repeating se ⁇ es of a neutral ammo acid (e g , glycine, alanine, valine, isoleucme or leucine) ranging in number from 1 to 10 or more (e g , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)
  • Another example of a spacer configuration can be a se ⁇ es of interspersed amino acids that may be neutral (e g , glycine alanme-glycme-alanine-glycme-alanme, or glycme-glycine-glycme-valme-valine-valme) or charged amm
  • a fusion protein can be produced in vitro by continuous peptide synthesis according to standard chemical methods know to those in the art Synthetic polypeptides can also be purchased from commercial sources
  • a fusion protein can also be produced by recombinant DNA techniques
  • Nucleic acid segments encoding the hgand can be operably linked in the same open reading frame to nucleic acid sequences encoding the immunogen in a vector that includes the requisite regulatory elements, e g , promoter sequences, transcription initiation sequences, and enhancer sequences, for expression in prokaryotic or eukaryotic cells
  • Methods well known to those skilled in the art can be used to construct expression vectors contaimng relevant coding sequences and approp ⁇ ate transcnptional/translational control signals
  • suitable vector systems can be purchased from commercial sources
  • nucleic acid segments encoding hgands and immunogens are readily available in the public domain
  • nucleic acid segments encoding hgands include, without limitation, the erythrocyte (glycopho ⁇ n A)-bmdmg antigen of Plasmodium falciparum EB A-173 (Bharara et al , MoI Biochem Parasitol 138 123-9, 2004), or mouse anti-human glycophonn A monoclonal antibody heavy chain (GenBank accession # AAZ67132) and corresponding light chain (Genbank accession # AAA21366))
  • nucleic acid segments encoding immunogens include, without limitation, Hepatitis virus C polyprotem (Genbank public gi number 2654998), influenza virus A conserved M2 protein ectodomarn peptide M2e (Genbank public gi number gi 78210829, HBV preSl protein (Genbank public gi number gi 92111469)
  • nucleic acid and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs
  • Polynucleotides can have any three-dimensional structure
  • a nucleic acid can be double-stranded or smgle- stranded (i e , a sense strand or an antisense strand)
  • Non-limitmg examples of polynucleotides include genes, gene fragments, exons, mtrons, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ⁇ bozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs
  • an isolated nucleic acid can be, for example, a naturally-occurring DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule m a naturally-occurring genome is removed or absent
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e g , a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or rest ⁇ ction endonuclease treatment)
  • An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote
  • an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hyb ⁇ d or fusion nucleic acid A nucle
  • Isolated nucleic acid molecules can be produced by standard techniques For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA Various PCR methods are desc ⁇ bed, for example, in PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds , Cold Spnng Harbor Laboratory Press, 1995 Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide p ⁇ mers that are identical or similar in sequence to opposite strands of the template to be amplified Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e g , using automated DNA
  • percent sequence identity refers to the degree of identity between any given query sequence and a subject sequence
  • a subject sequence typically has a length that is more than 80 percent, e g , more than 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120 percent, of the length of the query sequence
  • a query nucleic acid or ammo acid sequence can be aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1 83, default parameters), which allows alignments of nucleic acid or protein sequences to be earned out across their entire length (global alignment) Chenna et al , Nucleic Acids Res , 31(13) 3497-500 (2003) ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher bcm tmc edu/multi- ahgn/multi-align html)
  • exogenous nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not m its natural environment
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i e , a heterologous nucleic acid Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism
  • An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e g , non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where
  • nucleic acids can encode a polypeptide having a particular ammo acid sequence
  • the degeneracy of the genetic code is well known to the art, i e , for many amino acids, there is more than one nucleotide triplet that serves as the codon for the ammo acid
  • a "vector” is a rephcon, such as a plasmid, phage, or cosmid, mto which another DNA segment may be inserted so as to b ⁇ ng about the replication of the inserted segment
  • a vector is capable of replication when associated with the proper control elements
  • Suitable vector backbones include, for example, those routinely used m the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs
  • the term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors
  • An "expression vector” is a vector that includes a regulatory region
  • Suitable expression vectors include, without limitation, plasmids and viral vectors de ⁇ ved from, for example, bacteriophage, baculoviruses, and retroviruses Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto,
  • Vectors typically contain one or more regulatory regions
  • regulatory region refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transc ⁇ ptional start sites, termination sequences, polyadenylation sequences, and mtrons
  • the term "operably linked” refers to positioning of a regulatory region and a sequence to be transcribed m a nucleic acid so as to influence transcription or translation of such a sequence
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site
  • a promoter typically comprises at least a core (basal) promoter
  • a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR)
  • UAR upstream activation region
  • the vectors also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers
  • a marker gene can confer a selectable phenotype, e g , antibiotic resistance, on a cell hi addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e g , purification or localization) of the expressed polypeptide Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidme, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidme e-myc
  • hemagglutinin hemagglutinin
  • FlagTM tag FlagTM tag
  • the expression vectors disclosed herein containing the above described coding can be used, for example, to transfect or transduce either prokaryotic (e g , bacteria) cells or eukaryotic cells (e g , yeast, insect, or mammalian) cells Such cells can then be used, for example, for large or small scale in vitro production of the relevant fusion protein by methods known in the art In essence, such methods involve culturmg the cells under conditions which maximize production of the fusion protein and isolating the fusion protein from the cells or from the culture medium
  • the hgand and immnogen can be obtained separately, either through chemical synthesis or synthesis in vivo, purified and then linked non-covalently or covalently
  • a useful non-covalent linkage is a biotm-avidm linkage
  • the binding of biotm to avidm or streptavidm is essentially irreversible, with a reported Kd of 10 "15 M
  • biotin-avidm linkage refers to any linkage via biotm or a biotm denvative or biomimic (e g , Strep-Tag (IBA, St Louis, MO)) and avidm or an avidm denvative, streptavidm, or biotm-bmding fragments or subumts of avidm or streptavidm
  • biotmylated hgand can be linked to a biotinylated immunogen via avidm or streptavidm, or biotm binding fragments or subumts of avidm or streptavidm
  • avidm or streptavidm or biotm binding fragments or subumts of avidm or streptavidm
  • Methods for forming biotm-avidm linkages are well known to those in the art (See for example, Handbook of Affinity Chromatography, (Chromatographic Sciences Se ⁇ es, vol 63) ed T Kline, ISBN 0824789393 - Marcel Dekker (1993) Avidin and avidm derivatives are available from commercial sources (Pierce Biotechnology, Rockford, IL, Invitrogen, Carlsbad, CA)
  • the hgand and the immunogen can also be linked through a biotm- streptavidin linkage that includes an additional biotmylated immunoglobulin
  • a biotmylated hgand can be linked to an avidin molecule that is bound to a biotmylated antibody that specifically binds the immunogen
  • biotm-avidm heterocomplexes can be prepared to include 1 molecule of the hgand and 3 molecules of the immunogen, 2 molecules each of hgand and lmmmunogen, or 3 molecules of hgand and 1 molecule of immunogen Assembly of the biotin-avidm linkages can be performed in any order
  • the composition of the assembled immune conjugates can be validated by SDS-PAGE and western blotting and LC/MS methods
  • the hgand and the immunogen can also be synthesized as separate entities (by either chemical synthetic or recombinant methods) and then linked together by standard chemical methods known in the art
  • Chemical cross-linkmg agents can be homo-bifunctional (the same chemical reaction takes place at each end of the linker) or hetero-bifunctional (different chemical reactions take place at the ends of the linker)
  • the chemistries available for such linking reactions include, but are not limited to, reactivity with sulfhydryl, ammo, carboxyl, diol, aldehyde, ketone, or other reactive groups using electrophilic or nucleophilic chemistries, as well as photochemical cross-linkers using alkyl or aromatic azido or carbonyl radicals
  • An example of a targeted conjugate coupled via a homobifunctional cross-lmkmg reagent can be a complex of an anti-band 3 monoclonal antibody as the red blood cell-targeting component and anthrax protective antigen as the immunogen linked
  • Examples of chemical cross-linkmg agents include, without limitation, glutaraldehyde, carbodumides, bisdiazobenzidme, and N-maleimidobenzoyl-N- hydroxysuccimmide ester
  • Chemical cross-linkers are widely available from commercial sources (e g , Pierce Biotechnology (Rockford, IL), Invitrogen (Carlsbad, CA), Sigma- Ald ⁇ ch (St Louis, MO), and US Biological (Swampscott, MA))
  • the hgand and Hie immunogen can be connected through a linking polymer
  • linking molecules include, but are not limited to linear or branched polymers or co-polymers (e g , polyalkylene, poly(ethylene-lysme), polymethacrylate, polyammo acids, poly- or oligosaccharides, dendnmers)
  • the hgand and the immunogen can be attached to the linking molecule or microparticle through a non covalent high affinity linkage, e g , streptavidm-biotm high affinity binding or chemical cross-hnkmg techniques as desc ⁇ bed above
  • a polymer-supported targeted immunogen conjugate can be formed using a poly(ethylene-lysme) backbone
  • a poly(ethylene-lysme) backbone Such a linear copolymer backbone can be synthesized using bis(succimmidyl) poly(ethylene glyco ⁇ ooo) (Fluka Chemicals) to react with the ⁇ and ⁇ ammo groups of lysine
  • the available carboxyl termini of the lysines can be activated using (l-ethyI-3-[3-dimethylammopropyl] carbodnmide Hydrochlo ⁇ de) (EDC, Pierce Biotechnology) in preparation for coupling amme- contaimng compounds
  • EDC alkyimethylammopropyl] carbodnmide Hydrochlo ⁇ de
  • the total length of the co-polymer can be determined, in part, by the duration of the polymerization reaction
  • the targeting and IMG units can be combined in various ratios since there are up to 10 positions in
  • polymer-based targeted immunogen conjugate can be the addition of equimolar amounts of a targeting monoclonal antibody (e g , anti-CR2) and immunogen (e g- , influenza virus peptide M2e) to produce a complex with a co- polymenc scaffold studded with targeting antibody molecules as well as immunogen molecules
  • a targeting monoclonal antibody e g , anti-CR2
  • immunogen e g- , influenza virus peptide M2e
  • the hgand and the immunogen can be connected through a microparticle
  • linking microparticles include, but are not limited to, micelles, liposomes, fullerenes, nanotubes, or other colloidal complexes such as lipoproteins
  • Liposomes and micelles can be prepared by methods desc ⁇ bed m Lasic DD, 1998, TIBTech 16 307 Fullerenes and nanotubes can be purchased from American Dye Source (www adsdves com') Lipoproteins can be purchased from Biodesign International (www biodesien coin)
  • the ligand and the immunogen can be attached to the linking molecule or microparticle through a non-covalent high affinity linkage, e g , avidm-biotm high affinity binding or chemical cross-hnkmg techniques as desc ⁇ bed above
  • the hgand and/or the immunogen can be adsorbed or incorporated into a hydrophobic microparticle by hydrophobic affinity
  • a hgand and/or and immunogen with an available hydrophobic domain can spontaneously associate with a hydrophobic microparticle by hydrophobic partitioning
  • the hydrophobic domain on the ligand and/or immunogen can be a polyamino acid stretch comprised of repeating or mixed hydrophobic ammo acids (eg , poly- Ala, poly-Gly, poly-Leu, poly-Ile, or Ala-Gly-Leu-Ile (SEQ ID NO 5), etc ) or a bilayer-spanmng polypeptide from a known trans-membrane protein, such as membrane IgM), alkyl chains (e g , fatty acyl), or other hydrophobic structure (e g , steroid)
  • Such hydrophobic sequences can be naturally occurring sequences within the ligand and/or immunogen Alternatively,
  • the immune conjugates disclosed herein can include one or more of the same hgands or any combination of different ligands
  • the immune conjugate can also include one or more of the same immunogens or any combination of different immunogens
  • the immune conjugates can include immunogens that contain multiple copies (eg , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) of a single antigen or a single copy of multiple (e g , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) antigens or multiple copies (eg , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) of two or more antigens (e g , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more)
  • the immunogen can contain one or more copies of one or more peptide epitopes together with one or more copies of any of the non-peptide epitopes, e g , post transcriptional modifications, carbohydrates, or lipopolysacchandes
  • a targeted immune conjugate can include an immunogen that includes more than one polypeptide, or any combination of different polypeptides
  • each polypeptide m a composition can have an identical ammo acid sequence
  • the polypeptides in a composition can contain different amino acid segments, each of which can act as a defined immunogenic umt against which an immune response is desired
  • the polypeptides in a composition can contain different ammo acid segments that correspond to any region from a polypeptide including, without limitation, receptor binding regions, ligand binding regions, enzyme active sites, enzyme cleavage sites of polypeptide substrates, antigen-bindmg regions of antibodies, and epitopes recognized by antibodies
  • the administration of a polypeptide results in the formation of antibodies having specificity for an epitope or combination of epitopes formed by the ammo acid segments withm one or more of the polypeptides m the composition
  • the immune conjugates disclosed herein are generally useful for generating immune responses and as prophylactic vaccines or immune response- stimulating therapeutics
  • prophylactic vaccines or immune response- stimulating therapeutics can mean complete prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the seventy of subsequently developed disease symptoms
  • “therapy” can mean a complete abolishment of the symptoms of a disease or a decrease in the severity of the symptoms of the disease
  • the methods disclosed herem can be applied to a wide range of species, e g , humans, non human p ⁇ mates (e g , monkeys), horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, and mice Thus, they can be used, for example, as vaccines or therapeutic agents against infectious diseases, including diseases that can potentially result from bioterro ⁇ sm attacks
  • the immune conjugates can be used in the preparation of a medicament for treatment of an infectious disease
  • Infectious diseases can include diseases caused by any of the pathogens listed herein Examples include, without limitation, influenza, HTV-AIDS, hepatitis, botulism, plague, smallpox, tularemia, viral hemorrhagic fevers, brucellosis, gastrointestinal disease induced by pathogenic forms of £ coh, Salmonella and Shigella, glanders, melioidosis, psit
  • the immune conjugates can be useful as both prophylactics and therapeutics for cancer (e g , any of those recited above)
  • the immune conjugates can be employed to stimulate an immune response against cells in a cancer patient or can be admimstered in cases where a subject is at relatively high risk for a cancer (e g , lung cancer m a tobacco smoker or melanoma m a subject with multiple nevi)
  • the immune conjugates can also be useful in therapy or prophylaxis of neurodegenerative diseases
  • the immune conjugates can be admimstered to an individual with Alzheimer's disease or TSE or admimstered to an individual who is at risk for developing Alzheimer's disease or TSE
  • Immune conjugates disclosed herein can also be useful as a contraceptive vaccine, when the immunogen is a germ cell antigen
  • the immune conjugates disclosed herein can also be useful as prophylactics and therapeutics against medical conditions that result from exposure to toxms.
  • Such targeted immune conjugates that include non-toxic variants of toxic substances, e g , ncm, botulinum toxin, nicotine and other drugs, can be used to stimulate an immune response against the harmful form of the toxm, and thus protect against or mitigate the potential damage the toxm or drug may cause
  • the immune conjugates can be administered directly to a mammal
  • the immune conjugates can be used in the preparation of a medicament
  • the immune conjugates can be suspended m a pharmaceutically-acceptable carrier (e g , physiological salme)
  • a composition can be made by combining any of the immune conjugates provided herein with a pharmaceutically acceptable carrier
  • Such earners can include, without limitation, ste ⁇ le aqueous or non-aqueous solutions, suspensions, and emulsions
  • non-aqueous solvents include mineral oil, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, for example
  • Aqueous earners include, without limitation, water, alcohol, salme, and buffered solutions
  • Preservatives, flavonngs, and other additives such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like also may be present
  • any matenal descnbed herein that is to be administered to a mammal can
  • compositions descnbed herein can be administered to any part of the host's body
  • a composition can be delivered to, without limitation, the joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or pentoneal cavity of a mammal
  • a composition can be administered by intravenous, mtrapentoneal, intramuscular, subcutaneous, intramuscular, intrarectal, mtravagmal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, by inhalation, or by gradual perfusion over time
  • an aerosol preparation of a composition can be given to a host by inhalation
  • the dosage required depends on the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician Suitable dosages are in the range of 0 01 1,000 ⁇ g/kg Wide variations in the needed dosage are to be expected in view of the variety of immune conjugates available and the diffenng efficiencies of va ⁇ ous routes of administration Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art Administrations can be single or multiple (e g , 2- or 3-, A-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold) Encapsulation of the immune conjugate m a suitable delivery vehicle (e g , polymeric microparticles or implantable devices) may increase the efficiency of delivery
  • the duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e g , many years)
  • an immune conjugate can be administered once a month for three months or once a year for a pe ⁇ od often years
  • the frequency of treatment can be variable
  • an immune conjugate can be administered once (or twice, three times, etc ) daily, weekly, monthly, or yearly
  • the immune conjugates can be administered along with an adjuvant
  • adjuvant is an immunological compound that can enhance an immune response against a particular antigen such as a polypeptide
  • adjuvants include alum and other aluminum-based compounds (e g , AI 2 O 3 )
  • Aluminum- based compounds can be obtained from va ⁇ ous commercial suppliers
  • Other adjuvants include lmmuno-stimulatmg complexes (ISCOMs) that can contain such components as cholesterol and saponins, one or more additional immunostimulatory components, including, without limitation, muramyldipeptide (e g , N-acetyhnuramyl-L-alanyl-D- lsoglutarmne, MDP), monophosphoryl-hpid A (MPL), and formyl-mefhiomne containing t ⁇ peptides such as N-formyl-Met-Leu-Phe
  • ISCOMs lmmuno-stimulatmg complexes
  • compositions provided herein can contain any ratio of adjuvant to immune conjugate
  • the adjuvant immune conjugate ratio can be 50 50 (vol vol), for example Alternatively, the adjuvant immune conjugate ratio can be, without limitation, 90 10, 80 20, 70 30, 64 36, 60 40, 55 45, 40 60, 30 70, 20 80, or 90 10
  • an effective amount of any composition provided herem can be administered to a host
  • the term "effective" as used herem refers to any amount that induces a desired immune response while not inducing significant toxicity in the host Such an amount can be determined by assessing a host's immune response after administration of a known amount of a particular composition
  • the level of toxicity if any, can be determined by assessing a host's clinical symptoms before and after administering a known amount of a particular composition
  • the effective amount of a particular composition administered to a host can be adjusted according to a desired outcome as well as the host's response and level of toxicity Significant toxicity can vary for each particular host and depends on multiple factors including, without limitation, the host's disease state, age, and tolerance to pam
  • any method can be used to determine if a particular immune response is induced
  • antibody responses against a particular immunogen can be determined using an immunological assay (e g , ELISA or lymphocyte proliferation assay)
  • an immunological assay e g , ELISA or lymphocyte proliferation assay
  • the wells of a microtiter plate can be coated with the immunogen and incubated with serum from a mammal treated with the immune conjugate designed to produce antibodies against the corresponding immunogen in that mammal, and the presence or absence of antibodies against the immunogen can be determined by standard methods know to those in the art
  • clinical methods that can assess the degree of a particular disease state can be used to determine if a desired immune response is induced
  • a reduction m tumor burden can indicate a desired immune response in a patient treated with a composition designed to stimulate an immune response against a tumor antigen expressed on the patient's tumor
  • a polynucleotide containing a nucleic acid sequence encoding an immune conjugate of interest
  • liposomes prepared by standard methods
  • the vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies
  • Poly-L-lysme binds to a hgand that can bind to a receptor on target cells
  • Delivery of "naked DNA" ( ⁇ e , without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression
  • nucleic acid sequence encoding the fusion protein of interest with an initiator methionine and optionally a targeting sequence is operatively linked to a promoter or enhancer-promoter combination Promoters and enhancers are described above
  • Polynucleotides can be administered in a pharmaceutically acceptable earner
  • Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human or other mammalian subject, e g , physiological salme
  • a therapeutically effective amount is an amount of the polynucleotide which is capable of producing a medically desirable result (e g , a T cell response) in a treated mammal
  • the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently Dosages will vary, but a preferred dosage for administration of polynucleotide is from approximately 10 6 to 10 12 copies of the polynucleotide molecule This dose can be repeatedly admimstered, as needed Routes of administration can be any of those listed above
  • the immune conjugates provided herein can be admimstered in conjunction with other therapeutic modalities to an individual m need of therapy
  • the immune conjugates can be given p ⁇ or to, simultaneously with or after treatment with other agents Pn the case of infectious disease, the immune conjugates can be administered in conjunction with any antimicrobial agent, e g an antibiotic, e g including, without limitation, aminoglycosides, cephalosporins, macrohdes, penicillins, peptides, qmnolones, sulfonamides, tetracyclines, an antiviral, including without limitation, amantadine, rimantadine, zanamavir and oseltamivir, an anti-fungal, including, without limitation, echmocandm, caspofungm, anidulafungm, or anti-parasitic agent, including, without limitation, chlorqume, mebendazole, and clotrimazole
  • an antimicrobial agent e g
  • the immune conjugates can also be used in conjuction with standard anticancer therapies, including, without limitation, chemotherapy, e g , alkylating agents, anthracyclmes, cycloskeletal disruptors, topoisomerase inhibitors, nucleotide analogues, platinum based agents, retinoids, vmca alkaloids, radiation therapy, hormone ablation and surgery
  • standard anticancer therapies including, without limitation, chemotherapy, e g , alkylating agents, anthracyclmes, cycloskeletal disruptors, topoisomerase inhibitors, nucleotide analogues, platinum based agents, retinoids, vmca alkaloids, radiation therapy, hormone ablation and surgery
  • the immune conjugates can also be used m conjuction with other therapueitcs for neurodegenerative diseases, including donepezil, galantamine, memantine
  • articles of manufacture that can include immune conjugates as provided herein Components and methods for producing articles of manufacture are well known
  • An article of manufacture can include, for example, one or more immune conjugates
  • an article of manufacture further may include, for example, packaging materials, instructions for use, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required
  • Example 1 Assembly of tripartite conjugates of the biotmylated monoclonal antibody anti-TER-119, streptavidin and the biotinylated peptide M2e.
  • the peptide M2e (SLLTEVETPIRNEWGCRCNDSSD) (SEQ ID NO 1) was produced by peptide synthesis (BioWorld, Dublin OH) to contain a biotmylated lysine residue at its carboxy terminus
  • Anti-TER- 119 a rat anti-mouse IgG2b, ⁇ monoclonal antibody, was purchased biotmylated from BD-Pharmmgen, as was the isotype control, biotmylated rat IgG2b, ⁇
  • Streptavidin was purchased from Sigma- Aldrich
  • Biotmylated M2e peptide (b-M2e) (SEQ ID NO 1) was incubated with StAv in phosphate-buffered salme, 10 mM sodium phosphate, 140 mM NaCl, pH 74 (PBS) at ambient temperature at various molar ratios After 30 mm incubation biotmylated anti-TER-119 antibody (b-Ab) was added and the incubation was continued for an additional 30 mm with occasional agitation Reaction samples were analyzed by 3 to 8% gradient gel electrophoresis under native (or non-denaturmg) conditions (NDGE, Invitrogen, t ⁇ s-glycine running buffer) and visualized by Coomassie blue staining The results showed that mixtures of conjugates of different apparent molecular weights and stoichiometrics were formed caused by differential cross-hnkmg by StAv between the biotmylated peptide and the Ab The relative amounts of reagents used m the reactions are shown
  • Example 2 Analysis of anti M2e activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab - streptavidin - biotinylated M2e peptide
  • mice are challenged mtrape ⁇ toneally with an immune conjugate of biotinylated rat anti-mouse TER-119 Mab - streptavidm- biotinylated M2e peptide ((SEQ ID NO I)) and their serum antibody response to M2e (SEQ H) NO 1) is assayed
  • M2e peptide (SEQ EO NO 1) with a C-termmal addition of lysyl- ⁇ -N- biotm or PGGG (SEQ ID NO 6) is synthesized by solid phase peptide synthesis (Bio- World) This peptide is coupled to streptavidm (StAv) (Sigma- Ald ⁇ ch) at a peptide StAv molar ratio of 3 1 by mixing with b-Ab, as desc ⁇ bed m
  • StAv streptavidm
  • StAv streptavidm
  • Example 1 Identical tripartite conjugate is prepared using biotinylated rat IgG (BD Pharmmgen) as a control
  • TER-119-StAv and rat IgG-StAv conjugates the StAv is first reacted at a biotm StAv molar ratio of 3 1 to occupy 3 of the 4 biotin-bmdmg sites (BBSs) on each StA
  • mice Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are injected intravenously ( ⁇ v) in the tail vein with a range of free and conjugated peptide doses (0 01 to 10 ⁇ g equivalent of peptide m 50 ⁇ l PBS) Since StAv and TER-119 (a rat IgG) are also immunogenic m mice, Ab titers against them alone or in RBC-targeted conjugates are also evaluated Groups 1-6 are the minimum set to analyze the relative anti-M2e responses Groups 7-15 include additional controls to further analyze the anti-StAv and anti rat IgG responses, as well as the effect of simply mixmg the various components, instead of coupling them into targeted conjugates The expe ⁇ mental design is depicted in the table below
  • mice are boosted with a repeat i v injection after 2 and again after 4 weeks
  • Blood samples (-100 ⁇ l) are collected retro-orbitally prior to and every week after the primary injection for 5 weeks
  • Blood samples are chilled on ice and allowed to clot overnight
  • the tubes are cent ⁇ fuged and serum is collected and stored at -2O 0 C
  • Mouse IgG titers against all 3 antigens are determined by ELISA ELISA plates (hnmulon, VWR) are coated with 100 ⁇ l Ag at 10 ⁇ g/ml in sodium carbonate (Na 2 CO 3 ) buffer, 50 mM, pH 9 6 overnight at 4 0 C Plates are washed and blocked with 200 ⁇ l/well of 3% BSA in PBS containing 0 05% Tween-20 (PBST) for 1 5 hours at room temperature (RT) Wash 3X with 200 ⁇ l/well of PBST Dispense 100 ⁇ l/well of individual mouse sera at 1 50 dilution in PBST m duplicates and prepare senal dilutions in 1 3X increments Allow mouse antisera to bmd for 1 5 hours at RT and wash the plates as above Add 100 ⁇ l/well goat anti-mouse IgG conjugated with horseradish peroxida
  • Example 3 Analysis of anti M2e activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab-streptavidin- biotinylated M2e peptide (SEQ ID NO: 1): comparison of different routes of administration
  • Example 4 Analysis of anti-M2e fecal IgG and IgA activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab-streptavidin-biotinylated M2e peptide(SEQ ID NO: 1)
  • mice are dosed with TER-119 - StAv M2e conjugate and controls as depicted m Table 2 and following the dosmg schedule m Example 2
  • Example 5 Analysis of anti-M2e activity in mice challenged with the immune conjugate biotmylated rat anti-mouse CD21/CD35 Mab-streptavidin- biotinylated M2e peptide(SEQ ID NO: 1)
  • mice are injected with an immune conjugate of biotmylated rat anti-mouse CD21/CD35 Mab + streptavidm- biotmylated M2e peptide (influenza) and their serum antibody response to M2e is assayed
  • the anti-CD21/CD35 antibody is purchased from eBioscience
  • the conjugate is prepared according to the method desc ⁇ bed in Example 2
  • Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are injected intravenously (i v) with a range of free and conjugated peptide doses (0 01 to 10 ⁇ g equivalent of peptide m 50 ⁇ l PBS) according to the expenmental design in shown in Table 4 below
  • Example 6 Analysis of anti-M2e activity in mice challenged with the immune conjugate biotinylated Plasmodium falciparum EBA-175 peptide 1085-1096 (SEQ ID NO: 7) (pEBA) + streptavidin-biotinylated M2e peptide
  • Example 7 Analysis of anti-M2e activity in mice challenged with the synthetic peptide pEBA-Glycine-Glycine-Glycine-lVKe peptide.
  • mice are injected intravenously with a synthetic peptide of the sequence pEBA m -175 SEQ ID NO 7 linked via three glycine residues to the M2e peptide (SEQ ID NO 1) desc ⁇ bed m Example 1 above and according to the scheme depicted in Table 5 Each mouse receives a range of free and conjugated peptide doses (0 01 to 10 ⁇ g equivalent of M2e peptide m 50 ⁇ l PBS) intravenously and blood samples are collected and analyzed according to the schedule in Example 2
  • Example 8 Analysis of anti-HBV peptide (preSl amino acids 34-59 (SEQ ID NO:2), or pHBV) activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab + streptavidi ⁇ -bioti ⁇ ylated pHBV.
  • mice are challenged with immune conjugates in which the immunogen is an HBV (hepatitis B virus) peptide preSl ammo acids 34 59 (Hu WG, et al 2005 World J Gastroenterol 11 2088-2094 Identification of the immunogenic domains in HBsAg preS 1 region using overlapping preS 1 fragment fusion proteins) or pHBV coupled to
  • HBV hepatitis B virus
  • Example 9 Analysis of anti-M2e activity in mice challenged with the immune conjugate biotinylated mouse anti-mouse band 3 Mab-streptavidin- biotinylated M2e peptide (SEQ ID NO:1)
  • Mouse anti -mouse band 3 Mabs have been produced m several labs from NZB mice The immune conjugate, biotinylated mouse anti-mouse band 3 Mab- streptavidin-biotmylated M2e peptide, is produced according to the method in Example 1 with an IgG anti-mouse band-3 Mabs (e g , 34-3C or class-switched 4C8 (Fossati-Jrmack, 2002, J Autoimmun 18 17-25 Selective increase of autoimmune epitope expression on aged erythrocytes m mice implications in anti-erythrocyte autoimmune responses )), and is evaluated according to the protocol in Example 2
  • IgG anti-mouse band-3 Mabs e g , 34-3C or class-switched 4C8 (Fossati-Jrmack, 2002, J Autoimmun 18 17-25 Selective increase of autoimmune epitope expression on aged erythrocytes m mice implications in anti-erythrocyte autoimmune responses
  • the FluoReporter Biotm Quantitation Kit (Invitrogen) is used for estimating the molar ratio of biotm protein Mab anti-TER-119 is biotmylated using ammo-reactive (Pierce EZ-link NHS biotm) and carbohydrate-reactive (Pierce EZ-lmk hydrazide-biotm) reagents at various levels by adding a range of biotinylation molar excess factors (e g , 3X, 5X, 10X, 5OX, 250X) Add a 5X molar excess StAv per mole of biotmylated Mab to saturate all biotmylated sites Test functional integrity of the biotmylated Mab preparations by binding to mouse RBCs Use goat anti-rat IgG conjugated with fluoresceme isothiocyanate (FITC) to detect RBC-bound TER-119 using flow cytometry Select the condition that results in the highest degree of biotinylation of
  • Example 11 Determination of the optimal stoichiometry of Mab- StAv-IMG
  • Example 12 Comparison of monovalent, divalent and trivalent peptide-mediated targeting of IMG to RBC.
  • Biotmylated pEBA m (SEQ ID NO 7) (see Example 7) is used as the targeting hgand and biotmylated M2e is used as the IMG
  • the following constructs are prepared IpEBA 1n IStAv 3M2e, 2pEBA m IStAv 2M2e and 3pEBA m IStAv lM2e
  • IpEBA 1n IStAv 3M2e 2pEBA m IStAv 2M2e
  • 3pEBA m IStAv lM2e Inject mice with each of the constructs containing equal equivalents of M2e (1 0 ⁇ g) Evaluate the elicited immune response according to the methods m
  • Example 13 Evaluation of the efficacy of immunizing with a construct in which the targeting component and the IMG are covalently bound to a common polymer backbone
  • Example 14 Preparing targeted constructs using a microparticulate linking component.
  • Liposomes containing a biotmylated lipid in the bilayer are prepared
  • the exposed biotm groups are used to bind StAv and then the StAv's biotm binding sites are loaded with biotmylated targeting and IMG components at various ratios
  • the comparatively large liposomal surface permits adding many more units of each component without causing steric hindrance
  • pre-load 3 mole equivalents of either targeting or IMG units (or combinations of these) per mole of StAv Administer and analyze as in Example 2
  • Example 15 Preparing targeted constructs using a microparticulate linking component and a mixture of IMG's
  • Biotmylate a mixture of IMGs from a relevant source e g , the multiple strain variants of a multivalent pneumococcal polysaccharide vaccine or the various proteins or peptides from a viral vaccine
  • a suitable reactive biotm reagent m the case of oligosaccharides use a biotmylation reagent with a hydrazide reactive terminus (EZ-lmk hydrazide biotm, Pierce), and in the case of peptidic preparations use one with a terminus containing either a succimmidyl group for reaction with amines, a maleimidyl group for reaction with sulfliydryls, or an amine terminus reactive with carboxyls m the presence of ethylenediamme carbodiimide (EDC))
  • EDC ethylenediamme carbodiimide
  • Example 16 Using anti-band 3 Mab for targeting IMGs to senescent RBCs
  • Nabs pre-existmg natural IgG Abs
  • These Nabs have low affinity for band 3 They can only bind their target Ag on the RBC firmly if the band 3 molecules are clustered in the RBC membrane, through augmented avidity Band 3 cluste ⁇ ng occurs as a result of cumulative oxidative damage in the course of the life of the cells
  • the Mab can be a humanized IgG, for targeting IMGs to senescent RBCs
  • human anti-band 3 Mab To develop a human anti-band 3 Mab, immunize a mouse with human band 3 protein or with human red blood cells (which contain a senescent subpopulation) and fuse the spleen cells with a myeloma fusion partner (e g , SP2/0) Plate fused cells at 100,000 cells/well m hybndoma selection medium (HAT) After 3 weeks screen supernatants of wells with hybndoma clones by ELISA on wells coated with band 3 protein extracellular domain Subclone hybndomas that react specifically with human band 3 until the cultures are monoclonal
  • the resulting murine Mab can be "humanized" by methods familiar to those skilled m the art (e g , Recombinant Antibodies (1999), Breitlmg, F and Dubel, S (eds ), John Wiley & Sons) Alternately, human anti-band 3 Mabs can be produced by fusion of normal

Abstract

Disclosed herein are materials and methods related to vaccines. Materials and methods for delivery of immunogens to the reticuloendothelial system via non-circulating lymphoid cells are provided.

Description

TARGETED IMMUNE CONJUGATES
TECHNICAL FIELD
[0001] This invention relates to mateπals and methods involved m immunotherapy
BACKGROUND
[0002] Vaccination is one of the most important medical interventions for preventing disease The purpose of vaccination is to induce an optimal immune response that provides preventive or therapeutic benefit to the host Vaccines typically contain one or more immunogens (IMGs) that are harmless variants or derivatives of pathogens, which act to stimulate the immune system to mount defenses against the actual pathogen There are many types of immunogens (FMGs), ranging from attenuated or killed microorganisms, microbial extracts, whole proteins, polysaccharides, and peptides Some BVIGs are very effective inducers of the desired immune response, while others require the co-admmistration of non-specific immune stimulants, or adjuvants, or the coupling of the IMG to a carrier protein or microparticulate substances Still other IMGs are inherently poor at inducing effective immune responses, despite combination with adjuvants and repeated boosts Many of these inherently weak immunogens are involved m diseases e g , influenza and cancer, that remain major causes of human morbidity and mortality There is a continuing need for effective and more potent vaccines, particularly those with the capacity to stimulate a robust immune response against weakly immunogenic targets
SUMMARY
[0003] Disclosed herein are immune conjugates that are useful for inducing or enhancing an immune response against an antigen Accordingly, disclosed are immune conjugates that include a ligand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, and an immunogen coupled to the ligand, wherein the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen In one embodiment, the immune conjugate includes a
ligand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, wherein the molecule is selected from the group consisting of CR2, glycophorin A, band 3, Ter-119, blood group antigen H, blood group antigen A, blood group antigen B, CD41a, CD14, CD56, CD66d, CD83, CMKLRl, and BDCA-4, and an immunogen coupled to the ligand, where the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen
[0004] A circulating non- lymphoid cell can be any cell that circulates through the body of a mammal in the blood and/or lymph system, and is not a B lymphocyte or a T lymphocyte The circulating non-lymphoid cell can be, for example, a red blood cell, a platelet, a natural killer cell, a monocyte, a granulocyte or a plasmacytoid dendritic cell In one embodiment, the circulating non-lymphoid cell is a red blood cell In another embodiment, the circulating non-lymphoid cell is a platelet The circulating non- lymphoid cell can be derived from any mammal, e g , humans, a non-human primates, cattle, horses, pigs, sheep, goats, deer, elk, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, or mice
[0005] The cell-surface molecule can be any molecule that is differentially expressed on circulating non-lymphoid cells relative to the levels of the same molecule on a cell type that is not a circulating non-lymphoid cell The cell surface molecule can be a polypeptide, a carbohydrate, a phospholipid, or a glycohpid
[0006] In one embodiment, the cell surface molecule can be a molecule on the surface of a red blood cell, including, for example, complement receptor 2 (CR2), glycophonnA (CD235A), band 3 (CD233), TER-119, the ABO blood group antigens, e g , blood group antigen A, blood group antigen B, blood group antigen H, and phosphatidyl seπne In another embodiment, the cell surface molecule can be a molecule on the surface of a platelet, including, for example, gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectm) and CDl 51 In another embodiment, the cell surface molecule can be a molecule on the surface of a natural killer (NK) cell, including, for example, CD56 (NCAM, Leu-19, NKHl) In another embodiment, the cell surface molecule can be a molecule on the surface of a monocyte, including, for example, CD14 (LPS-receptor) In another embodiment, the cell surface molecule can be a molecule on the surface of a granulocyte, including, for example, CD66d (CGMl, a member of the CEA family) In another embodiment, the cell surface molecule can be a molecule on the surface of a plasmacytoid dendritic cell, including, for example, CMKLRl (serpentine chemokme- like receptor 1), BDCA-2 (CD303) and BDCA-4 (CD304)
[0007] The ligand can be any molecule capable of binding a specific cell surface molecule on a circulating non-lymphoid cell A ligand can include, for example, a polypeptide, a carbohydrate, glycolipid or biomimetic of a polypeptide, carbohydrate or glycohpid, as long as the ligand binds specifically to a cell surface molecule that is differentially expressed on a circulating non-lymphoid cell
[0008] A ligand can be a polypeptide, provided that it is not C3d, a heat shock protein, muramyl dipeptide, or muramyl tπpeptide
[0009] In one embodiment, a polypeptide ligand can be an antibody The antibody can be a monoclonal antibody, a polyclonal antibody, a holoantibody, a single chain variable fragment immunoglobulin, or a chimeric molecule that contains the constant region of an immunoglobulin and cell-binding sequences from a different source grafted in place of the immunoglobulin variable regions The antibody can be, for example, an antibody that recognizes targets on red blood cells eg , , complement receptor 2 (CR2), glycophoπn A (CD235A), band 3 (CD233), TER- 119, blood group antigen A, blood group antigen B, and blood group antigen H The antibody can be, for example, an antibody that recognizes targets on platelets, e g , gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectm) and CD151 The antibody can be, for example, an antibody that recognizes targets on monocytes, e g , CD 14 The antibody can be, for example, an antibody that recognizes targets on granulocytes, e g , CD66d The antibody can be, for example, an antibody that recognizes targets on NK cells, e g , CD56 The antibody can be, for example, an antibody that recognizes targets on plasmacytoid dendritic cells, e g , CMKLRl, BDCA-2 (CD303) and BDCA-4 (CD304)
[0010] A ligand can also be a polypeptide that is not an immunoglobulin, including for example, the erythrocyte-bmdmg antigen 175 (EBA- 175) of Plasmodium falciparum or a fragment of EBA- 175 that binds to a red blood cell, for example, SEQ ID NO 7, a complement fragment that binds to CR2, for example, C4b, C3b, iC3b, CIq, C3d or a peptide derived from these complement fragments, e g , C3 residues 1201-1214, or a
lectin, e g , & glycoprotein that recognizes blood group antigen A, blood group antigen B or blood group antigen H.
[0011] The polypeptide hgand can include post-translational modifications, e g , biotinylation, glycosylation, acetylation, alkylation, isoprenylation, hpoylation, phosphorylation
[0012] In another embodiment a hgand can also be a carbohydrate or glycolipid, including, for example, bacteπal lipopolysacchande or a fragment of it, microbial products bound by Toll-like receptors, bacteπal diacyl and tπacyl hpopeptides, lipoteichoic acid or zymosan
[0013] In another embodiment, a hgand can be a nucleic acid, including, for example, single- and double-stranded viral RNA and CpG DNA
[0014] The immunogen can be any molecule capable of eliciting a functional immune response in a mature T or a B lymphocyte or a precursor of a T or a B lymphocyte Irnrnunogens can include polypeptides, carbohydrates, glycolipids, haptens or biomimetics thereof The immunogen can be a molecule expressed or released by an infectious agent, including, for example, viruses, viroids, bactena, fungi, pπons or parasites An infectious agent can include for example, Orthomyxovmdae, e g , influenza viruses, including the strain A(H5N1), Rhadboviπdae, Hepadnavmdae, e g , hepatitis B, Picornavindae, e g , hepatitis A, Flavivindae, e g , hepatitis C, Retroviπdae, e g , human immunodeficiency viruses HFVl and HTV2, Togavindae, Bunyaviπdae, e g , hantavirus, Paramyxoviπdae, Herpesviπdae, Arenavindae, e g , lassa virus, Reoviπdae, Bacillus anthracis, Clostridium botuhnum, Salmonella entenditis, Escherichia coll, including E coll O157 H7, Streptococcus pneumoniae, Staphylococcus aureus, Aspergilllus, Stachybotrys, Candida, Cryptosporidium, Toxoplasma, and Plasmodium falciparum
[0015] Immunogens derived from pathogenic organisms can include, for example, influenza A M2 protein, hepatitis B surface antigen, HBV preSl protein, HIV tat, HIV gpl20, anthrax protective antigen, botuhnum toxm, and Streptococcus pneumoniae pneumococcal polysaccharides An influenza M2 protein antigen can be the ectodomam peptide M2e, for example SEQ ID NO 1, or a variant of the ectodomam
peptide M2e, for example, SEQ ID NO 3 or SEQ ID 4 An HBV preSl protein can include the preSl protein peptide 35-49, for example SEQ ID NO 2
[0016] The immunogen can also be a molecule expressed by a mammal For example, an immunogen can be a molecule whose expression is correlated with a particular disease for example, cancer or neurodegenerative disease The immunogen can be a tumor-associated antigen (TAA), including for example, MART-I, Muc-1, MAGE, RAGE, or CEA In another embodiment, the immunogen can be an antigen that is involved in the initiation or progression of neurodegenerative diseases, e g Alzheimer's disease and Transmissible Spongiform Encephalopathies (TSEs), including for example, beta-amyloid, tau protein, alpha synuclem, or a prion-related protem In another embodiment, the immunogen can be a germ cell antigen, including for example, sperm adhesion molecule 1 (SPAM-I), and human lntra-acrosomal protein In another embodiment, the immunogen can be a non-toxic variant of a toxic substance, including for example, ricm, botulmum toxins A, B, C, D, E, F and G, nicotine, or a drug of abuse such as an opiate or opiate derivative
[0017] The ligand and the immunogen are connected by a linker A linker can be any reagent, molecule or macromolecule that connects the ligand and the immunogen such that a) the immune complex is stable under physiological conditions, b) the connection between the linker and the ligand does not alter the ability of the ligand to bmd to its target on the surface of a circulating non- lymphoid cell, and c) the connection between the linker and the immunogen does not abolish the capacity of the immunogen to induce an effective immune response in a host against an infectious agent, cell or molecule on which the immunogen is naturally found
[0018] In one embodiment, a linker can be a peptide bond The ligand and the immunogen can be a fusion polypeptide comprising one or more ammo acid segments from the ligand and one or more ammo acid segments from the immunogen The ammo acid segments of the ligand can be contiguous with the ammo acid segments of the immunogen or they can be separated by ammo acids inserted as a structural spacer A spacer segment can be one or more ammo acids The one or more ammo acids can include ammo acids that are the same or that are different Also encompassed are nucleic acids composing a nucleotide sequence that encodes the fusion proteins, a vector (e g , s. vector that includes a transcriptional regulatory element (TRE) operably linked to the nucleotide sequence) containing the nucleic acid, and a cell (e g , a prokaryotic cell or a eukaryotic cell) containing the vector
[0019] hi another embodiment, the hgand and immunogen can be obtained separately, either through chemical synthesis or synthesis m vivo, purified and then linked non-covalently or covalently The non-covalent linkage can be a for example, a biotm-avidm (or streptavidm) linkage The covalent linkage can be through a chemical cross-linking agent, for example, a homobifunctional cross-lmkmg reagent or a heterobifunctional cross-lmkmg reagent In another embodiment, the hgand and the immunogen can be connected through a linking polymer, including, for example, linear or branched polymers or co-polymers (e g , polyalkylene, poly(ethylene-lysme), polymethacrylate, polyammo acids, poly- or oligosaccharides, or dendπmers)
[0020] In another embodiment, the hgand and the immunogen can be connected through a microparticle, including, for example, micelles, liposomes, fullerenes, nanotubes, or other colloidal complexes such as lipoproteins The hgand and the immunogen can be attached to the linking molecule or microparticle through a non- covalent high affinity linkage, e g , avidm-biotin high affinity binding, adsorbed or incorporated into a hydrophobic microparticle by hydrophobic affinity, or covalent chemical cross-lmkmg techniques
[0021] The immune conjugates provided herein can include one or more of the same hgands or any combination of different hgands The immune conjugates can also include one or more of the same immunogens or any combination of different immunogens
[0022] Also provided are methods and mateπals for inducing or enhancing an immune response m a mammal, the method comprising administering to the mammal an effective amount of a composition comprising an immune conjugate that includes a hgand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, and an immunogen coupled to the hgand, wherem the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen In one embodiment, the immune conjugate includes a hgand which bmds specifically to a cell surface molecule on a circulating non-lymphoid cell, wherem the molecule is selected from the group consisting of CR2, glycophorm A, band 3, Ter-119, blood group antigen H, blood group antigen A, blood group antigen B, CD41a, CD14, CD56, CD66d, CD83, CMKLRl, and BDCA-4, and an immunogen coupled to the hgand, where the immune conjugate, when administered to an individual, induces or enhances an immune response against the immunogen
[0023] The mammals can be, for example, humans, non-human pπmates, horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, and mice The mammal can have, be likely to have, or be at risk for having, an infectious disease, e g , a viral disease, a bacteπal disease, a protozooal disease, or a fungal disease Infectious diseases can include, for example, influenza, HTV-AIDS, hepatitis, botulism, smallpox, viral hemorrhagic fevers, gastrointestinal disease induced by pathogenic forms of E coh, Salmonella and Shigella, Staphylococcal infection, trypanosomiasis, and malaria Alternatively, the mammal can have, be likely to have, or be at nsk for having, a proliferative cell disease, e g , a cancer such as a neural tissue cancer, melanoma, breast cancer, lung cancer, a gastrointestinal cancer, ovarian cancer, testicular cancer, lung cancer, prostate cancer, cervical cancer, bladder cancer, vaginal cancer, liver cancer, renal cancer, bone cancer, a hematological cell cancer, or a vascular tissue cancer hi another embodiment, the mammal can have, be likely to have, or be at nsk for having, a neurodegenerative disease, e g , Alzheimer's disease or a Transmissible Spongiform Encephalopathy (TSE)
[0024] In another embodiment, methods and mateπals for inducing or enhancing an immune response in a mammal against a germ cell antigen are provided In a further embodiment, methods and mateπals for inducing or enhancing an immune response in a mammal against toxic substances, e g , ricm, botulinum toxm, nicotine and drugs of abuse are provided
[0025] Also provided are compositions featuring immune conjugates that include a hgand which binds specifically to a cell surface molecule on a circulating non- lymphoid cell, and an immunogen coupled to the hgand, wherem the immune conjugate, when administered to an individual, induces or enhances an immune response agamst the immunogen m a pharmaceutically acceptable earner or excipient hi another embodiment, the composition can include an adjuvant [0026] Also provided are articles of manufacture that can include immune conjugates as descπbed herein An article of manufacture can include, for example, one or more immune conjugates In addition, an article of manufacture further may include, for example, packaging mateπals, instructions for use, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required
[0027] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims
DESCRIPTION OF DRAWINGS
Figure 1 depicts the electrophoretic mobility of tripartite immune conjugates containing biotmylated antibody, streptavidm, and varying amounts of biotmylated M2e peptide (SEQ ID NO 1)
Figure 2 depicts the electrophoretic mobility of tripartite immune conjugates containing biotmylated antibody and a constant ratio of streptavidm, and biotmylated M2e peptide (SEQ ID NO 1)
DETAILED DESCRIPTION
[0028] The mammalian immune system mounts two different types of responses to immunogens (IMGs), humoral and cellular The humoral response, mediated by B lymphocytes, defends primarily against extracellular pathogens through the production of circulating antibodies that mark foreign cells and molecules for destruction by other specialized cells and proteins The cellular response, mediated by T lymphocytes, defends predominantly against intracellular pathogens and cancers by directly binding to and destroying the infected or cancerous cells Both responses depend upon specialized cells that internalize through endocytosis, pinocytosis or phagocytosis, and process IMGs, fragments of the IMGs are then presented to T lymphocytes, which m turn, help to trigger B-lymphoctye responses against the immunogens and/or the T cells to attack the target directly
[0029] The phagocytic cells that function as antigen-presentmg cells (APCs) are part of the reticuloendothelial system (RES) The RES is a diffuse system comprised of circulating and tissue-fixed cells including monocytes, macrophages, dendritic cells, Kupffer cells in the liver, Langerhans cells in the skin and microglial cells in the brain Several tissues and organs, by virtue of their wealth of phagocytic and specialized endothelial cells, comprise critical locations for the clearance and antigen presentation functions of the RES These include the liver, spleen, bone marrow and lymphatic tissues The RES plays an important role in clearing potentially harmful materials from the blood including micro-organisms, bacteπal endotoxins, immune complexes, tumor cells and senescent and damaged cells of the blood and lymph systems The cells that cycle through the RES, e g red blood cells (or erythrocytes), platelets, natural killer cells, monocytes, granulocytes, and plasmacytoid dendπtic cells, serve as earners of foreign materials, which may be shipped off in RES organs If the load of foreign material on the surface of the circulating cells is very dense, these cells themselves can be targets for phagocytic activity In addition, as circulating cells age, they acquire surface markers that cause them to be captured by the RES, removed from circulation and destroyed by phagocytosis, resulting in the degradation of the circulating cell components and their surface-bound mateπals Once these cells have been phagocytized, any fragments of macromolecules they originally contained, either endogenously expressed molecules or exogenous molecules that were opsonized to the cell surface can potentially be presented to T lymphocytes Thus, molecules bound to red blood cells, platelets, natural killer cells, monocytes, granulocytes, and plasmacytoid dendπtic cells can function as immunogens
[0030] Disclosed herein are mateπals and methods for the specific delivery of immunogens to the reticuloendothelial system via circulating non-lymphoid cells In particular, targeted immune conjugates are provided that specifically bind to circulating non-lymphoid cells, e g , red blood cells, platelets, natural killer cells, monocytes, granulocytes, and plasmacytoid dendπtic cells, that are cleared through the RES Thus, the circulating non-lymphoid cells act as vehicles for the specific delivery of IMGs to the RES and thereby to the immune system As used herein, the term "targeting immune conjugate" includes a hgand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell coupled to an IMG, where the targeted immune conjugate, when administered to an individual, induces or enhances an immune response against the IMG Also provided are methods of treatment using targeted immune conjugates Targeted immune conjugates provide injectable vaccines for efficient immunization against weakly immunogenic IMGs
11. Compositions
[0031] The targeting immune conjugates provided herein have the general formula
(L)y-X-(IMG)z
wherein L is a ligand which binds specifically to a cell surface molecule on a circulating non-lymphoid cell, X is a linker, BVIG is an immunogen, and y and z are integers having a value of one or greater than one
Circulating non-lymphoid cells and targets
[0032] As used herein, a circulating non-lymphoid cell can be any cell that a) circulates through the body of a mammal m the blood and/or lymph system, and b) is not a B lymphocyte or a T lymphocyte Thus, circulating non-lymphoid cells include erythrocytes, i e , red blood cells, platelets, natural killer cells, monocytes, granulocytes, i e , neutrophils, eosinophils, and basophils, and plasmcytoid dendritic cells
[0033] B and T lymphocytes are ultimately denved from hematopoietic stem cells and perform the pπncipal functions of the immune system T lymphocytes mature through the thymus and are generally identified by their expression of CD3 (which is associated with the T cell receptor) and either CD4 or CD8 CD8-expressing (or CD8+) T cells are principally involved with direct cell killing, or cytotoxicity CD4+ T cells are primarily regulatory cells which stimulate and suppress immune responses as needed B lymphocytes are characterized by their expression of CD19 or CD20, among other
surface markers, and they are responsible for antibody production B cells are also effective antigen presenting cells
[0034] Erythrocytes, also known as red blood cells, are the most abundant cell type in mammalian blood They are small disc-shaped, anucleated, biconcave cells whose primary function is to carry oxygen and carbon dioxide to and from the tissues Red blood cells express a distinctive complement of cell surface markers, including the human blood group antigens, glycophoπn, band 3 and the Lewis antigens
[0035] Platelets are denved from megakaryocytes, they are centrally involved in blood clotting, and can be identified by their surface expression of CD41a (or gpIIb/IIIa) Natural killer cells, also referred to as large granular lymphocytes, are denved from the bone marrow and do not express T-cell antigen receptors (TCR), the pan-T marker CD3 or surface immunoglobulins (Ig) B cell receptor, but typically express the surface markers CD 16 (FcγRIII) and CD56 Monocytes are denved from myeloid stem cells and are found pnmanly in the circulation They are competent phagocytes Upon their binding of pathogens and/or stimulation by various cytokines, monocytes mature mto macrophages, which are even more avid phagocytes and producers of many cytokines, degradative enzymes and other molecules that mediate inflammatory reactions Macrophages are generally found bound to vascular endothelium or within vanous tissues Monocytes (and macrophages) are charactenzed by the surface expression of CD 14, among other markers
[0036] Granulocytes are also denved from myeloid stem cells and are charactenzed by the presence of abundant granules in their cytoplasm, different classes of granulocytes, e g , eosinophils, basophils and neutrophils, are distinguished by their ability stain with eosin, basophilic dyes or neither, respectively Eosinophils are involved m defense against parasitic pathogens and allergens, basophils are also involved m allergic reactions Neutrophils are early and aggressive phagocytes at the site of infections and release products that induce inflammatory reactions
[0037] Plasmacytoid dendntic cells (pDC) are distmct from myeloid dendntic cells Both are found m the circulation and in tissues pDC are an important link between the innate and adaptive immune responses, in particular in mounting anti-viral
immune responses They produce abundant interferons and can be identified by the surface marker BDCA 2 (CD303)
[0038] The circulating non-lymphoid cells can be deπved from any mammal, e g , humans, a non-human primates, cattle, horses, pigs, sheep, goats, deer, elk, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, or mice
[0039] Any cell surface molecule that is differentially expressed on circulating non lymphoid cells relative to the levels of the same molecule on a cell type that is not a circulating non-lymphoid cell is a suitable target for the hgand The cell-surface molecule can be a polypeptide, a carbohydrate, or a glycohpid Full-length molecules, epitopes, analogs, mutants, and functional fragments thereof are encompassed by this definition A "functional fragment" of a molecule is a fragment of the molecule that is smaller (shorter where the molecule is a polypeptide) than the molecule per se but has at least 10% {e g , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99 5%, 100% or even more) of the hgand-bindmg activity of the molecule per se
[0040] Typically, a cell-surface molecule on a circulating non-lymphoid cell can be classified as being differentially expressed if the molecule is present at a level that is greater than the average level observed in cells that are not circulating non-lymphoid cells if the expression levels differ by at least 50% {eg , 50, 100, 200, 300% or more) Any method can be used to determine whether or not a specific gene product is expressed at a level that is greater or less than the average level of expression observed in control cells The level of expression of a cell surface polypeptide can be measured using any method such as immuno-based assays (e g , immunofluorescence, flow cytometry, ELISA), western blotting, or polyacrylamide gel electrophoresis with silver staining Levels of particular carbohydrates or lipids can be measured by immunodetection e g , ELISA, flow cytometry, or immunostammg using fluorochrome- or radioisotope-labeled antibodies or lectins In some embodiments, the level of expression from a particular gene can be measured by assessing the level of mRNA expression from the gene Levels of mRNA expression can be evaluated using, without limitation, northern blotting, slot blotting, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), or chip hybridization techniques Such methods can be used to determine simultaneously the relative expression levels of multiple mRNAs [0041] Examples of targets on red blood cells include, without limitation, complement receptor 2 (CR2), glycophoπn A (CD235A), band 3 (CD233), TER 119 (Kma et al , Br J Hematol 109 280-7, 2000), the ABO blood group antigens, e g , blood group antigen A, blood group antigen B, blood group antigen H, and phosphatidyl seπne (Hematology Basic Principles and Practice, R Hoffman, 2005, 4th ed New York Churchill-Livingstone) One useful red blood cell target is TER 119, a region selectively bound by the antibody, anti-TER-119 and which corresponds to the extracellular domain of glycophorm A Suitable platelet targets includes gplrb/illa (CD41a), CD42d, CD61, CD62P (P-selectin) and CD151 Natural killer cell targets include, for example, CD56 (NCAM, Leu-19, HNKl) Monocyte and granulocyte targets include, for example, CD14 (LPS-receptor) and CD66d (CGMl, a member of the CEA family), respectively Plasmacytoid dendritic cell targets include, for example, CMKLRl (serpentine chemokme like receptor 1), BDCA-2 (CD303) and BDCA-4 (CD304) (Kuby Immunology, J Kuby et al , edt , 2002, 5th edition, W H Freeman & Co )
Ligands
[0042] The term "ligand" as used herein refers to a molecule capable of binding a specific cell surface molecule on a circulating non lymphoid cell A ligand can be a polypeptide, a carbohydrate, glycolipid or biomimetic of a polypeptide, carbohydrate or glycolipid, as long as the ligand binds specifically to a cell surface molecule that is differentially expressed on a circulating non-lymphoid cell
[0043] As defined herein, ligands are defined to be "specifically binding" if 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross react with related target molecules The binding affinity of a ligand can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann NY Acad Sci 51 660-672, 1949) For example, a ligand disclosed herein can bind to its target with at least 1 5-fold, 2-fold, 5-fold, 10-fbld, 100-fold, 103- fold, 104-fold, 105-fold, 106-foldor greater affinity for the target than for a closely related or unrelated polypeptide A ligand can bind its target with high affinity (10"4M or less, 107M or less, 109M or less, or with subnanomolar affinity (0 9, 0 8, 0 7, 0 6, 0 5, 04, 0 3, 02, 0 1 nM or even less) Ligands can also be descnbed or specified m terms of their binding affinity to a target, for example, binding affinities include those with a Kd less than 5 x 102 M, 102 M, 5 x 103 M, 103 M, 5 x 10"4 M, 104 M, 5 x 105 M, 105 M, 5 x 106 M, 106 M, 5 x 107 M, 107 M, 5 x 108 M, 108 M, 5 x 109 M, 109 M, 5 x 10 10 M, 10 10 M, 5 x 10 " M, 10 u M, 5 x 10 n M, 10 n M, 5 x 10 13 M, 10 13 M, 5 x 10 14 M, 10 14 M, 5 x 10 15 M, or 10 15 M, or less
[0044] In some embodiments, the hgands disclosed herein do not bind to known related molecules In other embodiments, the hgands disclosed herein can bmd to orthologs, homologs, paralogs or variants, or combinations and subcombmations thereof, of their targets
[0045] As defined herein, a hgand that specifically binds to circulating non- lymphoid cells is a hgand the binds to circulating non-lymphoid cells and does not significantly bind to lymphoid cells For example, an immune conjugate that includes a hgand that specifically binds to red blood cells will bind to red blood cells and not significantly bind to other circulating cells, e g , lymphocytes, platelets, natural killer cells, monocytes, granulocytes or dendritic cells In another example, an immune conjugate that includes a hgand that specifically binds to platelets will bind to platelets and not significantly bind to other circulating cells, e g , lymphoid cells, red blood cells, natural killer cells, monocytes, granulocytes or dendritic cells
[0046] Ligands may be screened against known related target polypeptides to isolate a hgand that specifically binds the target For example, a hgand specific to a target will flow through an affinity chromatography column comprising other closely related target molecules adhered to insoluble matrix under appropriate buffer conditions Such screening allows isolation of ligands non-crossreactive to closely related targets (Antibodies A Laboratory Manual, Harlow and Lane (eds ), Cold Spπng Harbor Laboratory Press, 1988, Current Protocols in Immunology, Cooligan et al (eds ), National Institutes of Health, John Wiley and Sons, Inc , 1995) Screening and isolation of specific antibodies is well known m the art (see, Fundamental Immunology, W Paul (ed ), Raven Press, 1993, Getzoff et al , Adv in Immunol 43 1-98, 1988, Monoclonal Antibodies Principles and Practice, Goding, J W (ed ), Academic Press Ltd , 1996, Benjamin et al , Ann Rev Immunol 2 67-101, 1984) Representative examples of such assays include concurrent Immunoelectrophoresis, radioimmunoassay (RIA), radioimmunoprecipitation, flow cytometry, FACS, enzyme-linked immunosorbent assay (ELISA), dot blot or western blot assay, inhibition or competition assay, and sandwich assay
[0047] A hgand can be a polypeptide, provided that it is not C3d, a heat shock protein, muramyl dipeptide, or muramyl tπpeptide The term "polypeptide" as used herein refers to a compound of two or more subunit ammo acids, ammo acid analogs, or other peptidomimetics, regardless of post-translational modification, e g , phosphorylation or glycosylation The subumts may be linked by peptide bonds or other bonds such as, for example, dicysteme, ester or ether bonds The term "ammo acid" refers to natural and/or unnatural or synthetic ammo acids, including D/L optical isomers Full-length proteins, analogs, mutants, and fragments thereof are encompassed by this definition
[0048] The ammo acid sequence of the hgands disclosed herem can be identical to the wild-type sequences of appropπate components Alternatively, any of the components can contain mutations such as deletions, additions, or substitutions All that is required is that the variant hgand have at least 5% (e g , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the hgand containing only wild-type sequences to specifically bind the target on the circulating non- lymphoid cell Substitutions will preferably be conservative substitutions Conservative substitutions typically include substitutions within the following groups glycine and alanine, valine, lsoleucme, and leucine, aspartic acid and glutamic acid, asparagme, glutamme, seπne and threonine, lysine, mstidine and argmme, and phenylalanine and tyrosine
[0049] Any method can be used to make a polypeptide including, for example, expression by prokaryotic systems, expression by eukaryotic systems, and chemical synthesis techniques Any method can be used to purify a polypeptide including, without limitation, fractionation, centπfugation, and chromatography, e g , gel filtration, ion exchange chromatography, reverse-phase HPLC and immunoaffmity purification
[0050] A suitable polypeptide ligand can be an antibody In one embodiment, the antibody can be a monoclonal antibody, ; e , homogeneous antibodies of identical antigenic specificity produced by a smgle clone of antibody-producing cells In another embodiment, the antibody can be a polyclonal antibody, i e , heterogenous antibodies that can recognize different epitopes on the same antigen and that are produced by more than one clone of antibody producing cells
[0051] The antibody can include any class of immunoglobulin, e g , IgG, IgA, IgM, and can be derived from any species e g , humans, mice, rats, or can be a humanized version of a non-human antibody An antibody can include, without limitations, a holoantibody, i e , an antibody that includes one or more of an immunoglobulin monomer units of two heavy and two light chains, a single cham variable fragment immunoglobulin, or a chimeπc molecule that contains the constant region of an immunoglobulin and cell-binding sequences from a different source grafted in place of the immunoglobulin variable regions The cell-binding regions can be from a different antibody, a lectin, a cytokine, a microbial protein fragment or any other molecule that binds the target cell receptor molecule with specificity
[0052] Methods for producing antibodies are well know to those in the art, Antibodies can be purified by chromatographic methods known to those of skill m the art, including ion exchange and gel filtration chromatography (for example, Came et al , Protein Expr Punf (1996) 8(2) 159-166) Alternatively or in addition, antibodies can be purchased from commercial sources, for example, Invitrogen (Carlsbad, CA), MP Biomedicals (Solon, OH), Nventa Biopharmaceuticals (San Diego, CA) (formerly Stressgen), Serologicals Corp (Norcross, GA)
[0053] The antibody can be, for example, an antibody that recognizes targets on red blood cells, including for example, without limitation, glycophoπn A (CD235A), band 3 (CD233), TER-119, blood group antigen A, blood group antigen B, and blood group antigen H One useful antibody is anti-TER-119, which specifically binds to TER- 119, a region corresponding to the extracelllular domain of glycophoπn A Other suitable antibodies include, without limitation, antibodies that recognize targets on platelets, eg , gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectin) and CD151, monocytes, e g , CD14, NK cells, e g , CD56, granulocytes, e g , CD66d, and plasmacytoid dendritic cells, e g , CMKLRl,BDCA-2 (CD303) and BDCA-4 (CD304)
[0054] A ligand can also be a polypeptide that is not an immunoglobulin One non-immunoglobulm type ligand can be the erythrocyte-bindmg antigen 175 (EBA-175) of Plasmodium falciparum, which specifically hmds the red blood cell surface protein band 3, or a fragment of EBA-175 that binds to a red blood cell, for example EBA-175 peptide 1085-96, SEQ ED NO 7 Another polypeptide ligand can also be a complement fragment that binds to CR2, for example, C4b, C3b, iC3b, CIq or a peptide derived from the complement fragments, e g , C3 residues 1201-1214 (Tsokos et al , Journal of Immunol 144 1640-45, 1990) or residues 727 768 (Becherer, Biochemistry 31(6) 1787- 94 1992) A polypeptide ligand can also be a lectin, e g & glycoprotein that recognizes blood group antigen A, blood group antigen B or blood group antigen H
[0055] A ligand can also be a peptidomimetic, a small protem-hke chain containing non-peptidic structural elements that is capable of mimicking or antagonizing the biological action(s) of a natural parent peptide Peptidomimetic compounds are synthetic, non-peptide compounds having a three-dimensional conformation (; e , a "peptide motif) that is substantially the same as the three dimensional conformation of a selected peptide The peptide motif provides the peptidomimetic compound with the ability to bind the ligand in a manner qualitatively identical to that of the parent peptide from which the peptiomimetic was deπved Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased prolonged biological half-life
[0056] The peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based Several types of chemical bonds, eg , ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known m the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics
[0057] Any peptidomimetic that binds specifically and selectively to a cell surface molecule that is differentially expressed on a circulating non-lymphoid cell can be used Examples of useful peptidomimetics include those that mimic the ability of antibodies that recognize, for example, complement receptor 1 (CRl), complement receptor 2 (CR2), glycophorm A (CD235A), band 3 (CD233), TER-119, blood group antigen A, blood group antigen B, and blood group antigen H, gpIIb/IJIa (CD41a), CD42d, CD61, CD62P (P-selectm), CD151, CD14, CD56, CD66d, CMKLRl and BDCA-2
[0058] The polypeptide can include post-translational modifications, i e , chemical modification of the polypeptide after its synthesis Chemical modifications can be naturally occurring modifications made m vivo following translation of the mRNA encoding the polypeptide or synthetic modifications made in vitro A polypeptide can include one or more post-translational modifications, in any combination of naturally occurring, i e , in vivo, and synthetic modifications made in vitro Examples of post- translational modifications include, but are not limited to, biotinylation, e g , acylation of lysine or other reactive ammo acid residues with a biotm molecule, glycosylation, e g , addition of a glycosyl group to either asparagmes, hydroxylysme, seπne or threonine residues to generate a glycoprotein or glycopeptide, acetylation, e g , the addition of an acetyl group, typically at the N-termmus of a polypeptide, alkylation, eg , the addition of an alkyl group, isoprenylation, e g , the addition of an isoprenoid group, hpoylation, e g attachment of a hpoate moeity, phosphorylation, e g , addition of a phosphate group to seπne, tyrosine, threonine or histidme
[0059] A highly suitable post-translational modification can be biotmylation Biotm, also known as vitamin H or B7 is a water-soluble B-complex vitamin that binds avidm or streptavidm with very high affinity (10 15M) Both egg avidm and bacteπal streptavidm have 4 biotm-bmdmg sites and thus can serve to couple several biotinylated ligands or to couple biotinylated hgand(s) to other biotinylated molecules (e g , IMGs) Polypeptides can be convalently linked to one or more biotm molecules through primary amines, e g lysine and N-termmus), carboxyl groups found on aspartic- and glutamic- acid residues and at the C-termmus, sulfhydryl groups, or carbohydrate modifications on glycoproteins Methods for denvatizmg polypeptides with biotm are well known in the art and there are many commercial sources for such reagents (e g Pierce, Sigma- Aldπch) Any method of biotmylation can be used that preserves the ability of the hgand to bind to its target on the non-circulatmg lymphoid cell For example, desirable biotmlyation methods would modify residues in the Fc portion of the antibody without compromising the lmmunoreativity of the antibody Polypeptides derivatized with biotm can then be linked to immunogens through an avidm or streptavidm molecule [0060] A post-translational modification can be glycosylation, i e , the addition of saccharides Glycosylation is typically classified based on the ammo acid through which the saccharide linkage occurs and can include N linked glycosylation to the amide nitrogen of asparagmes side chains, 0-lmked glycosylation to the hydroxyl oxygen of serine and threonine side chains, and C-mannosylation
[0061 ] A ligand can also be a carbohydrate or glycolipid Examples of carbohydrate and glycolipid ligands, include, without limitation, bacteπal lipopolysacchande (LPS) or a fragment of it, microbial products bound by Toll-like receptors (TLRs), bacterial diacyl and tnacyl hpopeptides and lipoteichoic acid from bacteπa, and zymosan from yeast cell walls
[0062] A ligand can also be a nucleic acid Examples of nucleic acids include, without limitation, single- and double-stranded RNA from viruses, and CpG DNA from bacteπa or viruses
Immunogens
[0063] As defined herein, an immunogen is any molecule capable of eliciting a functional immune response (e g , a cytotoxic or helper T cell response or an antibody producing response) m a T or a B cell
[0064] As used herein, an "effector T lymphocyte" is a T lymphocyte having immunological activity Such immunological activity can be, without limitation, cytotoxic activity, helper activity, suppressive activity, immune-deviating activity, inflammatory activity, or pro-inflammatory activity As used herein, an "effector T lymphocyte precursor cell" is a T lymphocyte that, subsequent to activation, has any of the above immunological activities Activation can occur, without limitation, by recognition of a complex of the relevant immunogenic peptide epitope and the major histocompatibility complex (MHC) molecule by the T cell receptor (TCR) on the effector T lymphocyte or by a non-specific stimulus, e g , a T cell mitogen such as concanavalm A Thus, an effector T lymphocyte cell can be a "virgin" T lymphocyte that has never previously been activated or a "memory" T lymphocyte that has previously been activated or the progeny of such a memory T lymphocyte As used herein, a "cytotoxic T
lymphocyte" (CTL) is a T lymphocyte that can kill a target cell expressing on its surface a peptide epitope-MHC molecular complex for which the TCR of the CTL is specific
[0065] As used herein, a "CTL cell" is a T lymphocyte that can, subsequent to activation, kill a target cell expressing on its surface a peptide epitope-MHC molecular complex for which the TCR of the CTL is specific Activation can be, without limitation, by recognition of the relevant peptide epitope- MHC molecular complex by a TCR on the CTL or by a non-specific stimulus, e g , a T cell mitogen such as concanavalm A Thus, a CTL cell can be a "virgin" T lymphocyte that has never previously been activated or a "memory" T lymphocyte that has previously been activated or the progeny of such a memory T lymphocyte
[0066] As used herein, a "helper T lymphocyte" (Th) is a T lymphocyte that provides helper or regulatory activity in an immune response Such an immune response can be, for example, an antibody-producing response of a B lymphocyte, a response of a CTL precursor cell, or an inflammatory or pro-inflammatory response of a variety of leukocyte types As used herein, a "Th cell" is a T lymphocyte that, subsequent to activation, provides helper activity m an immune response such as those listed above for Th Activation can be as indicated above for CTL cells Furthermore, a Th cell can be a "virgin" T lymphocyte that has never previously been activated or a "memory" T lymphocyte that has previously been activated or the progeny of such a memory T lymphocyte
[0067] As used herein, a B cell is a B lymphocyte that, subsequent to activation, can produce antibody molecules Activation of a B cell can be, without limitation, by recognition of an antigen by an antigen specific immunoglobulin receptor on the B cell surface or by a non-specific stimulus, e g , a B cell mitogen such as hpopolysacchaπde or pokeweed mitogen Thus, a B cell can be a "virgin" B lymphocyte that has never previously been activated or a "memory" B lymphocyte that has previously been activated or the progeny of such a B lymphocyte
[0068] As used herein, "antigenic" means capable of being recognized by an effector lymphocyte or an antibody molecule Thus a substance is antigenic if it is recognized by an antigen specific receptor on, for example, a CTL, a Th, or a B
lymphocyte producing antibody molecules or by an antibody molecule physically unassociated with a B lymphocyte
[0069] An immunogen can be a polypeptide, carbohydrate, glycohpid, hapten or biomimetic thereof A polypeptide immunogen, as defined above, can include without limitation, a compound of two or more subumt ammo acids, ammo acid analogs, or other peptidomimetics, regardless of post-translational modification, e g , phosphorylation or glycosylation The subumts may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds The term "ammo acid" refers to natural and/or unnatural or synthetic ammo acids, including D/L optical isomers Full-length proteins, analogs, mutants, and fragments thereof are encompassed by this definition
[0070] The ammo acid sequence of the immunogens disclosed herein can be identical to the wild-type sequences of appropπate components Alternatively, any of the components can contain mutations such as deletions, additions, or substitutions All that is required is that the variant hgand have at least 5% (e g , 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the immunogen contaimng only wild-type sequences to induce an immune response against the naturally ocurπng wild-type immunogen Substitutions will preferably be conservative substitutions Conservative substitutions typically include substitutions withm the following groups glycine and alanine, valine, isoleucme, and leucine, aspartic acid and glutamic acid, asparagme, glutamme, serine and threonine, lysine, histidme and argmme, and phenylalanine and tyrosine
[0071] A polypeptide immunogen can include any peptide epitopes of a variety of lengths, for example, 7 - 50 (eg , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, or 50) ammo acid residues long A polypeptide immunogen can include one or more epitopes, for example, 1, 2, 4, 6, 10, 20, 30, 50 or more
[0072] Polypeptide immunogens can include one or more post-transcπptional modifications as descπbed above in the Ligands section An immunogen can be, without limitation, biotmylated, glycosylated, acetylated, alkylated, isoprenylated, hpoylated, or phosphorylated
[0073] An immunogen can also be a molecule that is not a protein, e g , a carbohydrate or glycolipid Examples of carbohydrate and glycolipid lmmunogens, include, without limitation, bacteπal hpopolysacchande (LPS) or a fragement of it, microbial products bound by various Toll like receptors (TLRs), bacterial diacyl and triacyl lopopeptides and lipoteichoic acid from bacteπa, and zymosan from yeast cell walls
[0074] The immunogen can be present m a killed or attenuated organism, m a crude cellular extract, a cell lysate or partially or substantially pure The term "substantially pure" with respect to a naturally-occurπng immunogen refers to an immunogen that has been separated from cellular components by which it is naturally accompanied, such that it is at least 60% (e g , 70%, 80%, 90%, 95%, or 99%), by weight, free from naturally-occurπng organic molecules with which it is naturally associated Methods for purifying lmmunogens are known to those in the art For example, in general, a substantially pure polypeptide will yield a single major band on a non-reducmg polyacrylamide gel
[0075] The immunogen can be a molecule expressed or released by any of a wide range of infectious agents, including, without limitation, viruses, viroids, bacteπa, fungi, pπons or parasites
[0076] For example, viral pathogens can include, without limitation, influenza viruses, including the strain A(H1N5), hepatitis viruses (e g, Hepatitis A, B, C and D), Arenaviruses, Bunyaviruses, Flavivimses, Filoviruses, Alphaviruses, (e g , Venezuelan equme encephalitis, eastern equme encephalitis, western equine encephalitis), Hantaviruses, human immunodeficiency viruses HIVl and HIV2, felme immunodeficiency virus, simian immunodeficiency virus, measles virus, rabies virus, rotaviruses, papilloma virus, respiratory syncytial virus, Variola, and viral encephalitides, (e g , West Nile Virus, LaCrosse, California encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, Kyasanur Forest Virus) Bacteπal pathogens can include, but are not limited to, Bacillus anthracis Yersinia pestis Yersinia enterocohtica, Clostridium botuhnum, Clostridium perfringens Francisella tularensis, Brucella species, Salmonella spp , including Salmonella entenditis, Escherichia coh, including E coli 0157 H7, Streptococcus pneumoniae, Staphylococcus aureus, Burkholdena mallei, Burkholdena pseudomallei, Chlamydia spp , Coxiella burnetii, Rickettsia prowazehi, Vibrio spp , Shigella spp Listeria monocytogenes, Mycobacteria tuberculosis, M leprae, Borreha burgdorferi, Actinobacillus pleuropneumoniae, Helicobacter pylori, Neisseria meningitidis Bordetella pertussis Porphyromonas gmgivahs, and Campylobacter jejuni
[0077] Fungal pathogens can include, without limitation, members of the genera Aspergilllus, Penecillium, Stachybotrys, Tnchoderma, mycoplasma, Histoplasma capsulatum, Cryptococcus neoformans, Chlamydia trachomatis, and Candida albicans
[0078] Pathogenic protozoa can include, for example, members of the genera Cryptosporidium, e g , Cryptosporidium parvum , Giardia lambha, Microsporidia and Toxoplasma e g, Toxoplasma brucei, Toxoplasma gondii, Entamoeba histolytica, Plasmodium falciparum, Leishmama major and Cyclospora cayatanensis
[0079] Examples of useful immunogens derived from pathogenic organisms include, for example, but are not limited to, influenza A M2 protein, hepatitis B surface antigen, HBV preSl protein, HTV tat, HIV gpl20, anthrax protective antigen, and botulmum toxin An influenza M2 protein antigen can be the ectodomam peptide M2e, for example, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO 1), or a variant of the ectodomam peptide M2e, for example, SEQ ID NO 3 or SEQ ID 4 An HBV preSl protein can include the preSl protein peptide 35-49, e g ,
FGANSNNPDWDFNPNKDHWPEANQVGA (SEQ ID NO 2) Examples of useful non- peptidic immunogens include the pneumococcal polysacchaπdes from Streptococcus pneumoniae
[0080] The immunogen can also be a molecule expressed by a mammal For example, an immunogen can be a molecule whose expression is correlated with a particular disease state, for example, cancer or neurodegenerative disease
[0081] Thus, the immunogen can be a tumor-associated antigen (TAA) As used herein, a TAA is a molecule (e g , a polypeptide, carbohydrate or lipid) that is expressed by a tumor cell and either (a) differs qualitatively from its counterpart expressed m normal cells, or (b) is expressed at a higher level m tumor cells than in normal cells Thus, a TAA can differ (e g , by one or more ammo acid residues where the molecule is a protein) from, or it can be identical to, its counterpart expressed m normal cells Preferably it is not expressed by normal cells Alternatively, it is expressed at a level at least two-fold higher (e g , a two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold, 100-fold, 500-fold, 1, 000-fold, 5,000-fold, or 15,000-fold higher) m a tumor cell than in the tumor cell's normal counterpart Examples of relevant cancers include, without limitation, hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such ovarian cancer, vaginal cancer, bladder cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, and vascular tumors Relevant TAAs include, without limitation, carcino embryonic antigen (CEA), RAGE, MART (melanoma antigen), MAGE (melanoma antigen) 1-4, 6 and 12, MUC (mucin) (e g , MUC-I, MUC-2, etc ), tyrosinase, Pmel 17 (gplOO), GnT-V mtron V sequence (N-acetylglucoaminyltransferase V mtron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catemn, MUM-I-B (melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, C-ERB2 (Her2/neu), EBNA (Epstem-Barr Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance protein (LRP) Bcl-2, prostate specific antigen (PSA), and Ki-67
[0082] Other immunogens that can be included in the immune conjugates disclosed herein are those derived from antigens that are involved in the initiation or progression of neurodegenerative diseases, e g Alzheimer's disease and Transmissible Spongiform Encephalopathies (TSEs), e g , human pπon diseases such as Creutzfeld- Jacob disease (CJD), variant CJD ("mad cow disease'), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal familial Insomnia (FFI), animal prion diseases such as Scrapie in sheep, bovme spongiform encephalopathy (BSE) in cows, transmissible mink encephalopathy (TME) in mink, chronic wasting disease (CWD) in elk and deer
[0083] As used herein, a "neurodegenerative antigen" is a molecule (e g , a polypeptide, carbohydrate or lipid) that is expressed by a neuronal cell in an individual with a neurodegenerative disease and either (a) differs qualitatively from its counterpart expressed in cells from an individual who does not have the neurodegenerative disease, e g , the molecule appears m abnormal locations within the body or is associated with other molecules not normally found with the antigen m healthy individuals who do not have the neurodegenerative disease, or (b) is expressed at a higher level m cells from an individual who does not have the neurodegenerative disease Thus, a neurodegenerative antigen can differ (e g , by one or more ammo acid residues where the molecule is a protem) from, or it can be identical to, its counterpart expressed in normal cells It is preferably not expressed by normal cells Alternatively, it is expressed at a level at least two-fold higher {e g , 3. two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold, 100- fold, 500 fold, 1, 000-fold, 5,000-fold, or 15,000-fold higher) in a tumor cell than in the tumor cell's normal counterpart
[0084] Examples of neurodegenerative antigens found in Alzheimer's disease include beta-amyloid, tau protein, alpha synuclem Other neurodegenerative disease antigens can be deπved from prions As defined herein, a prion is small protemaceous infectious particle that resists mactivation by procedures that modify nucleic acids Prions are encoded by the pπon-related protem gene (PrP) Mutant forms of the PrP protein aggregate as pπons which can lead to fatal neurodegenerative disease Thus, an immunogen can be a PrP polypeptide
[0085] Germ cell immunogens can be useful in the generation of immune responses that block the function of germ cells, thereby interfering with conception Germ cell antigens can include antigens on sperm cells Examples include, without limitation sperm adhesion molecule 1 (SPAM-I), and human intra-acrosomal protein
[0086] An immunogen can also be a non-toxic variant of a toxic substance (a "toxoid") that can be used to stimulate an immune response against the harmful form of the toxm A toxoid can be, without limitation, a toxm that has been rendered less toxic or completely non-toxic through treatment with high temperature, aggregation, chemical reaction (e g , formalin fixation), couplmg to a carrier molecule, or molecular alteration (e g , deletion, augmentation or substitution) A toxoid can be thus denved from a toxm such as, for example, πcm, anthrax or botuhnum toxm types A, B, C, D, E, F or G
[0087] An immunogen can also be a substance of abuse such as nicotine, or an opiate or opiate derivative Such an immunogen can induce antibodies capable of binding and neutralizing the corresponding substance of abuse
Forms of immune conjugates
[0088] The ligand and the immunogen are connected by a linker A linker can be any reagent, molecule or macromolecule that connects the hgand and the immunogen such that a) the immune complex is stable under physiological conditions, b) the connection between the linker and the hgand does not alter the ability of the hgand to bind to its target on the surface of a circulating non-lymphoid cell, and c) the connection between the linker and the immunogen does not substantially affect the capacity of the immunogen to induce an effective immune response m a host against an infectious agent, cell or molecule on which the immunogen is naturally found
Fusion proteins A linker can be a peptide bond That is, the ligand and the immunogen can be a fusion polypeptide comprising one or more ammo acid segments from the hgand and one or more ammo acid segments from the immunogen The term "ammo acid segment" as used herein refers to a contiguous stretch of ammo acids within a polypeptide For example, the ammo acid residues 30 to 40 withm a 100 ammo acid polypeptide would be considered an ammo acid segment An ammo acid segment can be a length greater than eight ammo acid residues (e g , greater than about nine, ten, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 500, 1000, or more ammo acid residues) In some embodiments, an ammo acid segment can have a length less than 1000 ammo acid residues (e g , less than 500, less than 400, less than 350, less than 300, less than 200, or less than 100 ammo acid residues) In other embodiments, an ammo acid segment can have a length from about 20 to about 200 ammo acid residues (e g , about 30 to about 180 ammo acid residues, or about 40 to about 150 ammo acid residues)
[0089] The ammo acid segments of the ligand can be contiguous with the ammo acid segments of the immunogen or they can be separated by ammo acids inserted as a structural spacer A spacer segment can be one or more ammo acids The one or more ammo acids can include amino acids that are the same or that are different For example, a spacer can be a repeating seπes of a neutral ammo acid (e g , glycine, alanine, valine, isoleucme or leucine) ranging in number from 1 to 10 or more (e g , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) Another example of a spacer configuration can be a seπes of interspersed amino acids that may be neutral (e g , glycine alanme-glycme-alanine-glycme-alanme, or glycme-glycine-glycme-valme-valine-valme) or charged ammo acids (e g , glutamate- glutamate-glutamate-argmme-arginine-argmme, or aspartate-lysme-aspartate-lysine- aspartate-lysme) or amino acids with other functional groups (e g , prolme-proline- prolme-serme-serme-senne or tyrosme-glutamine-cysteme-methiomne-tryptophan) ranging in number from 1 to 10 or more (e g , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) In another embodiment, a spacer configuration can be a sequence of ammo acids deπved from a naturally occurring protein such as the hinge region joining the heavy chain CHl and CH2 domains of immunoglobulin G
[0090] A fusion protein can be produced in vitro by continuous peptide synthesis according to standard chemical methods know to those in the art Synthetic polypeptides can also be purchased from commercial sources
[0091] A fusion protein can also be produced by recombinant DNA techniques Nucleic acid segments encoding the hgand can be operably linked in the same open reading frame to nucleic acid sequences encoding the immunogen in a vector that includes the requisite regulatory elements, e g , promoter sequences, transcription initiation sequences, and enhancer sequences, for expression in prokaryotic or eukaryotic cells Methods well known to those skilled in the art can be used to construct expression vectors contaimng relevant coding sequences and appropπate transcnptional/translational control signals Alternatively, suitable vector systems can be purchased from commercial sources
[0092] Nucleic acid segments encoding hgands and immunogens are readily available in the public domain Examples of nucleic acid segments encoding hgands include, without limitation, the erythrocyte (glycophoπn A)-bmdmg antigen of Plasmodium falciparum EB A-173 (Bharara et al , MoI Biochem Parasitol 138 123-9, 2004), or mouse anti-human glycophonn A monoclonal antibody heavy chain (GenBank accession # AAZ67132) and corresponding light chain (Genbank accession # AAA21366)) Examples of nucleic acid segments encoding immunogens include, without limitation, Hepatitis virus C polyprotem (Genbank public gi number 2654998), influenza virus A conserved M2 protein ectodomarn peptide M2e (Genbank public gi number gi 78210829, HBV preSl protein (Genbank public gi number gi 92111469)
[0093] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs Polynucleotides can have any three-dimensional structure A nucleic acid can be double-stranded or smgle- stranded (i e , a sense strand or an antisense strand) Non-limitmg examples of polynucleotides include genes, gene fragments, exons, mtrons, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, πbozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs The nucleic acid molecules can be synthesized (for example, by phosphoramidite based synthesis) or obtained from a biological cell, such as the cell of a mammal The nucleic acids can be those of mammal, e g , humans, a non-human pπmates, cattle, horses, pigs, sheep, goats, deer, elk, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, or mice
[0094] An "isolated" nucleic acid can be, for example, a naturally-occurring DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule m a naturally-occurring genome is removed or absent Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e g , a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restπction endonuclease treatment) An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybπd or fusion nucleic acid A nucleic acid existing among hundreds to millions of other nucleic acids withm, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restπction digest, is not to be considered an isolated nucleic acid
[0095] Isolated nucleic acid molecules can be produced by standard techniques For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA Various PCR methods are descπbed, for example, in PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds , Cold Spnng Harbor Laboratory Press, 1995 Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide pπmers that are identical or similar in sequence to opposite strands of the template to be amplified Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e g , using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a senes of oligonucleotides For example, one or more pairs of long oligonucleotides (e g , >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e g , about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be hgated into a vector Isolated nucleic acids disclosed herein also can be obtained by mutagenesis of, e g , a naturally occurring DNA
[0096] As used herein, the term "percent sequence identity" refers to the degree of identity between any given query sequence and a subject sequence A subject sequence typically has a length that is more than 80 percent, e g , more than 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120 percent, of the length of the query sequence A query nucleic acid or ammo acid sequence can be aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1 83, default parameters), which allows alignments of nucleic acid or protein sequences to be earned out across their entire length (global alignment) Chenna et al , Nucleic Acids Res , 31(13) 3497-500 (2003) ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher bcm tmc edu/multi- ahgn/multi-align html) and at the European Biomformatics Institute site on the World Wide Web (ebi ac uk/clustalw)
[0097] The term "exogenous" with respect to a nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not m its natural environment For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i e , a heterologous nucleic acid Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e g , non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found
[0098] It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular ammo acid sequence The degeneracy of the genetic code is well known to the art, i e , for many amino acids, there is more than one nucleotide triplet that serves as the codon for the ammo acid
[0099] A "vector" is a rephcon, such as a plasmid, phage, or cosmid, mto which another DNA segment may be inserted so as to bπng about the replication of the inserted segment Generally, a vector is capable of replication when associated with the proper control elements Suitable vector backbones include, for example, those routinely used m the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs The term "vector" includes cloning and expression vectors, as well as viral vectors and integrating vectors An "expression vector" is a vector that includes a regulatory region Suitable expression vectors include, without limitation, plasmids and viral vectors deπved from, for example, bacteriophage, baculoviruses, and retroviruses Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA)
[0100] Vectors typically contain one or more regulatory regions The term "regulatory region" refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcπptional start sites, termination sequences, polyadenylation sequences, and mtrons
[0101] As used herein, the term "operably linked" refers to positioning of a regulatory region and a sequence to be transcribed m a nucleic acid so as to influence transcription or translation of such a sequence For example, to bπng a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site A promoter typically comprises at least a core (basal) promoter A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR) The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectabihty, mducibihty, desired expression level, and cell- or tissue-preferential expression It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropπately selecting and positioning promoters and other regulatory regions relative to the coding sequence
[0102] The vectors also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers A marker gene can confer a selectable phenotype, e g , antibiotic resistance, on a cell hi addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e g , purification or localization) of the expressed polypeptide Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidme, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide Such tags can be inserted anywhere withm the polypeptide, including at either the carboxyl or ammo terminus
[0103] The expression vectors disclosed herein containing the above described coding can be used, for example, to transfect or transduce either prokaryotic (e g , bacteria) cells or eukaryotic cells (e g , yeast, insect, or mammalian) cells Such cells can then be used, for example, for large or small scale in vitro production of the relevant fusion protein by methods known in the art In essence, such methods involve culturmg the cells under conditions which maximize production of the fusion protein and isolating the fusion protein from the cells or from the culture medium
Coniugates In another embodiment, the hgand and immnogen can be obtained separately, either through chemical synthesis or synthesis in vivo, purified and then linked non-covalently or covalently A useful non-covalent linkage is a biotm-avidm linkage The binding of biotm to avidm or streptavidm is essentially irreversible, with a reported Kd of 10"15M The term "biotin-avidm linkage" as used herein refers to any linkage via biotm or a biotm denvative or biomimic (e g , Strep-Tag (IBA, St Louis, MO)) and avidm or an avidm denvative, streptavidm, or biotm-bmding fragments or subumts of avidm or streptavidm
[0104] Thus, the biotmylated hgand can be linked to a biotinylated immunogen via avidm or streptavidm, or biotm binding fragments or subumts of avidm or streptavidm Methods for forming biotm-avidm linkages are well known to those in the art (See for example, Handbook of Affinity Chromatography, (Chromatographic Sciences Seπes, vol 63) ed T Kline, ISBN 0824789393 - Marcel Dekker (1993) Avidin and avidm derivatives are available from commercial sources (Pierce Biotechnology, Rockford, IL, Invitrogen, Carlsbad, CA)
[0105] The hgand and the immunogen can also be linked through a biotm- streptavidin linkage that includes an additional biotmylated immunoglobulin For example, a biotmylated hgand can be linked to an avidin molecule that is bound to a biotmylated antibody that specifically binds the immunogen
[0106] Avidin and streptavidm have four biotm-bmdmg sites each By varying the relative ratios of the hgand and the immunogens used m the assembly of the targeted immune conjugates, it is possible to generate targeted immune conjugates with vaπous ratios of linker immunogen Thus, biotm-avidm heterocomplexes can be prepared to include 1 molecule of the hgand and 3 molecules of the immunogen, 2 molecules each of hgand and lmmmunogen, or 3 molecules of hgand and 1 molecule of immunogen Assembly of the biotin-avidm linkages can be performed in any order The composition of the assembled immune conjugates can be validated by SDS-PAGE and western blotting and LC/MS methods
[0107] Alternative docking pairs of molecules with ultra-high affinity (10"10M or more) may be used in place of biotm-avidm An example of such a pair is vitamin B 12 (cyanocobalamin), which is bound by vitamin B12-bmdmg protein with a Kd of 10 10M)
[0108] The hgand and the immunogen can also be synthesized as separate entities (by either chemical synthetic or recombinant methods) and then linked together by standard chemical methods known in the art Chemical cross-linkmg agents can be homo-bifunctional (the same chemical reaction takes place at each end of the linker) or hetero-bifunctional (different chemical reactions take place at the ends of the linker) The chemistries available for such linking reactions include, but are not limited to, reactivity with sulfhydryl, ammo, carboxyl, diol, aldehyde, ketone, or other reactive groups using electrophilic or nucleophilic chemistries, as well as photochemical cross-linkers using alkyl or aromatic azido or carbonyl radicals An example of a targeted conjugate coupled via a homobifunctional cross-lmkmg reagent can be a complex of an anti-band 3 monoclonal antibody as the red blood cell-targeting component and anthrax protective antigen as the immunogen linked by disuccimmidyl suberate (DSS, Pierce, Rockford, IL) An example of a targeted conjugate coupled via a heterobifunctional cross-lmkmg reagent can be an anti-glycophoπn A monoclonal antibody as the targeting component and a non-toxic fragment of botulmum neurotoxin A as the immunogen linked by N succinimidyl 3-[2 pyπdyldithio]-propionamido (SPDP) In this example, the antibody is first deπvatized at sulfhydryl groups with SPDP's pyπdyldithio reactivity, followed by the addition of the toxm, whose ammo residues react with SPDP's succinimidyl groups
[0109] Examples of chemical cross-linkmg agents include, without limitation, glutaraldehyde, carbodumides, bisdiazobenzidme, and N-maleimidobenzoyl-N- hydroxysuccimmide ester Chemical cross-linkers are widely available from commercial sources (e g , Pierce Biotechnology (Rockford, IL), Invitrogen (Carlsbad, CA), Sigma- Aldπch (St Louis, MO), and US Biological (Swampscott, MA))
[0110] In another embodiment, the hgand and Hie immunogen can be connected through a linking polymer Examples of linking molecules include, but are not limited to linear or branched polymers or co-polymers (e g , polyalkylene, poly(ethylene-lysme), polymethacrylate, polyammo acids, poly- or oligosaccharides, dendnmers) The hgand and the immunogen can be attached to the linking molecule or microparticle through a non covalent high affinity linkage, e g , streptavidm-biotm high affinity binding or chemical cross-hnkmg techniques as descπbed above
[0111] For example, a polymer-supported targeted immunogen conjugate can be formed using a poly(ethylene-lysme) backbone Such a linear copolymer backbone can be synthesized using bis(succimmidyl) poly(ethylene glyco^ooo) (Fluka Chemicals) to react with the α and ε ammo groups of lysine The available carboxyl termini of the lysines can be activated using (l-ethyI-3-[3-dimethylammopropyl] carbodnmide Hydrochloπde) (EDC, Pierce Biotechnology) in preparation for coupling amme- contaimng compounds The total length of the co-polymer can be determined, in part, by the duration of the polymerization reaction The targeting and IMG units can be combined in various ratios since there are up to 10 positions in a 2000 dalton co-polymer built with, e g , PEG units of 2,000 dalton Co-polymers using shorter PEG units (e g , 500 daltons) can also be synthesized
[0112) One example of a polymer-based targeted immunogen conjugate can be the addition of equimolar amounts of a targeting monoclonal antibody (e g , anti-CR2) and immunogen (e g- , influenza virus peptide M2e) to produce a complex with a co- polymenc scaffold studded with targeting antibody molecules as well as immunogen molecules
[0113] In another embodiment, the hgand and the immunogen can be connected through a microparticle Examples of linking microparticles include, but are not limited to, micelles, liposomes, fullerenes, nanotubes, or other colloidal complexes such as lipoproteins Liposomes and micelles can be prepared by methods descπbed m Lasic DD, 1998, TIBTech 16 307 Fullerenes and nanotubes can be purchased from American Dye Source (www adsdves com') Lipoproteins can be purchased from Biodesign International (www biodesien coin)
[0114] The ligand and the immunogen can be attached to the linking molecule or microparticle through a non-covalent high affinity linkage, e g , avidm-biotm high affinity binding or chemical cross-hnkmg techniques as descπbed above
[0115] Alternatively or in addition, the hgand and/or the immunogen can be adsorbed or incorporated into a hydrophobic microparticle by hydrophobic affinity A hgand and/or and immunogen with an available hydrophobic domain can spontaneously associate with a hydrophobic microparticle by hydrophobic partitioning The hydrophobic domain on the ligand and/or immunogen can be a polyamino acid stretch comprised of repeating or mixed hydrophobic ammo acids (eg , poly- Ala, poly-Gly, poly-Leu, poly-Ile, or Ala-Gly-Leu-Ile (SEQ ID NO 5), etc ) or a bilayer-spanmng polypeptide from a known trans-membrane protein, such as membrane IgM), alkyl chains (e g , fatty acyl), or other hydrophobic structure (e g , steroid) Such hydrophobic sequences can be naturally occurring sequences within the ligand and/or immunogen Alternatively, such sequences can be introduced into the native ammo acid sequence of the ligand or immunogen by standard recombinant DNA technology The recombinant protein can be expressed and puπfied as descπbed above
[0116] The immune conjugates disclosed herein can include one or more of the same hgands or any combination of different ligands The immune conjugate can also include one or more of the same immunogens or any combination of different immunogens Thus, the immune conjugates can include immunogens that contain multiple copies (eg , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) of a single antigen or a single copy of multiple (e g , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) antigens or multiple copies (eg , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) of two or more antigens (e g , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) The immunogen can contain one or more copies of one or more peptide epitopes together with one or more copies of any of the non-peptide epitopes, e g , post transcriptional modifications, carbohydrates, or lipopolysacchandes
[0117] Further, a targeted immune conjugate can include an immunogen that includes more than one polypeptide, or any combination of different polypeptides It is noted that each polypeptide m a composition can have an identical ammo acid sequence In addition, the polypeptides in a composition can contain different amino acid segments, each of which can act as a defined immunogenic umt against which an immune response is desired Thus, the polypeptides in a composition can contain different ammo acid segments that correspond to any region from a polypeptide including, without limitation, receptor binding regions, ligand binding regions, enzyme active sites, enzyme cleavage sites of polypeptide substrates, antigen-bindmg regions of antibodies, and epitopes recognized by antibodies Typically, the administration of a polypeptide results in the formation of antibodies having specificity for an epitope or combination of epitopes formed by the ammo acid segments withm one or more of the polypeptides m the composition
III. Methods of use
[0118] The immune conjugates disclosed herein are generally useful for generating immune responses and as prophylactic vaccines or immune response- stimulating therapeutics As used herein, "prophylaxis" can mean complete prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the seventy of subsequently developed disease symptoms As used herein, "therapy" can mean a complete abolishment of the symptoms of a disease or a decrease in the severity of the symptoms of the disease
[0119] The methods disclosed herem can be applied to a wide range of species, e g , humans, non human pπmates (e g , monkeys), horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats, mustehds, rabbits, guinea pigs, hamsters, rats, and mice Thus, they can be used, for example, as vaccines or therapeutic agents against infectious diseases, including diseases that can potentially result from bioterroπsm attacks The immune conjugates can be used in the preparation of a medicament for treatment of an infectious disease Infectious diseases can include diseases caused by any of the pathogens listed herein Examples include, without limitation, influenza, HTV-AIDS, hepatitis, botulism, plague, smallpox, tularemia, viral hemorrhagic fevers, brucellosis, gastrointestinal disease induced by pathogenic forms of £ coh, Salmonella and Shigella, glanders, melioidosis, psittacosis, Q fever, Staph infection, typhus fever, viral encephalitis, water and foodborne safety threats, cholera, diphtheria, endocarditis, Legionaire's disease, Listeriosis, periodontal disease, Aspergillosis, Blastomycosis, histoplasmosms, trypanosomiasis, malaria, Giardiasis, Schistosomiasis, toxoplasmosis, smallpox, west Nile virus
[0120] In addition, the immune conjugates can be useful as both prophylactics and therapeutics for cancer (e g , any of those recited above) The immune conjugates can be employed to stimulate an immune response against cells in a cancer patient or can be admimstered in cases where a subject is at relatively high risk for a cancer (e g , lung cancer m a tobacco smoker or melanoma m a subject with multiple nevi) Moreover, as descπbed above, the immune conjugates can also be useful in therapy or prophylaxis of neurodegenerative diseases Thus the immune conjugates can be admimstered to an individual with Alzheimer's disease or TSE or admimstered to an individual who is at risk for developing Alzheimer's disease or TSE
[0121] Immune conjugates disclosed herein can also be useful as a contraceptive vaccine, when the immunogen is a germ cell antigen
[0122] The immune conjugates disclosed herein can also be useful as prophylactics and therapeutics against medical conditions that result from exposure to toxms Such targeted immune conjugates that include non-toxic variants of toxic substances, e g , ncm, botulinum toxin, nicotine and other drugs, can be used to stimulate an immune response against the harmful form of the toxm, and thus protect against or mitigate the potential damage the toxm or drug may cause
[0123] The immune conjugates can be administered directly to a mammal The immune conjugates can be used in the preparation of a medicament Generally, the immune conjugates can be suspended m a pharmaceutically-acceptable carrier (e g , physiological salme) A composition can be made by combining any of the immune conjugates provided herein with a pharmaceutically acceptable carrier Such earners can include, without limitation, steπle aqueous or non-aqueous solutions, suspensions, and emulsions Examples of non-aqueous solvents include mineral oil, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, for example Aqueous earners include, without limitation, water, alcohol, salme, and buffered solutions Preservatives, flavonngs, and other additives such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like also may be present It will be appreciated that any matenal descnbed herein that is to be administered to a mammal can contain one or more pharmaceutically acceptable earners
[0124] Any composition descnbed herein can be administered to any part of the host's body A composition can be delivered to, without limitation, the joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or pentoneal cavity of a mammal In addition, a composition can be administered by intravenous, mtrapentoneal, intramuscular, subcutaneous, intramuscular, intrarectal, mtravagmal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, by inhalation, or by gradual perfusion over time In a further example, an aerosol preparation of a composition can be given to a host by inhalation
[0125] The dosage required depends on the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician Suitable dosages are in the range of 0 01 1,000 μg/kg Wide variations in the needed dosage are to be expected in view of the variety of immune conjugates available and the diffenng efficiencies of vaπous routes of administration Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art Administrations can be single or multiple (e g , 2- or 3-, A-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold) Encapsulation of the immune conjugate m a suitable delivery vehicle (e g , polymeric microparticles or implantable devices) may increase the efficiency of delivery
[0126] The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e g , many years) For example, an immune conjugate can be administered once a month for three months or once a year for a peπod often years It is also noted that the frequency of treatment can be variable For example, an immune conjugate can be administered once (or twice, three times, etc ) daily, weekly, monthly, or yearly
[0127] Alternatively or in addition the immune conjugates can be administered along with an adjuvant An "adjuvant" is an immunological compound that can enhance an immune response against a particular antigen such as a polypeptide Examples of adjuvants include alum and other aluminum-based compounds (e g , AI2O3) Aluminum- based compounds can be obtained from vaπous commercial suppliers Other adjuvants include lmmuno-stimulatmg complexes (ISCOMs) that can contain such components as cholesterol and saponins, one or more additional immunostimulatory components, including, without limitation, muramyldipeptide (e g , N-acetyhnuramyl-L-alanyl-D- lsoglutarmne, MDP), monophosphoryl-hpid A (MPL), and formyl-mefhiomne containing tπpeptides such as N-formyl-Met-Leu-Phe Such compounds are commercially available from Sigma Chemical Co (St Louis, MO), for example Other adjuvants can include CpG ohgodeoxynucleotides (Coley Pharmaceuticals), QS21 (Cambridge Biotech) and MF59 (Chiron)
[0128] The compositions provided herein can contain any ratio of adjuvant to immune conjugate The adjuvant immune conjugate ratio can be 50 50 (vol vol), for example Alternatively, the adjuvant immune conjugate ratio can be, without limitation, 90 10, 80 20, 70 30, 64 36, 60 40, 55 45, 40 60, 30 70, 20 80, or 90 10
[0129] An effective amount of any composition provided herem can be administered to a host The term "effective" as used herem refers to any amount that induces a desired immune response while not inducing significant toxicity in the host Such an amount can be determined by assessing a host's immune response after administration of a known amount of a particular composition In addition, the level of toxicity, if any, can be determined by assessing a host's clinical symptoms before and after administering a known amount of a particular composition It is noted that the effective amount of a particular composition administered to a host can be adjusted according to a desired outcome as well as the host's response and level of toxicity Significant toxicity can vary for each particular host and depends on multiple factors including, without limitation, the host's disease state, age, and tolerance to pam
[0130] Any method can be used to determine if a particular immune response is induced For example, antibody responses against a particular immunogen can be determined using an immunological assay (e g , ELISA or lymphocyte proliferation assay) In such an assay, the wells of a microtiter plate can be coated with the immunogen and incubated with serum from a mammal treated with the immune conjugate designed to produce antibodies against the corresponding immunogen in that mammal, and the presence or absence of antibodies against the immunogen can be determined by standard methods know to those in the art In addition, clinical methods that can assess the degree of a particular disease state can be used to determine if a desired immune response is induced For example, in a cancer patient, a reduction m tumor burden can indicate a desired immune response in a patient treated with a composition designed to stimulate an immune response against a tumor antigen expressed on the patient's tumor [0131] Alternatively, a polynucleotide containing a nucleic acid sequence encoding an immune conjugate of interest can be delivered to an appropriate cell of the animal This can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages For example, PLGA (poly-lacto-co-glycohde) microparticles approximately 1-10 μm m diameter can be used The polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide Once released, the DNA is expressed within the cell A second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation These polymeric particles should therefore be large enough to preclude phagocytosis (z e , larger than 5μm and preferably larger than 20μm)
[0132] Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods The vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L- lysine by electrostatic or covalent forces Poly-L-lysme binds to a hgand that can bind to a receptor on target cells Delivery of "naked DNA" (ι e , without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression
[0133] In the relevant polynucleotides (e g , expression vectors) the nucleic acid sequence encoding the fusion protein of interest with an initiator methionine and optionally a targeting sequence is operatively linked to a promoter or enhancer-promoter combination Promoters and enhancers are described above
[0134] Polynucleotides can be administered in a pharmaceutically acceptable earner Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human or other mammalian subject, e g , physiological salme A therapeutically effective amount is an amount of the polynucleotide which is capable of producing a medically desirable result (e g , a T cell response) in a treated mammal As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently Dosages will vary, but a preferred dosage for administration of polynucleotide is from approximately 106 to 1012 copies of the polynucleotide molecule This dose can be repeatedly admimstered, as needed Routes of administration can be any of those listed above
[0135] The immune conjugates provided herein can be admimstered in conjunction with other therapeutic modalities to an individual m need of therapy The immune conjugates can be given pπor to, simultaneously with or after treatment with other agents Pn the case of infectious disease, the immune conjugates can be administered in conjunction with any antimicrobial agent, e g an antibiotic, e g including, without limitation, aminoglycosides, cephalosporins, macrohdes, penicillins, peptides, qmnolones, sulfonamides, tetracyclines, an antiviral, including without limitation, amantadine, rimantadine, zanamavir and oseltamivir, an anti-fungal, including, without limitation, echmocandm, caspofungm, anidulafungm, or anti-parasitic agent, including, without limitation, chlorqume, mebendazole, and clotrimazole
[0136] The immune conjugates can also be used in conjuction with standard anticancer therapies, including, without limitation, chemotherapy, e g , alkylating agents, anthracyclmes, cycloskeletal disruptors, topoisomerase inhibitors, nucleotide analogues, platinum based agents, retinoids, vmca alkaloids, radiation therapy, hormone ablation and surgery The immune conjugates can also be used m conjuction with other therapueitcs for neurodegenerative diseases, including donepezil, galantamine, memantine
[0137] In vitro application of the immune conjugates can be useful, for example, m basic scientific studies of immune mechanisms or for production of activated T cells for use rn either studies on T cell function or, for example, passive immunotherapy
Articles of Manufacture
[0138] Also disclosed are articles of manufacture that can include immune conjugates as provided herein Components and methods for producing articles of manufacture are well known An article of manufacture can include, for example, one or more immune conjugates In addition, an article of manufacture further may include, for example, packaging materials, instructions for use, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required
[0139] Anumber of embodiments of the invention have been descπbed Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention Accordingly, other embodiments are withm the scope of the following claims
EXAMPLES
Example 1: Assembly of tripartite conjugates of the biotmylated monoclonal antibody anti-TER-119, streptavidin and the biotinylated peptide M2e.
[0140] Conjugates to deliver the immunogen M2e, to the mouse red blood cell (mRBC), were prepared by coupling the peptide with the rat anti-mouse monoclonal antibody anti-TER 119 and using streptavidin (StAv) as the linker
[01411 Preparation of tripartite immune coniugates The peptide M2e (SLLTEVETPIRNEWGCRCNDSSD) (SEQ ID NO 1) was produced by peptide synthesis (BioWorld, Dublin OH) to contain a biotmylated lysine residue at its carboxy terminus Anti-TER- 119, a rat anti-mouse IgG2b,κ monoclonal antibody, was purchased biotmylated from BD-Pharmmgen, as was the isotype control, biotmylated rat IgG2b,κ The extent of biotinylation of these products was not available from the manufacturer Streptavidin was purchased from Sigma- Aldrich
[0142] Biotmylated M2e peptide (b-M2e) (SEQ ID NO 1) was incubated with StAv in phosphate-buffered salme, 10 mM sodium phosphate, 140 mM NaCl, pH 74 (PBS) at ambient temperature at various molar ratios After 30 mm incubation biotmylated anti-TER-119 antibody (b-Ab) was added and the incubation was continued for an additional 30 mm with occasional agitation Reaction samples were analyzed by 3 to 8% gradient gel electrophoresis under native (or non-denaturmg) conditions (NDGE, Invitrogen, tπs-glycine running buffer) and visualized by Coomassie blue staining The results showed that mixtures of conjugates of different apparent molecular weights and stoichiometrics were formed caused by differential cross-hnkmg by StAv between the biotmylated peptide and the Ab The relative amounts of reagents used m the reactions are shown in the grids below the respective NDGE images
[0143] Characterization of tripartite immune comugates In the experiment depicted m Figure 1 the amounts of StAv and b-Ab were kept constant but the amount of b-M2e was varied as indicated Pre-mcubation of 100 or 200 pmol b-M2e with 20 pmol StAv, which is well m excess of the 80 pmol required to saturate the StAv biotin-bmdmg sites, resulted m formation of StAv(M2e)4 This conjugate migrated faster than free StAv and similarly to unreacted b-M2e (lanes 10-12 compared to lane 3)
[0144] Because the mobility of reagents in NDGE is not solely determined by their molecular weight but also by their conformation and charge, StAv migrated considerably faster than Ab, to a location on the gel that was slightly above the peptide b- M2e Addition of b-M2e peptide to StAv, while increasing its MW, caused a shift m the complex's migration downward, to the position of unreacted b-M2e (lanes 3 and 6) That this was the effect of binding of b M2e to StAv was demonstrated by the reaction m lane 4, in which excess non-biotmylated peptide M2e did not alter the mobility of StAv) When b-M2e + StAv mixtures containing an excess of b-M2e were combined with b-Ab there was no shift in the migration of the Ab (lanes 11 and 12, compared to lane 5)
[0145] The admixture of 50 pmol or less b-M2e, which was below the theoretical 80 pmol needed to saturate 20 pmol StAv, did shift the mobility of the added b-Ab upward indicating the presence OfAb-StAvn, where n represents the number of biotin residues per Ab molecule hi addition, the laddeπng pattern m lanes 7-10, indicated the presence of possible higher order complexes such as Ab-StAv-Ab, Ab- StAv2-Ab2, Ab-StAv-Ab-StAv-Ab, etc Useful tripartite complexes were those that did not include unreacted Ab, these complexes had a mobility that was shifted upward from that of the unconjugated antibody, but not to a degree that prevented the complexes from entering the gel, e g , m lanes 9-10, where the ratio of tetravalent StAv b-M2e was approximately 1 3
[0146] Conditions for the formation of the Ab-StAv-M2e tripartite immune conjugates were analyzed m the experiment shown m Figure 2 The concentration of b- Ab was held constant at 12 5 pmol, and different amounts of StAv-b-M2e conjugates pre- formed at a 1 3 ratio were added Complexes of vaπous stoichiometrics were seen in all combinations, with the higher order complexes seen where StAv was in excess over b-Ab (lanes 6 9) Figure 2 also showed that m the 1 3 StAv b-M2e mixtures there were some StAv molecules that had a minimum of 2 free biotm-bmdmg sites which were responsible for cross-lmkmg b-Ab molecules (lanes 5-9) This experiment also indicated that the antibody molecules contained more than one biotin residue If b Ab were mono- biotmylated, the reaction with free StAv would lead to a preponderance of Ab tetramers, with lesser amounts of tπmers, dimmers and monomers Lane 3 shows that this is not the case Instead there were more types of complexes without apparent preponderance of tetramers
[0147] One objective of these expeπments was to define conditions that produced tripartite conjugates with a maximum number of M2e peptides per Ab molecule The stoichiometric ratios of Ab StAv M2e m the expenments descπbed above are listed in Table 1.
Table 1. Molar ratios of the components in tripartite conjugate reactions depicted in Figures 1 and 2 (normalized to the concentration of Ab).
Figure imgf000045_0001
[0148] The conditions in which most of the Ab was shifted by the reaction, but the complexes formed were not too large to diffuse into the gel are found m Figure 1, lanes 9 and 10 and Figure 2, lanes 6, 7 and 8 The immune conjugates that included the highest proportion of peptides per Ab (6 or 7 2) were those shown m lane 7 or 8 of Figure
2
[0149] Example 2: Analysis of anti M2e activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab - streptavidin - biotinylated M2e peptide
[0150] Mice are challenged mtrapeπtoneally with an immune conjugate of biotinylated rat anti-mouse TER-119 Mab - streptavidm- biotinylated M2e peptide ((SEQ ID NO I)) and their serum antibody response to M2e (SEQ H) NO 1) is assayed
[0151] M2e peptide (SEQ EO NO 1) with a C-termmal addition of lysyl-ε-N- biotm or PGGG (SEQ ID NO 6) is synthesized by solid phase peptide synthesis (Bio- World) This peptide is coupled to streptavidm (StAv) (Sigma- Aldπch) at a peptide StAv molar ratio of 3 1 by mixing with b-Ab, as descπbed m Example 1 Identical tripartite conjugate is prepared using biotinylated rat IgG (BD Pharmmgen) as a control To prepare TER-119-StAv and rat IgG-StAv conjugates the StAv is first reacted at a biotm StAv molar ratio of 3 1 to occupy 3 of the 4 biotin-bmdmg sites (BBSs) on each StAv molecule In groups receiving uncoupled StAv, it is first reacted with a minimum of 4 molar equivalents of biotm to saturate all the BBSs M2e peptide conjugated to keyhole limpet hemocyanm (KLH) is produced by Bio-World
[0152] Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are injected intravenously (ι v) in the tail vein with a range of free and conjugated peptide doses (0 01 to 10 μg equivalent of peptide m 50 μl PBS) Since StAv and TER-119 (a rat IgG) are also immunogenic m mice, Ab titers against them alone or in RBC-targeted conjugates are also evaluated Groups 1-6 are the minimum set to analyze the relative anti-M2e responses Groups 7-15 include additional controls to further analyze the anti-StAv and anti rat IgG responses, as well as the effect of simply mixmg the various components, instead of coupling them into targeted conjugates The expeπmental design is depicted in the table below
Table 2: Experimental design: Example 2.
Figure imgf000047_0001
[0153] The mice are boosted with a repeat i v injection after 2 and again after 4 weeks Blood samples (-100 μl) are collected retro-orbitally prior to and every week after the primary injection for 5 weeks Blood samples are chilled on ice and allowed to clot overnight The tubes are centπfuged and serum is collected and stored at -2O0C
[0154] Mouse IgG titers against all 3 antigens (Ags), M2e, StAv and rat IgG, are determined by ELISA ELISA plates (hnmulon, VWR) are coated with 100 μl Ag at 10 μg/ml in sodium carbonate (Na2CO3) buffer, 50 mM, pH 9 6 overnight at 40C Plates are washed and blocked with 200 μl/well of 3% BSA in PBS containing 0 05% Tween-20 (PBST) for 1 5 hours at room temperature (RT) Wash 3X with 200 μl/well of PBST Dispense 100 μl/well of individual mouse sera at 1 50 dilution in PBST m duplicates and prepare senal dilutions in 1 3X increments Allow mouse antisera to bmd for 1 5 hours at RT and wash the plates as above Add 100 μl/well goat anti-mouse IgG conjugated with horseradish peroxidase (diluted per vendor instructions, Sigma), mcubate for 1 hour at RT, and wash as above Add 100 μl/well substrate (SureBlue, KPL) and read absorbance at 405 run Positive control Abs are Mouse Mab 14C2 which recognizes the N-terminal ectodomain epitope of M2 (Abeam), mouse Mab anti-StAv (Abeam) and mouse polyclonal anti-rat IgG (H & L chains) (Invitrogen)
[0155] Example 3: Analysis of anti M2e activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab-streptavidin- biotinylated M2e peptide (SEQ ID NO: 1): comparison of different routes of administration
[0156] The effect of the route of administration on the serum antibody response to M2e peptide in mice challenged with an immune conjugate of biotinylated rat anti-mouse TER-119 Mab + streptavidm- biotinylated M2e peptide is evaluated by comparing serum titers of anti-M2e antibodies m mice immunized mtrapentoneally, intravenously, subcutaneously and intramuscularly The full Mab-StAv-IMG conjugate is compared to the non-targeted StAv-IMG partial complex, as well as to PBS vehicle The dose will be 1 0 μg equivalent of M2e peptide (SEQ ED NO 1) Immunizations and ELISA analysis are performed as described in Example 2
[0157] Example 4: Analysis of anti-M2e fecal IgG and IgA activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab-streptavidin-biotinylated M2e peptide(SEQ ID NO: 1)
[0158] Mice are dosed with TER-119 - StAv M2e conjugate and controls as depicted m Table 2 and following the dosmg schedule m Example 2
Table 3. Experimental design: Example 4.
Figure imgf000049_0001
Feces are collected at the times descπbed in Example 2 Soybean trypsin inhibitor (0 1 mg/ml in PBS) is added to every 0 1 g of feces, which are vortexed in a mmi-beadbeater (Biospec Products) for 10 sec at 2500 rpm, and debns is removed by centπfugation at 9000xg at 40C for 15 mm The supernatant is assayed for anti-M2e IgG titer in the ELISA set-up descπbed in Example 2, but also for IgA by adding anti-IgA 2nd antibody (goat anti-mouse IgA conjugated with horseradish peroxidase (Sigma- Aldrich)
[0159] Example 5: Analysis of anti-M2e activity in mice challenged with the immune conjugate biotmylated rat anti-mouse CD21/CD35 Mab-streptavidin- biotinylated M2e peptide(SEQ ID NO: 1)
[0160] Mice are injected with an immune conjugate of biotmylated rat anti-mouse CD21/CD35 Mab + streptavidm- biotmylated M2e peptide (influenza) and their serum antibody response to M2e is assayed The anti-CD21/CD35 antibody is purchased from eBioscience The conjugate is prepared according to the method descπbed in Example 2 Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are injected intravenously (i v) with a range of free and conjugated peptide doses (0 01 to 10 μg equivalent of peptide m 50 μl PBS) according to the expenmental design in shown in Table 4 below
Table 4: Experimental design: Example 5.
Figure imgf000050_0001
Immunizations and ELISA analysis will be performed as descnbed in Example 2
[0161] Example 6: Analysis of anti-M2e activity in mice challenged with the immune conjugate biotinylated Plasmodium falciparum EBA-175 peptide 1085-1096 (SEQ ID NO: 7) (pEBA) + streptavidin-biotinylated M2e peptide
[0162] Since Plasmodium falciparum EBA-175 peptide 1085-1096 binds human, but not mouse RBCs, the mouse experiments are performed m human GphA-transgemc mice (see Auffrey, et al , 2001, Blood 97 2872-2878 Glycophorm A dimeπzation and band 3 interaction during erythroid membrane biogenesis in vivo studies in human glycophonn A transgenic mice) Alternately, the corresponding binding protein deπved from other species of Plasmodium, e g, P berghen or P yoeln yoeln, which bind mouse RBCs is used (referred to as pEBAm) Complexes are assembled and immunizations and ELISA analysis are performed as described m Example 2
[0163] Example 7: Analysis of anti-M2e activity in mice challenged with the synthetic peptide pEBA-Glycine-Glycine-Glycine-lVKe peptide.
[0164] Mice are injected intravenously with a synthetic peptide of the sequence pEBAm-175 SEQ ID NO 7 linked via three glycine residues to the M2e peptide (SEQ ID NO 1) descπbed m Example 1 above and according to the scheme depicted in Table 5 Each mouse receives a range of free and conjugated peptide doses (0 01 to 10 μg equivalent of M2e peptide m 50 μl PBS) intravenously and blood samples are collected and analyzed according to the schedule in Example 2
Table 5. Experimental Design: Example 7.
Figure imgf000051_0001
[0165] Example 8: Analysis of anti-HBV peptide (preSl amino acids 34-59 (SEQ ID NO:2), or pHBV) activity in mice challenged with the immune conjugate biotinylated rat anti-mouse TER-119 Mab + streptavidiπ-biotiπylated pHBV.
[0166] Mice are challenged with immune conjugates in which the immunogen is an HBV (hepatitis B virus) peptide preSl ammo acids 34 59 (Hu WG, et al 2005 World J Gastroenterol 11 2088-2094 Identification of the immunogenic domains in HBsAg preS 1 region using overlapping preS 1 fragment fusion proteins) or pHBV coupled to
biotmylated TER-119 Mab Experimental groups, immunizations and ELISA analysis are as descnbed in Example 2
[0167] Example 9: Analysis of anti-M2e activity in mice challenged with the immune conjugate biotinylated mouse anti-mouse band 3 Mab-streptavidin- biotinylated M2e peptide (SEQ ID NO:1)
[0168] Mouse anti -mouse band 3 Mabs have been produced m several labs from NZB mice The immune conjugate, biotinylated mouse anti-mouse band 3 Mab- streptavidin-biotmylated M2e peptide, is produced according to the method in Example 1 with an IgG anti-mouse band-3 Mabs (e g , 34-3C or class-switched 4C8 (Fossati-Jrmack, 2002, J Autoimmun 18 17-25 Selective increase of autoimmune epitope expression on aged erythrocytes m mice implications in anti-erythrocyte autoimmune responses )), and is evaluated according to the protocol in Example 2
[0169] Example 10: Optimization of the degree of biotinylation of the targeting Mabs
[0170] Establish the optimal number of StAv-EVIG units to couple to the biotmylated Mab Maximize IMG load while retaimng full target binding capacity of the Mab
[0171] The FluoReporter Biotm Quantitation Kit (Invitrogen) is used for estimating the molar ratio of biotm protein Mab anti-TER-119 is biotmylated using ammo-reactive (Pierce EZ-link NHS biotm) and carbohydrate-reactive (Pierce EZ-lmk hydrazide-biotm) reagents at various levels by adding a range of biotinylation molar excess factors (e g , 3X, 5X, 10X, 5OX, 250X) Add a 5X molar excess StAv per mole of biotmylated Mab to saturate all biotmylated sites Test functional integrity of the biotmylated Mab preparations by binding to mouse RBCs Use goat anti-rat IgG conjugated with fluoresceme isothiocyanate (FITC) to detect RBC-bound TER-119 using flow cytometry Select the condition that results in the highest degree of biotinylation of
the TER- 119 Mab while retaining maximum RBC-bmdmg Use un-biotmylated TER- 119 Mab and goat anti-rat IgG-FITC as the reference
[0172] Example 11: Determination of the optimal stoichiometry of Mab- StAv-IMG
[0173] The effect of loading 1, 2 or 3 IMGs per StAv and the effect of binding 1 or more of these StAv-IMG conjugates per targeting Mab is compared The optimal construct is identified based on the elicited response in vivo (see Example 2) Prepare anti-TER-119 + StAv + M2e conjugates at various ratios (e g , 1 1 1, 1 1 3, 1 2 2, 1 2 6, 1 3 3, 1 3 9) and administer them to mice as in Example 2 (always administer 1 0 μg equivalent of M2e) Compare the resulting anti-M2e titers
[0174] Example 12: Comparison of monovalent, divalent and trivalent peptide-mediated targeting of IMG to RBC.
[0175] Biotmylated pEBAm (SEQ ID NO 7) (see Example 7) is used as the targeting hgand and biotmylated M2e is used as the IMG The following constructs are prepared IpEBA1n IStAv 3M2e, 2pEBAm IStAv 2M2e and 3pEBAm IStAv lM2e Inject mice with each of the constructs containing equal equivalents of M2e (1 0 μg) Evaluate the elicited immune response according to the methods m Example 2
[0176] Example 13: Evaluation of the efficacy of immunizing with a construct in which the targeting component and the IMG are covalently bound to a common polymer backbone
[0177] Synthesize each component peptide (e g , pEBAm (SEQ ID NO 7) and M2e (SEQ ID NO I)) with an added Lys whose α-carboxyl group is joined to the peptide's C terminus, leaving the α and ε ammo groups of Lys free Co-polymenze various ratios of the components with bis-succmimidyl poly(ethylene glycol), or BS-PEG (mean molecular weight 2,000 da) The free α and ε ammo groups of Lys are linked to the reactive PEG units to create a co-polymer that has the pattern PEG-P-PEG-P-PEG-P- PEG , where P is either peptide pEBAm or M2e in a random distribution The ratio of pEBAm to M2e is a function of the ratio of added peptides to the polymerization reaction Prepare co-polymer backbone targeted IMG constructs m which the Mab IMG ratios are 0 10 (un-targeted multivalent MG control), 1 9, 3 7, 5 5, 7 3 and 9 1 Evaluate their efficacy by injecting 1 0 μg equivalents of M2e as descπbed in Example 2
[0178] Example 14: Preparing targeted constructs using a microparticulate linking component.
[0179] Liposomes containing a biotmylated lipid in the bilayer (e g , phosphatidyl ethanolamme reacted with succimmidyl biotm) are prepared The exposed biotm groups are used to bind StAv and then the StAv's biotm binding sites are loaded with biotmylated targeting and IMG components at various ratios The comparatively large liposomal surface permits adding many more units of each component without causing steric hindrance In order to prevent StAv from cross-linking the exposed biotm groups on the liposomal surface, pre-load 3 mole equivalents of either targeting or IMG units (or combinations of these) per mole of StAv, and then add this conjugate to the liposomes Administer and analyze as in Example 2
[0180] Example 15: Preparing targeted constructs using a microparticulate linking component and a mixture of IMG's
[0181] Biotmylate a mixture of IMGs from a relevant source (e g , the multiple strain variants of a multivalent pneumococcal polysaccharide vaccine or the various proteins or peptides from a viral vaccine) using a suitable reactive biotm reagent (m the case of oligosaccharides use a biotmylation reagent with a hydrazide reactive terminus (EZ-lmk hydrazide biotm, Pierce), and in the case of peptidic preparations use one with a terminus containing either a succimmidyl group for reaction with amines, a maleimidyl group for reaction with sulfliydryls, or an amine terminus reactive with carboxyls m the presence of ethylenediamme carbodiimide (EDC)) Add such a mixture of biotmylated IMGs to a targeting conjugate consisting of StAv bound to a biotmylated Mab, a biotmylated targeting peptide, such that the StAvs have remaining unoccupied biotm- bmdmg sites These are produced by reacting a large molar excess of StAv with the biotmylated targeting component The biotmylated IMG mixture can also be reacted with liposomes bearing surface StAv molecules bound to lipid-bound biotm (e g , biotmylated phosphatidyl ethanolamme), with sufficient unoccupied biotm-bmdmg sites remaining on the bound StAv molecules Administer and analyze as in Example 2
[0182] Example 16: Using anti-band 3 Mab for targeting IMGs to senescent RBCs
[0183] Approximately 1% of RBCs are senescent and destined for removal from the circulation by phagocytosis in the RES Their clearance is mediated by pre-existmg natural IgG Abs (Nabs) These Nabs have low affinity for band 3 They can only bind their target Ag on the RBC firmly if the band 3 molecules are clustered in the RBC membrane, through augmented avidity Band 3 clusteπng occurs as a result of cumulative oxidative damage in the course of the life of the cells Use a slightly higher affinity anti-band 3 Mab, sufficiently high to out-compete the Nabs, but not so high as to bind all RBCs The Mab can be a humanized IgG, for targeting IMGs to senescent RBCs
[0184] To develop a human anti-band 3 Mab, immunize a mouse with human band 3 protein or with human red blood cells (which contain a senescent subpopulation) and fuse the spleen cells with a myeloma fusion partner (e g , SP2/0) Plate fused cells at 100,000 cells/well m hybndoma selection medium (HAT) After 3 weeks screen supernatants of wells with hybndoma clones by ELISA on wells coated with band 3 protein extracellular domain Subclone hybndomas that react specifically with human band 3 until the cultures are monoclonal The resulting murine Mab can be "humanized" by methods familiar to those skilled m the art (e g , Recombinant Antibodies (1999), Breitlmg, F and Dubel, S (eds ), John Wiley & Sons) Alternately, human anti-band 3 Mabs can be produced by fusion of normal human donor B cells, among which are found the cells producing anti-band 3 Nabs Enrich B cells (the CD19+ or CD20+ subpopulation) from the peripheral blood mononuclear fraction by immunomagnetic purification (StemCell Sciences) Fuse the B cells by electrofusion (CytoPulse) to a suitable fusion partner myeloma or heteromyeloma cell line (e g , K6H6/A5 or A6 (ATCC) or SP2 IL6-TERT (Dessain SK, et al , 2004, J Immunol Methods 291 109-122 High efficiency creation of human monoclonal antibody-producing hybπdomas )) to form hybndomas Plate fused cells at 20,000 cells/well in hybπdoma selection medium (HAT) After 3 weeks screen supernatants of wells with hybndoma clones by ELISA on wells coated with band-3 protein extracellular domain Subclone band 3-specific IgG hybndomas Purify the antibodies and use them to prepare an immune conjugate as described in Example 1 Use biotmylated influenza M2e peptide as the IMG and couple to the Mab with a StAv bridge Assay in mice according to the method in Example 2

Claims

WHAT IS CLAIMED IS:
1. An immune conjugate compnsing
(a) a ligand that binds specifically to a cell surface molecule on a circulating non- lymphoid cell of a mammal, wherein said molecule is selected from the group consisting of glycophonn A, band 3, Ter-119, blood group antigen H, blood group antigen A, blood group antigen B, CD41a, CD14, CD56, CD66d, CD83, CMKLRl, and BDCA-4, and
(b) an immunogen coupled to said ligand, wherein said immune conjugate, when administered to an individual, induces or enhances an immune response against said immunogen
2 The immune conjugate of claim 1, wherein the ligand is an antibody
3 The immune conjugate of claim 2, wherein the antibody is an anti-TER-119 antibody, an anti-glycophoπn A antibody, an anti-band 3 antibody, an anti-blood group antigen A antibody, an anti-blood group antigen B antibody, an anti-blood group antigen H antibody, an anti-CD41a antibody, an anti-CD14 antibody, an anti-CD56 antibody, an anti-CD66d antibody, an anti-CD83 antibody, an anti CMKLRl antibody, or an anti- BDCA-4 antibody
4 The immune conjugate of claim 3 wherem the antibody is an anti-TER-119 antibody
5 The immune conjugate of claim 1, wherein the ligand is erythrocyte-bmding antigen
175 (EBA 175) or a fragment of EBA-175 that binds to a red blood cell
6 The immune conjugate of claim 1, wherein the ligand is biotmylated, glycosylated, acetylated, alkylated, isoprenylated, lipoylated, or phosphorylated
7 The immune conjugate of claim 1, wherein the ligand selectively binds blood group antigen A, blood group antigen B or blood group antigen H
The immune conjugate of claim 1, wherein the cell-surface molecule is on a red blood cell
The immune conjugate of claim 8, wherein the cell-surface molecule is glycophoπn A
(CD235A), band 3 (CD233), TER-119, blood group antigen A, blood group antigen B, or blood group antigen H
The immune conjugate of claim 1, wherein the cell-surface molecule is on aplatelet
The immune conjugate of claim 10, wherein the cell surface molecule is CD41a
The immune conjugate of claim 1, wherein the cell-surface molecule is on a monocyte
The immune conjugate of claim 12, wherein the cell-surface molecule is CD14
The immune conjugate of claim 1, wherem the cell-surface molecule is on a granulocyte
The immune conjugate of claim 14, wherein the cell-surface molecule is CD66d
The immune conjugate of claim 1, wherem the cell-surface molecule is on a plasmcytoid dendritic cell
The immune conjugate of claim 16 wherem the cell-surface molecule is CD83, CMKLRl or BDCA-4
The immune conjugate of claim 1, wherem the circulating non-lymphoid cell is a red blood cell
The immune conjugate of claim 1 wherem the circulating non-lymphoid cell is a platelet
20 The immune conjugate of claim 1, wherein the mammal is human
21 The immune conjugate of claim 1, wherein the immunogen is a molecule expressed or released by an infectious agent
22 The immune conjugate of claim 21, wherein the infectious agent comprises a virus, viroid, bacterium, fungus, prion or parasite
23 The immune conjugate of claim 22, wherein the infectious agent is a virus
24 The immune conjugate of claim 23, wherein the virus is an influenza virus
25 The immune conjugate of claim 24, wherein the influenza virus is strain A
26 The immune conjugate of claim 22, wherein the infectious agent is a bacterium
27 The immune conjugate of claim 22, wherein the infectious agent is a fungus
28 The immune conjugate of claim 22, wherein the infectious agent is a parasite
29 The immune conjugate of claim 21 , wherein the immunogen is influenza A M2 protein or hepatitis B virus preS 1 protein
30 The immune conjugate of claim 29, wherein the immunogen is influenza A M2 protein or a fragment of influenza A M2 protein
31 The immune conjugate of claim 30, wherein the fragment of influenza A M2 comprises the ectodomain peptide M2e
32 The immune conjugate of claim 30, wherein the fragment of influenza A M2 is SEQ ID NO 1
33 The immune conjugate of claim 21, wherein the immunogen is hepatitis B virus preSl protein or a fragment of hepatitis B virus preSl protein
34 The immune conjugate of claim 33, wherem the fragment of hepatitis B virus preS 1 protein is SEQ ID NO 2
35 The immune conjugate of claim 1, wherem the immunogen is biotinylated, glycosylated, acetylated, alkylated, isoprenylated, hpoylated, or phosphorylated
36 The immune conjugate of claim 1, wherem the immunogen is coupled to the ligand via a covalent bond
37 The immune conjugate of claim 1 , wherem the immunogen and the ligand compπse a fusion protein
38 A nucleic acid sequence encoding the fusion protein of claim 37
39 An expression vector comprising the nucleic acid sequence of claim 38
40 A host-cell comprising the expression vector of claim 39
41 The immune conjugate of claim 1, wherem the immunogen is coupled to the ligand via a non-covalent bond
42 The immune conjugate of claim 41, wherem the non-covalent bond is a biotm-avidm linkage
43 The immune conjugate of claim 1, wherem the immune conjugate comprises two or more of said hgands
44 The immune conjugate of claim 43, wherein the two or more hgands bind different cell surface molecules
45 The immune conjugate of claim 1, wherein the immune conjugate compnses two or more of said immunogens
46 The immune conjugate of claim 45, wherein the two or more immunogens are not the same
47 The immune conjugate of claim 1, wherein the hgand is an anti-TER-119 antibody and the immunogen is the ectodomam peptide of M2e
48 A composition comprising the immune conjugate of claim 1 and an adjuvant
49 A method of inducing or enhancing an immune response against an immunogen in a mammalian subject, the method comprising administering a therapeutically effective amount of the immune conjugate of claim 1, wherein an immune response to the immunogen is induced in the mammalian subject
50 The method of treatment of claim 1, wherein the mammalian subject is identified as suffering from or being at risk for an infectious disease
51 The method of treatment of claim 1 , wherein the mammalian subj ect is identified as suffering from or being at risk for exposure to a toxin
52 Use of the immune conjugate of any of claims 1 -48 in the preparation of a medicament
53 Use of the immune conjugate of any of claims 1-47 m the preparation of a medicament for treatment of an infectious disease
54 An article of manufacture comprising a measured amount of one or more immune conjugates and one or more items selected from the group consisting of packaging material, a package insert compπsing instructions for use, a steπle fluid, and a sterile container
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