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Timestamp: 2015-04-28 00:39:35
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Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 09', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'art. 100']

Patent US6528051 - Targeted delivery system comprising at least one component-specific ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe methods and compositions of the present invention are directed to enhancing an immune response and increasing vaccine efficacy through the simultaneous or sequential targeting of specific immune system components. More particularly, specific immune components, such as macrophages, dendritic cells,...http://www.google.com/patents/US6528051?utm_source=gb-gplus-sharePatent US6528051 - Targeted delivery system comprising at least one component-specific immunostimulating molecule bound to a platform.Advanced Patent SearchPublication numberUS6528051 B2Publication typeGrantApplication numberUS 09/935,062Publication dateMar 4, 2003Filing dateAug 22, 2001Priority dateNov 10, 1997Fee statusPaidAlso published asUS6407218, US7790167, US20020071826, US20030180252Publication number09935062, 935062, US 6528051 B2, US 6528051B2, US-B2-6528051, US6528051 B2, US6528051B2InventorsLawrence Tamarkin, Giulio F. PaciottiOriginal AssigneeCytimmune Sciences, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (62), Non-Patent Citations (20), Referenced by (19), Classifications (32), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetTargeted delivery system comprising at least one component-specific immunostimulating molecule bound to a platform.
US 6528051 B2Abstract
We claim: 1. A method for affecting an immune response, comprising, administering a composition comprising colloidal metal particles, an antigen, and at least one component-specific immunostimulating agent, wherein the component-specific immunostimulating agent is interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), Flt-3 ligand, interleukin-6 (IL-6), heat killed Mycobacterium butyricum, or interleukin-2 (IL-2).
2. The method of claim 1, wherein the antigen comprises interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin B, Type I Interferon, Type II Interferon, tumor necrosis factor a (TNF-α), transforming growth factor-β (TGF-β), lymphotoxin migration inhibition factor, granulocyte-macrophage colony-stimulating factor (CSF), monocyte-macropahge CSF, granulocyte CSF, vascular epithelial growth factor (VEGF), angiogenin, transforming growth factor (TGF-α), heat shock proteins (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factor, nucleotides, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof.
3. The method of claim 1, wherein administering the composition comprises sequentially activating a primer phase and an immunization phase of the immune response.
4. The method of claim 3, wherein activating the primer phase comprises the stimulation of antigen presenting cells selected from a group consisting of macrophages, dendritic cells and B cells.
5. The method of claim 3, wherein activating the primer phase comprises the activation of macrophages and dendritic cells.
6. The method of claim 3, wherein activating the immunization phase comprises the stimulation of lymphocytes selected from a group consisting of B cells and T cells.
7. The method of claim 3, wherein activating the primer phase and the immunization phase comprise a simultaneous activation of macrophages and dendritic cells followed by a simultaneous activation of B cells and T cells.
8. The method of claim 1, wherein the method stimulates the immune response.
9. The method of claim 1, wherein the method suppresses the immune response.
10. A method for enhancing vaccine efficacy comprising administering a composition comprising colloidal metal particles, an antigen, and at least one component-specific immunostimulating agent, wherein the component-specific immunostimulating agent is interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), Flt-3 ligand, interleukin-6 (IL-6), heat killed Mycobacterium butyricum, or interleukin-2 (IL-2).
11. The method of claim 10, wherein the antigen comprises interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin B, Type I Interferon, Type II Interferon, tumor necrosis factor α (TNF-α), transforming growth factor-β (TGF-β), lymphotoxin migration inhibition factor, granulocyte-macrophage colony-stimulating factor (CSF), monocyte-macropahge CSF, granulocyte CSF, vascular epithelial growth factor (VEGF), angiogenin, transforming growth factor (TGF-α), heat shock proteins (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factor, nucleotiudes, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof.
12. The method of claim 10, wherein the administering of component-specific immunostimulating agents comprises sequential administration of component-specific immunostimulating agents.
13. The method of claim 10, wherein the colloidal metal particles are of different sizes.
14. The method of claim 13, wherein administering the component-specific stimulating agent, the antigen, and the colloidal metal particles of different sizes comprises simultaneously administering the component-specific stimulating agent, the antigen and the colloidal metal particles of different sizes as a single dose.
15. The method of claim 10, wherein administering the component-specific stimulating agent, colloidal metal particles, and an antigen comprises oral, intramuscular, or parenteral administration.
16. The method of claim 10, wherein administering the component-specific immunostimulating agent, colloidal metal particles, and an antigen comprises administration with a carrier molecule.
17. The method of claim 10, wherein the carrier molecule comprises liposomes, microcapsules, microspheres or a combination thereof.
18. The method of claim 10, wherein the method is used to stimulate or suppress immune components in the treatment of disease, wherein the disease is cancer, allergic rhinitis, eczema, urticaria, anaphylaxis, transplant rejection, systemic lupus erthymatosus, rheumatoid arthritis, seronegative spondyloarthritides, Sjogren's syndrome, systemic sclerosis, polymyositis, dermatomyositis, Type I Diabetes Mellitus, Acquired Immune Deficiency Syndrome, Hashimoto's thyroiditis, Graves' disease, Addison's disease, polyendocrine autoimmune disease, hepatitis, sclerosing cholangitis, primary biliary cirrhosis, pernicious anemia, coeliac disease, antibody-mediated nephritis, glomerulonephritis, Wegener's granulomatosis, microscopic polyarteritis, polyarteritis nodosa, pemphigus, dermatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain-Barre syndrome, Myasthenia Gravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, myeloma, x-linked hyper IgM syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimrune neutropenia, Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocytic leukemia, or non-Hodgkin's lymphoma.
This application is a continuation of U.S. application Ser. No. 09/189,748, filed on Nov. 10, 1998, now U.S. Pat. No. 6,407,218 further claims the benefit of U.S. Provisional Application No. 60/107,455, filed Nov. 6, 1998, U.S. Provisional Application No. 60/075,811, filed Feb. 24, 1998, and of U.S. Provisional Application No. 60/065,155 filed Nov. 10, 1997.
One of the most widely employed aspects of the immune response capabilities is the production of monoclonal antibodies. The advent of monoclonal antibody (Mab) technology in the mid 1 970s provided a valuable new therapeutic and diagnostic tool. For the first time, researchers and clinicians had access to unlimited quantities of uniform antibodies capable of binding to a predetermined antigenic site and having various immunological effector functions. Currently, the techniques for production of monoclonal antibodies is well known in the art.
Vaccines may be generally divided into two types, whole and subunit vaccines. Whole vaccines may be produced from viruses or microorganisms which have been inactivated or attenuated or have been killed. Live attenuated vaccines have the advantage of mimicking the natural infection enough to trigger an immune response similar to the response to the wild-type organism. Such vaccines generally provide a high level of protection, especially if administered by a natural route, and some may only require one dose to confer immunity. Another advantage of some attenuated vaccines is that they provide person-to-person passage among members of the population. These advantages, however, are balanced with several disadvantages.
Some attenuated vaccines have a limited shelf-life and cannot withstand storage in tropical environments. There is also a possibility that the vaccine will revert to the virulent wild-type of the organism, causing harmful, even life-threatening, illness. The use of attenuated vaccines is contraindicated in immunodeficient states, such as AIDS, and in pregnancy.
Production of subunit vaccines require knowledge about the epitopes of the microorganism or cells to which the vaccine should be directed. Other considerations in designing subunit vaccines are the size of the subunit and how well the subunit represents all of the strains of the microorganism or cell. The current focus for development of bacterial vaccines has shifted to the generation of subunit vaccines because of the problems encountered in producing whole bacterial vaccines and the side effects associated with their use. Such vaccines include a typhoid vaccine based upon the Vi capsular polysaccharide and the Hib vaccine to Haemophilus influenzae. Other vaccines which have been developed include combination vaccines and DNA vaccines. An example of a combination vaccine is the Bordetella pertussis toxin and its surface fimbrial hemaglutinin. In DNA vaccination, the patient is administered nucleic acids encoding a protein antigen that is then transcribed, translated and expressed in some form to produce strong, long-lived humoral and cell mediated immune responses to the antigen. The nucleic acids may be administered using viral vectors or other vectors, such as liposomes.
In addition to the typical use of vaccines for protection against disease, vaccination is being used to fight cancer. The idea of non-specifically stimulating the immune system to reject tumors is nearly a century old. Coley, an early reseacher in the field, used bacterial filtrates with considerable success. Attempts to vaccinate against cancer with purified cytokines and immunostimulants have had only limited success and have been effective for only a few types of tumors.
Many diseases, in addition to cancer, are mediated by the immune system. The diseases include allergies, eczema, rhinitis, urticaria, anaphylaxis, transplant rejection, such as kidney, heart, pancreas, lung, bone, and liver transplants; rheumatic diseases, systemic lupus erthematosus, rheumatoid arthritis, seronegative spondylarthritides, sjogren's syndrome, systemic sclerosis, polymyositis, dermatomyositis, type I diabetes mellitus, acquired immune deficiency syndrome, Hashimoto's thyroiditis, Graves' disease, Addison's disease, polyendocrine autoimmune disease, hepatitis, sclerosing cholangitis, primary biliary cirrhosis, pernicious anemia, coeliac disease, antibody-mediated nephritis, glomerulonephritis, Wegener's granulomatosis, microscopic polyarteritis, polyarteritis nodosa, pemphigus, dermatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia, Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
The present invention comprises methods and compositions for targeted delivery of component-specific immunostimulating molecules to individual immune cells. These component-specilic immunostimulating molecules bind and stimulate specific immune cells because of specific receptors on the cells. Thus, in a mixture of different cell types, the component-specific immunostimulating molecules are bound only by cells having the selected receptor, and cells lacking the receptor are unaffected. In some populations of immune cells, only one cell type contains receptors that bind a given component-specific immunostimulating agent. In other cell populations, multiple immune cells may contain the receptor that binds the component-specific immunostimulating agent. It is also possible for a given cell type to contain receptors for multiple component-specific immunostimulating agents.
In another embodiment, the present invention comprises methods and compositions for the production of antigen-specific, speciespecific monoclonal antibodies. These methods and compositions rely upon the conversion of immune cells. In a preferred embodiment, the methods and compositions comprise thein vitro conversion of circulating immune cells. These cells mount a primary response to the antigen, resulting in the production of antigen specific antibody. These selected primary clones are then immortalized to produce cells that secrete antibodies comprised entirely of protein from the selected species.
Another object of the present invention is to provide compositions for using simultaneous/sequential component-specific agents to initiate an immune response to a primary cancer capable of not only enhancing the immune response to the primary tumor but also mounting a systemic immune response to residual disese.
FIG. 8 illustrates that the gold stain is associated with frefloating clusters of activated B-cells, not macrophages or dendritic cells.
The inventors have found that they could use certain component-specific immunostimulating agents provide a specific stimulatory, up regulation, effect on individual immune components. For example, Interleukin-1β (IL-1β) specifically stimulates macrophages, while TNF-α (Tumor Necrosis Factor alpha) and Flt-3 ligand specifically stimulate dendritic cells. Heat killed Mycobacterium butyricum and Interleukin-6 (IL-6) are specific stimulators of B cells, and Interleukin-2 (IL-2) is a specific stimulator of T cells. Compositions comprising such component-specific immunostimulating agents provide for specific activation of macrophages, dendritic cells, B cells and T cells, respectively. For example macrophages are activated when a composition comprising the component-specific immunostimulating agent IL-1β, is administered. A preferred composition is IL-1β in association with colloidal metal, and a most preferred composition is IL-1β in association with colloidal metal and an antigen to provide a specific macrophage response to that antigen.
The methods and compositions of the present invention can also be used to treat diseases in which an immune response occurs, by stimulating or suppressing components that are a part of the immune response. Examples of such diseases include, but are not limited to, Addison's disease, allergies, anaphylaxis, Bruton's syndrome, cancer, including solid and blood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, type 1 diabetes mellitus, acquired immune deficiency syndrome, transplant rejection, such as kidney, heart, pancreas, lung, bone, and liver transplants, Graves' disease, polyendocrine autoimmune disease, hepatitis, microscopic polyarteritis, polyarteritis nodosa, pemphigus, primary biliary cirrhosis, pernicious anemia, coeliac disease, antibody-mediated nephritis, glomerulonephritis, rheumatic diseases, systemic lupus erthematosus, rheumatoid arthritis, seronegative spondylarthritides, rhinitis, sjogren's syndrome, systemic sclerosis, sclerosing cholangitis, Wegener's granulomatosis, dernatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, urticaria, transient hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia, Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
The present application claims priority to, and herein incorporates by reference, in their entirety, U.S. Application No. 09/189,748, filed on Nov. 10, 1998, and to U.S. Provisional Application No. 60/107,455, filed Nov. 6, 1998, and to U.S. Provisional Application No. 60/075,811, filed Feb. 24, 1998, and to U.S. Provisional Application No. 60/065,155 filed Nov. 10, 1997.
The methods and compositions of the present invention can further be used to produce antigen-specific, species-specific monoclonal antibodies that enhance immune response. These antibodies are produced, for example, by contactingin vitro an antigen, antigen presenting cells (APCs), immune cells, such as B cells, and optionally one or more component-specific immunostimulating agents. Once antigen-specific antibodies are detected, the activated immune cells are immortalized, for example, by fusing with human immortalized cancer cells. The resulting hybridomas can then be screened for specific antibody secretion and a single monoclonal antibody producing cell may then be isolated.
The antigen, APCs, immune cells, and component-specific immunostimulating agent may all be introduced into the in vitro culture at the same time. Optionally, these various components may be added sequentially in any order or combination. The antigen and component-specific immunostimulating agent may be two distinct molecules, or may be present in the form of a complex. For example, an antigen may be complexed with different cytokines, which when added in a sequential fashion would stimulate specific cells in the culture in a predictable stepwise fashion.
The compositions of the present invention comprise component-specific immunostimulating agents. Such a composition may comprise one component-specific immunostimulating agent or multiple component-specific immunostimulating agents. In one preferred embodiment, the composition comprises component-specific immunostimulating agents in association with colloidal metals. More preferably the compositions comprise component-specific immunostimulating agents in association with colloidal metals and other elements for specifically targeting the effect of the component-specific immunostimulating agents, including, but not limited to, antigens, receptor molecules, nucleic acids, pharmaceuticals, chemotherapy agents, and carriers.
The term �colloidal metal� as used herein, includes any water-insoluble metal particle or metallic compound as well as colloids of non-metal origin such as collodial carbon dispersed in liquid or water (a hydrosol). Examples of colloidal metals which can be used in the present invention include, but are not limited to, metals in groups IIA, IB, IIB and IIIB of the periodic table, as well as the transition metals, especially those of group VIII. Preferred metals include gold, silver, aluminum, ruthenium, zinc, iron, nickel and calcium. Other suitable metals may also include the following in all of their various oxidation states: lithium, sodium, magnesium, potassium, scandium, titanium, vanadium, chromium, manganese, cobalt, copper, gallium, strontium, niobium, molybdenum, palladium, indium, tin, tungsten, rhenium, platinum, and gadolinium. The metals are preferably provided in ionic form (preferably derived from an appropriate metal compound), for example, the Al3+, Ru3+, Zn2+, Fe3+, Ni2+ and Ca2+ ions. A preferred metal is gold, particularly in the form of Au3+. An especially preferred form of colloidal gold is HAuCl4 (E-Y Laboratories, Inc., San Mateo, Calif.). Another preferred metal is silver, particularly in a sodium borate buffer, having the concentration of between approximately 0.1% and 0.001%, and most preferably as approximately a 0.01% solution. The color of such a colloidal silver solution is yellow and the colloidal particles range from 1 to 40 nanometers. Such metal ions may be present in the complex alone or with other inorganic ions.
Any antigen may be used in the present invention. Examples of antigens useful in the present invention include, but are not limited to, Interleukin-1 (�IL-1�), Interleukin-2 (�IL-2�), Interleukin-3 (�IL-3�), Interleukin-4 (�IL-4�), Interleukin-5 (�IL-5�), Interleukin-6 (�IL-6�), Interleukin-7 (�IL-7�), Interleukin-8 (�IL-8�), Interleukin-10 (�IL-10�), Interleukin-11 (�IL-11�), Interleukin-12 (�IL-12�), Interleukin-13 (�IL-13�), lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin B and other toxins, Type I Interferon, Type II Interferon, Tumor Necrosis Factor (�TNF-α�), Transforming Growth Factor-β (�TGF-β�)Lymphotoxin, Migration Inhibition Factor, Granulocyte-Macrophage Colony-Stimulating Factor (�CSF�), Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelial growth factor (�VEGF�), Angiogenin, transforming growth factor (�TGF-α�), heat shock proteins, carbohydrate moieties of blood groups, Rh factors, fibroblast growth factor, and other inflammatory and immune regulatory proteins, nucleotides, DNA, RNA, mRNA, sense, antisense, cancer cell specific antigens; such as MART, MAGE, BAGE, and heat shock proteins (HSPs); mutant p53; tyrosinase; autoimmune antigens; immunotherapy drugs, such as AZT; and angiogenic and antiangiogenic drugs, such as angiostatin, endostatin, and basic fibroblast growth factor, and vascular endothelial growth factor (VEGF).
The following is the general experimental protocol that was followed for binding a molecule, whether antigen or component-specific immunostimulating agent, to colloidal gold. The molecule was reconstituted in water. 200 μg of the molecule was incubated with 25 mL colloidal gold for 24 hours. The molecule/colloidal gold complex solution was then centrifuged at 14,000 rpm for 20 minutes in a micro centrifuge at room temperature. The supernatant was then removed from the pellet.
50 μg of epidermal growth factor (EGF) was bound to 25 ml of 40 nm colloidal gold particles at a pH of 11.0. The solution rocked on rocking platform for 24 hours. 50 μg (added as 50 μl) of targeting cytokine (i.e., IL-1β to target macrophages, IL-2 to target T cells, IL-6 to target B cells, and either TNF alpha or Flt-3 Ligand to target dendritic cells) was added to the EGF/Au solution and rocked for an additional 24 hours. To separate colloidal gold bound and unbound material the solution was then centrifuged at 14,000 rpm. The supernatant was removed and the pellet was reconstituted in 1 ml of water containing 1% human serum albumin.
EGF was bound to colloidal gold (CG) using the procedure in Example 2. Tumor Necrosis Factor-α (TNF-α) was then bound to the EGF/CG complex using the procedure of Example 1 to produce an EGF/CG/TNF-α chimera.
EGF was bound to colloidal gold (CG) using the procedure in Example 2. Interleukin-2 (IL-2) was then bound to the EGF/CC complex using the procedure of Example 1 to produce an EGF/CG/IL-2 chimera.
The buffy coat was separated from a sample of whole blood as is well known in the art. 100-500 mL of whole blood was collected on heparin. The blood was carefully layered onto a 50% (v/v) ficoll-hypaque solution and centrifuiged at 2700 rpm for 7 minutes. The buffy coat, the collection of white blood cells at the serum/ficoll interface, was collected with a Pasteur pipette and placed into 10 mL of PBS containing 0.5 mg/mL heparin. The were centrifuged at 1500 rpm and thepellet washed and recentrifuged. The cells were washed 2� in the PBS solution and centrifuged once again.
As shown in FIG. 1, only macrophages internalized the EGF/CG/I-1β chimera, while only dendritic cells internalized the EGF/CG/TNF-α chimera (FIG. 2). Similarly, only B cells internalized the EGF/CG/IL-6 chimera (FIG. 3), and only T cells internalized the EGF/CG/IL-2 chimera (FIG. 4).
For this example staphyloccal enterotoxin B was used as the putative antigen/vaccine molecule, since there is evidence that binding the toxin to colloidal gold reduces its toxicity. 500 μg of the toxin was initially bound to 250 ml of 40 nm colloidal gold particles. The colloidal solution was then aliquotted. 50 ug of a targeting cytokine (IL-1β, IL-2, IL-6 and TNFα) was added to one of the aliquots and re-incubated for 24 hours. The toxin-AU-cytokine colloid was centrifuged at 14,000 rpm and the supernatant removed. The pellet was reconstituted to 1 ml of water. The pellet was assayed for cytokine concentration by either sandwich or competitive ELISA. This was done to determine the amount of neat cytokine (unbound) that was to be injected in control animals receiving saline or toxin alone.
Neat TNFβ
The two aliquots were then centrifuged at 14,000 rpm for 20 minutes. The supernatant was then removed from the pellet. The pellets were blocked by reconstitution with 10 ml of a 1% solution of human serum albumin (HSA) in water at apH of 11.
Generation of Human-Anti-Human INF-α Antibodies
The buffy coat was separated from peripheral blood by ficollation and washed with PBS containing 0.5 mg/ml heparin and EDTA. The cells were placed into 10-T-75 culture flasks. The cells were cultured for two weeks in RPMI with ten percent (10%) heat inactivated fetal bovine serum, ten percent (10%) ORIGEN� and 100 ng/ml of cytokine cocktail which is composed of the TNF-α/colloidal gold complex of Example 8 along with the following cytokines: IL-4, IL-6, IL-7, IL-10, IL-11, stem cell factor (�SCF�), GMCSF, and GSF, both alone and bound to colloidal gold.
ELISA Assay for Human-Anti-Human TNF-α Antibodies
Volume of a PEG added (dropwise)
Effect of Colloidal Gold Bound TNF-α on Cell Surface Markers Determined by Flow Cytometry
Each well contained 2 ml of either (1) media alone, (2) 0.5 ug/ml of the mitogen phytohemaglutinin (PHA) (for the induction of a T cell response), (3) 1.0 ug/ml of the mitogen lipopolysaccharides (L,PS) (for the induction of an inflammatory response), or (4) a combination of the mitogens LPS & PHA each at a final concentration of 0.5 & 1.0 ug/mI, respectively. Note that it is possible to use other mitogens, such as Pokeweed mitogens, as well as other agents including superantigens, such as staphylococcal enterotoxin A and B in this assay. The ills were stimulated with either mitogens (PHA or LPS) alone, or mitogens in the presence of either gold/TNF-α which has been stabilized with polyethylene glycol (PEG) in HSA (human serum albumin) or gold/TNF-α which has been treated with HSA alone. The culture plates were harvested for flow cytometric analysis of the cell surface, cell activation markers, and cytokine expressions.
Although flow cytometric analysis did not reveal any significant changes in CD4, CD8, or CD19 cell populations within 24-48 hours after stimulation, trafficking of the colloidal gold bound TNF-α into several cell types was observed. While control cells had a normal transparent phenotype, the stabilized and unstabilized gold treated cells had concentrations of the gold stain in several bcations on the cells as well as cell clusters. The distribution of the gold was varied from a central intracellular location to the cell surface. Also, the material appeared in multiple cell types, including rounded as well as dendritic cells. In rounded cells the localization of the gold material was either in the nucleus or on one side of the cell surface. Although not identified by cell surface markers, the rounded cells are thought to be differentiating monocytes/macrophages because of their ability to form giant cells. (FIGS. 6a and 6 b) The colloidal gold stain disappeared with time and, therefore, does not appear to be permanent. This indicates that the colloidal gold/TNF-α mixture was being metabolized once it entered the cell. However, the cells retained their ability to uptake colloidal gold since the stain reappeared upon restimulation with colloidal gold bound TNF-α.
As illustrated in FIG. 8, the gold stain was no longer associated with either the macrophage or dendritic cells, but was associated with frefloating clusters of cells, which may be activated B-cells. Phenotypying studies are currently underway to confirm this hypothesis.
Streptavidin bound to colloidal gold exhibited saturable binding kinetics. For this experiment 500 μg streptavidin was bound to 50 ml of 32 nm colloidal gold for 1 hour. Subsequently, 5 ml of a stabilizing solution (5% PEG 1450,0.1% BSA) was added to the tube and allowed to mix for an additional 30 mm. The sol was centrifuged to remove unbound streptavidin and washed 2 times with 5 ml of the stabilizing solution. After a final spin, the pellet was reconstituted to a volume of 5 ml with the stabilizing solution. 1 ml aliquots were distributed to microfuge tubes. To these tubes increasing amounts of biotinylated human TNF alpha were added. The biotinylated cytokine was incubated with the streptavidin gold for 1 hour. The material was centrifuged at 10,000 rpms for 10 mm. The resultant supernatant was collected and saved for TNF determinations. The pellets from each tube were washed 1 time with stabilizing solution and recentrifuged. The supernatant from this spin was discarded. The pellet was reconstituted to 1 ml with stabilizing solution and both the pellet and initial supernatant were assayed for TNF concentrations using our CYTELISA� TNF kit. One can see that greater than 90% of the biotinylated TNF immunoreactivity was found in the pellet (FIG. 10.) indicating that the biotinylated TNF was captured by the streptavidin bound gold.
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