Source: http://www.google.com/patents/US7790167?dq=6,202,008
Timestamp: 2017-11-20 07:06:11
Document Index: 33349202

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'art. 100', 'Application No. 02729092', 'art 1']

Patent US7790167 - Methods and compositions for enhancing immune response and for the ... - Google Patents
The 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/US7790167?utm_source=gb-gplus-sharePatent US7790167 - Methods and compositions for enhancing immune response and for the production of in vitro Mabs
Publication number US7790167 B2
Application number US 10/325,485
Also published as US6407218, US6528051, US20020071826, US20030180252
Publication number 10325485, 325485, US 7790167 B2, US 7790167B2, US-B2-7790167, US7790167 B2, US7790167B2
Original Assignee Cyt Immune Sciences, Inc.
Patent Citations (114), Non-Patent Citations (70), Referenced by (4), Classifications (26), Legal Events (4)
US 7790167 B2
The 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, B cells and T cells, are individually activated by component-specific immunostimulating agents. One such component-specific immunostimulating agent is an antigen-specific, species-specific monoclonal antibody. The invention is also directed to a method for the in vitro production of the antigen-specific, species-specific monoclonal antibodies which relies upon the in vitro conversion of blood-borne immune cells, such as macrophages and lymphocytes. Vaccine efficacy is enhanced by the administration of compositions containing component-specific immunostimulating agents and other elements, such as antigens or carrier particles, such as colloidal methods, such as gold.
1. An immune component-stimulating composition comprising at least one antigen and at least one independent component specific immunostimulating agent bound to a colloidal metal particle, wherein the at least one antigen or the at least one independent component specific immunostimulating agent or both are bound to the colloidal metal particle by binding pairs, and wherein the antigen is not TNF-α or lymphotoxin.
2. The composition of claim 1, wherein the independent component specific immunostimulating agent is selected from the group consisting of adjuvants, receptor molecules, nucleic acids, immunogenic proteins, pharmaceuticals, chemotherapy agents, and accessory cytokines; and wherein the accessory cytokines are selected from the group consisting of interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), 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, 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 (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factors, nucleotides, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof.
3. The composition of claim 1, wherein the antigen is selected from the group consisting of, nucleic acids, interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), 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, 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 (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factors, nucleotides, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof.
4. The composition of claim 1, wherein the immune component-stimulating composition is used to stimulate or suppress immune components in the treatment of a disease wherein the disease is selected from the group consisting: cancer, 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, Grave's 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 lymphocyte leukemia, and non-Hodgkin's lymphoma.
6. The composition of claim 5, wherein the adjuvant comprises liposomes, microcapsules and microspheres.
7. An immune component-stimulating composition comprising at least one component-specific immunostimulating agent, an antigen and a colloidal metal; wherein the colloidal metal is bound to the at least one component specific immunostimulating agent and the antigen, and wherein the component specific immunostimulating agent comprises interleukin- 1β (IL-1β), Flt-3 ligand, interleukin-6 (IL-6), heat killed Mycobacterium butyricum, or interleukin-2 (IL-2), wherein the at least one antigen or the at least one component specific immunostimulating agent or both are bound to the colloidal metal by binding pairs, and wherein the antigen is not TNF-α or lymphotoxin.
This application is a continuation of U.S. patent application Ser. No. 09/935,062, filed Aug. 22, 2001 now U.S. Pat. No. 6,528,051 which is a continuation of Ser. No. 09/189,748, now U.S. Pat. No. 6,407,218, filed Nov. 10, 1998 which claims benefit of U.S. Provisional Patent Application No. 60/065,155, filed Nov. 10, 1997, U.S. Provisional Patent Application No. 60/075,811, filed Feb. 24, 1998 and U.S. Provisional Patent Application No. 60/107,455, filed Nov. 6, 1998.
The present invention relates generally to immunology. More specifically, the invention relates to methods and compositions for the enhancement of an immune response in a human or animal. Such enhancement may result in stimulation or suppression of the immune response. The invention also relates to targeted component-stimulating compositions that easily and efficiently present antigenic components to particular immune cells to enhance an immune response in a human or animal. The present invention further relates to the use of such methods and compositions for the production of antigen-specific, species-specific monoclonal antibodies and the in vitro methods for production of such antibodies.
The introduction of desired agents into specific target cells has been a challenge to scientists for a long time. The challenge of specific targeting of agents is to get an adequate amount of the agent or the correct agent to the target cells of an organism without providing too much exposure of the rest of the organism. A very desired target for delivery of specific agents is the selective control of the immune system. The immune system is a complex response system of the body that involves many different kinds of cells that have differing activities. Activation of one portion of the immune system usually causes many different responses due to unwanted activation of other related portions of the system. Currently, there are no methods or compositions for producing the desired response by targeting the specific components of the immune system.
One method that has been used with limited success is the targeting of cells that bear a specific receptor and providing an antibody to that receptor that acts as a carrier for an agent. The agent could be a pharmaceutical agent that is a cell stimulant or the therapeutic agent could be a radioactive moiety that causes cell death. The problems inherent in this techniques are the isolation of the specific receptor, the production of an antibody having selective activity for that receptor and no cross reactivities with other similar epitopes, and attachment of the agent to the antibody. A problem attendant to such limited delivery is that the agent may never be released internally in the targeted cell, the agent is not releasably bound to the antibody and therefore, may not be fully active or capable of any activity once it is delivered to the site.
One of the better known aspects of the immune system is its ability to respond to foreign antigens presented by invading organisms, cellular changes within the body, or from vaccination. Some of the first kinds of cells that respond to such activation of the immune system are phagocytes and natural killer cells. Phagocytes include among other cells, monocytes, macrophages, and polymorphonuclear neutrophils. These cells generally bind to the foreign antigen, internalize it and may. destroy it. They also produce soluble molecules that mediate other immune responses, such as inflammatory responses. Natural killer cells can recognize and destroy certain virally-infected embryonic and tumor cells. Other factors of the immune response include both complement pathways which are capable of responding independently to foreign antigens or acting in concert with cells or antibodies.
One of the aspects of the immune system that is important for vaccination is the specific response of the immune system to a particular pathogen or foreign antigen. Part of the response includes the establishment of “memory” for that foreign antigen. Upon a secondary exposure, the memory function allows for a quicker and generally greater response to the foreign antigen. Lymphocytes in concert with other cells and factors, play a major role in both the memory function and the response.
Generally, it is thought that the response to antigens involves both humoral responses and cellular responses. Humoral immune responses are mediated by non cellular factors that are released by cells and which may or may not be found free in the plasma or intracellular fluids. A major component of a humoral response of the immune system is mediated by antibodies produced by B lymphocytes. Cell mediated immune responses result from the interactions of cells, including antigen presenting cells and B lymphocytes (B cells) and: T lymphocytes (T cells).
The response is initiated by the recognition of foreign antigens by various kinds of cells, principally macrophages or other antigen presenting cells. This leads to activation of lymphocytes, in particular, the lymphocytes that specifically recognize that particular foreign antigen and results in the development of the immune response, and possibly, elimination of the foreign antigen. Overlaying the immune response directed at elimination of the foreign antigen are complex interactions that lead to helper functions, stimulator functions, suppresser functions and other responses. The power of the immune system's responses must be carefully (ntrolled at multiple sites for stimulation and suppression or the response will either not occur, over respond or not cease after elimination.
In the immune response to antigens, immune cells interact with each other by direct cell to cell contact or indirect cell to cell (factor mediated) communication. For example, interactions between T cells, macrophages, dendritic cells, and B cells are necessary for an effective immune response. B and T cells are activated by signals from dendritic cells or macrophages, which are antigen presenting cells (APC) that present antigens and deliver activation signals to resting cells. Activated T cells help control immune responses and participate in the removal of foreign organisms. Helper T cells cause cells to become better effector cells, such as helping cytotoxic T cell precursors, to develop into killer cells, helping B cells make antibodies, and helping increase functions of other cells like macrophages. Activated B cells divide and produce antigen specific antibodies and memory B cells. The cells involved in the immune response also secrete cellular factors or cytokines, which enhance the functions of phagocytes, stimulate inflammatory responses and effect a variety of cells.
The biggest drawback to the use of monoclonal antibodies is the fact that nonhuman monoclonal antibodies are immunogenic when injected into a human patient. After injection of a foreign antibody, the immune response mounted by a patient can be quite strong. The immune response causes the quick elimination of the foreign antibody, essentially eliminating the antibody's therapeutic utility after an initial treatment. Unfortunately, once the immune system is primed to respond to foreign antibodies, later treatments with the same or different nonhuman antibodies can be ineffective or even dangerous because of crossreactivity.
Mice can be readily immunized with foreign antigens to produce a broad spectrum of high affinity antibodies. However, the introduction of murine antibodies into humans results in the production of a human-anti-mouse antibody (HAMA) response due to the presentation of a foreign protein in the body. Use of murine antibodies in a patient is generally limited to a term of days or weeks Longer treatment periods may result in anaphylaxis. Moreover, once HAMA has developed in a patient, it often prevents the future use of murine antibodies for diagnostic or therapeutic purposes.
Recombinant molecular biological techniques have been used to create chimeric antibodies. Recombinant DNA technology was used to construct a gene fusion between DNA sequences encoding mouse antibody variable light and heavy chain domains and human antibody light chain (LC) and heavy chain (HC) constant domains to permit expression of chimeric antibodies. These chimeric antibodies contain a large number of nonhuman amino acid sequences and are immunogenic to humans. Patients exposed to these chimeric antibodies produce human-antichimera antibodies (HACA). HACA is directed against the murine V region and can also be directed against the novel V-region/C-region (constant region) junctions present in recombinant chimeric antibodies.
To overcome some of the limitations presented by the immunogenicity of chimeric antibodies, molecular biology techniques are used to created humanized or reshaped antibodies. The DNA sequences encoding the antigen binding portions or complementarity determining regions (CDRs) of murine monoclonal antibodies are grafted, by molecular means, on the DNA sequences encoding the frameworks of human antibody heavy and light chains. The humanized Mabs contain a larger percentage of human antibody sequences than do chimeric Mabs. The end product, which comprises approximately 90% human antibody and 10% mouse antibody, contains a mouse binding site on an human antibody. It also contains certain amino acid substitutions from the mouse Mab into the framework of the humanized Mab in order to retain the correct shape, and thus, binding affinity for thetarget antigen.
In an effort to avoid the immune response to foreign proteins, a variety of approaches are being developed to make human Mabs that contain only human antibody components. One approach is to isolate a human B cell clone that naturally makes antibody to the desired antigen and grow it in a trioma cell culture system. Because human antibodies are made only against antigens that are foreign to thehost, none of the human B cells will make antibodies against human antigens. Therefore, this approach is not useful to produce Mabs against antigens that are human proteins.
All of the current technologies for producing human or human-like Mabs are insufficient to provide a species specific antibody that is antigen specific for a described antigen. Chimeric antibodies have the advantages of retaining the specificity of the murine antibody and stimulating human Fe dependent complement fixation and cell-mediated cytotoxicity. However, the murine variable regions of these chimeric antibodies can still elicit a HAMA response, thereby limiting the value of chimeric antibodies as diagnostic and therapeutic agents.
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.
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 1 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 macroglobulinem.ia, 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-specific 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.
Additionally, it is contemplated in the. present invention that the compositions and methods described herein can be used forstimulation of an immune response or the suppression of an immune response. Administration of component-specific immunostimulating agents for the suppression of immune responses can be used to control autoimmune diseases or organ rejection.
In another embodiment, the present invention comprises methods and compositions for the production of antigen-specific, species-specific 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.
In a preferred embodiment of the invention the antibodies produced are wholly human monoclonal antibodies which are produced through the in vitro culturing of human peripheral blood lymphocytes. A key element to this invention is the antigenic recognition of “self” molecules. Such self molecules include those molecules that are native or naturally occurring in an individual, as well as any molecule having a structure which is the same as that which occurs naturally in a particular species. This recognition reduces immunogenicity because the antibodies contain protein from only one species.
The methods and compositions of the present invention provide a novel and versatile approach to systems for the targeted stimulation of an immune response. In one disclosed embodiment, the present invention comprises component-stimulating compositions. In a preferred embodiment, the component-stimulating compositions comprise component-specific immunostiinulating agents. In another preferred embodiment, the component-stimulating compositions comprise component-specific immunostimulating agents in association with colloidal metal. In yet another preferred embodiment, such compositions comprise an antigen in combination with a component-specific immunostimulating agent, and in a further preferred embodiment an antigen and a component-specific immunostimulating agent are bound to a colloidal metal, such as colloidal gold, and the resulting chimeric molecule is presented to the immune component.
One embodiment of such a composition comprises a delivery structure or platform with a member of a binding group reversibly bound to it. A preferred embodiment of the present invention comprises colloidal gold as a platform that is capable of binding a member of a binding group to which component-specific immunostimulating molecules and antigen/vaccine molecules are bound to create a component-stimulating composition. In a more preferred embodiment, the binding group is streptavidin/biotin and the component-specific immunostimulating molecule is a cytokine. Embodiments of the present invention may also comprise binding the component-specific. immunostimulating molecules or antigen/vaccine in a less specific method such as by using polycations.
This patent contains at least one color photograph. Copies of this patent with the color photographs will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
FIG. 6 a is a 200× bright field micrograph illustrating the giant cell formation induced by this long term incubation of isolated human lymphocytes with colloidal gold/TNF-α. FIG. 6 b is a 200× phase contrast micrograph bright field monograph of the same cells.
Enhancement of an Immune Response
The present invention relates to compositions and methods for enhancing an immune response and increasing vaccine efficacy through the simultaneous or sequential targeting of specific immune components. More particularly, specific immune components including, but not limited to, antigen presenting cells (APCs), such as macrophages and dendritic cells, and lymphocytes, such as B cells and T cells, are individually effected by one or more component-specific immunostimulating agents. An especially preferred embodiment provides for activation of the immune response using a specific antigen in combination with the component-specific immunostimulating agents. As used herein, component-specific immunostimulating agent means an agent, that is specific for a component of the immune system, and that is capable of effecting that component, so that the component has an activity in the immune response. The agent may be capable of effecting several different components of the immune system, and this capability may be employed in the methods and compositions of the present invention. The agent may be naturally occurring or can be generated and manipulated through molecular biological techniques or protein receptor manipulation.
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 be used to enhance the effectiveness of any type of vaccine. The present methods enhance vaccine effectiveness by. targeting specific immune components for activation. Compositions comprising component-specific immunostimulating agents in association with colloidal metal and antigen are used for increasing the contact between antigen and specific immune component. Examples of diseases for which vaccines are currently available include, but are not limited to, cholera, diphtheria, Haemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid, Varicella-zoster, whooping cough, and yellow fever.
The antigen/component-specific immunostimulating agent/metal-complex is slowly released from the liposome and is recognized by the immune system as foreign and the specific component to which the component-specific immunostimulating agent is directed activates the immune system. The cascade of immune response is activated more quickly by the presence of the component-specific immunostimulating agent and the immune response is generated more quickly and more specifically.
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, dermatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis, Lambert-Eaton syndrome, sciera, 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. patent application Ser. No. 09/935,062, filed Aug. 22, 2001, U.S. Pat. No. 6,407,218, filed Nov. 10, 1998, U.S. Provisional Patent Application No. 60/065,155, filed Nov. 10, 1997, U.S. Provisional Patent Application No. 60/075,811, filed Feb. 24, 1998 and U.S. Provisional Patent Application No. 60/107,455, filed Nov. 6, 1998.
Production of In Vitro Monoclonal Antibodies
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 contactingiin 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 bound colloidal metal composition can be produced by the method described below. The antigen bound colloidal metal may be added to the buffy coat or APCs alone, or in the presence of adjuvants, immunogenic proteins, nucleotides, or accessory cytokine/immuostimulators which aid in the development of a Th2/B-cell, response. Optionally, these adjuvants, immunogenic proteins, nucleotides, and accessory cytokine/immuostimulators may be bound to the colloidal metal in a manner similar to that by which the antigen was bound prior to incubation of the colloidal metal bound antigen with the buffy coat or APCs.
Although immortaliztion of the primary clones may be accomplished in any manner, the following is one preferred method. Immortalized cancer cells are added directly to the vessel containing the seroconverted cells. After incubation, the cells are washed in serum free DMEM (Delbecco's Minimum Essential Medium), PBS (Phosphate Buffered Saline), or any serum free physiologic buffer. The cells may then be fused, for example, using a 40% to 100% PEG solution diluted in serum free DMEM. The fused cells may then be washed and the pellet reconstituted in a 50% DMEM/RPMI media containing 10% fetal bovine serum (FBS), 10% Origen™, the antigen cocktail mentioned above and a selective media, such as the hybridoma selecting agent HAT at a final concentration of 10%. The cells are seeded into 96 well tissue culture plates in 150 μl aliquots. To increase the proliferation of clones, the cells may, optionally, be stimulated by the addition of the initial antigen or antigen/component-specific immunostimulating agent mixture such as those used in the initial immunizations.
The cells may be grown in HAT (hypozanthine, aminopterin, thymidine) containing medium for approximately two weeks. Then a nonselective media, such as HT (hypozanthine, thymidine) is substituted for the HAT as a selection drug. After another incubation of about two weeks, the cells are grown in a growth media, such as 50% DMEM/RPMI supplemented with the antigen cocktail, 10% Origen™, and 10% FBS.
In another embodiment of the present invention, the buffy coat or APCs may be incubated simultaneously with the colloidal metal bound antigen and optionally an adjuvant. This type of incubation has been found to change the type of immunoresponse elicited from a Thl-like response, in which the colloidal metal antigen is associated with the APCs which may or may not contain cellular elements, to a Th2-type response in which the colloidal metal bound antigen is associated with the free-floating clusters of B cells.
Component-Stimulating Compositions
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 a ssociation 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.
In another embodiment, the component-specific immunostimulating molecules of the present invention comprise a delivery structure or platform. The component-specific immunostimulating molecule and/or the antigen/vaccine may be bound directly to the platform or may be bound to the platform throug members of a binding group. A preferred embodiment of the present invention comprises a colloidal metal as a platform that is capable of binding a member of a binding group to which component-specific immunostimulating agents and putative antigen/vaccines are bound to create a targeted immune-enhancing agent. In a most preferred embodiment, the binding group is streptavidin/biotin and the component-specific immunostimulating agent is a cytokine. Embodiments of the present invention may also comprise binding the antigen/vaccine in a less specific method, without the use of binding partners, such as by using polycations or proteins.
The present invention comprises methods and compositions for targeted delivery of component-specific immunostimulating molecules that use colloidal metals as a platform. Such colloidal metals bind, either reversibly or irreversibly, molecules that interact with either an antigen/vaccine or component-specific immunostimulating agents or antigen/vaccine. The interacting molecules may either be specific binding molecules, such as members of a binding pair, or may be rather nonspecific interacting molecules that bind less specifically. The present invention contemplates the use of interacting molecules such as polycationic elenents known to those skilled in the art including, but not limited to, polylysine, protamine sulfate, histones or asialoglycoproteins.
Method for Binding of Composition Components to Platform
Each of the elements of the compositions may be bound, separately or in combinations, to the colloidal metal by any method. However, a preferred method for binding the elements to the colloidal metal is as follows. In this example, the composition comprises an antigen and a component-specific immunostimulating agent, though the method is not limited to this embodiment. The antigen is reconstituted in water. Approximately 50 to 100 μg of antigen is then incubated with colloidal metal.
The pH of the colloidal antigen mixture may have to be adjusted so that it is 1-3 pH unites above the pl of the component specific agent. Subsequently, 50-100 μg of the component specific agent is added to the antigen colloidal mixture and incubated for an additional 24 hours. During this time, the targeting component specific agent becomes incorporated into the antigen gold complex resulting in an immune component targeted antigen delivery system. The inventors have successfully performed such experiments and in fact have linked up to 3 different moieties on the same colloidal metal particle.
After the binding of the component specific agent to the antigen/Au the mixture is stabilized by the addition of a 1% v/v solution of 1-100% polyethylene glycol. Other stabilizing agents may include Brij 58 and cysteine, other sulfhydryl containing compounds, phospholipids, polyvinylpyrolidone, poly-L-lysine and/or poly-L-proline. The mixture is stabilized overnight and subsequently centrifuged to separate the bound antigen and component specific agent from unbound material. The mixture is centrifuged at 14,000 rpms for 30 min., the supernatant removed and the pellet resuspended in water containing 1% albumin. This procedure has a relatively high efficiency of coupling the antigen and targeting component since 75% to 95% of both moieties are bound. Furthermore, free material which is not bound to the colloid is separated by centrifugation.
Exemplified Components
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 HAuCI4 (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, lnterleukin-I (“IL-I”), 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 component-specific immunostimulating agent may be any molecule or compound which increases the APC's ability to stimulate the B cell's production of antibodies. Examples of component-specific immunostimulating agents include, but are not limited to, antigens, colloidal metals, adjuvants, recqptor moelcules, nucleic acids, immunogenic proteins, and accessory cytokine/immuostimulators, pharmaceuticals, chemotherapy agents, and carriers. These component-specific immunostimulating agents may be employed separately, or in combinations. They may be employed in a free state or in complexes, such as in combination with a colloidal metal.
Accessory cytokine/immuostimulators include, but are not limited to, Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3 (“IL-3”), Interleukin-4 (“IL4”), 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 HSPs; flt3 ligand/receptor system; B7 family of molecules and receptors; CD 40 ligand/receptor; and immunotherapy drugs, such as AZT; and angiogenic and anti-angiogenic drugs, such as angiostatin, endostatin, and basic fibroblast growth factor, and vascular endothelial growth factor (VEGF).
Methods and compositions, other than the use of colloidal metal, can be used to deliver the component-specific immunostimulating agents, alone or in combination with antigens or other elements. For example, the compositions may be encapsulated in a liposome or microsphere or may be delivered by means of other cell delivery vehicles, such as a viral vector. Additional combinations are colloidal gold particles studded with viral particles which are the active vaccine candidate or are packaged to contain DNA for a putative vaccine. The gold particle would also contain a cytokine which could then be used to target the virus to specific immune cells. Furthermore. one could create a fusion protein vaccine targets two or more potential vaccine candidates and generate a vaccine for two or more applications. The particles may also include immunogens which have been chemically modified by the addition of polyethylene glycol which may release the material slowly.
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 40nm 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 EGFICG/TNF-o chimera.
EGF was bound to colloidal gold (CG) using the procedure in Example 2. Interleukin-6 (IL-6) was then bound to the EGF/CG complex using the procedure of Example 1 to produce an EGF/CG/IL-6 chimera.
EGF was bound to colloidal gold (CG) using the procedure in Example 2. Interleukin-2 (IL-2) was then bound to the EGF/CG 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 centrifuged 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/IlL-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.
The immunization strategy involved simultaneous or sequential administration of neat toxin/cytokine mixture (as composition controls) or the toxin-Au-cytokine chimera. 5 mice/group were injected on days 1, 5 & 9 with either 2.5 ug neat toxin or the same dose of toxin/cytokine mixture bound to colloidal gold. During the 14 day immunization period two additional groups of mice received the neat toxin/cytokine or toxin-Au-cytokine following the schedule provided in Table 1.
Day Group type Treatment Injected
1 Control Neat toxin + Neat IL-1β +
Gold Toxin-Au-IL-1β +
5 Control Neat toxin + Neat IL-6
Gold Toxin-AU-IL-6
9 Control Neat toxin + Neat IL-2
Gold Toxin-AU-IL-2
All groups were rechallenged with 1 μg of neat toxin alone on day 30. Protective immunization was demonstrated by the reduced or lack of ability of the neat toxin to induce morbidity. The key observation is that the toxin bound to colloidal gold greatly reduced the toxicity of the toxin. Secondly, serum antibody titers to the toxin were 10× higher than those receiving neat treatment alone. However, the serum antibodies of animals receiving the sequential treatment were 100 times greater than the animals receiving the neat treatment. Finally, upon the rechallenge with the neat toxin 100% of the animals treated with toxin died whereas only 20% fatality was observed in the simultaneous group.
Binding of Cytokine to Colloidal Gold
Generation of Human-Anti-Human TNF-α Antibodies
The bufty 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 Assayfor Human-Anti-Human TNF-α Antibodies
1 ml aliquots were taken from three of the flasks of cells treated as shown in Example 9 and centrifuged at 1,500 rpm for 15 minutes. The supernatant was collected and stored at −20° C.
Recombinant human TNF was coated onto the wells of a microtiter plate in a carbonate/bicarbonate buffer. The plate was washed four times with TBS having 2.0 ml/l-Tween 20. 100 μl of the supernatant was added to each well. Control wells received unused growth medium. The samples were incubated overnight at room temperature.
Cell Fusion and Hybridoma Selection
Once in vitro seroconversion of the cells in Example 9 was confirmed, 106-107 K6H6/B5 myeloma cells were added directly into the vessel in which the seroconverted cells were detected. The cells were gently mixed, collected and centrifuged at 1,200 rpm for 15 minutes. The supernatant was removed, and the pellet washed in serum free DMEM. The cells were centrifuged one last time, and the supernatant completely removed. The pellet was gently tapped loose, and the cells fused by the addition of a 53% PEG 1450 solution according to the strategy described in the table below.
The PEG solution was added to the cells using the following method and incubations, while shaking the cells at 37° C.:
Time Volume of PEG added (dropwise)
0 min 0.5 ml over 30 seconds and wait 30 seconds
1 min 0.5 ml over 30 seconds and wait 30 seconds
2 min 1.0 ml over 60 seconds and wait 60 seconds
Time Volume of DMEM added (dropwise)
0 min 1.0 ml over 30 seconds and wait 30 seconds
4 min 1.0 ml over 30 seconds and Wait 30 seconds
5 min 8.0 ml over 60 seconds and wait 60 seconds
7 min 15.0 ml over 60 seconds and incubate 1 minute
The cells were subsequently centrifuged at 1,200 rpm for 15 minutes. The supematant was removed, and the pellet was reconstituted in a 50% DMEM/RPMI media containing 10% FBS, 10% Origen™, the cytokine cocktail mentioned above and the hybridoma selecting agent HAT at a final concentration of 10%. The cells were initially seeded into five 96 well tissue culture clusters in 150 μl aliquots. To increase the proliferation of clones, the cells were also stimulated with 25 μl colloidal gold bound TNF-α used in the initial immunizations.
The cells were grown in HAT containing medium for two weeks, after which HT was-substituted for the HAT as a selection drug. Following two weeks of growth, the cells were grown in 50% DMEM/RPMI media supplemented with the cytokine cocktail, 10% Origen™, and 10% FBS.
Testing of Supernatants for Positive Antibody Function
The presence of TNF-α-specific antibodies in the samples in Example 13 was tested during all phases of the growth. The supernatants were initially tested by direct EIA and then by an in vitro assay which measures the inhibition of proliferation of WEHI cells in a dosedependent manner by TNF-α. Positive clones were scaled-up from original 96 well plates to 6 well plates. Subsequently, all clones testing positive were scaled-up for cryopreservation, as well as the generation of 5 ml of ascites in pristine primed mice. The ascites were purified, and the antibody tested for its ability to prevent the inhibition of proliferation of WEHI cells by TNF-α. The ability of the purified antibody to block bioactivity indicated its neutralizing activity.
Effect of Colloidal Gold Bound TNF-α on Cell Surface Markers Determined by Flow Cytometty
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 (LPS) (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 cells 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 CDl9 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 roundud 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. 6 a 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.
Lymphocytes were isolated from the buffy coats of human peripheral blood obtained from the American Red Cross. The lymphocytes were treated with either (1) colloidal gold alone, (2) colloidal gold bound with human serum albumin (HSA), or (3) colloidal gold bound with TNF-α. Each group was divided into two aliquots. One aliquot was blocked with 1% PEG, and the other remained untreated.
This result is indicative of receptor mediated binding of the colloidal gold bound TNF-α. Additionally, the requirement of TNF-α for the differentiation of dendritic cells suggests that the colloidal gold bound TNF-α retained its biologic activity.
This experiment was designed to determine the effect of adjuvant components on the uptake of colloidal gold by isolated lymphocytes. The experiment was performed in the same manner as Examples 6 and 7, except that an additional group of cells was included. These cells received 100 μl of a 1.0 mg/ml suspension of heat killed Mycobacterium Butyricum. This bacteria is routinely used in adjuvant preparation for antibody generation.
As illustrated in FIG. 8, the gold stain was no longer asociated with either the macrophage or dendritic cells, but was associated with frelfloating 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 supematant 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.
This experiment was to evaluate the feasibility of the streptavidin gold complex as a targeted drug delivery system. In order for this occur the streptavidin conjugated colloidal gold must bind both a biotinylated targeting ligand as well as a biotinylated therapeutic. To investigate this, we performed the following experiment.
100 ml of a 32 nm colloidal gold solution was bound with a saturating concentration of streptavidin. After 1 hour the sol was centrifuged and washed as described above. The colloidal gold bound streptavidin was then bound with sub-saturating concentrations of biotinylated cytokine. The material was vortexed and incubated for 1 hour at room temperature. Afterwards the sol was centrifuged and the pellet incubated with a solution of biotinylated polylysine. After a 1 hour incubation, the sol was re-centrifuged and washed. After a final spin and resuspension (the final volume of the sol was approximately 1 ml) 50 μg of the Ogalactosidase reporter gene was incubated with the concentrated streptavidin/biotinylated cytokine/polylysine chimera for 1 hour. The material was centrifuged to remove unbound plasmid DNA. The final construct (biotin EGF-SAP-Au-biotin polylysine-DNA) was centrifuged at 14,000 rpms. The supernatant was assayed for the presence of DNA by determining its OD at 260 nm. We observed a decrease in the supernatant OD @ 260 nm from 0.95 to 0.25 after the incubation of the plasmid DNA with the biotin EGF-SAP-Au-biotin polylysine construct. The DNA was bound by the biotin EGF-SAP-Au-biotin polylysine-DNA and was centrifuged out of the sol into the pellet. These data show that a new drug delivery system was developed using avidin binding to colloidal gold. Biotinylation of the targeting and delivery payload was then used as the method for binding these molecules to the colloidal gold based drug/gene delivery system.
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U.S. Classification 424/184.1, 424/85.2, 424/278.1, 424/604, 424/649, 424/85.1
International Classification C07K16/24, C07K16/18, A61K47/48
Cooperative Classification A61K47/64, A61K47/665, A61K47/645, C07K16/241, A61K47/6923, B82Y5/00, Y10S530/806, Y10S530/808, A61K2039/55522, A61K2039/55516, C07K2317/77, A61K2039/55505
European Classification A61K47/48R6D, A61K47/48R2, A61K47/48W8B, A61K47/48R2T, C07K16/24B
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