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Matched Legal Cases: ['Application No. 60', 'Application No. 2004311630', 'Application No. 2004311630', 'Application No. 02729092', 'Application No. 02729092', 'art 1', 'art 1', 'Application No. 200480041234', 'Application No. 200480041234']

Patent US7951614 - Methods and compositions for the production of monoclonal antibodies - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsThe present invention comprises compositions and methods for making monoclonal antibodies. The present invention further comprises vectors that replicate the immune system components, particularly an antigen-presenting cell (APC) element of the immune synapse. Additionally, the present invention may...http://www.google.com/patents/US7951614?utm_source=gb-gplus-sharePatent US7951614 - Methods and compositions for the production of monoclonal antibodiesAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS7951614 B2Publication typeGrantApplication numberUS 12/549,207Publication dateMay 31, 2011Filing dateAug 27, 2009Priority dateDec 2, 2003Fee statusPaidAlso published asCA2548179A1, CN1925843A, EP1694301A2, EP1694301A4, US8435801, US20050175583, US20100068261, US20110195456, WO2005065121A2, WO2005065121A3Publication number12549207, 549207, US 7951614 B2, US 7951614B2, US-B2-7951614, US7951614 B2, US7951614B2InventorsLawrence Tamarkin, Giulio F. PaciottiOriginal AssigneeCytimmune Sciences, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (130), Non-Patent Citations (122), Referenced by (2), Classifications (37), Legal Events (2) External Links: USPTO, USPTO Assignment, EspacenetMethods and compositions for the production of monoclonal antibodies
US 7951614 B2Abstract
The present invention comprises compositions and methods for making monoclonal antibodies. The present invention further comprises vectors that replicate the immune system components, particularly an antigen-presenting cell (APC) element of the immune synapse. Additionally, the present invention may further comprise synthetic T-cells.
This application is a divisional of U.S. patent application Ser. No. 11/004,623 filed Dec. 2, 2004 now abandoned which claims priority to U.S. Provisional Application No. 60/526,360 filed Dec. 2, 2003.
The present invention relates generally to immunology. The present invention further relates to methods and compositions for the production of monoclonal antibodies and 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 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 a variety of responses due to unwanted activation of other related portions of the system. Currently, there are no satisfactory methods or compositions for producing a specifically desired response by targeting the specific components of the immune system.
Transgenic mice are also being used to create “human” antibodies. The transgenic mice are created by replacing mouse immunoglobulin gene loci with human immunoglobulin loci. This approach may provide advantages over phage display technologies because it takes advantages of mouse in vivo affinity maturation machinery.
The present invention comprises compositions and methods for making species-specific antigen-specific monoclonal antibodies, preferably IgG monoclonal antibodies. The present invention further comprises vectors that replicate elements of the immune system, particularly the antigen-presenting cell (APC) element of the immune synapse. A preferred vector optionally comprises binding an antigen-loaded major histocompatibility (MHC) class II protein, the co-stimulatory protein B7, and the structural protein intracellular adhesion protein (I-CAM) onto the surface of colloidal metal vectors. Such vectors replicate the 3-D orientation of the APC (FIG. 3) generating a synthetic antigen-presenting cell (sAPC) capable of activating CD4+ T-cells to mature the antibody response of immunized B-cells.
FIG. 1 provides a schematic representation of the immune synapse.
The present invention may be understood more readily by reference to the following detailed description of specific embodiments included herein. Although the present invention has been described with reference to specific details of certain embodiments, thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention. The entire text of the references mentioned herein are hereby incorporated in their entireties by reference, including U.S. Provisional Application Ser. No. 60/526,360.
The most common procedures require that the production of monoclonal antibodies start with the immunization of an animal. Antigen, draining into a local lymph node or spleen, activates naïve B-cells to produce IgM antibodies. These activated B cells are then presented with antigen-activated CD4+ T cells to induce class switching. Class switching is characterized by a change in the production of antibody type from IgMs to IgGs (Kuby, J., Immunology Third Edition 1997. eds Allen D., pp-205-213). Antibody secreting B cell lymphocytes are isolated from the lymph node or spleen of the immunized animal, and are fused with species-specific myeloma cells. The fused cells are then allowed to grow to produce antigen specific IgG antibodies. During the screening process, positive fusion clones are selected for their therapeutic potential.
The first barrier to in vitro antibody production is the relatively low conversion rate of naïve human B cell lymphocytes to activated B cells. In the past resolving this challenge proved difficult even when recall antigens, such as Tetanus toxin (Butler et. al., J. Immuol. 1983. volume 130: pp-165), were used to induce a primary antibody response from human peripheral blood B cell lymphocytes. The present invention comprises methods for making vectors that activate pathways that lead to antibody generation. The present invention also comprises compositions of naturally occurring or synthetic vectors. Such vectors comprise colloidal gold platforms with multiple B cell ligands associated.
Generation of a primary antibody response from naïve human B cells in vitro represents only the first step in the in vitro reconstruction of the human antibody response. The primary antibody response from immunized human B cells results in the secretion of IgM antibodies. A second class of lymphoid cells, known as antigen presenting cells (APCs), also internalizes the antigen. Once internalized these cells process the protein antigen into fragments, which are then expressed on the cell's surface bound to one of two major histocompatibility complexes (MHCs). These cells are important for antibody class switching.
The methods and compositions of the present invention comprising synthetic antigen-presenting cells (sAPC) comprise compositions that are readily available and can be “pulled out of the refrigerator” and used to manipulate the human antibody response. Thus the present invention comprises methods of treatment of diseases and immune related dysfunctions and pathologies. The colloidal metal compositions provide control over the variables that are responsible for initiating, maintaining and regulating the immune response (either down-regulating or up-regulating), such as particle size, the amount of protein bound per particle, the flexibility of protein movement on the particle, as well as the 3-D assembly of the particles, ensures reproducible control of the sAPC.
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 colloidal 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 HA, 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 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. Another preferred metal is gold, particularly in the form of Au3+. An especially preferred form of colloidal gold is HAuCl4 (OmniCorp, South Plainfield, N.J.). The color of such a colloidal silver solution is yellow and the colloidal particles may range from 1 to 100 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, Pertussis toxin, Tetanus toxin and other toxins, Type I Interferon, Type II Interferon, Tumor Necrosis Factor (TNF-α or b), 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, Epidermal growth factor (“EGF”), 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; mucines, such as Muc-1, PSA, TSH, autoimmune antigens; immunotherapy drugs, such as AZT; and angiogenic and anti-angiogenic drugs, such as angiostatin, endostatin, basic fibroblast growth factor, and vascular endothelial growth factor, prostate specific antigen and thyroid stimulating hormone.
Examples of component-specific immunostimulating agents 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”), Flt-3 ligand, 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 anti-angiogenic drugs, such as angiostatin, endostatin, basic fibroblast growth factor, vascular endothelial growth factor (VEGF) and prostate specific antigen and thyroid stimulating hormone.
After diafiltration, cryoprotectants, such as a compositions of mannitol, 20 mg/ml; and/or human serum albumin, 5 mg/ml, are added and the samples frozen at −80° C. The samples are lyophilized to dryness and sealed under a vacuum, subsequently reconstituted and analyzed for the amount of free and colloidal gold bound TNF present in the reconstituted samples.
Manufacture of Colloidal Gold
Colloidal gold sols are manufactured using the reaction described by Frens and Horisberger (Frens, G. Nature Phys. Sci. 1972, 241, 20-22, and Horsiberger, M. Biol. Cellulaire. 1979. 36: 253-258). In this reaction ionic gold, in the form of HAuCl4, is reduced to nanoparticles of Au0 by the addition of sodium citrate. Typically, 2.5 ml of a 4% chloroauric acid (in water) solution is added to 1 L of deionized water. The solution is vigorously stirred and heated to a rolling boil. The reduction reaction is initiated by the addition of a 1% sodium citrate solution. The size of the particle is controlled by the amount of citrate added to the reaction. For example, 17, 32, and 64 nm particles are formed by the addition of 40, 15, and 7.5 ml of the citrate solution, respectively. After the addition of citrate, the solution is allowed to boil and mix for an additional 45 minutes. Upon cooling, the sol is filtered through a 0.22 μm sterilization filter and stored at room temperature until used.
Increasing the Number of Immune Competent B Cells
To increase the number of immune competent B cells for immunization, MHC class II restricted-surface IgM+/sIgD+ human B cells are isolated from units of whole blood or buffy coats. Magnetic beads coated with anti-IgM, anti-IgD and anti-CD19 antibodies separate the B cell populations. Treating sIgM+/sIgD− immature B cells with the cytokine interleukin-7 is used to recruit additional B cells (Sudo, T., Ito, M., Ogawa, Y., Iizuka, M., Kodoma, H., Kunisasa, T., Hayashi, S. C., Ogawa, M., Sakai, K., Nishikawa, S., Nishkawa, S. C. J. Exp. Med. 1989. 170: 333-338). This treatment has been shown to mature these B cells as signaled by the phenotype conversion of sIgM+/sIgD− B cells to sIgM+/sIgD+ B cells. These isolated cells are purified to near homogeneity using FACS separation.
Differentiation of the Primary Antibody Response
Critical to the production of a therapeutic antibody is the process of class switching. The primary antibody response from immunized human B cells results in the secretion of IgM antibodies. A second class of lymphoid cells, known as antigen presenting cells (APCs), also internalizes the antigen. Once internalized these cells process the protein antigen into fragments, which are then expressed on the cell's surface bound to one of two major histocompatibility complexes (MHCs).
Creation of sAPC/sTc/sGC with Spacer Arms
This sAPC is built on streptavidin colloidal gold particles that are used to bind biotinylated forms of the MHC, B7, and ICAM proteins. This single particle sAPC has a greater degree of flexibility, since the constituent proteins are bound to the colloidal gold particle indirectly through biotinylated spacer arms that form a biotin-avidin bridge. Similarly, the sTc and sGCs may be generated using a similar strategy for tethering their respective components to the colloidal metal.
Self-Assembling APCs/sTcs/sGCs
Self-assembling synthetic APCs are developed. Binding each APC protein to a different colloidal gold particle creates a complex matrix of immune synapse proteins. To direct the assembly of this sAPC, site directed molecular scaffolds are made to better orient the various particles in 3-D. Shown in FIG. 5 is a representation of this self-assembling sAPC. The formulation of each particle subunit allows for a single particle to bind multiple reagents. For illustration purposes the MHC class II molecule is bound to a 32 nm colloidal gold particle that is also bound with streptavidin. The remaining two subunits of the sAPC, the B7 and ICAM, are bound to 17 nm particles. Like the MHC particle the ICAM subunit contains streptavidin-docking sites. To assemble this particle biotinylated human serum albumin is used to join the ICAM and MHC particles together. To complete the assembly of the vector, dithiolated polyethylene glycol is used to link the MHC and B7 particles together.
In this model, the formation of the immune synapse occurs through T-cell receptor/membrane rearrangements. This vector may also be bound to a solid support stage such as an EIA plate. These scaffolds allow both colloidal gold-targeted antigens and sAPCs present in the same matrix. As a result, upon immunization of the naïve B-cell the sAPC may activate the CD4 cell to express CD40 ligand and as a result induce class switching.
Binding of Proteins to Colloidal Gold Particles
The binding of proteins to colloidal gold particles is influenced by the pH of the colloidal gold sol and protein solutions. At an optimal pH, proteins bind to the surface of colloidal gold particles and prevent their precipitation by salts. Salt-induced precipitation of the colloidal gold is easily documented by the changes in the color of the sol from red to black. The pH binding optimum is determined for each protein described, including the MHC, B7, ICAM, IL-6 and the KLH:TNF antigen. As an example, the procedure described below outlines the method for binding the MHC molecule to the colloidal gold particles. A similar procedure will be used to determine the binding conditions for each of the other proteins
Quantification of the Mass of the MHC Protein Bound
To quantify the mass of the MHC protein bound per particle of gold, quantitative EIAs are developed for the measurement of the MHC and B7 proteins. EIAs for ICAM are already commercially available. The MHC and B7 proteins are quantitatively measured by developing a competitive binding EIA for each protein. Commercially available antibodies to B7 and MHC proteins (both antibodies are available from Research Diagnostics, Inc.) are coated onto EIA plates using a carbonate/bicarbonate buffer at pH 9.6. MHC and B7 reference standards are generated to provide a dose range of 1.56 ng/ml to 500 ng/ml. These standards are added to the EIA plate containing specific antibodies for either the MHC or B7 protein. The colloidal gold bound samples are added to other designated wells in the EIA plate.
Binding Multiple Proteins to the Same Particle
To increase the efficiency and specificity of the in vitro immunization multiple chemically distinct proteins need to be bound onto the surface of a single colloidal gold particle. The binding of three different protein cytokines (IL-1, IL-6 and TNF) to the same particle of colloidal gold is demonstrated. Each cytokine binds to colloidal gold at a specific pH.
Targeting of Chimeric Vectors to Specific Cells
EGF and streptavidin were bound to the same 32 nm particle of colloidal gold. The sample was divided into three aliquots for the binding of secondary/targeting molecules. One sample was bound with biotinylated IL-1, another biotinylated GM-CSF, and the third with biotinylated IL-6. After binding the biotinylated ligands, the samples were centrifuged to remove any free reagents and the colloidal gold pellets were added to Ficoll-separated human white blood cells. After 8 days in culture the uptake of the various colloidal gold vectors was documented by digital photography.
Immunization of Human Lymphocytes
These vectors can be used to generate a primary immune response from isolated lymphocytes. White blood cells were collected from whole blood by density centrifugation. These cells were treated with a thyroglobulin conjugated TNF/IL-6 colloidal gold vector. The cells received pulses of the colloidal gold vector every 2 days for a total of eight days. After the final pulse, the cells were cultured for another 5 days. The supernatants were collected and tested for the presence of human anti-human TNF (IgM/IgD and IgG combination) antibodies using a direct EIA. As can be seen in FIG. 8, the chimeric cAU thyroglobulin TNF had the highest immunodensity.
Immunization of Human B-Cells and Dendritic Cells for Class II MHC Expression
Two different approaches to increase the efficiency of in vitro human lymphocyte immunization are used. First, coupling TNF to immunogenic carriers, such as Thyroglobulin, Keyhole Limpet Hemocyanin or Murine Serum Albumin enhances TNF's immunogenicity. Carrier:TNF conjugations are performed using standard EDC/NHS and gluteraldehyde methods. Second, coupling them to particles of colloidal gold, containing cell-specific targeting agents increases the specificity of these antigens. To target the delivery of the antigen to B cells the carrier:antigen complex is bound to particles of colloidal gold particles containing IL-6. To target the delivery of the carrier antigen to dendritic cells the carrier:antigen complex is bound to colloidal gold particles containing GM-CSF.
These vectors are initially used to immunize naïve MHC restricted human B cells and dendritic cells for the generation of the class II MHC antigen.
These same vectors are used at a later time to induce the primary antibody response from a new or replicate set of naïve B cells. The immunization scheme involves the sequential immunization of B cells and dendritic cells with the various vectors. As a result the B cells and dendritic cells see the carrier once and the TNF antigen three times.
Generation of Class II MHC Protein by B Cells
To cause human B-cells to produce class II MHC protein, 106 surface IgM+/IgD+ human B-cells are plated in 24-well plates and cultured in 1.5 ml of AIM V media. Twenty four hours after plating, the cells are pulsed with the THYRO:TNF antigen bound to an IL-6 targeted colloidal gold vector. Two days later the cells are pulsed with the KLH:TNF carrier targeted by the IL-6 vector. After an additional two days in culture the cells are immunized with the third carrier:TNF antigen, MSA:TNF. The cells are incubated for an additional three to seven days and tested for the presence of Class II MHC expression by FACS analysis. Alternatively, the cells may be simultaneously pulsed with the colloidal gold antigens.
Method Development for the Isolation of the MHC Class II Antigen
The method for the isolation of the MHC uses “generic”-non-MHC compatible blood samples. These MHC molecules are used to define the pH and saturation optima for the protein on colloidal gold particles. Once defined, these methods are adapted to purify antigen loaded MHC from immunized MHC restricted blood pools.
The isolation of generic and antigen loaded human class II MHC is done using the method described by Sette (Sette et al., J. Immunol. 1992. 148: 844). Briefly the buffy coats from non-HLA matched human whole blood are frozen at a minimum density of 108 cells/ml and sonicated to disrupt the cells. These cells are suspended in a buffer of 50 mM TRIS-HCl, pH 8.5 with 2% Renex, 150 mM NaCl, 5 mM EDTA and 2 mM PMSF. Large particulates including the nuclei are removed by centrifugation (10000×g for 20 minutes). The cell lysate is then fractionated on an affinity column made by binding murine antibodies to the human class II MHC molecule (Research Diagnostics Inc.) to protein A/G sepharose beads. The lysate is passed through the column at least 5 times to maximize the binding of the MHC protein to the immobilized antibody. The column is washed with 10 column volumes of a buffer containing 10 mM TRIS-HCl pH 8.0/0.1% Renex followed by an additional wash of 5 column volumes of PBS with 1% n-ocytlglucoside. The MHC class II protein is eluted from the column using a buffer of 50 mM diethylamine in saline with 1% n-ocytlglucoside at a pH 11.5. Upon elution each fraction is immediately neutralized with the addition of 2 M glycine, pH 2.0. The fractions containing the MHC II molecules are aliquoted and lyophilized in 25 μg aliquots.
Generation of Human B7.1 Molecule
The human co-stimulatory molecule B7.1 is made by recombinant DNA technology. The gene is supplied as part of a commercially available transient expression vector system (InVivogen Inc.). The construct is provided with the appropriate restriction sites allowing for the separation of the active gene from the plasmid construct. The human B-7.1 gene is isolated from the pORF host plasmid using the restriction enzyme NcoI and NheI. This double digestion results in the formation of two linearized pieces of DNA. One of the gene fragments consists of the B-7.1 gene (893 bp) while the other fragment (3210 bp) constitutes the accessory genes of the p-ORF plasmid. The gene fragments are fractionated on a 1% agarose gel and visualized by ethidium bromide staining. The bands are cut from the gel and purified using QuiaQuick gel extraction resin. The purified linearized gene is inserted into a baculovirus expression system (CloneTech Inc.) under the control of the strong CMV promoter. The baculovirus incorporated genes are transfected into the SF9 insect cell line according to the manufacturers specifications and conditions. 106 B7 transfected NOS cells will be expanded in bioreactors. The incubation media and cell lysates are processed by affinity chromatography using a murine monoclonal antibody against the human B7.1 protein (Research Diagnostics Inc.) previously immobilized to a protein A/G sepharose column.
Generation of the Synthetic Antigen Presenting Cell
The Single Particle sAPC
To mature the primary antibody response the sAPC capable must induce the CD4 T-cell/B-cell interactions that result in antibody class switching. The first sAPC is developed by binding the proteins of the immune synapse on a single particle of colloidal gold. This vector as well as one built on a streptavidn colloidal gold core are tested for their ability to activate CD4+ T-cells.
Generation of a Self-Assembling sAPC
The multiparticle sAPC will have the flexibility of self orientation during immune synapse formation. The flexibility is a direct result of assembling the moieties used to join the particles together. Linkers can be alkane, protein, and polyethylene glycol (PEG) to allow for the greatest vector functionality.
The MHC, B7 and ICAM proteins are bound to different particles of colloidal gold as previously described. The particles are physically joined by a variety of scaffolding molecules. The function of the “joining” molecules is to provide greater flexibility of the individual particles of colloidal gold in the formation of the immune synapse. This flexibility occurs whether the sAPC is provided as an independent particle or as part of a matrix bound to a solid surface.
Stimulation of CD4+T-cells by sAPC to Express CD4+ Ligand
Single particles and self-assembling sAPCs are tested for their ability to induce the expression of CD40 ligand from MHC restricted CD4+ T-cells. Subsequently, 0.1 to 10 ug of antigen loaded MHC (present on the sAPC) are added to 106 class II restricted CD4+ T cells growing in AIM V media. The stimulation occurs in the presence of IL-4 and IL-10, which drives the production of the TH2 subset of CD4+ T cells. After 4, 12 and 24 hours of sAPC stimulation the CD4 cells are collected and stained with a FITC labeled mouse anti human CD40 ligand antibody and analyzed by FACS.
Antibody Detection and Immortalization of B Cells
All of the cells from positive wells are combined, centrifuged once, washed with PBS and combined with 2×106 mouse/human heteromyeloma K6H6/B5 cells. The heteromyeloma cell line, K6H6/B5 (available through the ATCC), is an ideal fusion partner for these human lymphocytes because these cancer cells are non-secretors of antibody and are available with no patent restrictions. The human and myeloma cells are fused using standard fusion protocols with PEG. Successfully fused cells are selected using traditional HAT/HT selection protocols. A direct ELISA is used to test growing clones for the production of TNF specific human IgG antibody. Those clones that show antigen recognition are scaled-up in T-75 flasks, at which point all clones are cryopreserved and their supernatants tested for neutralizing antibody activity as described below.
Neutralization of TNF Biologic Activity
The ability of the TNF antibodies to neutralize the biologic activity of TNF is tested using the well-characterized WEHI 164 bioassay. Briefly, TNF dose-dependently inhibits the in vitro proliferation of these cells.
Effect of Ionic Strength on the Lyophilization Stability of Colloidal Gold Bound TNF
The colloidal gold binding apparatus, shown in FIG. 11, was used to bind TNF to colloidal gold nanoparticles as previously described. After binding, 30K PEG-Thiol was added to the solution at 50 μg/ml in deionized water, pH 9.
To test the effect of ionic strength on the stability of the TNF-colloidal gold bond various amounts of salt (in the form of 1× normal phosphate buffered saline; PBS) were added to the container holding the TNF solution. Final concentrations of PBS varied from 0 to 0.325% of normal PBS. After binding and diafiltration, cryoprotectants (mannitol, 20 mg/ml; human serum albumin, 5 mg/ml) were added to the samples. The samples were subsequently aliquoted into 1 ml samples and frozen at ±80° C. After freezing the samples were lyophilized to dryness and sealed under a vacuum.
Effect of Increasing Ionic Strength on the Stability of a Lyophilized Colloidal Gold-TNF Drug
The solution of TNF, which was previously diluted in a 3 mM TRIS solution to a final concentration of TNF of 0.5 μg/ml, was modified by adding 0.25× solution (77.25 milli-osmol/kg) of normal phosphate buffered saline. The solution was bound as was described above. After binding, 30K PEG-Thiol and the cryoprotectants described above were added and the samples frozen at −80° C. The samples were lyophilized as described above, subsequently reconstituted and analyzed for the amount of free and colloidal gold bound TNF present in the reconstituted samples. The data from this study are presented in FIG. 12.
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N., Colloidal Gold and Biotin-Avidin Conjugates as Ultrastructural Markers for Neural Antigens, Quarterly Journal of Experimental Physiology, vol. 69, pp. 1-33, Jan. 1, 1984.120Van Rensen et al., Liposomes with Incorporated MHC Class II/Peptide Complexes as Antigen Presenting Vesicles for Specific T Cell Activation, Pharmaceutical Research, vol./Iss: 16 (2), pp. 198-204, Jan. 1, 1999.121Vidal et al., Steric Stabilization of Polystyrene Colloids Using Thiol-ended Polyethylene Oxide, Polymers for Advanced Technologies, vol./Iss: 6, pp. 473-479, Nov. 15, 1994.122Walden et al., Induction of Regulatory T-lymphocyte Responses by Liposomes Carrying Major Histocompatibility Complex Molecules and Foreign Antigen, Nature, vol./Iss: 315, pp. 327-329, May 23, 1985.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS9152988Nov 19, 2013Oct 6, 2015Open Invention NetworkMethod and device utilizing polymorphic data in E-commerceUS20120134956 *Oct 26, 2011May 31, 2012Cytimmune Sciences, Inc.Colloidal metal compositions and methods* Cited by examinerClassifications U.S. Classification436/525, 530/811, 424/278.1International ClassificationC07K16/24, A61K31/28, A01N55/02, A61K45/00, A61K47/00, A61K39/395, G01N33/553, A61K47/48, A61K48/00, A61K9/14, A61K38/16Cooperative ClassificationY10S530/811, A61K47/48861, A61K47/48884, A61K47/4833, B82Y5/00, A61K33/38, A61K31/28, A61K33/26, A61K45/06, A61K33/06, C07K16/241, A61K33/24European ClassificationA61K47/48R6D, C07K16/24B, A61K33/38, B82Y5/00, A61K33/26, A61K33/24, A61K45/06, A61K47/48W8B, A61K31/28, A61K33/06, A61K47/48W14BLegal EventsDateCodeEventDescriptionDec 16, 2009ASAssignmentOwner name: CYTIMMUNE SCIENCES, INC.,MARYLANDFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TAMARKIN, LAWRENCE;PACIOTTI, GIULIO F.;REEL/FRAME:023662/0231Effective date: 20050128Owner name: CYTIMMUNE SCIENCES, INC., MARYLANDFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TAMARKIN, LAWRENCE;PACIOTTI, GIULIO F.;REEL/FRAME:023662/0231Effective date: 20050128Nov 7, 2014FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services