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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/515—CD3, T-cell receptor complex
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
  • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers

Definitions

• This invention relates to a biodegradable device for activating, expanding and differentiating T-cells for use in cell therapy treatment protocols.
• TEL tumor infiltrating lymphocyte
• cytotoxic T-cells see U.S. Patent No. 6,255,073 issued to Cai, et al.; and U.S. Patent No. 5,846,827 issued to Celis, et al.
• expanded tumor draining lymph node cells see U.S. Patent No. 6,251,385 issued to Terman
• various other lymphocyte preparations see U.S. Patent No.
• T-cells must be activated in order to proliferate, perform effector functions and produce cytokines (Liebowitz, Lee et al. 1998).
• T-cells require direct contact with antigen presenting cells ("APC") for activation.
• APC antigen presenting cells
• APC convert protein antigens to peptides and then present peptide-MHC complexes in a form that can be recognized by T-cells.
• the interaction of the peptide-MHC complex on the APC and the T-cell receptor (“TCR”) on the surface of the T- cell usually provides the first of the two signals required for activation.
• the second of the two signals required for activation is usually provided by membrane-bound or secreted products of the APC.
• T-cells Due to the difficulty in maintaining large numbers of natural APC in cultures and in identifying disease-associated antigens and controlling the processing and presentation of these antigens to T-cells by natural APC, alternative methods have been sought for ex-vivo activation of T-cells for use in cell therapy.
• One method is to by-pass the need for the peptide-MHC complex on natural APC by instead stimulating the TCR with polyclonal activators, such as immobilized or cross-linked anti-CD3 monoclonal antibodies (mAbs) to provide the first signal to T-cells.
• polyclonal activators such as immobilized or cross-linked anti-CD3 monoclonal antibodies (mAbs)
• mAbs monoclonal antibodies
• Other methods take advantage of the secondary T-cell activation pathway to provide the first signal, such as the use of immobilized or cross-linked anti-CD2 mAb.
• anti-CD3 mAb first signal
• anti-CD28 mAb second signal
• the signals provided by anti-CD3 and anti-CD28 mAbs are best delivered to T-cells when the antibodies are immobilized on a solid surface such as plastic plates (Baroja, Loire et al. 1989; Damle and Doyle 1989) or sepharose beads (Anderson, Blue et al. 1988) (see also US Pat No 6,352,694 issued to June, et al.).
• paramagnetic beads with immobilized mAbs for expansion of T-cells in cell therapy protocols requires the separation and removal of the beads from the T-cells prior to patient infusion. This is a very labor-intensive process and results in cell loss, cell damage, increased risk of contamination and increased cost of processing. Because of the tight association of the immobilized mAbs on the beads with the corresponding ligands on the surface of the target T-cells, the removal of the beads from the T-cells is difficult. The beadxell conjugates are often separated by waiting until the T-cells internalize the target antigens and then by using mechanical disruption techniques to separate the beads from the T-cells.
• This technique can cause damage to the T- cells and can also cause the ligated antigens on the T-cells to be removed from the cell surface for a period of time (Rubbi, Patel et al. 1993).
• highly activated T-cells are most desirable for use in cell therapy protocols and T-cells often lose this desirable property during the 24-72 hour waiting time for the T-cells to internalize the target antigens.
• the process of removing the paramagnetic beads after separation from the T-cells requires the passing of the cell/bead solution over a magnet. This process can greatly reduce the quantity of beads remaining with the T-cells, but does not completely eliminate the beads. This incomplete bead removal results in some beads being infused in patients which can cause toxic effects.
• the magnetic bead removal process also reduces the number of T-cells available for therapy, as many T-cells remain associated with the paramagnetic beads even after the waiting time and mechanical disassociation, and are thus removed with the beads in the magnetic field. Some cell loss also occurs when T-cells that may not be bound to the beads become entrapped by beads pulled to the surface next to the magnetic source.
• the present invention includes a biodegradable device for activating T- cells including a biodegradable support and a binder attached to the biodegradable support, the binder having reactivity to one or more agents capable of binding to a T-cell surface antigen.
• the present invention also includes a method for activating T-cells, the method comprising attaching one or more T-cell activators to a population of T- cells and mixing the T-cells with a biodegradable support with an attached binder having reactivity to the T-cell activators.
• the present invention utilizes a biodegradable support material coated with a first material capable of immobilizing or cross-linking one or more second materials with reactivity for structures on the surface of a T-cell.
• the biodegradable nature of the support material eliminates the need to employ a process to separate and remove the support from the T-cells prior to infusion into a patient.
• Biodegradable materials have been used for encapsulation of proteins for vaccination and controlled drug release (see for example US Pat No 6,572,894 issued to Rossling, et al.).
• Biodegradable materials have also been formulated for use as sutures (see e.g., Bezemer at al., 6,500,193), and have been used in tissue engineering applications (see e.g., Vert et al. in U.S. Pat. No. 4,279,249 and Slivka, et al in US Pat No. 6,511,511) and used as implants (see e.g., Leatherbury, et al. in US Pat. No. 6,514,286).
• biodegradable material for use in these prior art applications differ significantly from the requirements of the present invention.
• Prior art applications require slow, controlled degradation and encapsulation of active ingredients and/or high tensile strength and stability.
• the application of the present invention requires rapid degradation which does not need to be at a controlled rate and also does not require high tensile strength.
• the present invention does not require encapsulation of an active ingredient as in most prior art methods using biodegradable microspheres.
• the biodegradable material selected for use in the present invention must be non-toxic and non-antigenic in humans, and preferably must be capable of being delivered to humans parenterally, preferably intravenously.
• the biodegradable material can be derived from natural or synthetic materials that degrade in biological fluids. It is preferable that degradation occur using non- enzymatic means.
• biological fluids include cell culture media and blood.
• the biodegradable material must degrade rapidly (i.e., within a month, preferably within 2 weeks, more preferably within 1 week, and most preferably within 3 days).
• the biodegradable material degradation products must produce non-toxic by-products that can be metabolized and/or excreted via normal physiological pathways.
• biodegradable materials used in formulating the device of the present invention do not utilize organic solvents in the manufacturing process, as these solvents pose a health risk on long-term exposure in humans.
• a preferred embodiment is the formulation of microspheres from synthetic polymers of which virtually all fabrication processes require use of an organic solvent such as dichloromethane. If organic solvents are utilized in the manufacture of the biodegradable supports, attempts should be made to reduce the amount of residual solvent in the final formulation. Acceptable residual solvent concentrations are determined by regulatory agencies. For example, ICH (International Conference on Harmonization) guidelines set the maximal permissible dichloromethane levels in the blood at 6mg/day.
• suitable natural materials for use as biodegradable supports include proteins such as collagen, gelatin and albumen and polysaccharides such as starch, dextran, inulin, cellulose and hyaluronic acid.
• Examples of synthetic materials for use as biodegradable supports include aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides. These materials have been widely used as biodegradable polymers in medical applications. Synthetic polymers in general offer greater advantages over natural materials in that they can be tailored to give a wider range of properties and have more predictable lot-to-lot uniformity. The factors which affect the physical properties and performance of biodegradable polymers are well known. These factors include monomer selection, initiator selection, process conditions and the presence of additives.
• polymers should be selected for high hydrophilicity, polymers may be either semicrystalline or amorphous, preferably amorphous, with a glass transition temperature that is preferably well above 37° C, allowing the polymer to act more like a glass than a rubber at body temperature. Polymers should have a low molecular weight distribution and low inherent viscosity for accelerated degradation, and the preference is for random over blocky compositions for the same reason.
• Biodegradable polymers can be formulated into various shapes, such as films, strips, fibers, gels, meshes, sponges and spheres (such as nanospheres or microspheres). They can also be extruded, injection molded, compression molded, or solvent or spun cast. The primary processing may also be followed by subsequent machining into final parts. The choice of shape is dependent on the cell therapy application. For example, if the biodegradable material is to be used only to culture T-cells ex- vivo, but will be degraded so as not to be infused into a patient, the formulation of a matrix with high surface area is preferred. Such a matrix would preferably simulate the structure of dendritic cells in the lymph nodes, providing interconnecting star-burst like structures or honey-combed structure shapes.
• Microspheres are a preferred formulation because of the simplicity of manufacture and the spherical shape allows an increased surface area for interaction with cellular receptors. Small microsphere particle sizes of 1 to about 500 ⁇ m enables direct injection into the body by conventional methods. Thus the spherical shaped device can be used both for the cell culture and infusion steps of a cell therapy protocol. Nanospheres can also be utilized, however, nanospheres do not provide enough cross-linking to activate naive T- cells and thus can only be used with previously activated T-cells. In preferred embodiments, microspheres that range in size from l ⁇ m to 10 ⁇ m are formulated.
• the biodegradable support is first formulated into a shape, such as a microsphere.
• the biodegradable support is then coated with a first material providing a reactive surface which is capable of binding to one or more second materials.
• the second materials have a reactive surface which permits binding to surface structures on a cell.
• second materials are capable of transducing a signal to a cell through interaction with a surface expressed cellular receptor.
• the second materials can be first bound to the first material on the biodegradable support and then mixed with the target T- cells, whereby the second materials bind to surface structures on the T-cells.
• the second materials can be first bound to the surface structures of the T-cells and the T-cells with the bound second material then mixed with the biodegradable support coated with the first material.
• the final mixture contains a biodegradable support coated with a first material, such first material which is bound to one or more second materials, and such second materials which are bound to surface structures on a T-cell.
• the first material can be attached to the biodegradable support by means of absorption, reactive groups, or by means of a coupling agent or linker.
• the terms "coupling agent” or “linker” as used herein refer to bifunctional crosslinking or coupling agents such as molecules containing two reactive groups which may be separated by a spacer.
• Suitable first materials are any biocompatible material capable of binding to a portion of the second material. Examples of suitable first materials include polyclonal or monoclonal antibodies, or fragments thereof, and bioactive substances such as Protein A, avidin or biotin.
• suitable first materials are polyclonal antibodies with specificity for the mouse immunoglobulins, such as sheep or goat-derived anti-mouse polyclonal antibodies or anti-mouse monoclonal antibodies such as rat-derived anti-mouse Fc antibodies.
• a suitable first material is avidin or an antibody specific for biotin.
• a suitable first material is biotin or an anti-avidin antibody.
• the first materials can be agents with high affinity for IgG, such as Protein G or Protein A.
• First materials can be chemically coupled to the biodegradable support with glutaraldehyde or other di-aldehyde with or without the first attachment of diaminoheptane spacer groups to the biodegradable support.
• Covalent bonding by nucleophilic displacement to biodegradable supports activated with tosyl groups (p-toluenesulfonyl), through cyanogens bromide activation or other similar methods can also be used.
• the biodegradable support could also be coated directly with avidin or biotin to interact with a second material such as a mitogenic protein coated with the opposite corresponding biotin or avidin.
• Suitable second materials are biocompatible materials which are capable of binding to a cell surface structure.
• the binding of the second material to the T-cell surface will transduce a signal to the T-cell when the second agent is immobilized or cross-linked by the first material.
• Signal transduction will have the effect of causing the target T-cell to perform a function desirable in cell therapy applications, such as proliferate, produce cytokines, differentiate and/or express effector molecules such as FasL, TRAIL and CD40L.
• suitable second materials for use in the present invention include agents such as synthesized compounds, nucleic acids and proteins, including polyclonal or monoclonal antibodies, and fragments or derivatives thereof, and bioengineered proteins, such as fusion proteins.
• the second materials are mitogenic proteins. Mitogenic proteins are two or more proteins that are able to deliver the requisite minimum of two signals to T-cells in order to cause the T-cells to become activated.
• mitogenic proteins are anti-CD3 and anti-CD2 mAbs, in combination with a co-stimulatory protein such as and including proteins specific for one or more of the following T-cell surface molecules: CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-I, OX40, CD27L (CD70), 4- IBBL, CD30L and LIGHT, including the corresponding ligands to these surface structures, or fragments thereof.
• a co-stimulatory protein such as and including proteins specific for one or more of the following T-cell surface molecules: CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-I, OX40, CD27L (CD70), 4- IBBL, CD30L and LIGHT, including the corresponding ligands to these surface structures, or fragments thereof.
• suitable second materials include agents capable of delivering a signal to T-cells through cytokine receptors such as IL-2R, IL- 12R, IL-IR, IL- 15R, EFN-gammaR, TNF-alphaR, EL-4R, and IL-IOR, including mAbs to these receptors, fusion proteins with a reactive end specific for these receptors and the corresponding ligands to these receptors or fractions thereof.
• Other suitable second materials include any agent capable of binding to cellular adhesion molecules on T-cells such as mAbs, fusion proteins and the corresponding ligands or fractions thereof to adhesion molecules in the following categories: cadherins, ICAM, integrins, and selectins.
• adhesion molecules on T-cells are: CD44, CD31, CD18/CDl la (LFA-I), CD29, CD54 (ICAM-I), CD62L (L-selectin), and CD29/CD49d (VLA-4).
• suitable second materials include any agents capable of binding to chemokine receptors, including those in the C-C and C-X-C categories.
• chemokine receptors associated with T-cell function include CCRl, CCR2, CCR3, CCR4, CCR5, and CXCR3.
• the biodegradable support material is constructed from a linear polyester polymer containing a mixture of lactic acid and glycolic acid.
• PLGA polymers that meet the requirements of biocompatibiliry and biodegradation into harmless end products. These polymers, hereinafter referred to as PLGA, are degraded by ester hydrolysis into lactic acid and glycolic acid. PLGA has been shown to possess excellent biocompatibility. The innocuous nature of PLGA can be exemplified by the approval by the regulatory authorities, including the U.S. Food and Drug Administration, of several parenteral delayed release preparations based on these polymers. Parenterally administrable delayed release products currently on the market and based on PLGA include DecapeptyTM (Ibsen Biotech), Prostap S ® . (Lederle), Decapeptyl ® , Depot (Ferring) and Zoladex ® (Zeneca).
• DecapeptyTM Ibsen Biotech
• Prostap S ® . Lederle
• Decapeptyl ® Depot
• Zoladex ® Zoladex ®
• Copolymers of DL-lactate and glycolide, rather than L-lactate and glycolide, are preferred because they are amorphous when DL-lactate is a major component, as opposed to semicrystalline when L-lactate is a major component. This property decreases the degradation time of the polymer.
• the inherent viscosity (abbreviated as "LV. "; units are in deciliters/gram) of the polymer is a measure of its molecular weight.
• the inherent viscosity of the polymer is from about 0.10 dL/g to about 1.0 dL/g (as measured in chloroform), more preferably from about 0.10 dL/g to about 0.50 dL/g and most preferably from 0.10 to 0.30 dL/g.
• Suitable biodegradable polymer material is a 50/50 mixture of poly(DL- lactide co-glycolide).
• the polymer can be purchased from commercial suppliers such as Birmingham Polymers, Inc (Birmingham, AL) under the trade name Lactel ® .
• the 50/50 DL-PLG product number 50DG020 with a inherent viscosity of 0.15 to 0.25 dl/g is a preferred material for use in the present invention.
• Another preferred material is 50/50 DL-PLG with an inherent viscosity of 0.32 to 0.44 dl/g manufactured by Boehringer Ingelheim (Ingelheim, Germany) under the trade name Resomer ® RG 503.
• Lactel ® 50/50 DL-PLG product number 50D040 (Birmingham Polymers) with a 0.26 to 0.54 inherent viscosity.
• polymer end groups can be added to the biodegradable polymers, such as monofunctional alcohol, water or alpha-hydroxy acid, or PEG in order to increase the hydrophilicity of the polymer and thus increase the degradation time and provide active groups for covalent binding of first materials to the polymer.
• the 50/50 DL-PLG is formulated into microspheres.
• Microspheres can be prepared by various known methods, including solvent evaporation, phase separation, spray-drying, or solvent extraction at low temperature. The process selected should be simple, reproducible and scalable. The resulting microspheres should be free-flowing and not aggregates in order to produce a uniform syringeable suspension. The microspheres must also be sterile. This can be ensured by a terminal sterilization step and/or through aseptic processing.
• the solvent evaporation method is utilized to produce the microspheres (McGinity and O'Donnell 1997).
• the hydrophobic 50/50 DL-PLG polymer is dissolved in a water-immiscible organic solvent to give a polymer solution.
• the solution is then added into an aqueous solution of a surfactant to form an emulsion system and stirred.
• the faster the stirring speed the smaller the size of the microspheres.
• Microspheres are obtained by subsequently evaporating the solvent by continuous stirring, which can be under vacuum or heat.
• the water-miscible organic solvents of the present invention need to be non-toxic to the body.
• Typical examples of organic solvents are members selected from the group consisting of acetic acid, lactic acid, formic acid, acetone, acetonitrile, dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide, dioxane, and N-methyl pyrrolidone and mixtures thereof.
• the water-miscible organic solvent is a member selected from the group consisting of acetic acid, lactic acid, N-methyl pyrrohdone, or a mixture thereof.
• the water-miscible organic solvent may be used alone or in a mixture with water.
• the aqueous phase can contain an emulsion stabilizer that is preferably soluble in water and alcohol, is capable of increasing viscosity of the suspending medium (water-miscible alcohol) when dissolved in the medium, is non-toxic to the body and causes no environmental problems.
• emulsion stabilizer solutions are: water-soluble synthetic polymers such as polyvinylpyrrolidone, poly(ethylene glycol), and poloxamer; cellulose derivatives such as hydroxypropyl cellulose and hydroxypropylmethyl cellulose, and preferably, polyvinylpyrrolidone and hydroxypropyl cellulose.
• the content of emulsion stabilizer in the water-miscible alcohol is preferably within the range of 0.1 to about 50% (w/v), and more preferably within the range of 0.2 to about 20% (w/v).
• the content of emulsion stabilizer can be varied according to the viscosity of the water-miscible alcohol needed.
• the water-miscible alcohol, wherein the emulsion stabilizer is dissolved is stirred at a temperature of 10 about 8O 0 C, preferably from 20 to about 6O 0 C, and most preferably at room temperature at a speed of 200 to about 20,000 rpm, preferably at a speed of 800 to 1000 rpm.
• the polymer solution is slowly added to the water-miscible alcohol wherein the emulsion stabilizer is dissolved, and the mixture is stirred from 5 minutes to about 60 minutes. Stirring can be continued for up to 5 hours to allow evaporation of the organic solvent.
• the resulting microspheres can then collected by centrifugation and washed extensively. The washed microspheres are then ready for attachment of the first material.
• the diameter of the microspheres prepared should preferably be within the range from 0.01 to 300 urn, and more preferably within the range from 0.1 to 100 um. and most preferably between 1 and 10 um.
• the particle size (diameter of the microspheres) can be controlled by adjusting the stirring speed during processing, the viscosity of the water-miscible alcohol, and the viscosity of the polymer solution.
• Post-coating of the biodegradable support with the first material can be accomplished by a variety of standard methods depending upon the nature of the first material. For most applications where the first material is a protein, passive absorption techniques are adequate for protein attachment to the biodegradable support.
• Other applications may require direct covalent attachment or covalent attachment through a linking group, such as when using first materials with low affinity for the biodegradable support, or use of first materials such as DNA, lectins, enzymes and drugs, or in applications where the biodegradable device is used in an environment where a material is used that will displace the passively absorbed first material.
• Various schemes of modification to the surface of the biodegradable support can be used to introduce applicable functional groups for covalent protein immobilization including: hydrolysis to form carboxylic groups (the immobilization is carried out through the protein's amino groups using condensing agents), hydrazinolysis to form hydrazide groups (immobilization through the aldehyde groups of the glycoprotein's carbohydrate fragments oxidized with periodate), aminolysis with bifunctional amines (condensation with the protein's carboxylic groups), modification with glutaric aldehyde (immobilization through the amino and sulfhydryl groups of a protein) (Ertl, B., F. Heigl, et al. (2000).
• first material After coating a first material to the surface of the biodegradable support directly or through a linker, it is desirable to block non-specific adsorption of proteins that may be present during cell culture or upon infusion to a patient. Any innocuous protein may be used for this purpose. Bovine or human serum albumin are desired blocking agents. In cases where the large size of the albumin obscures the activity of smaller active. first material proteins, glycine or small polypeptides can be used as alternative blocking agents. If the biodegradable supports are formulated into particles of less than
• first materials that are proteins can be bond to the biological support material by adsorption with standard known methods.
• One method for adsorbing a protein to the biodegradable support where the support is formulated into microspheres is to suspend the microspheres in 0.1 M Borate buffer at pH 8.5, spin down and resuspend the microspheres 2 or 3 times.
• the first material protein is then suspended in the borate buffer and added to the microspheres.
• the mixture is mixed end-to-end for at least 4 hours and for up to 24 hours.
• the mixing is preferably conducted at 4° C.
• the microspheres are spun and the supernatant removed and analyzed for protein determination.
• the coated microspheres are then resuspended in a physiological buffer, such as phosphate buffered saline containing a blocking agent, such as 1- 5% bovine serum albumen and/or 0.05% w/v Tween 20.
• coated biodegradable supports can then be combined with the desired second materials or the second materials can be bound to the target T- cells and then mixed with the first material-coated biodegradable supports.
• Drying can also be accomplished by vacuum drying or drying in a recirculating air dryer. Care must be taken when drying polymers above room temperature, as some amorphous compositions may fuse when the drying temperature exceeds the glass transition temperature.
• the biodegradable devices are best packaged in small aliquots so that the material is used quickly once the package is opened. Packaging should be in desiccated moisture proof bags.
• the devices can be sterilized by a variety of methods such as storage in alcohol, gamma radiation or ethylene oxide gas. Biodegradable devices should not be sterilized by autoclave as the high temperatures can cause degradation.
• the devices of the present invention can also be stored by flash freezing and then stored in liquid nitrogen and can also be lyopbilized prior to storage.
• Example #1 Microsphere Preparation The solvent evaporation method was used for preparation of microspheres. Lactel ® (Birmingham Polymers, Birmingham, AL) 50/50 DL- PLG product number 50DG020 with a inherent viscosity of 0.15 to 0.25 dl/g was used as the polymer. The DL-PLG powder was dissolved in 20ml of methylene chloride to a final 5% DL-PLG w/v ratio.
• Polyclonal goat anti-mouse polyclonal antibody was suspended in 30 ml of PBS solution with 5% human serum albumen (HSA) at a concentration of 10 ug/ml. This solution was used to resuspend the dried microspheres at a concentration of approximately 2 x 10 8 particles per ml. The microspheres and the polyclonal antibody were mixed end over end at 4 0 C for 8 hours. The microspheres were then washed 3 times in PBS with HSA, filtered and dried.
• HSA human serum albumen
• second materials were added directly to the goat anti-mouse antibody coated microspheres.
• a 50/50 mixture of anti-human CD3 mAb and anti-human CD28mAbs at a final concentration of 10 ug/ml were prepared in PBS containing 5% HSA. This solution was then used to resuspend the coated microspheres at a final concentration of 2 x 10 particles per ml. The mixture was vigorously mixed end to end for 4 hours at room temperature, washed 3 times, filtered and dried overnight.
• microspheres In order to determine the size distribution of the microspheres, a aliquot of the spheres was analyzed by laser diffraction (Shimadzu Laser Diffraction Type Particle Analyzer) and by phase contrast microscopy. For laser diffraction studies, the microspheres were suspended in PBS containing 0.2% Tween as a wetting agent. The mixture was sonicated for 1 min and analyzed under stirred conditions to minimize aggregate formation. The distribution (after eliminating aggregates) indicated spheres ranging in size from 4 to 24 microns with a mean of 7 microns.
• microspheres absorption coated with goat anti-mouse polyclonal antibody was suspended in PBS containing 1% HSA and stained with a FITC-conjugated mouse IgG mAb.
• FITC-conjugated mouse IgG mAb As a control, non-coated microspheres were stained under the same conditions. The beads were then analyzed by flow cytometry. The coated beads showed intense staining indicating successful coating with the first material.
• CD4+ T-cells were purified by positive selection using anti-CD4 microbeads (Miltenyi Biotech, Germany). Cells were cultured in X- Vivo 15 (BioWhittiker) supplemented with glutamine.
• Purified CD4+ cells were placed in 24 well plates and were incubated with either goat anti-mouse coated microspheres coated directly with anti-CD3 and anti-CD28 mAbs in a 50:50 ratio (direct method) or the cells were first labeled with the anti-CD3 and anti-CD28 mAbs and then incubated with the coated microspheres.
• unlabelled cells were incubated with polyclonal goat anti-mouse coated microspheres.
• As a positive control cells were incubated with CD3/CD28 coated Dynabeads. All groups were adjusted to a beadxell ratio of 3 : 1.
• Purified CD4+ cells were placed in the wells at cell densities of 0.5 x 10 6 per ml. Concentrations of cytokines in the cell-free supernatants after 72 hours was measured by ELISA.
• the cytokine data represents the mean +/- SD of six different blood samples.
• Example #3 Proliferation CD4+ cells were prepared as described in the example above except that the cultures were continued for 9 days. Fresh beads and/or antibodies were added every three days when the cultures were split to a concentration of 0.5-1 x 10 6 cells/ml. Cells were seeded in triplicate at the beginning of each experiment.

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