WO2005007116A2

WO2005007116A2 - Artificial antigen presenting cell devices

Info

Publication number: WO2005007116A2
Application number: PCT/US2004/022919
Authority: WO
Inventors: Michael L. Gruenberg
Original assignee: Valeocyte Therapies Llc
Priority date: 2003-07-18
Filing date: 2004-07-16
Publication date: 2005-01-27
Also published as: WO2005007116A3

Abstract

Provided herein are methods and compositions relating to an artificial Antigen Presenting Cell device (“aAPC” devices). The disclosed aAPC devices include a linking agent that dramatically enhances the efficacy of the bound agonists to stimulate and expand a population of T cells that are high producers of pro-inflammatory cytokines such as IFN-Ϝ. Such cells are useful compositions in adoptive immunotherapy protocols.

Description

ARTIFICIAL ANTIGEN PRESENTING CELL DEVICES

Field of the Invention [0001] The methods and compositions disclosed herein relate to the preferential expansion of T cells producing pro-inflammatory cytokines, e.g., IFN-γ, using an artificial antigen presenting cell ("aAPC") system. The methods are useful in expanding T cells in sufficient numbers for use in adoptive immunotherapy.

Background [0002] Adoptive immunotherapy confers immunity through the transfer of activated and/or memory lymphocytes to a naive or immunocompromised recipient. This therapy involves the removal of lymphocytes from the patient, the processing and activating of the lymphocytes outside the body, i.e., ex vivo, and the subsequent reinfusion of the activated lymphocytes as a therapeutic regimen. Adoptive immunotherapy has been used to enhance the host immune response to tumors, viruses and bacterial pathogens and typically involves the ex vivo expansion of T cells. Examples of these type of treatments include the use tumor infiltrating lymphocytes (TIL) cells, cytotoxic T cells, expanded tumor draining lymph node cells, various lymphocyte preparations, and regulatory T cells to initiate or augment cellular immune responses against tumors and pathogens. [0003] Thl cells play a critical role in the development and sustainment of most cellular immune responses. Thl cells are CD4+ T cells with a characteristic profile of cytokine production, namely, high IFN-γ and IL-2 and low to no IL-4 production. IFN-γ is considered the prototypical Thl cytokine and is a key player in the host immune defenses against tumors and viruses. IFN-γ mediates its antitumor effects through the inhibition of tumor cell growth, the enhancement of immunogenicity via the upregulation of MHC class I, MHC class II and co-stimulatory molecules, and the recruitment and activation of granulocytes, NK cells and macrophages to tumor sites. For example, in viral infection, IFN-γ stimulates the processing and presentation of viral epitopes at the surface of virally-infected cells, effecting their elimination by lymphocytes and other immune effectors. In murine tumor models, IFN-γ correlates with therapeutic efficacy of adoptively transferred T-cells. Turtle et al. (1993) Can. Res. 55:833-39; Nagoshi et al. (1998) J Immunol. 160:334-44; Barth et al. (1991) J. Exp. Med. 173:647-58. [0004] Because IFN-γ as well as other pro-inflammatory cytokines have a significant role in the cellular immune response, it is desirable to enhance the amount of IFN-γ and/or other pro-inflammatory cytokines produced by T cells expanded for use in adoptive immunotherapy protocols. The methods and compositions disclosed herein provide expanded T cells that produce high levels of IFN-γ and/or other pro-inflammatory cytokines.

Summary [0005] The methods and compositions provided herein relate to the expansion of IFN-γ producing T cells in sufficient numbers to use in adoptive immunotherapy protocols. [0006] Provided herein is a method to induce and expand a population of IFN-γ producing T cells comprising: contacting a population of T cells with a solid phase support comprising: a) a linking agent immobilized on the solid phase support to which a first and a second agonist are bound; b) the first agonist for a first stimulatory molecule on the T cell; and c) the second agonist for a second stimulatory molecule on the T cell; whereby contacting the T cells with the solid phase support induces and expands the population of T cells that produce IFN-γ, which results in the expanded T cells producing more IFN-γ than T cells expanded by the first and second agonist immobilized on the solid phase support in the absence of the linking agent. In one embodiment, the T cells are contacted with the solid phase support a minimum of three times at two to four day intervals. In another embodiment, the method further comprises repeated contact with the agonists on the solid phase support to result in the production of a polyclonal Thl CD4⁺ memory cell population. [0007] The T cells to be expanded can be purified CD3⁺ T cells or purified CD3⁺, CD4⁺ T cells. In one embodiment, the T cells to be expanded are CD3⁺, CD4⁺, CD45RO^' T cells. The T cells can be purified by positive selection. [0008] Further provided herein is an artificial antigen presenting cell (aAPC) comprising a) a linking agent immobilized on the solid phase support to which a first agonist and a second agonist are bound; b) the first agonist for a first stimulatory molecule on the T cell; and c) the second agonist for a second stimulatory molecule on the T cell. [0009] In one embodiment, the solid phase support comprises polyacrylamide, latex gel, dextran, rubber, silicon, plastics, nitrocellulose, cellulose, natural sponge, polypropylene, glass, polytetrafluoroethylene, polyethylene, polystyrene, polyester, ceramic, and composites thereof. In a particular embodiment, the solid phase support is a bead, more particularly a magnetic immunobead. In another embodiment, the solid phase support is a microtiter plate. [0010] In one embodiment, the linking agent is an antibody. In a particular embodiment, the linking agent is an anti-IgG antibody. The linking agent can be immobilized to the solid phase support by direct or indirect linkage. In one embodiment, the direct linkage is a covalent linkage. In another embodiment, the indirect linkage is a biotin-avidin linkage. The first agonist can be an anti-CD3 antibody or an anti-CD2 antibody. The second agonist can be an anti-CD28 antibody, B7-1, or B7-2. In one embodiment, the anti-CD3 antibody binds human CD3 and the anti-CD28 antibody bind human CD28. [0011] Also provided are combinations of a bioreacter, e.g., a hollow fiber bioreactor, and the aAPC devices; and combinations of syringe and such devices. Kits containing combinations and optionally instructions for activating T-cells are also provided.

Detailed Description [0012] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong. All patents, applications, published applications and other publications referred to throughout the disclosure herein are incorporated by reference in their entirety. [0013] The artificial antigen presenting cell (hereinafter "aAPC") provides an effective and reliable method to expand pro-inflammatory cytokine (e.g., IFN-γ) producing T cells for use in adoptive immunotherapy. The aAPC is based on the two signal model for T cell activation. In vivo the T cell response to a pathogen or tumor is initiated by the antigen presenting cell (hereinafter "APC"). The antigen presenting cell presents antigenic epitopes from the pathogen or tumor in the context of MHC class I and MHC class II molecules. Additionally, the APC bears a number of co-stimulatory molecules such as B7-1(CD80), B7- 2 (CD86), and LICOS. When the T cell encounters the APC laden with the antigen for which the T cell is specific, the T cell receptor (TCR) binds the antigen and a complex cascade of signaling events is initiated through the TCR/CD3 complex and the surrounding costimulatory molecules, such as CD28, CTL4, and ICOS. The resulting signaling cascade stimulates the T cell to differentiate and to proliferate. The aAPC disclosed herein seeks to replicate these initiating signals using agonists for particular stimulatory molecules to stimulate T cells to produce IFN-γ and/or other proinflammatory cytokines. [0014] To provide the first signal, cells can be activated with an agonist, such as a monoclonal antibody (mAb) to the CD3/TCR complex or via stimulation of the CD2 surface protein, but other suitable signals, such as, but not limited to, antigens, super antigens, polyclonal activators, anti-CD2 and anti-TCR antibodies, can be used. Other suitable agents can be empirically identified. Immobilized or cross-linked anti-CD3 mAb, such as OKT3 or 64.1, can activate T-cells in a polyclonal manner. See Tax, et al. (1983) Nature 304:445. Other polyclonal activators, however, such as phorbol myristate acetate also can be used. See, e.g., Hansen, et al. (1980) Immunogenetics 10:247. [0015] Similarly, stimulatory forms of anti-CD2 antibodies are known and available. Stimulation through CD2 with anti-CD2 antibodies is typically accomplished using a combination of at least two different anti-CD2 antibodies. Stimulatory combinations of anti- CD2 antibodies, include, but are not limited to: the Tl 1.3 antibody in combination with the Tl 1.1 or Tl 1.2 antibody (Meuer et al. (1983) Cell 36:897^906) and the 9.6 antibody (which recognizes the same epitope as Tl 1.1) in combination with the 9-1 antibody (Yang et al. (1986) J Immunol. 757:1097-1100). Other antibodies that bind to the same epitopes as any of the above described antibodies also can be used. Additional antibodies, or combinations of antibodies, can be prepared and identified by standard techniques. [0016] Monovalent anti-CD3 mAb in the soluble phase can be used to activate T-cells. See Tamura et al. (1992) J Immunol. 148:2370. Soluble heteroconjugates of anti-CD3 and anti-T-cell surface antigen mAb can preferentially activate a particular T-cell subset. See, e.g., Ledbetter, et al. (1988) Eur. S. Immunol. 18:525. Anti-CD2 mAb can also activate T- cells. See Huet, et al. (1986) J Immunol. 737:1420. Anti-MHC class II mAb can have a synergistic effect with anti-CD3 in inducing T-cell proliferation. See Spertini et al. (1992) J. Immunol. 149:65. Anti-CD44 mAb can activate T-cells in a fashion similar to anti-CD3 mAb. See Galandrini, et al. (1993) J Immunol. 150:4225. [0017] A primary activation signal also can be delivered to a T-cell using other agonists, for example, through the use of a combination of a protein kinase C (PKC) activator, such as, but are not limited to, a phorbol ester, such as phorbol myristate acetate, and a calcium ionophore, such as ionomycin, which raises cytoplasmic calcium concentrations. The use of such agents bypasses the TCR/CD3 complex but delivers a stimulatory signal to T-cells. [0018] In order to induce an activated population of T-cells to proliferate in the absence of exogenous growth factors or stimulatory cells, an stimulatory molecule on the surface of the T-cell, such as CD28, is stimulated with a ligand that binds the stimulatory molecule. In one embodiment, stimulation of the stimulatory molecule CD28 and activation occurs simultaneously by contacting a population of T-cells with a ligand that binds CD3 and a ligand that binds CD28. Activation of the T-cells with, for example, an anti-CD3 antibody and stimulation of the CD28 stimulatory molecule results in selective proliferation of CD4+ T-cells. Representative co-stimulatory molecules that can provide the second signal for T cells include, but are not limited to CD28, CD134 (OX40), ICOS, and CD137 (4-1BB) or adhesion molecules on T-cells such as CD54 (ICAM-1), CD1 la/CD18 (LFA-1) and CD49d/CD29 (NLA-4). [0019] T-cells can be obtained from a number of sources, including peripheral blood leukocytes, bone marrow, lymph node tissue, spleen tissue, and tumors. Peripheral blood leukocytes are obtained from an individual by leukopheresis. To isolate T-cells, if desired, from peripheral blood leukocytes, it can be necessary to lyse the red blood cells and separate peripheral blood leukocytes from monocytes by, for example, centrifugation through a PERCOLL^® gradient. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640) that can contain factors necessary for proliferation and viability, including animal serum (e.g., fetal bovine serum) and antibiotics (e.g., penicillin streptomycin). The T-cells are maintained under conditions necessary to support growth, for example an appropriate temperature (e.g., 37°C.) and atmosphere (e.g., 5% CO ). [0020] Therefore, the source of T cell useful for adoptive immunotherapy can include any T-cells, autologous, syngeneic, or allogeneic from a normal or disease-bearing subject. Examples of such cells include but are not limited to, Thl cells (U.S. application Serial No. 09/957,194), co-stimulated T-cells (Lums, et al. (2001) J Immunother 25:408), polyclonal and antigen-specific CTL (Maus et al. (2002) Nat. Biotechnol. 20:143), co-stimulated CD4+ cells (Levine et al. (2002) Nat. Med 8:47), CML-specific T-cells (Muller et al. (2002) J Immunother. 24:482), soluble tumor antigen induced CTL (Li et al. (2001) Zhonghua Wai Ke Za Zhi 59:619), anti-cervical cancer CTL (Chiriva-Internati et al. (2002) Eur. J. Immunol. 52:30), tumor associated lymphocytes (Schuler et al. (2001) J. Exp. Med. 194:1161), EBN- specific T-cells (Savoldo et al. (2002) J. Immunol. 168:909; Hague et al. (2001) Transplantation 72:1399), CML-specific T-cells (Muller et al. (2001) J. Immunother. 24:482), CTL against lung cancer (Hiraki et al. (2001) Anticancer Res.21:2561; So et al. (2001) Jap JClin. Oncol. 31:311), anti-leukemia CTL (Montagna et al. (2001) Blood 98:3359), ex-vivo activated lymph node cells (Plautz et al. (2001) Cancer Chemother Biol Response Modif 19:327), interferon-γ enhanced T-cells (Becker et al. (2001) Nat Med. 7:1159), pharmacologically-activated lymph node cells (Bear et al. (2001) Cancer Immunol Immunother. 50:269), γ-δ T-cells (Chen et al. (2001) Int. Arch. Allergy Immunol. 125:256), CMV-specific CTL (Szmania et al. (2001) Blood 98:505; Cho et al. (2001) J Immunother. 24:242), activated T-cells (Chin et al (2001) JSurgRes PS: 108), pre-immunized effector cells (Morecki et al. (2001) J. Immunother 24:114), cytotoxic T-cells (U.S. Patent No. 6,255,073; U.S. Patent No. 5,846,827), expanded tumor draining lymph node cells (U.S. Patent No. 6,251,385), various preparations of lymphocytes (U.S. Patent No. 6,194,207; U.S. Patent No. 5,443,983; U.S. Patent No. 6,040,180; U.S. Patent No. 5,766,920; U.S. Patent No. 6,204,058), CD8+ TIL cells (Figlin et al. (1997) Journal of Urology 158:140), CD4+ T- cells activated with anti-CD3 monoclonal antibody in the presence of IL-2 (Nishimura (1992) J Immunol. 148:285), T-cells co-activated with anti-CD3 and anti-CD28 in the presence of IL-2 (Garlie et al. (1999) Journal of Immunotherapy 22:336), antigen-specific CD8+ CTL T- cells produced ex-vivo and expanded with anti-CD3 and anti-CD28 monoclonal antibodies (mAb) in the presence of IL-2 (Oelke et al. (2000) Clinical Cancer Research 6:1997), and the first injection of irradiated autologous tumor cells admixed with Bacille Calmette-Guerin (BCG) to vaccinate subjects followed seven days later by recovery of draining lymph node T- cells which are activated with anti-CD3 mAb followed by expansion in IL-2 (Chang et al. ' (1997) Journal of Clinical Oncology 15:196). [0021] A specific subpopulation of T-cells, such as T cells expressing CD28, CD4, CD8, CD45RA, and CD45RO, can be further isolated by positive or negative selection techniques. For example, negative selection of a T-cell population can be accomplished with a combination of antibodies directed to surface markers unique to the cells negatively selected. One method is cell sorting via negative magnetic immunoadherence using a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to isolate CD4 cells, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, GDI lb, CD16, HLA-DR, and CD8. The process of negative selection results in an essentially homogenous population of CD28, CD4 or CD8 T- cells. Likewise, the T cells can be purified using positive selection using routine methods known in the art. [0022] The methods provided herein expand a population of T cells that are producers inflammatory cytokines. As used herein, the term "pro-inflammatory cytokines" refers to cytokines that cell-mediated inflammatory reactions, support macrophage activation, immunoglobulin (Ig) isotype switching to IgG2a and activate cytotoxic function and they include, but are not limited to, IFN-γ, IL-1, IL-2, IL-6, IL-8, TNF- and GM-CSF. Preferably, these T cells are CD4+ Thl cells. As used herein, the term "Thl cell" refers to a CD4+ T cell that produce IFN-γ, and little to no IL-4 upon stimulation with an antigen, an aAPC, or other suitable stimulus. Cells that produce IL-4, and little to no IFN-γ, are Th2 cells. [0023] Flow cytometric analysis using intracellular cytokine staining techniques permits the identification of T cell populations are Thl or Th2. For example, a method for identifying Thl cells in a population of cells is to stain the cells intercellularly for IFN-γ, while Th2 cells are commonly identified by intercellular staining for IL-4. In normal (i.e., subjects not exhibiting overt disease) individuals, generally only about 12 -16% of the CD4+ cells stain positive for intracellular IFN-γ after activation; less than 1% stain positive for IFN- γ prior to activation. It is rare for a T-cell population to stain greater than 35% IFN-γ positive. The cells resulting from a method described herein, stain greater than 70% positive and often greater than 90% positive for IFN-γ. A pure or highly pure population of Thl cells is a CD3+, CD4+ T cell population that stains greater than 70% positive for intracellular IFN-γ and does not produce greater than about 26 pg/ml/10 cells of IL-4 in a 24 hour period. In most instances, they do not produce greater than about 6 pg/ml/10⁶ cells of IL-4 in a 24 hour period. As used herein, highly pure means greater than about 70%, generally greater than 75% and can be as pure as 85%, 90% or 95% or higher in purity. IFN-γ can also be detected by ELISA in Thl cell supernatants. It is generally in excess of 1 ng/ml/10⁶ cells and in the range of 1 ng/ml to 26 ng/ml per 10⁶ cells, but can be greater than 26 ng/ml per 10⁶ cells. [0024] Activated T-cells can be identified phenotypically, most commonly, by the expression of CD25. T cells that are CD25+ (i.e., T -2 receptor+) are referred to herein as "activated." A pure or highly pure population of activated cells typically express greater than 85% CD25+. [0025] Memory T cells can be expanded using the disclosed methods and compositions. As used herein, the term "memory T cell" is a T-cell that is CD45RO+, CD45RA-. A pure or highly pure population of memory cells expresses greater than 70%, generally greater than 80%o, and even greater than 90% or 95% CD45RO+. T cells with an activated, memory phenotype, i.e., CD25+, CD45RO+ can also be expanded using these methods. Such cells have the ability to traffic to a tumor or other site of inflammation upon infusion. Typically, these cells also express adhesion molecules, such as CD44. A pure or highly pure population of memory cells with the ability to traffic to a tumor or other site of inflammation upon infusion is greater than 70%, generally greater than 90% or 95% CD44+, and less than about 25%, including less than 5%, CD62L+. [0026] The methods include the steps of contacting T-cells with an artificial antigen presenting cell (aAPC) device under conditions in which the T cells are activated and proliferate. The aAPC device includes a solid support, a linking agent, and at least two agonists. The linking agent is a binding agent that coats at least one surface of the support. The agonists are at least two proteins or agents that activate the T-cells and stimulate an stimulatory molecule of the surface of the T-cells. The two agonists are bound to the linking agent. The resulting Thl cells produce higher levels of pro-inflammatory cytokines than Thl cells that are produced using aAPC devices in which the two agonists are bound directly to the solid support. Generally, the contacting is effected under conditions whereby the T-cells are directed to differentiate into Thl cells. Similarly, to stimulate proliferation of CD8 T- cells, an anti-CD3 antibody and the monoclonal antibody ES5.2D8 (ATCC) can be used as can other methods known in the art (Berg et al, Transplant Proc. 30(8):3915-3911, 1998; Haanen et αE J. Exp. Med. 190(9):1319-1328, 1999; Garland et al, J. Immunol Meth. 227(1- 2):53-63, 1999). [0027] Any suitable linking agent may be used with the methods provided herein. As used herein, a linking agent refers to any molecule that has an affinity for a given agonist or a with a defined sequence of amino acids and that can be used to coat directly or via a linker, via any interaction that results in stable attachment, such as covalent and ionic interactions, at least one surface or a portion thereof of a solid support. The linking agent additionally must bind to the agonist providing the signals for T-cell activation and proliferation and present the agonist to the cells such that the level of pro-inflammatory cytokines, such as IFN-γ, produced by the T-cells is greater in the presence of the linking agent than in its absence. [0028] Linking agents can be naturally-occurring or synthetic molecules, and include any molecule, including nucleic acids, small organics, proteins and complexes that bind to the agonist. Linker agents can be receptors and also can be referred to in the art as anti-ligands. Linking agents can be used in their unaltered state or as aggregates with other species. They can be attached or in physical contact with, covalently or noncovalently, a binding member, either directly or' indirectly via a specific binding substance or linker. Examples of linking agents, include, but are not limited to: antibodies, including polyclonal antibodies and monoclonal antibodies and fragments thereof, cell membrane receptors surface receptors and internalizing receptors, monoclonal antibodies and antisera reactive or isolated components thereof with specific antigenic determinants (such as on viruses, cells, or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. For purposes herein, the linking agent is the coated on the solid surface and is one to which the stimulatory agonists bind to permit the activation and proliferation of the T-cells. Typical linking agents include polyclonal antibodies, such as anti-IgG molecules, particularly in embodiments in which the stimulatory and co-stimulatory molecules are monoclonal antibodies of the IgG class. They also can be anti-IgG, IgM, IgA, IgD and IgE antibodies. One preferred embodiment is an anti-IgG antibody. [0029] The linking agent can be coupled to the solid phase support by any suitable means. Likewise, the linking agent can bind the agonist by any suitable means. As used herein, the term "coupled" or "coupling" includes a chemical, enzymatic or other means, such as for example, antibody, avidin/streptavidin-biotin) by which a linking agent, such as an IgG or other molecule to which an anti-CD3 monoclonal antibody and/or anti-CD28 monoclonal antibody binds, is linked to a solid surface such that the signals for T-cell proliferation, such as anti-CD3 and/or anti-CD28 monoclonal antibody, binds and is presented such that they trigger activation and proliferation of T-cells. For example, protein A coated solid surfaces can be used to bind capture agent, or the linking agent can be immobilized on the surface by chemical means such as crosslinking to the solid surface using commercially available crosslinking reagents (Pierce, Rockford IL) or other means. [0030] The amount of a particular linking agent to the solid phase surface can be readily determined, such as by FACS analysis if the solid surface is that of beads or by ELISA if the solid phase surface is that of a tissue culture dish. [0031] Any suitable agonist may be used with the methods provided herein. As used herein, the term "agonist" includes any molecule that interacts with a T cell to induce proliferation, activation, and/or differentiation. The agonist may be a binding peptide, antibody, or the ligand for the desired stimulatory molecule. Preferrably, the agonist interacts with a stimulatory molecule on the surface of the T cell, such as CD3, TCR, CD28, and the like. Therefore, as used herein, the term "agonist" includes those proteins that are the "natural" ligand for the cell surface protein, such as a B7 molecule for CD28, and synthetic or artificial ligands, such as antibodies that specifically bind (i.e. have an affinity of at least about 10^"7 M) directed to the cell surface protein. In one embodiment, the agonist is an antibody specific for a stimulatory molecule. A variety of agonists singly or in combination can provide the second signal for T-cell activation. For example, immobilized mAb or fusion proteins which interact with co-stimulatory molecules such as CD28, CD134 (OX40) and CD137 (4-1BB) or adhesion molecules on T-cells such as CD54 (ICAM-1), CD1 la/CD 18 (LFA-1) and CD49d/CD29 (NLA-4) singly or in combination can provide second signals for activation. Such antibodies and fragments thereof can be produced by methods well known in the art, such as hybridoma synthesis, panning using phage display libraries, recombinant DΝA techniques and protein synthesis. Antibodies include polyclonal, monoclonal antibodies, humanized and chimeric, and can be an intact molecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab' and F(ab)'2 fragments), or multimers or aggregates of intact molecules and/or fragments; and can occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering. Suitable mitogenic mAbs, or agonists, induce T-cell doubling times of 24 h to 48 h. [0032] In some embodiments, the agonist are mouse anti-human CD3 and anti-human

CD28 mAbs, for example in a ratio of 1 to 1, in other embodiments a ratio of 3 to 1 is used.

Adding mouse anti-CD3 and anti-CD28 mAbs in a 50:50 ratio to 4.5 micron paramagnetic beads coated with sheep anti-mouse polyclonal antibody creates an aAPC with enhanced capacity to stimulate T-cells to produce pro-inflammatory Thl cytokines, such as IFΝ-γ. The amount of IFΝ-γ production from cells stimulated with the aAPC is 2-5 fold higher than the amount produced when the agonists are bound directly to the solid support. The addition of the linking agent between the solid support and the agonists provides a means to create many permutations of orientation of the agonists bound thereto, providing enhanced interaction and activation of the T-cells. [0033] The aAPCs are prepared by coating a solid support, such as a microparticle with a diameter, for example, of greater than 1.5 microns and less than 15 microns. Suitable solid support material can be any described herein or known to those of skill in the art, such as plastic or polymer grafted materials. As described, solid support can take the form of inert particles or beads, flat surfaces, rod-shaped fibers, multi-finned star burst particles. Contemplated solid supports, include, microtiter places, the walls of a reaction vessel or bioreactor or the internal or external wall of a hollow fiber. It can be advantageous to select and design supports that maximized the surface area thereof. [0034] To prepare the aAPC, a solid support is coated with a linking agent providing a reactive surface onto which to bind agonists. Agonists are two or more molecules that are able to deliver the requisite two signals to T-cells in order to cause them to become activated. The techniques for binding a linking agent to a solid support are well known. The linking agent can be directly attached to the solid support by means of reactive groups or alternatively, by means of a coupling agent or linker. [0035] The methods and compositions provided herein can be used in any suitable culture format. In one embodiment, a hollow fiber bioreactor is used. A hollow fiber bioreactor is a device for growth in cells that includes an outer shell casing that is suitable for the growth of mammalian cells, a plurality of semi-permeable hollow fibers encased within the shell that are suitable for the growth of mammalian cells on or near them, and the ECS, which contains the cells and the ECS cell supernatant. The interior of the hollow fibers is called the lumen and the area between the outside of the capillaries to the inside of the outer housing is called the extracapillary space [ECS]. In the hollow fiber bioreactor, tissue culture medium perfuses through the fiber lumens and is also included within the shell surrounding said fibers. The tissue culture medium, which may differ in these two compartments, contains diffusible components that are capable of sustaining and permitting proliferation of immune cells. The medium is provided in a reservoir from which it is pumped through the fibers. The flow rate can be controlled varied by the varying the applied pressure. The walls and/or surfaces of the lumens exposed to the cells can serve as an aAPC device provided herein. Alternatively, aAPC devices, such as the particular devices, including the dendritic devices, provided herein can be included inside the bioreactor for contacting with the cells therein. [0036] Any culture conditions may be used in the methods provided herein. As used herein, the term "culture medium" includes any medium suitable for supporting the viability, growth, and/or differentiation of mammalian cells ex-vivo. Examples of culture medium include, but are not limited to, X-Nivol5 (BioWhittaker), RPMI 1640, DMEM, Ham's F12, McCoys 5 A and Medium 199. The medium can be supplemented with additional ingredients including serum, serum proteins, growth suppressing, and growth promoting substances, such as mitogenic monoclonal antibodies and selective agents for selecting genetically engineered or modified cells. [0037] To maintain long term stimulation of a population of T-cells following the initial activation and stimulation, it can be necessary to separate the T-cells from the stimulus after a period of about 12 to about 14 days. The rate of T-cell proliferation is monitored periodically (e.g., daily) by, for example, examining the size or measuring the volume of the T-cells, such as with a Coulter Counter. In this regard, a resting T-cell has a mean diameter of about 6.8 microns, and upon initial activation and stimulation, in the presence of the stimulating ligand, the T-cell mean diameter will increase to over 12 microns by day 4 and begin to decrease by about day 6. When the mean T-cell diameter decreases to approximately 8 microns, the T- cells can be reactivated and re-stimulated to induce further proliferation of the T-cells. Alternatively, the rate of T-cell proliferation and time for T-cell re-stimulation can be monitored by assaying for the presence of cell surface molecules, such as B7-1, B7-2, which are induced on activated T-cells. [0038] Any conditions and cognate method for producing Thl cells can be used, including exposure to appropriate cytokines and other factors, such as activation in the presence of γ-interferon, anti-IL-4 antibody, anti-TGF- antibody, or IL-12 (see, e.g., Sedar et al. (1993) Proc. Natl. Acad. Set U.S.A. C: 10188); and other methods known to those of skill in the art. In addition, the T-cells can be treated by repeated activation of the cells with the aAPC devices to produce Thl cells. The methods include the steps of: (i) the purification of T-cells from source material; and the frequent (every 2-3 days) activation of the purified T-cells and typically repeated (a minimum of 3 times). [0039] The methods provided herein support the expansion of T cells to a clinically relevant number for adoptive immunotherapy. As used herein, a composition containing a clinically relevant number or population of T cells is a composition that contains at least 10⁹, typically greater than 10⁹, at least 10¹⁰ cells, and generally more than 10¹⁰ cells. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cell. For example, if Thl cells that are specific for a particular antigen are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90- 95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 mis or less, even 250 mis or 100 mis or less. Hence the density of the desired cells is typically greater than 10⁶ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10⁹, 10¹⁰ or 10^π cells. [0040] As used herein, a clinically relevant number of activated polyclonal Thl memory cells is a composition containing a clinically relevant number or population of immune cells where a substantial portion, greater than at least about 70%, typically more than 80%, 90%), and 95%), of the lymphocytes are activated polyclonal Thl memory cells. The cells can be polyclonal in antigen specificity, T cell subclass (e.g., Thl, Th2, Th3, Tel, Tc2, etc), lymphocytic origin (e.g., T cell, B cell, NK cell), or some combination thereof. As used herein, the term "immune cells" include any cell of lymphocytic or monocytic origin that participates in a immune response. A population of cells that is predominantly of that subclasse is greater than about 50% of the identified subclass. [0041] As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as flow cytometry, used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as biological activities, of the substance. Methods for purification of the immune cells to produce substantially pure populations are known to those of skill in the art. A substantially pure cell population, can, however, be a mixture of subtypes; purity refers to the activity profile of the population. In such instances, further purification might increase the specific activity of the cell population. [0042] The recipient of the cells generated using the methods disclosed herein are any subject with a disease or disorder benefited from an initiation or enhancement of a Thl- mediated immune response. The cells can be administered to reverse the impact of an immunosuppressive local or system environment in vivo. In one embodiment, the cells are administered to a subject having an immunosuppressive tumor environment. An immunosuppressive tumor environment is the microenvironment created by cytokine production from tumor cells and infiltrating mononuclear cells. The sum total of cytokines are create an environment that is capable of suppressing the effector functions of immune cells. Examples of immunosuppressive cytokines in a tumor microenvironment include IL- 10, IL-6 and TGF-beta. The cells can be administered as a treatment for a disease characterized by a lack of Thl cytokines. As used herein, a disease characterized by a lack of Thl cytokine activity refers to a state, disease or condition where the algebraic sum of cytokines in a specific microenvironment in the body or in a lesion(s) or systemically is less than the amount of Thl cytokines present normally found in such microenvironment or systemically (i.e., in the subject or another such subject prior to onset of such state, disease or condition). The cytokines to assess include IFN-γ, IL-2, and TNF-alpha. The precise amounts and cytokines to assess depend upon the particular state, disease or condition. Thus, the diseases for which the cells have therapeutic application include, but are not limited to, cancer, infectious diseases, allergic diseases and diseases characterized by overactive humoral immunity (such as in systemic lupus erythematosus). 0043] The cells expanded using the method herein are also useful in the treatment of diseases dominated by pathogenic Th2 responses. As used herein, diseases characterized by a Th2-dominated immune response are characterized by either a suppressed cellular immune response or excessive humoral response. In one embodiment, a disease characterized by an excess of Th2 cytokine activity refers to a state, disease or condition where the algebraic sum of cytokines in a specific microenvironment in the body or in a lesion(s) or systemically is predominantly of the Th2 type, dominated by IL-4 and/or IL-10 and/or TGF- . Diseases, states or conditions that exhibit enhanced Th2 responses include infectious diseases such as, but are not limited to, chronic hepatitis C virus infection, leprosy toxoplasmosis infection and AIDS. Imbalance in favor of Th2 cells also occurs in asthma and lupus and other diseases that exhibit suppressed cellular immunity. [0044] The methods provided herein can further be used to generate cellular compositions useful in restoration of immune balance in a subject. As used herein, immune balance refers to the normal ratios, and absolute numbers, of various immune cells and their cytokines that are associated with a disease free state. Restoration of immune balance refers to restoration to a condition in which treatment of the disease or disorder is effected whereby the ratios of regulatory immune cell types or their cytokines and numbers or amounts thereof are within normal range or close enough thereto so that symptoms of the treated disease or disorder are ameliorated. The amount of cells to administer can be determined empirically, or, such as by administering aliquots of cells to a subject until the symptoms of the disease or disorder are reduced or eliminated. Generally a first dosage will be at least 10⁹-10¹⁰ cells. In addition, the dosage will vary depending upon treatment sought. As intended herein, about 10⁹ is from about 5 x 10⁸ up to about 5 x 10⁹; similarly about 10¹⁰ is from about 5 x 10⁹ up to about 5 10 , and so on for each order of magnitude. Dosages refer to the amounts administered in one or in several infusions. [0045] As used herein, a subject is a mammal, typically a human, including humans in need of treatment for a disease or disorder (i.e., a patient). Treatment of other mammals, such as domesticated animals, including cats and dogs, and farm and zoo animals, such as cows, pigs, sheep, goats, camels, llamas, gorillas, chimpanzees other primates, dolphins and other whales, mice, rats, and transgenic species thereof. [0046] As used herein, therapeutically effective refers to an amount of cells that is sufficient to ameliorate, or in some manner reduce the symptoms associated with a disease. When used with reference to a method, the method is sufficiently effective to ameliorate, or in some manner reduce the symptoms associated with a disease. Amelioration of the symptoms of a particular disorder by administration of a particular composition is any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition of expanded cells. [0047] As used herein, a therapeutically effective number is a clinically relevant number of immune cells that is at least sufficient to achieve a desired therapeutic effect, when such cells are used in a particular method. Typically such number is at least 10⁹, and generally 10¹⁰ or more. The precise number will depend upon the cell type and also the intended target or result and can be determined empirically. [0048] The cells can be administered by any suitable methods. As used herein, infusion medium is an isotonic solution suitable for intravenous infusion. Any such medium known to those of skill in the art can be used. Examples of infusion medium include, but are not limited to, normal saline (NS), 5% dextrose (D5W), Ringer' s Lactate, Plasma-Lyte and Normosol and any other commercially available medium. As used herein, formulating for infusion is the process of removing or harvesting the cells to be used in adoptive immunotherapy from a culture environment, then subsequently washing, concentrating and re-suspending the cells in infusion medium or in plasma as provided herein. [0049] The activated T-cells, activated and gene modified T-cells, and/or activated and otherwise modulated T-cells resulting from use of the aAPC devices provided herein can be administered either alone, or as a pharmaceutical composition, such as in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, mannose, sucrose and dextrans, mannitol, proteins, polypeptides, amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and preservatives. Compositions are generally formulated for intravenous administration. [0050] Any suitable solid phase support may be used with methods provided herein. A support (also referred to as a matrix support, a matrix, an insoluble support or solid support) can be any solid or semisolid or insoluble support to which a molecule of interest, typically a biological molecule, organic molecule or biospecific ligand is linked or contacted. Such materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. The matrix herein can be particulate or can be a be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, the walls of a bioreactor in contact with the cells, or other such materials. When particulate, typically the particles have at least one dimension in the 5-10 mm range or smaller. The support material is selected so that it is biologically compatible and does not interfere with T-cell activation. [0051] A matrix or support particles includes matrix materials that are in the form of discrete particles. The particles have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm or less, 50 μm or less and typically have a size that is 100 mm³ or less, 50 mm³ or less, 10 mm³ or less, and 1 mm³ or less, 100 μm³ or less and can be order of cubic microns; typically the particles have a diameter of about 1.5 microns and less than 15 microns, such as about 4-6 microns. Such particles, referred collectively herein as "beads", are often, but not necessarily, spherical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical "beads", particularly microspheres that can be used in the liquid phase, are also contemplated. The "beads" can include additional components, such as magnetic or paramagnetic particles (see, e.g., Dyna beads (Dynal, Oslo, Norway)) for separation using magnets, as long as the additional components do not interfere with the methods and analyses herein. Thus, a "bead" refers to a microparticle capable of having agents immobilized thereon. Exemplary beads include, but are not limited to, commercially available beads such as DYNABEADS®, Dynal Corporation. [0052] In exemplary embodiments, a microsphere with a diameter of 4.5 microns is used as a solid support. Commercially available microspheres suitable for use can be purchased from 3M Scotchlite Glass Bubbles, Dynal M-450 Dynabeads (Oslo, Norway), Biosphere Medical (Rockland, MA), Luminex microspheres (Austin, TX), Spherotech, Inc. (Libertyville, IL) and Structure Probe, Inc. (West Chester, PA). Other particulate supports, such as dendritic and starburst shaped supports are contemplated. [0053] The support also can be a relatively inert polymer, which can be grafted by ionizing radiation to permit attachment of a coating of polystyrene or other such polymer that can be derivatized and used as a support. Radiation grafting of monomers allows a diversity of surface characteristics to be generated on supports (see, e.g., Maeji et al. (1994) Reactive Polymers 22:203-212; and Berg et al. (1989) J Am. Chem. Soc. 111:8024-8026). For example, radiolytic grafting of monomers, such as vinyl momomers, or mixtures of monomers, to polymers, such as polyethylene and polypropylene, produce composites that have a wide variety of surface characteristics. These methods have been used to graft polymers to insoluble supports for synthesis of peptides and other molecules. [0054] The supports are typically insoluble substrates that are solid, porous, deformable, or hard, and have any required structure and geometry, including, but not limited to: beads, pellets, disks, capillaries, hollow fibers, needles, solid fibers, random shapes, thin films and membranes, and generally, form solid surfaces with addressable loci. The supports can also include an inert strip, such as a teflon strip or other material to which the capture agents antibodies and other molecules do not adhere, to aid in handling the supports, and can include an identifying symbology. [0055] The preparation of and use of supports are well known to those of skill in this art; there are many such materials and preparations thereof known. For example, naturally- occurring materials, such as agarose and cellulose, can be isolated from their respective sources, and processed according to known protocols, and synthetic materials can be prepared in accord with known protocols. These materials include, but are not limited to, inorganics, natural polymers, and synthetic polymers, including, but are not limited to: cellulose, cellulose derivatives, acrylic resins, glass, silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the like (see, Merrifield (1964) Biochemistry 5:1385-1390), polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides, rubber, silicon, plastics, nitrocellulose, celluloses, natural sponges, and many others. Selection of the supports is governed, at least in part, by their physical and chemical properties, such as solubility, functional groups, mechanical stability, surface area swelling propensity, hydrophobic or hydrophilic properties and intended use. [0056] The choice of surface type and configuration can be a matter of design choice or can be selected to improve a desired result. Exemplary supports that are contemplated for use herein include supports that contain cellulose, agarose, polyacrylamide, acrolein, dextran, any number of plastics, glass, derivatized glass and other such surfaces. The surface can be in any suitable configuration, including continuous surfaces such as microplates, the inside walls of a bioreactor and/or the fiber surfaces; and particulate surfaces, such as beads, including but are not limited to, Sepharose beads ( Pharmacia Fine Chemicals, Sweden); paramagnetic beads, such as DYNABEADS®, Dynal Inc., New York; Purabeads®, Prometic Biosciences®) and other such materials are commercially available for this purpose. When beads are used, the bead can be of any size that effectuates T-cell expansion, such as about 2.5 μm to about 500 μm in diameter, typically about 2.8 μm to about 50 μm. Of particular interest herein, are particulate supports in the shape of a starburst shape or dendritic shaped- particule intended to simulate the shape of a dendritic cell. [0057] Naturally-occurring supports include, but are not limited to agarose, other polysaccharides, collagen, celluloses and derivatives thereof, glass, silica, and alumina.

Methods for isolation, modification and treatment to render them suitable for use as supports is well known to those of skill in this art (see, e.g., Hermanson et al (1992) Immobilized

Affinity Ligand Techniques, Academic Press, Inc., San Diego). Gels, such as agarose, can be readily adapted for use herein. Natural polymers such as polypeptides, proteins and carbohydrates; metalloids, such as silicon and germanium, that have semiconductive properties, also can be adapted for use herein. Also, metals such as platinum, gold, nickel, copper, zinc, tin, palladium, silver can be adapted for use herein. Other supports of interest include oxides of the metal and metalloids such as Pt-PtO, Si-SiO, Au-AuO, TiO2, Cu-CuO, and the like. Also compound semiconductors, such as lithium niobate, gallium arsenide and indium-phosphide, and nickel-coated mica surfaces, as used in preparation of molecules for observation in an atomic force microscope (see, e.g., Ill et al. (1993) Biophys J. 64:919) can be used as supports. Methods for preparation of such matrix materials are well known. [0058] For example, U.S. Patent No. 4,175,183 describes a water insoluble hydroxyalkylated cross-linked regenerated cellulose and a method for its preparation. A method of preparing the product using near stoichiometric proportions of reagents is described. Use of the product directly in gel chromatography and as an intermediate in the preparation of ion exchangers is also described. [0059] There are innumerable synthetic supports and methods for their preparation known to those of skill in this art. Synthetic supports typically produced by polymerization of functional matrices, or copolymerization from two or more monomers from a synthetic monomer and naturally occurring matrix monomer or polymer, such as agarose. [0060] Synthetic matrices include, but are not limited to: acrylamides, dextran-derivatives and dextran co-polymers, agarose-polyacrylamide blends, other polymers and co-polymers with various functional groups, methacrylate derivatives and co-polymers, polystyrene and polystyrene copolymers (see, e.g., Merrifield (1964) Biochemistry 5:1385-1390; Berg et al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459; Berg et al (1989) in Pept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198; Berg et al (1989) J. Am. Chem. Soc. 111 :8024-8026; Kent et al. (1979) Isr. J. Chem. 17:243-241; Kent et al. (1918) J. Org. Chem. ^5:2845-2852; Mitchell et al. (1976) Tetrahedron Lett. 42:3195-3798; U.S. Patent No. 4,507,230; U.S. Patent No. 4,006,117; and U.S. Patent No. 5,389,449). Methods for preparation of such support matrices are well-known to those of skill in this art. [0061] Synthetic support matrices include those made from polymers and co-polymers such as polyvinylalcohols, acrylates and acrylic acids such as polyethylene-coacrylic acid, polyethylene-co-methacrylic acid, polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate, polypropylene-coacrylic acid, polypropylene-co-methyl-acrylic acid, polypropylene- co-ethylacrylate, polypropylene-co-methyl acrylate, polyethylene-co-vinyl acetate,' polypropylene-co-vinyl acetate, and those containing acid anhydride groups such as polyethylene-co-maleic anhydride, polypropylene-co-maleic anhydride and the like. Liposomes have also been used as solid supports for affinity purifications (Powell et al (1989) Biotechnol. Bioeng. 55:173). [0062] For example, U.S. Patent No. 5,403,750, describes the preparation of polyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plant growth medium containing a hydrophilic polyurethane gel composition prepared from chain-extended polyols; random copolymerization can be performed with up to 50% propylene oxide units so that the prepolymer is a liquid at room temperature. U.S. Pat. No. 3,939,123 describes lightly crosslinked polyurethane polymers of isocyanate terminated prepolymers containing poly(ethyleneoxy) glycols with up to 35% of a poly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producing these polymers, an organic polyamine is used as a crosslinking agent. Other supports and preparation thereof are described in U.S. Patent Nos. 4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992, 5,334,640, 5,328,603. [0063] U.S. Patent No. 4,162,355 describes a polymer suitable for use in affinity chromatography, which is a polymer of an aminimide and a vinyl compound having at least one pendant halo-methyl group. An amine ligand, which affords sites for binding in affinity chromatography is coupled to the polymer by reaction with a portion of the pendant halo-methyl groups and the remainder of the pendant halo-methyl groups are reacted with an amine containing a pendant hydrophilic group. A method of coating a substrate with this polymer is also described. An exemplary aminimide is 1,1 -dimethyl- 1- (2-hydroxyoctyl)amine methacrylimide and vinyl compound is a chloromethyl styrene. [0064] U.S. Patent No. 4,171,412 describes specific supports based on hydrophilic polymeric gels, generally of a macroporous character, which carry covalently bonded D-amino acids or peptides that contain D-amino acid units. The basic support is prepared by copolymerization of hydroxyalkyl esters or hydroxyalkylamides of acrylic and methacrylic acid with crosslinking acrylate or methacrylate comonomers are modified by the reaction with diamines, amino acids or dicarboxylic acids and the resulting carboxyterminal or amino terminal groups are condensed with D-analogs of amino acids or peptides. The peptide containing D-amino acids also can be synthesized step wise on the surface of the carrier. [0065] U.S. Patent No. 4,178,439 describes a cationic ion exchanger and a method for preparation thereof. U.S. Patent No. 4,180,524 describes chemical syntheses on a silica support. [0066] Immobilized Artificial Membranes (IAMs; see, e.g., U.S. Patent Nos. 4,931,498 ^' and 4,927,879) also can be used. IAMs mimic cell membrane environments and can be used to bind molecules that preferentially associate with cell membranes (see, e.g., Pidgeon et al. (1990) Enzyme Micr oh. Technol. 12:149). [0067] Any suitable format for a kit containing the aAPCs can be used. As used herein, packaging material means any material known to those of skill in the art that can be used for packaging pharmaceutical products. Exemplary packaging material includes, but is not limited to, containers, vials, blister packs, bottles, tubes, inhalers, pumps, bags, tubes, bioreacters, syringes and any containing means. A combination of kit components may include two or more component items, such as compositions or mixtures, that are intended for use either together or sequentially. The combination can be provided as a mixture of the components or as separate components packaged or provided together, such as in a kit.

[0068] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Examples

Example 1 Plate bound Agonists [0069] PBL were isolated by Percoll gradient centrifugation from leukopacks obtained by apheresis of healthy donors. 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. [0070] Purified CD4+ cells were placed in 24 well plates either coated directly with anti- CD3 and anti-CD28 mAbs in a 50:50 ratio or first coated with a polyclonal goat anti-mouse antibody and next coated with mouse anti-CD3 and anti-CD28 mAbs in a 50:50 ratio (indirect). As a control, a plate coated with polyclonal goat anti-mouse alone was prepared. [0071] Antibodies were attached to the plates by dissolving at a concentration of 10 μg/ml in a sodium borate buffer solution at pH 9.5 and incubation at 4 degrees C overnight. The plates were vigorously washed prior to use. [0072] Purified CD4+ cells were placed in the wells at cell densities of 0.5 x 10⁶ per ml. Concentrations of cytokines in the cell-free supernatants after 72 hours was measured by ELISA. In the control group the cells were first labeled with anti-CD3 and anti-CD28 mAbs and placed on goat anti-mouse coated wells. The cytokine data represents the mean +/- SD of six different blood samples.

[0073] These data show that the indirect methods provided herein enhance the Thl cytokine production from primary T-cells.

Example 2 Artificial Antigen Presenting Cell Devices and Use thereof to produce activated T-cells with enhanced pro-inflammatory cytokine production [0074] Thl cytokine production in cells activated with the devices as described herein and in accord with the methods herein was compared to Thl cytokine production in cells activated with the same signals bound directly to supports. The following experiment was conducted. [0075] Pure Thl cells were prepared according the repeated and frequent activation method described in co-pending U.S. application Serial No. U.S. application Serial No. 09/957,194. Briefly, peripheral blood from 5 advanced cancer patients was collected and PBL were isolated by density gradient centrifugation. CD4+ cells were purified by positive selection with CD4 microbeads (Mitenyi, Frankfurt, Germany). The purified CD4+ cells were suspended in X Vivo 15 media supplemented with glutamine and stimulated aAPC at a 3:1 bead to cell ratio on day 1 and incubated at 0.5 x 10⁶ cells per/ml in 24 well plates. The cultures were split every 48 hours and provided with fresh media. Every 3 days, additional aAPC were added at a 1 : 1 bead:cell ratio. On day 12, the cells were activated with aAPC for the last time. On day 14, the cells were harvested and separated from the aAPC. On day 15, the cells were reactivated with aAPC at a 1:1 bead to cell ratio and the amount of IFN-γ produced in the cell-free supernatant measured by ELISA after 48 hours. [0076] The cancer patient CD4+ cell samples were split into two identically treated groups with the first group activated with the TCE beads commercially available from Dynal and the second group activated with aAPC devices as provided herein, in which sheep anti- mouse coated M-450 Dynabeads were then further coated with anti-CD3 and anti-CD28 at a 50:50 ratio prior to use.

[0077] With the exception of Patient #5 in the TCE group, none of the groups produced IL-4 at levels above the detectable limits of the assay (25 pg/ml). The TCE Patient #5 group produced IL-4 at 37 pg/ml. These data demonstrate that the use of the aAPC devices as provided herein causes T-cells to produce enhanced amounts of IFN-γ, indicating unexpected result that the presence of the linking agent dramatically increased the IFN-γ level in the expanded T cells. [0078] Slightly higher IFN-γ production can be achieved with aAPC devices prepared using sheep anti-mouse beads coated with anti-CD3 and anti-CD28 at 3:1 ratio instead of a 1 : 1 ratio for the first 48 hours after activation. At 72 hours the production from the aAPC devices with antibodies coated at a 1 : 1 ratio is almost always higher.

Claims

Claims What is claimed is:

1. A method to induce and expand a population of IFN-γ producing T cells comprising: contacting a population of T cells with a solid phase support comprising: a) a linking agent immobilized on the solid phase support to which a first and second agonist are bound; b) the first agonist for a first stimulatory molecule on the T cell; and c) the second agonist for a second stimulatory molecule on the T cell; whereby contacting the T cells with the solid phase support induces and expands the population of T cells that produce IFN-γ results in the expanded T cells producing more IFN-γ than T cells expanded by the first and second agonist immobilized on the solid phase support in the absence of the linking agent.

2. The method of claim 1, wherein the T cells are purified.

3. The method of claim 1 , wherein the T cells are purified CD4⁺ T cells.

4. The method of claim 3, wherein CD4⁺ T cells are purified by positive selection.

5. The method of claim 4, wherein the CD4⁺ T cells are further purified to eliminate CD45RO⁺ T cells.

6. The method of claim 1 , wherein the solid phase support comprises polyacrylamide, latex gel, dextran, rubber, silicon, plastics, nitrocellulose, cellulose, natural sponge, polypropylene, glass, polytetrafluoroethylene, polyethylene, polystyrene, polyester, ceramic, and composites thereof.

7. The method of claim 1 , wherein the solid phase support is a bead.

8. The method of claim 7, wherein the bead is a magnetic immunobead.

9. The method of claim 1, wherein the solid phase support is a microtiter plate.

10. The method of claim 1 , wherein the linking agent is an antibody.

11. The method of claim 10, wherein the linking agent is an anti-IgG antibody.

12. The method of claim 1, wherein the first agonist is an anti-CD3 antibody or an anti-CD2 antibody.

13. The method of claim 12, wherein the anti-CD3 antibody binds human CD3.

14. The method of claim 1, wherein the second agonist is selected from the group consisting of an anti-CD28 antibody, B7-1, and B7-2.

15. The method of claim 14, wherein the anti-CD28 antibody binds human CD28.

16. The method of claim 1 , wherein the linking agent is immobilized by direct linkage to the solid support.

17. The method of claim 16, wherein the direct linkage is a covalent linkage to the solid support.

18. The method of claim 1 , wherein the linking agent is immobilized to the solid phase support by an indirect linkage.

19. The method of claim 18, wherein the indirect linkage is a biotin-avidin linkage.

20. The method of claim 1, wherein the T cells are contacted with the solid phase support a minimum of three times at two to four day intervals.

21. The method of claim 20, wherein repeated contact with the solid phase support^' results in the production of a polyclonal Thl CD4 memory cell population.

22. The method of claim 1, which method further comprises contacting the T cells with the solid phase support in the presence of IFN-γ, anti-IL-4 antibody, or anti-TGF-β antibody.

23. An artificial antigen presenting cell (aAPC), comprising a) a linking agent immobilized on the solid phase support to which a first agonist and a second agonist are bound; b) the first agonist for a first stimulatory molecule on the T cell; and c) the second agonist for a second stimulatory molecule on the T cell.

24. The artificial antigen presenting cell of claim 23, wherein the solid phase support comprises polyacrylamide, latex gel, dextran, rubber, silicon, plastics, nitrocellulose, cellulose, natural sponge, polypropylene, glass, polytetrafluoroethylene, polyethylene, polystyrene, polyester, ceramic, and composites thereof.

25. The artificial antigen presenting cell of claim 23, wherein the linking agent is an antibody.

26. The artificial antigen presenting cell of claim 23, wherein the first agonist is selected from the group consisting of anti-CD3 antibodies and anti-CD2 antibodies.

27. The artificial antigen presenting cell of claim 23, wherein the second agonist is selected from the group consisting of anti-CD28 antibodies, B7-1, and B7-2.

28. The artificial antigen presenting cell of claim 23, wherein the cell has dendritic protrusions.

29. A kit comprising the artificial antigen presenting cell of claim 23.

30. The kit of claim 29, wherein the artificial antigen presenting cell is in a syringe.

31. The kit of claim 29, wherein the artificial antigen presenting cell is in a hollow fiber bioreactor.