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WO2005007116A2 - Systemes de cellules presentant l'antigene artificielles - Google Patents

Systemes de cellules presentant l'antigene artificielles Download PDF

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WO2005007116A2
WO2005007116A2 PCT/US2004/022919 US2004022919W WO2005007116A2 WO 2005007116 A2 WO2005007116 A2 WO 2005007116A2 US 2004022919 W US2004022919 W US 2004022919W WO 2005007116 A2 WO2005007116 A2 WO 2005007116A2
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cells
cell
agonist
solid phase
antibody
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WO2005007116A3 (fr
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Michael L. Gruenberg
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VALEOCYTE THERAPIES LLC
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VALEOCYTE THERAPIES LLC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the methods and compositions disclosed herein relate to the preferential expansion of T cells producing pro-inflammatory cytokines, e.g., IFN- ⁇ , using an artificial antigen presenting cell (“aAPC”) system.
  • the methods are useful in expanding T cells in sufficient numbers for use in adoptive immunotherapy.
  • 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.
  • TIL tumor infiltrating lymphocytes
  • cytotoxic T cells cytotoxic T cells
  • expanded tumor draining lymph node cells various lymphocyte preparations
  • regulatory T cells to initiate or augment cellular immune responses against tumors and pathogens.
  • 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- ⁇ and IL-2 and low to no IL-4 production. IFN- ⁇ is considered the prototypical Thl cytokine and is a key player in the host immune defenses against tumors and viruses.
  • IFN- ⁇ 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.
  • IFN- ⁇ stimulates the processing and presentation of viral epitopes at the surface of virally-infected cells, effecting their elimination by lymphocytes and other immune effectors.
  • IFN- ⁇ correlates with therapeutic efficacy of adoptively transferred T-cells. Turtle et al. (1993) Can. Res. 55:833-39; Nagoshi et al. (1998) J Immunol.
  • IFN- ⁇ as well as other pro-inflammatory cytokines have a significant role in the cellular immune response
  • the methods and compositions disclosed herein provide expanded T cells that produce high levels of IFN- ⁇ and/or other pro-inflammatory cytokines.
  • a method to induce and expand a population of IFN- ⁇ 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- ⁇ , which results in the expanded T cells producing more IFN- ⁇ than T cells expanded by the first and second agonist immobilized on the solid phase support in the absence of the linking agent.
  • the T cells are contacted with the solid phase support a minimum of three times at two to four day intervals.
  • 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.
  • the T cells to be expanded can be purified CD3 + T cells or purified CD3 + , CD4 + T cells.
  • the T cells to be expanded are CD3 + , CD4 + , CD45RO ' T cells.
  • the T cells can be purified by positive selection.
  • an artificial antigen presenting cell 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.
  • 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.
  • the solid phase support is a bead, more particularly a magnetic immunobead. In another embodiment, the solid phase support is a microtiter plate.
  • 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.
  • the anti-CD3 antibody binds human CD3 and the anti-CD28 antibody bind human CD28.
  • 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.
  • the artificial antigen presenting cell (hereinafter "aAPC") provides an effective and reliable method to expand pro-inflammatory cytokine (e.g., IFN- ⁇ ) 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.
  • the T cell receptor TCR
  • 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- ⁇ and/or other proinflammatory cytokines.
  • 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.
  • 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.
  • 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. Seespertini 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.
  • 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.
  • PLC protein kinase C
  • phorbol ester such as phorbol myristate acetate
  • a calcium ionophore such as ionomycin
  • an stimulatory molecule on the surface of the T-cell such as CD28
  • a ligand that binds the stimulatory molecule is stimulated with a ligand that binds the stimulatory molecule.
  • 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.
  • 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.
  • 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).
  • animal serum e.g., fetal bovine serum
  • antibiotics e.g., penicillin streptomycin
  • 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.
  • T-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.
  • 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
  • T-cells co-activated with anti-CD3 and anti-CD28 in the presence of IL-2 Garlie et al.
  • 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).
  • BCG Bacille Calmette-Guerin
  • T-cells such as T cells expressing CD28, CD4, CD8, CD45RA, and CD45RO
  • T-cells can be further isolated by positive or negative selection techniques.
  • 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.
  • 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.
  • the T cells can be purified using positive selection using routine methods known in the art.
  • the methods provided herein expand a population of T cells that are producers inflammatory cytokines.
  • 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- ⁇ , IL-1, IL-2, IL-6, IL-8, TNF- and GM-CSF.
  • these T cells are CD4+ Thl cells.
  • Thl cell refers to a CD4+ T cell that produce IFN- ⁇ , 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- ⁇ are Th2 cells.
  • 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- ⁇ , while Th2 cells are commonly identified by intercellular staining for IL-4.
  • CD4+ cells stain positive for intracellular IFN- ⁇ after activation; less than 1% stain positive for IFN- ⁇ prior to activation. It is rare for a T-cell population to stain greater than 35% IFN- ⁇ positive.
  • the cells resulting from a method described herein stain greater than 70% positive and often greater than 90% positive for IFN- ⁇ .
  • 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- ⁇ and does not produce greater than about 26 pg/ml/10 cells of IL-4 in a 24 hour period.
  • Activated T-cells can be identified phenotypically, most commonly, by the expression of CD25.
  • T cells that are CD25+ are referred to herein as "activated.”
  • a pure or highly pure population of activated cells typically express greater than 85% CD25+.
  • Memory T cells can be expanded using the disclosed methods and compositions.
  • 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.
  • 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.
  • 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.
  • the contacting is effected under conditions whereby the T-cells are directed to differentiate into Thl cells.
  • an anti-CD3 antibody and the monoclonal antibody ES5.2D8 ATCC
  • ATCC monoclonal antibody
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a linking agent such as an IgG or other molecule to which an anti-CD3 monoclonal antibody and/or anti-CD28 monoclonal antibody binds
  • 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.
  • 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.
  • Any suitable agonist may be used with the methods provided 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.
  • 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.
  • 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.
  • 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.
  • co-stimulatory molecules such as CD28, CD134 (OX40) and CD137 (4-1BB) or adhesion molecules on T-cells
  • IAM-1 CD1 la/CD 18
  • NLA-4 CD49d/CD29
  • 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 IF ⁇ - ⁇ .
  • the amount of IF ⁇ - ⁇ 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.
  • 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.
  • a solid support material can be any described herein or known to those of skill in the art, such as plastic or polymer grafted materials.
  • 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.
  • 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.
  • 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].
  • 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.
  • 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.
  • Any culture conditions may be used in the methods provided herein.
  • the term "culture medium" includes any medium suitable for supporting the viability, growth, and/or differentiation of mammalian cells ex-vivo.
  • culture medium examples 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cell surface molecules such as B7-1, B7-2, which are induced on activated T-cells.
  • 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 ⁇ -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.
  • 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).
  • the methods provided herein support the expansion of T cells to a clinically relevant number for adoptive immunotherapy.
  • a composition containing a clinically relevant number or population of T cells is a composition that contains at least 10 9 , typically greater than 10 9 , at least 10 10 cells, and generally more than 10 10 cells. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cell.
  • the population will contain greater than 70%, generally greater than 80%, 85% and 90- 95% of such cells.
  • 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.
  • the density of the desired cells is typically greater than 10 6 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 9 , 10 10 or 10 ⁇ cells.
  • 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.
  • T cell subclass e.g., Thl, Th2, Th3, Tel, Tc2, etc
  • lymphocytic origin e.g., T cell, B cell, NK cell
  • immune cells include any cell of lymphocytic or monocytic origin that participates in a immune response.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ , IL-2, and TNF-alpha. The precise amounts and cytokines to assess depend upon the particular state, disease or condition.
  • 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).
  • the cells expanded using the method herein are also useful in the treatment of diseases dominated by pathogenic Th2 responses.
  • diseases characterized by a Th2-dominated immune response are characterized by either a suppressed cellular immune response or excessive humoral response.
  • 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.
  • 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.
  • a first dosage will be at least 10 9 -10 10 cells.
  • the dosage will vary depending upon treatment sought.
  • about 10 9 is from about 5 x 10 8 up to about 5 x 10 9 ; similarly about 10 10 is from about 5 x 10 9 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.
  • a subject is a mammal, typically a human, including humans in need of treatment for a disease or disorder (i.e., a patient).
  • therapeutically effective refers to an amount of cells that is sufficient to ameliorate, or in some manner reduce the symptoms associated with a disease.
  • 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.
  • 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 10 9 , and generally 10 10 or more. The precise number will depend upon the cell type and also the intended target or result and can be determined empirically.
  • the cells can be administered by any suitable methods.
  • infusion medium is an isotonic solution suitable for intravenous infusion.
  • infusion medium examples 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.
  • 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.
  • 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.
  • 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.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose and dextrans, mannitol
  • proteins such as glucose, mannose, sucrose and dextrans
  • proteins such as glucose, mannose, sucrose and dextrans
  • proteins such as glucose, mannose, sucrose and dextrans
  • proteins such as glucose, mannose, sucrose and dextrans
  • proteins such as glucose, mannose, sucrose and dextrans
  • proteins such as glucose, mannose, sucrose and dextrans
  • 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.
  • 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.
  • the particles typically 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.
  • 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 ⁇ m or less, 50 ⁇ m or less and typically have a size that is 100 mm 3 or less, 50 mm 3 or less, 10 mm 3 or less, and 1 mm 3 or less, 100 ⁇ m 3 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • monomers such as vinyl momomers, or mixtures of monomers
  • polymers such as polyethylene and polypropylene
  • 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.
  • 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.
  • 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.
  • inorganics 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
  • supports are 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.
  • 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.
  • the bead can be of any size that effectuates T-cell expansion, such as about 2.5 ⁇ m to about 500 ⁇ m in diameter, typically about 2.8 ⁇ m to about 50 ⁇ m.
  • Naturally-occurring supports include, but are not limited to agarose, other polysaccharides, collagen, celluloses and derivatives thereof, glass, silica, and alumina.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • kits containing the aAPCs can be used.
  • 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.
  • Example 1 Plate bound Agonists 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.
  • 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).
  • a plate coated with polyclonal goat anti-mouse alone was prepared.
  • Antibodies were attached to the plates by dissolving at a concentration of 10 ⁇ g/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.
  • 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. 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.
  • Example 2 Artificial Antigen Presenting Cell Devices and Use thereof to produce activated T-cells with enhanced pro-inflammatory cytokine production
  • 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.
  • 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 10 6 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- ⁇ produced in the cell-free supernatant measured by ELISA after 48 hours.
  • 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.

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Abstract

L'invention a trait à des procédés et à des compositions concernant un système de cellules présentant l'antigène artificielles (système </= AAPC >/= ). Les systèmes aAPC selon l'invention renferment un agent de liaison qui améliore considérablement l'efficacité des agonistes liés, de façon à stimuler et à élargir une population de lymphocytes T qui sont de grands producteurs de cytokines pro-inflammatoires telles que IFN- gamma . De telles cellules sont des compositions utiles pour des protocoles d'immunothérapie adoptive.
PCT/US2004/022919 2003-07-18 2004-07-16 Systemes de cellules presentant l'antigene artificielles Ceased WO2005007116A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1736482A1 (fr) * 2005-06-20 2006-12-27 INSERM (Institut National de la Santé et de la Recherche Medicale) Ligand recombinant trimeric de 4-1BB
EP2025747A1 (fr) * 2007-08-14 2009-02-18 TumorTec GmbH Procédé d'amplification in vitro de lymphocytes T-régulateurs
US20150118207A1 (en) * 2011-12-22 2015-04-30 Mogam Biotechnology Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
US11766456B2 (en) 2014-11-26 2023-09-26 GC Cell Corporation Method for culturing natural killer cells using T cells
US12203065B2 (en) 2017-05-26 2025-01-21 GC Cell Corporation Method for culturing natural killer cell, using transformed T cell
US12398372B2 (en) 2018-11-14 2025-08-26 GC Cell Corporation Method for culturing cord blood-derived natural killer cells using transformed T-cells

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Publication number Priority date Publication date Assignee Title
DK0700430T3 (da) * 1993-06-04 2005-08-15 Us Navy Fremgangsmåder til selektivt af stimulere proliferation af T-celler
JP2001520509A (ja) * 1995-07-25 2001-10-30 セルセラピー・インコーポレイテツド 自己免疫細胞療法：細胞組成物、方法およびヒト疾患の治療への応用

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1736482A1 (fr) * 2005-06-20 2006-12-27 INSERM (Institut National de la Santé et de la Recherche Medicale) Ligand recombinant trimeric de 4-1BB
WO2007000675A3 (fr) * 2005-06-20 2007-03-15 Inst Nat Sante Rech Med 4-1bbl trimeriques recombines
EP2025747A1 (fr) * 2007-08-14 2009-02-18 TumorTec GmbH Procédé d'amplification in vitro de lymphocytes T-régulateurs
WO2009021995A1 (fr) * 2007-08-14 2009-02-19 Tumortec Gmbh Procédé d'amplification in vitro de lymphocytes t régulateurs
US20150118207A1 (en) * 2011-12-22 2015-04-30 Mogam Biotechnology Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
US9834753B2 (en) * 2011-12-22 2017-12-05 Green Cross Labcell Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
US11766456B2 (en) 2014-11-26 2023-09-26 GC Cell Corporation Method for culturing natural killer cells using T cells
US12203065B2 (en) 2017-05-26 2025-01-21 GC Cell Corporation Method for culturing natural killer cell, using transformed T cell
US12398372B2 (en) 2018-11-14 2025-08-26 GC Cell Corporation Method for culturing cord blood-derived natural killer cells using transformed T-cells

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