US20040224402A1 - Generation and isolation of antigen-specific T cells - Google Patents

Generation and isolation of antigen-specific T cells Download PDF

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US20040224402A1
US20040224402A1 US10/742,622 US74262203A US2004224402A1 US 20040224402 A1 US20040224402 A1 US 20040224402A1 US 74262203 A US74262203 A US 74262203A US 2004224402 A1 US2004224402 A1 US 2004224402A1
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cells
antigen
antibody
cancer
cell
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Mark Bonyhadi
Dale Kalamasz
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Life Technologies Corp
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Xcyte Therapies Inc
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Publication of US20040224402A1 publication Critical patent/US20040224402A1/en
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Assigned to INVITROGEN CORPORATION reassignment INVITROGEN CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: XCYTE THERAPIES, INC.
Priority to US11/674,304 priority patent/US20070212767A1/en
Priority to US12/251,224 priority patent/US7977095B2/en
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Assigned to Life Technologies Corporation reassignment Life Technologies Corporation CORRECTIVE ASSIGNMENT TO CORRECT THE APPLICATION NO 09452626 PREVIOUSLY RECORDED ON REEL 023882 FRAME 0551. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER SHOULD NOT HAVE BEEN RECORDED AGAINST THIS PATENT APPLICATION NUMBER. Assignors: INVITROGEN CORPORATION
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Definitions

  • the present invention relates generally to methods for generating, isolating, and expanding antigen-specific T cells.
  • the present invention also relates to compositions of antigen-specific T cells.
  • APC antigen presenting cells
  • IL-2 interleukin-2
  • IL-2 has been used together with an anti-CD3 antibody to stimulate T-cell proliferation, predominantly expanding the CD8 + subpopulation of T-cells.
  • APC signals are thought to be required for optimal T-cell activation, expansion, and long-term survival of the T-cells upon re-infusion.
  • MHC-matched APCs as accessory cells presents a significant problem for long-term culture systems because APCs are relatively short-lived.
  • APCs must be continually obtained from a source and replenished.
  • the necessity for a renewable supply of accessory cells is problematic for treatment of immunodeficiencies in which accessory cells are affected.
  • the cells may contaminate the entire T-cell population during long-term culture.
  • antigen e.g. tumor/viral antigen
  • pulsing of antigen-presenting cells with antigens followed by infusion of cells.
  • Expansion of antigen-specific T cells to generate large numbers of antigen-specific T cells often requires labor intensive and expensive cloning, and/or multiple rounds of activation/expansion to achieve therapeutically relevant T cell numbers.
  • the present invention provides methods to generate an increased number of highly responsive antigen-specific T cells that have surface receptor and cytokine production characteristics that are more more desirable than other expansion methods.
  • the instant invention does not require knowledge of a particular antigen (although known antigens can be used in the context of this invention) and provides for a single, or double, round of expansion to achieve a therapeutically relevant dose of antigen-specific T cells, both of the CD4 and CD8 lineage (and either may be selected if desired).
  • the present invention relates to methods for activating, stimulating and isolating antigen-specific T cells.
  • the present invention also relates to compositions of antigen-specific T cells and methods of their use in the treatment and prevention of cancer, infectious diseases, autoimmune diseases, immune disfunction related to aging, or any other disease state where antigen-specific T cells are desired for treatment.
  • a method for expanding a population of antigen-specific T cells comprising contacting a population of cells wherein at least a portion thereof comprises antigen-specific T cells, with a surface, wherein said surface has attached thereto a first agent and a second agent, wherein said first agent ligates a CD3/TCR complex on said T cells and said second agent ligates an accessory molecule on said T cells, and wherein said ligation by said first and second agent of said T cells induces proliferation of antigen-specific T cells and wherein said surface is present in a ratio of surface to T cells of 1:2 or less. In certain embodiments the ratio of surface to T cells is between about 1:1 and about 1:50 and any ratio therebetween.
  • the ratio of surface to T cells is from about 1:2, 1:2.5, 1:5, 1:10, 1:25, 1:50, 1:75, 1:100, or lower.
  • the surface includes but is not limited to paramagnetic beads, lipids, and cell surfaces.
  • the surface comprises paramagnetic beads conjugated to one or more antibodies.
  • the surface can have 1, 2, 3, 4, or more antibodies or natural ligands conjugated thereto.
  • Another aspect of the present invention provides a method for generating antigen-specific T cells comprising exposing a first population of cells wherein at least a portion thereof comprises antigen presenting cells (APC) to a surface wherein said surface has antigen attached thereto, such that said surface with antigen attached thereto is ingested by said APC; exposing a second population of cells wherein at least a portion thereof comprises T cells to the population of cells in part (a); thereby generating antigen-specific T cells.
  • APC antigen presenting cells
  • Antigen may be attached or coupled to, or integrated into a surface by a variety of methods known and available in the art and described herein.
  • the antigen is crosslinked to said surface.
  • the attachment to said surface is by covalent or noncovalent, electrostatic, or hydrophobic and may be accomplished by a variety of attachment means, including for example, chemical, mechanical, enzymatic, electrostatic, or other means whereby the antigen(s) is capable of stimulating the cells.
  • the antibody to an antigen first may be attached to a surface, or avidin or streptavidin may be attached to the surface for binding to a biotinylated antigen.
  • the antibody to the ligand may be attached to the surface via an anti-idiotype antibody.
  • Another example includes using protein A or protein G, or other non-specific antibody binding molecules, attached to surfaces to bind an antibody.
  • antigen may be attached to the surface by chemical means, such as cross-linking to the surface, using commercially available cross-linking reagents (Pierce, Rockford, Ill.) or other means.
  • antigens are covalently bound to the surface.
  • commercially available tosyl-activated DYNABEADSTM or DYNABEADSTM with epoxy-surface reactive groups are incubated with the polypeptide antigen of interest according to the manufacturer's instructions. Briefly, such conditions typically involve incubation in a phosphate buffer from pH 4 to pH 9.5 at temperatures ranging from 4 to 37 degrees C.
  • the APC are in direct contact with the antigen-specific T cells.
  • the APC that are in direct contact with said antigen-specific T cells are isolated by exposing said APC to a magnetic field, wherein said surface comprises a paramagnetic, magnetic, or magnetizable component.
  • the antigen-specific T cells are expanded by exposing said T cells to a surface wherein said surface has attached thereto a first agent that ligates a first T cell surface moiety of a T cell, and the same or a second surface has attached thereto a second agent that ligates a second moiety of said T cell, wherein said ligation by the first and second agent induces proliferation (expansion) of said antigen-specific T cells.
  • at least one agent is an antibody or an antibody fragment.
  • the first agent is an antibody or a fragment thereof
  • the second agent is an antibody or a fragment thereof.
  • the first and the second agents are different antibodies.
  • the first agent is an anti-CD3 antibody, an anti-CD2 antibody, or an antibody fragment of an anti-CD3 or anti-CD2 antibody and the second the second agent is an anti-CD28 antibody or antibody fragment thereof.
  • the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody.
  • the anti-CD3 antibody and the anti-CD28 antibody are present at a ratio of about 1:1 to about 1:100.
  • the antigen-specific T cells are expanded by exposing said antigen-specific T cells to a mitogen, such as phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), and superantigen.
  • a mitogen such as phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), and superantigen.
  • the antigen of the present invention includes but is not limited to protein, glycoprotein, peptides, antibody/antigen complexes, whole tumor or virus-infected cells, fixed tumor or virus-infected cells, heat-killed tumor or virus-infected cells, tumor lysate, virus lysate, non-soluble cell debris, apoptotic bodies, necrotic cells, whole tumor cells from a tumor or a cell line that have been treated such that they are unable to continue dividing, allogeneic cells that have been treated such that they are unable to continue dividing, irradiated tumor cells, irradiated allogeneic cells, natural or synthetic complex carbohydrates, lipoproteins, lipopolysaccharides, transformed cells or cell line, transfected cells or cell line, transduced cells or cell line, and virally infected cells or cell line.
  • antigen is attached to said surface by an antibody/ligand interaction.
  • An antibody/ligand interaction includes but is not limited to an interaction between an antibody/ligand pair selected from the group consisting of anti-MART-1 antibody/MART-1 antigen, anti-WT-1 antibody/WT-1, anti-PR1 antibody /PR1, anti-PR3 antibody /PR3, anti-tyrosinase antibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1 antigen, anti-MUC-1 antibody/MUC-1 antigen, anti- ⁇ -fetoprotein antibody/ ⁇ -fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis C antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA 3B antigen, and anti-
  • the antigen is chemically attached to a surface.
  • the attachment of said antigen to said surface comprises a biotin-avidin interaction.
  • the population of cells wherein at least a portion thereof comprises APC is derived from a source selected from the group consisting of a leukapheresis product, peripheral blood, lymph node, tonsil, thymus, tissue biopsy, tumor, spleen, bone marrow, cord blood, CD34 + cells, monocytes, and adherent cells.
  • Another aspect of the present invention provides a method for generating and expanding antigen-specific T cells comprising exposing a first population of cells wherein at least a portion thereof comprises antigen presenting cells to antigen such that said antigen is taken up by said APC; exposing a second population of cells wherein at least a portion thereof comprises T cells to the population of cells in part (a); thereby generating antigen-specific T cells; and exposing said antigen-specific T cells of part (b) to a surface wherein said surface has attached thereto a first agent that ligates a first T cell surface moiety of a T cell, and the same or a second surface has attached thereto a second agent that ligates a second moiety of said T cell, wherein said ligation by the first and second agent induces proliferation (expansion) of said antigen-specific T cells.
  • At least one agent is an antibody or an antibody fragment.
  • the first agent is an antibody or a fragment thereof
  • the second agent is an antibody or a fragment thereof.
  • the first and the second agents are different antibodies.
  • the first agent is an anti-CD3 antibody, an anti-CD2 antibody, or an antibody fragment of an anti-CD3 or anti-CD2 antibody and the second the second agent is an anti-CD28 antibody or antibody fragment thereof.
  • the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody.
  • the anti-CD3 antibody and the anti-CD28 antibody are present at a ratio of about 1:1 to about 1:100.
  • said antigen-specific T cells are isolated by contacting said T cells with antibodies specific for T cell activation markers.
  • said antibodies are selected from the group consisting of anti-CD25, anti-CD54, anti-CD69, anti-CD38, anti-CD45RO, anti-CD49d, anti-CD40L, anti-CD137, anti-CD62L, and anti-CD134.
  • a further aspect of the present invention provides a population of antigen-specific T cells generated according to any one of the methods described herein.
  • An additional aspect of this invention is a composition comprising the antigen-specific T cells according to any of the methods described herein and a pharmaceutically acceptable excipient.
  • a further aspect of the present invention provides methods for stimulating an immune response in a mammal comprising, administering to the mammal compositions comprising the antigen-specific T cells of the present invention.
  • An additional aspect of the invention provides for reducing the presence of cancer cells in a mammal comprising, exposing the cancer cells to the compositions comprising antigen-specific T cells.
  • the cancer cells are from a cancer selected from the group consisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL).
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • One aspect of the present invention provides a method for inhibiting the development of a cancer in a mammal, comprising administering to the mammal the composition comprising antigen-specific T cells fo the present invention.
  • the cancer cells are from a cancer selected from the group consisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL).
  • ALL acute lymphoblastic leukemia
  • AML acute my
  • a further aspect of the present invention provides a method for ameliorating an immune response dysfunction in a mammal comprising administering to the mammal the compositions comprising antigen-specific T cells generated using any one of the methods described herein.
  • an infectious organism can include but is not limited to a virus, a single-stranded RNA virus, a single-stranded DNA virus, a double-stranded DNA virus, Human Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, a bacterium, M. tuberculosis, Pneumocystis carinii, Candida, Aspergillus.
  • HIV Human Immunodeficiency Virus
  • HSV Herpes Simplex Virus
  • HPV Human Papilloma Virus
  • CMV Cytomegalovirus
  • EBV Epstein-Barr virus
  • An additional aspect of the present invention provides a method for inhibiting the development of an infectious disease in a mammal, comprising administering to the mammal the compositions comprising antigen-specific T cells generated using any one of the methods described herein.
  • an infectious disease can be caused by an infectious organism including but not limited to a virus, an RNA virus, a DNA virus, Human Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, a bacterium, M. tuberculosis, Pneumocystis carinii, Candida, Aspergillus.
  • HIV Human Immunodeficiency Virus
  • HPV Human Papilloma Virus
  • CMV Cytomegalovirus
  • EBV Epstein-Barr virus
  • FIG. 1 is a photograph showing the tight association of antigen-specific T cells and bead-loaded antigen presenting cells (APC) post magnetic separation.
  • FIG. 2 is a plot showing upregulation of CD25 in re-stimulated memory CD8 CMV tetramer+ T cells expanded ex vivo.
  • Panel A is a negative control from an HLA-A2+, CMV ⁇ donor.
  • Panel B is a negative control showing uncoated bead stimulation from an HLA-A2+, CMV+ donor.
  • Panel C shows CMV antigen-coated bead stimulation of cells from an HLA-A2+, CMV+ donor.
  • FIG. 3 is a plot showing the effect of varying bead:cell ratio on expansion or deletion of CMV-specific T cells.
  • FIG. 4 panels A and B is a bar graph showing the effect on T cell expansion of sequential bead addition at varying bead:cell ratios at varying times during cutlure.
  • Panel A shows a comparison of total T cell expansion over 15 days, comparing standard static cuture (beads at day 0 at either 1:2.5 or 1:5 bead to cell ratio) or additional beads added at day 5, 7, or 9 at 1:10, 1:25, 1:50 or 1:100 bead to cell ratios.
  • Panel B shows CMV-specific T cell expansion under the same experimental conditions as Panel A.
  • FIG. 5 is a graph showing the effect on T cell expansion of low bead:T cell ratio and sequential addition of beads on Melanoma gp100(M)-specific T cells.
  • FIG. 6 is a graph depicting the fold increase of T-cells over time following stimulation with anti-CD3 and anti-CD28 co-immobilized beads with varying ratios of anti-CD3:CD28 antibodies attached thereto.
  • FIG. 7 is a graph depicting the fold increase of CMVpp65-specific T-cells over time following stimulation with anti-CD3 and anti-CD28 co-immobilized beads with varying ratios of anti-CD3:CD28 antibodies attached thereto.
  • biocompatible refers to the property of being predominantly non-toxic to living cells.
  • stimulation refers to a primary response induced by ligation of a cell surface moiety.
  • such stimulation entails the ligation of a receptor and a subsequent signal transduction event.
  • stimulation of a T-cell refers to the ligation of a T-cell surface moiety that in one embodiment subsequently induces a signal transduction event, such as binding the TCR/CD3 complex.
  • the stimulation event may activate a cell and upregulate or downregulate expression or secretion of a molecule, such as downregulation of TGF- ⁇ .
  • ligation of cell surface moieties may result in the reorganization of cytoskeletal structures, or in the coalescing of cell surface moieties, each of which could serve to enhance, modify, or alter subsequent cell responses.
  • activation refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change.
  • T-cells such activation, refers to the state of a T-cell that has been sufficiently stimulated to induce cellular proliferation.
  • Activation of a T-cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process.
  • target cell refers to any cell that is intended to be stimulated by cell surface moiety ligation.
  • an “antibody”, as used herein, includes both polyclonal and monoclonal antibodies; primatized (e.g., humanized); murine; mouse-human; mouse-primate; and chimeric; and may 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 may occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering; an “antibody fragment,” as used herein, refers to fragments, derived from or related to an antibody, which bind antigen and which in some embodiments may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. This includes, e.g., F(ab), F(ab)′ 2 , scFv, light chain variable region (V L ), heavy chain
  • protein includes proteins, polypeptides and peptides; and may be an intact molecule, a fragment thereof, or multimers or aggregates of intact molecules and/or fragments; and may occur in nature or be produced, e.g., by synthesis (including chemical and/or enzymatic) or genetic engineering.
  • agent refers to a molecule that binds to a defined population of cells.
  • the agent may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population.
  • the agent may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), or the like.
  • antibodies are used as a prototypical example of such an agent.
  • agent that binds a cell surface moiety and “cell surface moiety”, as used herein, are used in the context of a ligand/anti-ligand pair. Accordingly, these molecules should be viewed as a complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affinity (an affinity constant, K a , of about 10 6 M ⁇ 1 or tighter).
  • Antigen-presenting cell refers to those cells that normally initiate the responses of na ⁇ ve and/or memory T cells to antigen.
  • APC refers to any cell capable of antigen presentation.
  • APCs include, but are not limited to, dendritic cells, monocytes, macrophages, and B cells.
  • An APC may express high levels of MHC class II, ICAM-1 and B7-2.
  • a “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T-cell proliferation.
  • a “ligand/anti-ligand pair”, as used herein, refers to a complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affinity (an affinity constant, K a , of at least about 10 6 M ⁇ 1 ,).
  • affinity constants of the ligand/anti-ligand pairs useful in the context of the present invention might be lower or in some cases higher.
  • the streptavidin on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower.
  • the dissociation constant was determined to be 1.3 ⁇ 10( ⁇ 8)M.
  • ligand/anti-ligand pairs enzyme/inhibitor hapten/antibody, lectin/carbohydrate, ligand/receptor, and biotin/avidin or streptavidin.
  • receptors and other cell surface moieties are anti-ligands, while agents (e.g., antibodies and antibody fragments) reactive therewith are considered ligands.
  • Separatation includes any means of substantially purifying one component from another (e.g., by filtration, magnetic attraction, etc.).
  • “Quiescent”, as used herein, refers to a cell state wherein the cell is not actively proliferating.
  • a “surface”, as used herein, refers to any surface capable of having an agent attached thereto and includes, without limitation, metals, glass, plastics, co-polymers, colloids, lipids, cell surfaces, and the like. Essentially any surface that is capable of retaining an agent bound or attached thereto.
  • a prototypical example of a surface used herein, is a particle such as a bead. As such, the terms “surface” and “particle” are used herein interchangeably.
  • Immuno response or responsiveness refers to activation of cells of the immune system, including but not limited to, T-cells, such that a particular effector function(s) of a particular cell is induced. Effector functions may include, but are not limited to, proliferation, secretion of cytokines, secretion of antibodies, expression of regulatory and/or adhesion molecules, and the ability to induce cytolysis.
  • Stimulating an immune response refers to any stimulation such that activation and induction of effector functions of cells of the immune system are achieved.
  • Immuno response dysfunction refers to the inappropriate activation and/or proliferation, or lack thereof, of cells of the immune system, and/or the inappropriate secretion, or lack thereof, of cytokines, and/or the inappropriate or inadequate induction of other effector functions of cells of the immune system, such as expression of regulatory, adhesion, and/or homing receptors, and the induction of cytolysis.
  • preventing or “inhibiting” the development of a cancer or cancer cells” as used herein, refers to the occurrence of the cancer being prevented or the onset of the cancer being delayed.
  • treating means that the cancer growth is inhibited, which is reflected by, e.g., tumor volume or numbers of malignant cells.
  • Tumor volume may be determined by various known procedures, e.g., obtaining two dimensional measurements with a dial caliper.
  • Preventing or inhibiting the development of an infectious disease means the occurrence of the infectious disease is prevented or the onset of the infectious disease is delayed, or the spread of an existing infection is reversed.
  • “Ameliorate” as used herein, is defined as: to make better; improve (The American Heritage College Dictionary, 3 rd Edition, Houghton Mifflin Company, 2000).
  • Particles may include a colloidal particle, a microsphere, nanoparticle, a bead, or the like.
  • commercially available surfaces such as beads or other particles, are useful (e.g., Miltenyi Particles, Miltenyi Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADSTM, Dynal Inc., Oslo, Norway; PURABEADSTM, Prometic Biosciences, magnetic beads from Immunicon, Huntingdon Valley, Pa., microspheres from Bangs Laboratories, Inc., Fishers, Ind.).
  • Paraamagnetic particles refer to particles, as defined above, that localize in response to a magnetic field.
  • Antigen refers to any molecule 1) capable of being specifically recognized, either in its entirety or fragments thereof, and bound by the “idotypic” portion (antigen-binding region) of a mAb or its derviative; 2) containing peptide sequences which can be bound by MHC and then, in the context of MHC presentation, can specifically engage its cognate T cell antigen receptor.
  • To “load” an APC with antigen refers to exposing an APC to antigen or antigenic peptide for a period of time sufficient for the APC to uptake, process, and present the antigen, bound by MHC molecules, to T cells.
  • the antigen, especially peptide can be bound by MHC molecules and presented to T cells without being taken up and processed by the APC.
  • animal or “mammal” as used herein, encompasses all mammals, including humans.
  • the animal of the present invention is a human subject.
  • exposing refers to bringing into the state or condition of immediate proximity or direct contact.
  • lysate refers to the supernatant and non-soluble cell debris resulting from lysis of cells.
  • lysis buffers known in the art may be used (see for example Current Protocols in Immunology, John Wiley & Sons, New York. N.Y.). Cell lysis may also be carried out by freeze-thaw procedures or other means (e.g. sonication, etc.).
  • apoptotic body as used herein, is defined as the smaller, intact, membrane-bound fragments that result from apoptotic cells.
  • proliferation means to grow or multiply by producing new cells.
  • infectious disease refers to any disease that is caused by an infectious organism.
  • Infectious organisms may comprise viruses, (e.g., RNA viruses, DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV), cytomegalovirus (CMV) Epstein-Barr virus (EBV), human papilloma virus (HPV)), parasites (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria, in particular, M.
  • viruses e.g., RNA viruses, DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV), cytomegalovirus (CMV) Epstein-Barr virus (EBV), human papilloma virus (HPV)
  • fungi e.g., Candida species, Aspergillus species
  • Pneumocystis carinii fungi
  • prions known prions infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats, as well as bovine spongiform encephalopathy (BSE), or “mad cow disease”, and feline spongiform encephalopathy of cats.
  • BSE bovine spongiform encephalopathy
  • prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI)).
  • CJD Creutzfeldt-Jakob Disease
  • GSS Gerstmann-Straussler-Scheinker Disease
  • FFI fatal familial insomnia
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen tissue, or any other lymphoid tissue, and tumors.
  • T cells can be obtained from T cell lines and from autologous or allogeneic sources.
  • T cells may also be obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
  • cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis or leukapheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions.
  • a semi-automated “flow-through” centrifuge for example, the Cobe 2991 cell processor, Baxter
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca ++ /Mg ++ free PBS.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and by centrifugation through a PERCOLLTM gradient.
  • a specific subpopulation of T cells such as CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques.
  • CD3 + , CD28 + T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • a preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses 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, CD11b, CD16, HLA-DR, and CD8.
  • Another method for preparing T cells for stimulation is to freeze the cells after the washing step, which does not require the monocyte-removal step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population.
  • the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to ⁇ 80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • HSA human serum albumin
  • the source of antigen-presenting cell is typically a tissue source comprising APC or APC precursors that are capable of proliferating and maturing in vitro into professional APC (pAPC) when loaded with antigen and/or treated with the necessary cytokines or factors.
  • “Professional APC” (pAPC) or “antigen-presenting cell” (APC), as used herein, refers to those cells that normally initiate the responses of na ⁇ ve and/or memory T cells to antigen.
  • Professional APCs include, but are not limited to, DC, macrophages, and B cells.
  • pAPC may express high levels of MHC class II, ICAM-1 and B7-2.
  • APC precursor cells are capable of proliferating and maturing in vitro into dendritic cells (DC). While many tissue sources may be used, typical tissue sources comprise spleen, thymus, tissue biopsy, tumor, afferent lymph, lymph nodes, bone marrow, apheresis or leukapheresis product, and/or peripheral blood. In certain embodiments, apheresis product, bone marrow and peripheral blood are preferred sources. Fetal tissue, fetal or umbilical cord blood, which is also rich in growth factors may also be used as a source of blood for obtaining APC and/or precursor APC. Exemplary precursor cells may be, but are not limited to, embryonic stem cells, CD34 + cells, monocyte progenitors, monocytes, and pre-B cells.
  • APC may be derived from precursor cells comprising monocytes or CD34 + cells.
  • the source of APC and/or precursor APC is an apheresis or leukapheresis product.
  • Cells are collected using apheresis procedures known in the art. See, for example, Bishop et al., Blood, vol. 83, No. 2, pp. 610-616 (1994). Briefly, cells are collected using conventional devices, for example, a Haemonetics Model V50 apheresis device (Haemonetics, Braintree, Mass.).
  • Apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Gambro BCT, Lakewood, Colo.) according to the manufacturer's instructions.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
  • a variety of biocompatible buffers such as, for example, Ca-free, Mg-free PBS.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • blood leukocytes may be obtained using conventional methods that maintain their viability.
  • blood is diluted into medium (preferably RPMI) that may or may not contain heparin (about 100 U/ml) or other suitable anticoagulant.
  • the volume of blood to medium is about 1 to 1.
  • Cells are concentrated by centrifugation of the blood in medium at about 1000 rpm (150 g) at 4° C. Platelets and red blood cells are depleted by resuspending the cells in any number of solutions known in the art that will lyse erythrocytes, for example ammonium chloride.
  • the mixture may be medium and ammonium chloride (at a final concentration of about 0.839 percent) at about 1:1 by volume.
  • Cells may be concentrated by centrifugation and washed in the desired solution until a population of leukocytes, substantially free of platelets and red blood cells, is obtained, typically about two times.
  • Any isotonic solution commonly used in tissue culture may be used as the medium for separating blood leukocytes from platelets and red blood cells. Examples of such isotonic solutions are phosphate buffered saline, Hanks balanced salt solution, or complete growth media including for example RPMI 1640, DMEM, MEM, HAMS F-12, X-Vivo 15, or X-Vivo 20.
  • APC and/or APC precursor cells may also purified by elutriation, using, for example, a Beckman J6ME centrifuge equipped with a J5.0 rotor and a 40 ml elutriation chamber.
  • isolation of APC and/or precursor APC is performed by preincubating ficolled whole blood or apheresed peripheral blood with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles (approx. 1 vial of beads or 4 ⁇ 10 9 beads to one batch of cells (typically from about 5 ⁇ 10 8 to about 2 ⁇ 10 10 cells) for about 30 minutes to 2 hours at 22 to 37 degrees C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles.
  • Such separation can be performed using standard methods available in the art.
  • any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)).
  • DYNAL® Magnetic Particle Concentrator DYNAL MPC®
  • Assurance of isolation can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of cells before and after said isolation.
  • APC obtained from treatment of the tissue source may be cultured to form a primary culture in an appropriate culture container or vessel in an appropriate culture medium.
  • the culture medium is supplemented with one or more cytokines.
  • the appropriate culture container or vessel may be any container with tissue culture compatible surface. Examples include various bags (e.g., Lifecell culture bags), flasks, roller bottles, petri dishes and multi-well containing plates made for use in tissue culture. Surfaces treated with a substance, for example collagen or poly-L-lysine, or antibodies specific for a particular cell type to promote cell adhesion may also be used provided they allow for the differential attachment of cells as described below. Surfaces may be also be chemically treated, for example by ionization. Cells are plated at an initial cell density from about 10 5 to 10 7 cells/cm 2 . In one aspect, cells are plated at 10 6 cell s/cm 2 .
  • the primary cultures from the selected tissue source are allowed to incubate at about 37° C. under standard tissue culture conditions of humidity, CO 2 , and pH until a population of cells has adhered to the substrate sufficiently to allow for the separation of nonadherent cells.
  • Some immature APC in blood initially are nonadherent to plastic, particularly immature DC, in contrast to monocytes, so that the precursors can be separated after overnight culture.
  • Monocytes and fibroblasts are believed to comprise the majority of adherent cells and usually adhere to the substrate within about 30 minutes to about 24 hours.
  • nonadherent cells are separated from adherent cells between about 1 to 16 hours. Nonadherent cells may be separated at about 1 to 2 hours. Any method which does not dislodge significant quantities of adherent cells may be used to separate the adherent from nonadherent cells.
  • the cells are dislodged by simple shaking or pipetting. Pipetting is most preferred.
  • Adherent cells comprising precursor APC (e.g., monocytes) isolated according to the methods of the invention are allowed to incubate at about 37° C. under standard tissue culture conditions of humidity, CO 2 , and pH until a population of cells has reached an immature APC stage.
  • adherent cells are allowed to incubate for a period of between 4 hours and 7 days.
  • incubation times and conditions may vary.
  • Immature APC refers to an intermediate differentiation state of an APC wherein the APC has the capacity to endocytose or phagocytose antigen, foreign bodies, necrotic and/or apoptosing tissue and/or cells.
  • Immature APC may be CD14 ⁇ or CD14 + depending on the origin of the precursor cells.
  • Immature APC may also express CD1a, CD40, CD86, CD54, and intermediate levels of MHC class II (levels of marker expression on sample cells can be compared by flow cytometric analysis to levels of expression on MHC class II-negative cells and cells known to express high levels of MHC class II).
  • Immature APC typically do not express CCR7.
  • PBMC comprising APC and T cells can be exposed to antigen as described herein and the resulting antigen-specific T cells further expanded as described herein.
  • the APCs or the T cells described herein be derived from an autologous source.
  • the APC and T cells can be obtained from a matched or unmatched donor, or from a cell line, a T cell line, or other cells grown in vitro. Methods for matching haplotypes are known in the art.
  • the APC and T cells or supernatant therefrom may be obtained from a xenogeneic source, for example, mouse, rat, non-human primate, and porcine cells may be used.
  • the source of antigen may be, but is not limited to, protein, including glycoprotein, peptides (including pools of overlapping peptides), superantigens (e.g., SEA, SEB, TSST-1) antibody/antigen complexes, tumor lysate, viral lysate (e.g., CMV lysate and the like), non-soluble cell debris, apoptotic bodies, necrotic cells, whole cells which are live, fixed, irradiated, heat-killed or otherwise manipulated, whole tumor cells from a tumor or a cell line that have been treated such that they are unable to continue dividing, allogeneic cells that have been treated such that they are unable to continue dividing, irradiated tumor cells, irradiated allogeneic cells, natural or synthetic complex carbohydrates, lipoproteins, lipopolysaccharides, RNA or a translation product of said RNA, and DNA or a polypeptide encoded by said DNA.
  • protein including glycoprotein, peptid
  • Non-transformed cells are typically irradiated with gamma rays in the range of about 3000 to 3600 rads, more preferably at about 3300 rads.
  • Lymphoblastoid or tumor cell lines are typically irradiated with gamma rays in the range of about 6000 to 10,000 rads, more preferably at about 8000 rads.
  • Necrotic and apoptotic cells may be generated by physical, chemical, or biological means. Necrotic cells are typically generated by freeze-thawing, while apoptotic cells are generated using UV irradiation. UV and gamma irradiation, and freeze-thawing procedures are well known in the art and are described, for example, in Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York. N.Y.
  • Antigen source may also comprise non-transformed, transformed, transfected, or transduced cells or cell lines.
  • Cells may be transformed, transfected, or transduced using any of a variety of expression or retroviral vectors known to those of ordinary skill in the art that may be employed to express recombinant antigens. Expression may also be achieved in any appropriate host cell that has been transformed, transfected, or transduced with an expression or retroviral vector containing a DNA molecule encoding recombinant antigen(s). Any number of transfection, transformation, and transduction protocols known to those in the art may be used, for example those outlined in Current Protocols in Molecular Biology, John Wiley & Sons, New York.
  • recombinant vaccinia vectors and cells infected with said vaccina vectors may be used as a source of antigen.
  • Recombinant antigen may include any number of defined tumor antigens described below.
  • antigen may comprise viral antigens such as CMV pp65, HIV pg120, and the like.
  • antigen may comprise defined tumor antigens such as the melanoma antigen Melan-A (also referred to as melanoma antigen recognized by T cells or MART-1), melanoma antigen-encoding genes 1, 2, and 3 (MAGE-1, -2, -3), melanoma GP100, carcinoembryonic antigen (CEA), the breast cancer angtigen, Her-2/Neu, serum prostate specific antigen (PSA), Wilm's Tumor (WT-1), PR1, PR3 (antigens implicated in the graft-versus-leukemia (GVL) effect in chronic myeloid leukemia), mucin antigens, MUC-1, -2, -3, -4, B cell lymphoma idotypes, and the like.
  • melanoma antigen also referred to as melanoma antigen recognized by T cells or MART-1
  • bead:cell ratios can lead to different outcomes with respect to expansion of antigen-specific T cells.
  • bead:cell ratios can be varied to selectively expand or delete antigen-specific (memory) T cells.
  • the particular bead:cell ratio used selectively expands antigen-specific T cells.
  • antigen-specific T cells are activated by direct contact of a population of cells wherein at least a portion thereof comprises T cells (e.g., a leukaphersis product from an individual, blood sample, tumor biopsy, etc.), with a surface, wherein said surface has attached thereto a first agent that ligates a first T cell surface moiety of a T cell, and the same or a second surface has attached thereto a second agent that ligates a second moiety of said T cell, wherein said ligation by the first and second agent induces proliferation (expansion) of antigen-specific T cells present within the population of cells.
  • T cells e.g., a leukaphersis product from an individual, blood sample, tumor biopsy, etc.
  • the antigen-specific T cells are sensitized to further stimulation.
  • the quantity of the CD3/TCR (and CD28) receptors that are bound by ligands determines the signal strength.
  • stimulation with high bead:cell ratios provides a high concentration of stimulating antibody (i.e., “strong signal”), leading to over-stimulation of antigen-specific T cells, causing them to die, either by apoptosis or other mechanisms.
  • Using lower bead:cell ratios provides a stimulation signal to antigen-specific T cells that does not over-stimulate, but rather induces rapid proliferation of these cells.
  • antigen-specific T cells are activated by culturing T cells isolated as described herein above, with APC that have been loaded with antigen.
  • suitable APC are plated in culture dishes and exposed to a source of antigen as described herein, in a sufficient amount and for a sufficient period of time to allow the antigen to bind and/or be taken up by the APC.
  • antigen is exposed to the APC for a period of time between 24 hours and 4 days.
  • the antigen is exposed to the APC for 36, 48, or 72 hours.
  • the antigen is exposed to the APC for 2.5, 3, 3.5, or 4 days.
  • antigen may be exposed to the APC for periods longer than 4 days, for example 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 days.
  • the amount and time necessary to achieve binding and uptake of the antigen by the APC may differ depending on the source and type of antigen and may be determined by those of ordinary skill in the art by immunoassay or binding assay. Other methods known to those of skill in the art may be used to detect the presence of antigen in the context of MHC on the APC following their exposure to antigen.
  • PBMC e.g., from blood, a leukapheris product, etc.
  • PBMC from a subject are cultured directly in the presence of antigen, as described herein, to load APC with the antigen and to activate/stimulate antigen-specific T cells present in the PBMC.
  • PBMC may be collected from an individual, contacted with an antigen of interest, such as a tumor antigen, or a viral lysate, etc.
  • an antigen of interest such as a tumor antigen, or a viral lysate, etc.
  • the APC present in the PBMC are loaded with the antigen, which is then presented to the T cells present in the sample.
  • the antigen-specific T cells of the present invention may be stimulated with peptide-MHC tetramers, see for example Altman, et al., Science 1998 Jun. 19; 280(5371):1821.
  • the APC of the present invention may be loaded with antigen through genetic modification.
  • Genetic modification may comprise RNA or DNA transfection using any number of techniques known in the art, for example electroporation (using e.g., the Gene Pulser II, BioRad, Richmond, Calif.), various cationic lipids, (LIPOFECTAMINETM, Life Technologies, Carlsbad, Calif.), or other techniques such as calcium phosphate transfection as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
  • RNA or DNA in 500 ⁇ l of Opti-MEM can be mixed with a cationic lipid at a concentration of 10 to 100 ⁇ g, and incubated at room temperature for 20 to 30 minutes.
  • suitable lipids include LIPOFECTINTM, LIPOFECTAMINETM.
  • the resulting nucleic acid-lipid complex is then added to 1-3 ⁇ 10 6 cells, preferably 2 ⁇ 10 6 , antigen-presenting cells in a total volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37° C. for 2 to 4 hours.
  • the APC may also be transduced using viral transduction methodologies as described below.
  • APC are loaded with antigen attached to, coated on, or otherwise immobilized on particles, such as beads.
  • particles such as beads.
  • commercially available beads or other particles are useful, e.g., Miltenyi Particles, Miltenyi Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADSTM, Dynal Inc., New York.
  • paramagnetic particles or beads are particularly suitable. Such paramagnetic beads or particles are commercially available, for example, those produced by Dynal AS under the trade name DynabeadsTM. Exemplary DynabeadsTM in this regard are M-280, M-450, and M-500.
  • whole cells which are live, fixed, irradiated, heat-killed or ohterwise manipulated, are immobilized to ingestable beads, via for example antibody/ligand specific means or chemical means.
  • tumor cell or virus-infected cell lysates, or antigen-preparations can be attached or otherwise immobilized to the beads (which may be paramagnetic or otherwise selectable).
  • coated or antigen/cell/lysate-attached beads can be mixed with human or other animal peripheral blood preparations (or other compositions containing some percentage of antigen-presenting cells (particularly those capable of ingesting particles and then processing and presenting antigens associated with the particles).
  • Phagocytic cells will ingest the beads/particles, process antigens associated with the particles, and present them to T cells in the cell mix. As noted elsewhere herein, only T cells with specificity for the variety of presented antigens will interact in a positive manner with the APC. APC containing paramagnetic or otherwise selectable beads can then be isolated carrying with them antigen-specific T cells.
  • the particles of the present invention comprise a cell surface, such as described in U.S. patent application Ser. No. 10/336,224, PCT/US03/00339.
  • antigen can be attached to the cells via antibody/ligand specific means as described herein or through genetic modification.
  • Any number of transfection, transformation, and transduction protocols known to those in the art may be used, for example those outlined in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y., or in numerous kits available commercially (e.g., Invitrogen Life Technologies, Carlsbad, Calif.). Such techniques may result in stable transformants or may be transient.
  • electroporation which may be performed on a variety of cell types, including mammalian cells, yeast cells and bacteria, using commercially available equipment.
  • Optimal conditions for electroporation are experimentally determined for the particular host cell type, and general guidelines for optimizing electroporation may be obtained from manufacturers.
  • Other suitable methods for transfection will depend upon the type of cell used (e.g., the lithium acetate method for yeast), and will be apparent to those of ordinary skill in the art.
  • cells may be maintained in conditions that promote expression of the polynucleotide within the cell. Appropriate conditions depend upon the expression system and cell type, and will be apparent to those skilled in the art.
  • Antigen may be attached to the particles, such as beads, by antibody/ligand specific means, e.g. through particles, such as beads, conjugated to an antibody or antibodies.
  • Suitable antibody/ligand pairs may include, but are not limited to anti-MART-1 antibody/MART-1 antigen, anti-WT-1 antibody/WT-1, anti-PR1 antibody /PR1, anti-PR3 antibody/PR3, anti-tyrosinase antibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1 antigen, anti-MUC-1 antibody/MUC-1 antigen, anti- ⁇ -fetoprotein antibody/ ⁇ -fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis C antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV
  • antigen binding interactions may be suitable for attaching antigen to particles, such as beads, for example, receptor/ligand interactions may be utilized.
  • the antigen/protein is attached to the particles, such as beads by chemical means, e.g. antigen/protein can be bound through non-covalent association of the antigen and bead, simply by incubating/contacting the two together for a time and under conditions sufficient for association to occur.
  • antigen may be attached to the particles, such as beads by a biotin/avidin or streptavidin interaction.
  • hydrophobic “naked” beads with p-toluenesulphonyl (tosyl) reactive groups are used.
  • Proteins are adsorbed hydrophobically on initial coupling with covalent binding of primary amine groups (NH 2 ) and sulphydryl groups (SH) occurring overnight. Coupling reactions can be performed at neutral pH however high pH and incubation at 37° C. can promote covalent binding.
  • primary amine groups NH 2
  • SH sulphydryl groups
  • T cells isolated from a tissue source are exposed to antigen-loaded APC described herein for a time sufficient for T cells specific for a given antigen to be activated, for example as described in U.S. Pat. No. 5,827,642, or as described in Riddell, et al., 1990, J. Immunol. Methods, 128:189-201.
  • T cells are exposed to antigen-loaded APC for a period of between about several hours to about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 days.
  • the T cells are exposed to antigen, or antigen-loaded APC as described herein in vivo.
  • antigen or antigen-loaded APC may be administered to an individual in order to stimulate and activate the T cells in vivo.
  • the T cells may then be expanded either in vivo or ex vivo using the methods as described herein, such as with anti-CD3/anti-CD28 beads.
  • the quantity and frequency of administration will be determined by such factors as the condition of the individual, and the type and severity of disease, although appropriate dosages may be determined by clinical trials.
  • the T cells are exposed to antigen in vivo in an individual prior to onset of a disease or prior to treatment with other known therapies.
  • the antigen-specific T cells are generated and then isolated and expanded and preserved for later use.
  • isolation of antigen-specific T cells in direct contact with APC loaded with antigen immobilized on particles, such as beads is performed by magnetic isolation of cells which have attached to or engulfed paramagnetic particles.
  • Such separation can be performed using standard methods available in the art.
  • any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®), MACS, Miltenyi Biotec, Germany).
  • DYNAL® Magnetic Particle Concentrator DYNAL MPC®
  • MACS Miltenyi Biotec
  • APC containing paramagnetic (or otherwise selectable) beads can then be isolated (via magnet or otherwise) carrying with them antigen-specific T cells.
  • These antigen-specific T cells can then be activated/expanded by a variety of means, such as via XCELLERATETM technologies as described herein and U.S. patent application Ser. Nos. 10/350,305; 10/187,467; 10/133,236; 09/960,264; 09/794,230; PCT/US01/06139; and PCT/US02/28161.
  • antigen-specific T cells are isolated by positive selection. Such isolation can be carried out on T cells freshly isolated from a subject or on T cells that have been exposed to antigen or antigen-loaded APC as described herein. Numerous immunoselection methods known to skilled artisans may be used. Such techniques are described, for example, in Current Protocols in Immunology, John Wiley & Sons, New York. N.Y. Markers that may be useful for the positive selection of antigen-specific cells include, but are not limited to, CD25, CD54, CD69, CD38, CD45RO, CD49d, CD40L, CD137, CD62L, and CD134. In one embodiment, fluorescence activated cell sorting may also be used to isolate desired antigen-specific T cells. In an additional embodiment, antigen-specific T cells may be isolated using peptide-MHC tetramers, see for example Altman, et al., Science 1998 Jun. 19; 280(5371):1821.
  • antigen-specific T cells may be genetically modified. Genetic modification may comprise RNA or DNA transfection using any number of techniques known in the art, for example electroporation (using e.g., the Gene Pulser II, BioRad, Richmond, Calif.), various cationic lipids, (LIPOFECTAMINETM, Life Technologies, Carlsbad, Calif.), or other techniques such as calcium phosphate transfection as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
  • electroporation using e.g., the Gene Pulser II, BioRad, Richmond, Calif.
  • various cationic lipids (LIPOFECTAMINETM, Life Technologies, Carlsbad, Calif.)
  • calcium phosphate transfection as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
  • RNA or DNA in 500 ⁇ l of Opti-MEM can be mixed with a cationic lipid at a concentration of 10 to 100 ⁇ g, and incubated at room temperature for 20 to 30 minutes.
  • suitable lipids include LIPOFECTINTM, LIPOFECTAMINETM.
  • the resulting nucleic acid-lipid complex is then added to 1-3 ⁇ 10 6 cells, preferably 2 ⁇ 10 6 , antigen-presenting cells in a total volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37° C. for 2 to 4 hours.
  • the APC may also be transduced using viral transduction methodologies as described below.
  • the retroviral vector may be an amphotropic retroviral vector, preferably a vector characterized in that it has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV).
  • LTR long terminal repeat sequence
  • MoMLV Moloney murine leukemia virus
  • MPSV myeloproliferative sarcoma virus
  • MMV murine embryonic stem cell virus
  • MSCV murine stem cell virus
  • SFFV spleen focus forming virus
  • AAV adeno-associated virus
  • retroviral vectors are derived from murine retroviruses.
  • Retroviruses adaptable for use in accordance with the present invention can, however, be derived from any avian or mammalian cell source. These retroviruses are preferably amphotropic, meaning that they are capable of infecting host cells of several species, including humans.
  • the gene to be expressed replaces the retroviral gag, pol and/or env sequences.
  • a number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al.
  • genetically modified antigen-specific T cells can be isolated by any one of numerous immunoselection methods known to skilled artisans using antibodies or other receptors/ligands specific for the protein or proteins expressed from the transgene. Such techniques are known in the art, for example, in Current Protocols in Immunology, John Wiley & Sons, New York. N.Y.
  • the antigen-specific T cells may be genetically modified to express a suicide gene, e.g. the herpes simplex virus thymidine kinase (HSV-TK) as described in Bonini, et al., 1997 Science, 276(5319):1719-24, and/or other surface markers (e.g., truncated nerve growth factor (dNGFR)) for in vivo tracking and/or control of infused antigen-specific T cells.
  • HSV-TK herpes simplex virus thymidine kinase
  • dNGFR truncated nerve growth factor
  • the antigen-specific T cells may be genetically modified to express a protein for targeting the T cells to a particular tissue of interest.
  • CMOS complementary metal-oxide-semiconductor
  • a bioreactor e.g., CellCube (Corning Science Products) or CELL-PHARM, (CD-Medical, Inc. of Hialeah, Fla.)
  • petri dishes e.g., petri dishes and multi-well containing plates made for use in tissue culture, or any container capable of holding cells, preferably in a sterile environment.
  • a bioreactor is also useful.
  • Suitable complete growth media for the culture of the APC and antigen-specific T cells of the present invention include for example RPMI 1640, DMEM, MEM, ⁇ -MEM, AIM-V, HAMS F-12, X-Vivo 15, or X-Vivo 20.
  • the media can comprise a cytokine, such as IL-2, IFN- ⁇ , IL-4, GM-CSF, IL-10, IL-12, TGF ⁇ , and TNF- ⁇ , or a vitamin.
  • the medium comprises surfactant, an antibody, plasmanate or a reducing agent (e.g. N-acetyl-cysteine, 2-mercaptoethanol).
  • the growth medium for the cells at each step of the method of the invention should allow for the survival of the APC and/or the antigen-specific T cells.
  • Any growth medium typically used to culture cells may be used according to the method of the invention provided the medium is supplemented with the appropriate cytokines, serum, antibiotics, vitamins, amino acids or other necessary additives.
  • the cytokines may be, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), or IL-13.
  • cytokines and growth factors that may be added to the growth medium include but are not limited to interleukin 1 ⁇ (IL-1 ⁇ ) and ⁇ (IL-1 ⁇ ), IL-2, tumor necrosis factor alpha (TNF- ⁇ ), interleukin 3 (IL-3), monocyte colony stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), interleukin 6 (IL-6), interleukin 15 (IL-15), and Flt3-ligand.
  • IL-1 ⁇ interleukin 1 ⁇
  • IL-1 ⁇ tumor necrosis factor alpha
  • IL-3 interleukin 3
  • M-CSF monocyte colony stimulating factor
  • G-CSF granulocyte colony-stimulating factor
  • SCF stem cell factor
  • IL-6 interleukin 6
  • IL-15 interleukin 15
  • Flt3-ligand Flt3-ligand.
  • Preferred media include RPMI 1640, AIM-V, DMEM, MEM, ⁇ -MEM, F-12, X-Vivo 15, and X-Vivo 20, with added amino acids and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and an amount of cytokine(s) sufficient to support the expansion of the antigen-specific T cells.
  • the preferred media comprises 1 liter of X-Vivo 15, BioWhittaker; with 50 ml heat inactivated pooled human serum, 20 ml 1 M Hepes, 10 ml 200 mM L-glutamine with or without about 100,000 I.U. IL-2.
  • media may include lipids and/or sources of protein.
  • RPMI 1640 supplemented with 1-5% human AB serum preferred.
  • Mixtures of cytokines may also be used.
  • Cells may also be adapted to grow in other sera, such as fetal calf (bovine) serum (FCS/FBS), at other concentrations of serum, or in serum-free media.
  • FCS/FBS fetal calf serum
  • serum-free medium supplemented with hormones is also suitable for culturing the APC precursors.
  • Media may, but does not necessarily, contain antibiotics to minimize growth of bacteria in the cultures. Penicillin, streptomycin or gentamicin or combinations containing them are preferred.
  • the medium, or a portion of the medium, in which the cells are cultured should be periodically replenished to provide fresh nutrients including GM-CSF, IL-4, IL-13, IL-15 and/or other cytokines.
  • Expansion of the antigen-specific T cells of the present invention is carried out by cell surface moiety ligation that re-stimulates the antigen-specific T cells to proliferate.
  • the antigen-specific T cells are first isolated by methods described herein following exposure to antigen loaded APC.
  • the antigen-specific T cells are expanded directly from the culture with antigen-loaded APC present without an isolation step.
  • antigen-specific T cells are activated and expanded using XCELLERATETM processes as described herein and in U.S. patent application Ser. Nos. 10/350,305; 10/187,467; 10/133,236; 09/960,264; 09/794,230, with no addition of antigen or antigen-coated particles.
  • antigen-specific T cells that have been previously stimulated or activated in vivo (e.g.
  • memory T cells are expanded by an agent providing a primary activation signal such as an anti-CD3 antibody and an agent providing a co-stimulatory signal, such as an anti-CD28 antibody, with both agents co-immobilized to the same surface, such as a paramagnetic bead.
  • an agent providing a primary activation signal such as an anti-CD3 antibody
  • an agent providing a co-stimulatory signal such as an anti-CD28 antibody
  • both agents co-immobilized to the same surface such as a paramagnetic bead.
  • varying the bead:cell ratios during this expansion phase in particular using low bead:cell ratios, favors expansion of antigen-specific T cells.
  • bead to cell ratios of 1:200, 1:150, 1:125, 1:110, 1:100, 1:75, 1:50, 1:25, 1:20, 1:15, 1:10, 1:5 or 1:2.5 are used to expand antigen-specific T cells.
  • a particular advantage of this aspect of the present invention is that it is not
  • expansion is carried out by re-stimulating a population of antigen-specific T cells and simultaneously stimulating an accessory molecule on the surface of the antigen-specific T cells with a ligand which binds the accessory molecule, as described for example, in U.S. patent application Ser. Nos. 10/350,305, 10/187,467, 10/133,236, 09/960,264, 09/794,230, 08/253,694, 08/403,253, 08/435,816, 08/592,711, 09/183,055, 09/350,202, and 09/252,150, and U.S. Pat. Nos. 5,858,358; 6,352,694; and 5,883,223.
  • re-stimulation may be accomplished by cell surface moiety ligation, such as through the T cell receptor (TCR)/CD3 complex or the CD2 surface protein.
  • TCR T cell receptor
  • a number of anti-human CD3 monoclonal antibodies are commercially available, exemplary are, clone BC3 (XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, Wash.), OKT3, prepared from hybridoma cells obtained from the American Type Culture Collection, and monoclonal antibody G19-4.
  • 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 that have been described include the following: the T11.3 antibody in combination with the T11.1 or T11.2 antibody (Meuer et al., Cell 36:897-906, 1984), and the 9.6 antibody (which recognizes the same epitope as T11.1) in combination with the 9-1 antibody (Yang et al., J. Immunol. 137:1097-1100, 1986).
  • Other antibodies that bind to the same epitopes as any of the above-described antibodies can also be used.
  • Additional antibodies, or combinations of antibodies can be prepared and identified by standard techniques. Re-stimulation may also be achieved through contact with antigen, peptide, protein, peptide-MHC tetramers (see Altman, et al Science 1996 Oct.
  • superantigens e.g., Staphylococcus enterotoxin A (SEA), Staphylococcus enterotoxin B (SEB), Toxic Shock Syndrome Toxin 1 (TSST-1)
  • endotoxin or through a variety of mitogens, including but not limited to, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), T cell mitogen, and IL-2.
  • PHA phytohemagglutinin
  • PMA phorbol myristate acetate
  • LPS lipopolysaccharide
  • T cell mitogen and IL-2.
  • the antigen-specific cell population may be stimulated or restimulated as described herein, such as by contact with an anti-CD3 antibody or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used.
  • a population of CD4 + cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T-cells.
  • an anti-CD3 antibody and the anti-CD28 antibody B-T3, XR-CD28 (Diaclone, Besançon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).
  • a co-stimulatory or accessory molecule on the surface of the T cells such as CD28
  • a ligand that binds the accessory molecule can be used to stimulate T cells.
  • Exemplary anti-CD28 antibodies or fragments thereof useful in the context of the present invention include monoclonal antibody 9.3 (IgG2 a ) (Bristol-Myers Squibb, Princeton, N.J.), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM), clone B-T3 (XR-CD28; Diaclone, Besan wonder, France) and EX5.3D10 (IgG2 a ) (ATCC HB11373).
  • Exemplary natural ligands include the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86) (Freedman et al., J. Immunol.
  • activation of a T-cell population may be enhanced by co-stimulation of other T-cell integral membrane proteins.
  • binding of the T-cell integrin LFA-1 to its natural ligand, ICAM-1 may enhance activation of cells.
  • Another cell surface molecule that may act as a co-stimulator for T-cells is VCAM-1 (CD106) that binds very-late-antigen-4 (VLA-4) on T-cells.
  • VCAM-1 CD106
  • VLA-4 very-late-antigen-4
  • Ligation of 4-1BB (CD137), a co-stimulatory receptor expressed on activated T cells, and/or NKG2D may also be useful in the context of the present invention to amplify T-cell mediated immunity. It should be noted that more than one costimulatory molecule as described herein may be stimulated at a time, and in any combination, such that desired expansion of the T cells occurs.
  • binding homologues of a natural ligand can also be used in accordance with the present invention.
  • Other agents may include natural and synthetic ligands. Agents may include, but are not limited to, other antibodies or fragments thereof, a peptide, polypeptide, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens.
  • the primary stimulatory signal and the co-stimulatory signal for the T-cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation).
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.”
  • the agent providing the primary activation signal is an anti-CD3 antibody and the agent providing the co-stimulatory signal is an anti-CD28 antibody; and both agents are co-immobilized to the same surface, such as a bead, in equivalent molecular amounts.
  • a 1:1 ratio of each antibody bound to the beads for CD4 + T-cell expansion and T-cell growth is used.
  • One aspect of the present invention stems from the surprising finding that using lower ratios of anti-CD3:anti-CD28 antibodies bound to the beads results in improved expansion of T cells, including antigen-specific T cells.
  • a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one particular embodiment an increase of from about 0.5 to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between.
  • more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e. the ratio of CD3:CD28 is less than one.
  • the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1.
  • a 1:100 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1:75 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1:50 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1:30 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1:10 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1:3 CD3:CD28 ratio of antibody bound to the beads is used.
  • a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.
  • Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T-cells.
  • the ratio of particle to cells may dependant on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many.
  • the ratio of particles to cells ranges from 1:100 to 100:1 and any integer values in-between and in further embodiments the ratio comprises 1:9 to 9:1 and any integer values in between, can also be used to stimulate T-cells.
  • the ratio of anti-CD3- and anti-CD28-coupled particles to T-cells that result in T-cell stimulation and expansion can vary as noted above, however in certain embodiments the ratio may be 1:150 or lower. Certain preferred ratios include 1:150, 1:100, 1:75, 1:50, 1:40, 1:30, 1:25, 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, and 20:1 with one preferred ratio being 1:1 particles per T-cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used.
  • a preferred particle:cell ratio is 1:2.5 or 1:5.
  • the ratio of particles to cells can be varied depending on the day of stimulation.
  • the ratio of particles to cells is from 1:5, 1:2.5, 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1, 1:5, 1:20, 1:25, 1:50, or 1:100 (based on cell counts on the day of addition).
  • the ratio of particles to cells is 1:2.5, 1:5, or 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation.
  • the ratio of particles to cells is 1:2.5, 1:5, or 1:1 on the first day of stimulation and adjusted to 1:10, 1:20, 1:25, 1:50, or 1:100 at day 5, 7, or 9.
  • particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation.
  • the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation.
  • particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation.
  • ratios may be suitable for use in the present invention.
  • bead:cell ratios can be varied to selectively expand or delete antigen-specific (memory) T cells.
  • the particular bead:cell ratio used selectively deletes antigen-specific T cells.
  • the particular bead:cell ratio used selectively expands antigen-specific T cells.
  • bead to cell ratios of 1:100, 1:50, 1:25, 1:5 or 1:2.5 and the like are used to expand antigen-specific T cells.
  • Low bead:cell ratio can help preserve and promote expansion of memory (antigen-specific) T cells.
  • compositions and methods described herein can be used to expand specific populations of T cells, or to delete specific populations of T cells, for use in any variety of immunotherapeutic settings described herein.
  • the particle:cell ratios described herein can be used in any combination with the various ratios of antibodies bound on the beads.
  • beads containing about 1:5 to 1:10 ratio of anti-CD3/anti-CD28 antibodies bound thereto can be used at a ratio of about 1:5 to 1:10 particles:cell.
  • beads containing a 1:1 ratio of anti-CD3/anti-CD28 antibodies bound thereto can be used at a ratio of about 1:5 particles:cell, etc.
  • the ratio of anti-CD3:anti-CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between and such beads can be used at a ratio of particle:cell of anywhere from about 1:500 to 500:1 and any integer values in between, in any combination.
  • 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 T-cells When the mean T-cell diameter decreases to approximately 8 microns, the T-cells may 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, CD154, CD54, CD25, CD137, CD134, which are induced on activated T-cells.
  • T-cell stimulation is performed with anti-CD3 and anti-CD28 antibodies co-immobilized on beads (3 ⁇ 28 beads), for a period of time sufficient for the cells to return to a quiescent state (low or no proliferation) (approximately 8-14 days after initial stimulation).
  • the stimulation signal is then removed from the cells and the cells are washed and infused back into the patient.
  • the cells at the end of the stimulation phase are rendered “super-inducible” by the methods of the present invention, as demonstrated by their ability to respond to antigens and the ability of these cells to demonstrate a memory-like phenotype, as is evidence by the examples.
  • the activated T-cells demonstrate a robust response characterized by unique phenotypic properties, such as sustained CD154 expression and increased cytokine production.
  • the cells such as T-cells
  • the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, resulting in cell surface moiety ligation, thereby inducing cell stimulation.
  • the time of exposure to stimulatory agents such as anti-CD3/anti-CD28 (i.e., CD3xCD28)-coated particles, such as beads, may be modified or tailored to obtain a desired T-cell phenotype.
  • stimulatory agents such as anti-CD3/anti-CD28 (i.e., CD3xCD28)-coated particles, such as beads
  • T H helper T-cells
  • CD4 + T-cells express important immune-regulatory molecules, such as GM-CSF, CD40L, and IL-2, for example.
  • CD4-mediated help a method, such as that described herein, which preserves or enhances the CD4:CD8 ratio could be of significant benefit.
  • the T cell activation approaches described herein can also be utilized, by for example, pre-selecting for CD8 + cells prior to stimulation and/or culture. Such situations may exist where increased levels of IFN- ⁇ is preferred.
  • T H 1-type cells versus T H 2-type cells (or vice versa), or supernatants therefrom.
  • regulatory T cells e.g., Autoimmun Rev. 2002 August;1(4):190-7; Curr Opin Immunol. 2002 December; 14(6):771-8.
  • times of cell surface moiety ligation that induces re-stimulation may be varied or pulsed. For example expansion times may be varied to obtain the specific phenotype of interest and/or different types of stimulatory agents may be used (e.g., antibodies or fragments thereof, a peptide, polypeptide, MHC/peptide tetramer, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens).
  • stimulatory agents e.g., antibodies or fragments thereof, a peptide, polypeptide, MHC/peptide tetramer, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens.
  • the stimulation and/or expansion time may be four weeks or less, 2 weeks or less, 10 days or less, or 8 days or less (four weeks or less includes all time ranges from 4 weeks down to 1 day (24 hours)). In some embodiments, stimulation and expansion may be carried out for 6 days or less, 4 days or less, 2 days or less, and in other embodiments for as little as 24 or less hours, and preferably 4-6 hours or less (these ranges include any integer values in between).
  • the population of T-cells may not increase in number as dramatically, but the population will provide robust and healthy activated antigen-specific T-cells that can continue to proliferate in vivo and more closely resemble the natural effector T-cell pool.
  • the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention the beads and the T-cells are cultured together for about eight days. In another embodiment, the beads and T-cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more.
  • Conditions appropriate for T-cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (BioWhittaker)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, GM-CSF, IL-10, IL-12, TGF ⁇ , and TNF- ⁇ . or any other additives for the growth of cells known to the skilled artisan.
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, AIM-V, DMEM, MEM, ⁇ -MEM, F-12, X-Vivo 15, and X-Vivo 20, with added amino acids and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T-cells.
  • Antibiotics e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2 ).
  • Feeder cells can encompass a variety of cell types, including, irradiated peripheral blood lymphocytes (autologous or allogeneic) alone or in combination with EBV-transformed B cell lines (autologous or allogeneic), immortalized or non-immortalized cell lines of the myelomoncytic lineage, such as macrophges, dentritic cells, red blood cells, B-cells, tumor cell lines such as U937, Jurkat, Daudi, MOLT-4, HUT, CEM, Colo 205, HTB-13, and HTB-70.
  • Feeder cells need not be of human origin as long as they provide feeder function, e.g. the ability to facilitate the survival and growth of primary T cells and ther derived antigen-specific clones.
  • compositions of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as ethylenediaminetetraacetic acid (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 or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as ethylenediaminetetraacetic acid (EDTA) or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • a related embodiment of the present invention further provides a pharmaceutical composition comprising the antigen-specific T cells, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier should be sterilized by techniques known to those skilled in the art.
  • compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • the present invention also provides methods for preventing, inhibiting, or reducing the presence of a cancer or malignant cells in an animal, which comprise administering to an animal an anti-cancer effective amount of the subject antigen-specific T cells.
  • the cancers contemplated by the present invention against which the immune response is induced, or which is to be prevented, inhibited, or reduced in presence, may include but are not limited to melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), low-grade lymphoma, and other neoplasms known in the art.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous le
  • compositions as described herein can be used to induce or enhance responsiveness to pathogenic organisms, such as viruses, (e.g., single stranded RNA viruses, single stranded DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B, and C virus, herpes simplex virus (HSV), cytomegalovirus (CMV) Epstein-Barr virus (EBV), Human Papilloma Virus (HPV)), parasites (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria, Salmonella, Streptococci, E. coli, Staphylococci ), fungi (e.g., Candida species, Aspergillus species) and Pneumocystis carinii.
  • viruses e.g., single stranded RNA viruses, single stranded
  • the methods of the present invention can be used in conjunction with the generation of T regulatory cells for specific immunosuppression in the case of inflammatory disease, autoimmunity, and foreign graft acceptance.
  • Regulatory T cells can be generated and expanded using the methods of the present invention.
  • the regulatory T cells can be antigen-specific and/or polyclonal.
  • Regulatory T cells can be generated using art-recognized techniques as described for example, in Woo, et al., J. Immunol. 2002 May 1;168(9):4272-6; Shevach, E. M., Annu. Rev. Immunol. 2000, 18:423; Stephens, et al., Eur. J. Immunol.
  • T cells of the present invention can be used for the treatment of autoimmune diseases such as, but not limited to, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes, Addison's disease, celiac disease, chronic fatigue syndrome, inflammatory bowel disease, ulcerativecolitis, Crohn's disease, Fibromyalgia, systemic lupus erythematosus, psoriasis, Sjogren's syndrome, hyperthyroidism/Graves disease, hypothyroidism/Hashimoto's disease, Insulin-dependent diabetes (type 1), Myasthenia Gravis, endometriosis, scleroderma, pernicious anemia, Goodpasture syndrome, Wegener's disease, glomerulonephritis, aplastic anemia, par
  • the immune response induced in the animal by administering the subject compositions of the present invention may include cellular immune responses mediated by cytotoxic T cells, capable of killing tumor and infected cells, and helper T cell responses.
  • Humoral immune responses mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may also be induced.
  • a variety of techniques may be used for analyzing the type of immune responses induced by the compositions of the present invention, which are well described in the art; e.g., Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc. (1994).
  • an immunologically effective amount When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient. It can generally be stated that a pharmaceutical composition comprising the subject antigen-specific T cells, may be administered at a dosage of 10 4 to 10 7 APC/kg body weight, preferably 10 5 to 10 6 APC/kg body weight, including all integer values within those ranges. Antigen-specific T cells compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • antigen-specific T cells are administered approximately at 2 ⁇ 10 9 to 2 ⁇ 10 11 cells to the patient.
  • lower numbers of cells in the range of 10 6 /kilogram (10 6 -10 11 per patient) may be administered.
  • T cells are administered at 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 2 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 1 ⁇ 10 11 , 5 ⁇ 10 11 , or 1 ⁇ 10 12 cells to the subject.
  • T cell compositions may be administered multiple times at dosages within these ranges.
  • the antigen-specific T cells may be autologous or heterologous to the patient undergoing therapy.
  • the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1 ⁇ , etc.) as described herein to enhance induction of the immune response.
  • mitogens e.g., PHA
  • lymphokines e.g., IL-4, IL-13, Flt3-L, RANTES, MIP1 ⁇ , etc.
  • chemokines e.g., GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1 ⁇ , etc.
  • compositions of the subject pharmaceutical compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions of the present invention may be administered to a patient subcutaneously, intradermally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the antigen-specific T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.
  • the antigen-specific T cell compositions of the present invention are preferably administered by i.v. injection.
  • the compositions of antigen-specific T cells may be injected directly into a tumor or lymph node.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, 1990, Science 249:1527-1533; Sefton 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980; Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).
  • polymeric materials can be used (see Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
  • a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release, 1984, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., vol. 2, pp. 115-138).
  • the antigen-specific T cell compositions of the present invention may also be administered using any number of matrices.
  • Matrices have been utilized for a number of years within the context of tissue engineering (see, e.g., Principles of Tissue Engineering (Lanza, Langer, and Chick (eds.)), 1997.
  • the present invention utilizes such matrices within the novel context of acting as an artificial lymphoid organ to support, maintain, or modulate the immune system, typically through modulation of T cells. Accordingly, the present invention can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering. Accordingly, the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices.
  • compositions and devices set forth by U.S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized.
  • Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host. Matrices may be formed from both natural or synthetic materials. The matrices may be non-biodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant; or biodegradable.
  • the matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles.
  • matrices can be designed to allow for sustained release seeded cells or produced cytokine or other active agent.
  • the matrix of the present invention is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.
  • a matrix is used herein as an example of a biocompatible substance.
  • the current invention is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi-permeable membrane or used in conjunction with a particular semi-permeable substance.
  • the cells of the present invention are administered to a patient in conjunction with (e.g. before, simulataneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
  • agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxin, fludaribine, cyclosporin, FK506,
  • the cell compositions of the present invention are administered to a patient in conjunction with (e.g.
  • T-cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g. Rituxan.
  • the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.
  • the dose for CAMPATH for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days.
  • the preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used
  • CMV cytomegalovirus
  • CMV lysate prepared using standard techniques was mixed at room temperature for 1-2 hours with Dynabead M-450 while rotating. Beads were then washed once, and added to PBMC. Within hours, the beads were phagocytosed in the APC. Within 72 hours, CMVpp65-HLA-A2 tetramers detected CD25-high (activated) T cell specific for CMV pp65. Magnetic selection of the bead-loaded APC with the associated antigen-specific T cells was carried out at day 5, thereby enriching for CMV-specific T cells. As shown in FIG. 1, following magnetic separation, CMV-specific T cells were still tightly associated with bead-loaded APC. It should be noted that magnetic separation can be carried out anywhere from about day 1 to about day 10.
  • antigen-coated beads were used to activate CMV-specific CD8 + T cells ex vivo.
  • PBMC from CMV pp65 tetramer-positive and tetramer-negative donors were stimulated with paramagnetic Dynal M-450 beads coated with CMV lysate.
  • CMV pp65 tetramer-negative PBMC were cultured with CMV-lysate coated beads (FIG. 2, panel A)
  • CMV pp65 tetramer-positive PBMC were cultured with “naked” beads (no CMV antigen) (FIG. 2, panel B).
  • CMV pp65 tetramer-positive PBMC were cultured with CMV-lysate coated beads (FIG. 2, panel C).
  • CMV-specific T cells Following stimulation, activation of CMV-specific T cells was measured on Day 10 by CMV pp65 HLA-A2 tetramer stain and CD25 expression as an indicator of activation. As shown in FIG. 2, up-regulation of CD25 was observed in memory CD8 CMV tetramer+T cells expanded ex vivo using antigen-coated beads.
  • Antigen-coated beads can be used to activate and stimulate antigen-specific T cells. These antigen-specific T cells can then be enriched as described in Example 1 and elsewhere herein. These antigen-specific T cells can be further expanded as described herein and in U.S. patent application Ser. Nos. 10/350,305, 10/187,467, 10/133,236, 09/960,264, and 09/794,230.
  • the antigen-specific T cells of the present invention can be used in any number of immunotherapeutic settings as described herein.
  • This example shows that the bead:cell ratio can have a profound effect on expansion of different populations of T cells.
  • a high bead:cell ratio (3:1-10:1, 20:1 and higher) tends to induce death in antigen-specific T cells while a lower bead:cell ratio (1:1-1:10, 1:20, 1:30, 1:40, 1:50 or lower) leads to expansion of antigen-specific T cells.
  • the data described below show that lower bead:cell ratios lead to improved cell expansion in polyclonal cell populations as well.
  • this example shows that lower bead:cell ratios improve overall cell expansion.
  • the cells and beads are processed over a MaxSep Magnetic Separator in order to remove the beads and any monocytic/phagocytic cells that are attached to the beads.
  • a volume containing a total of 5 ⁇ 10 8 CD3 + T-cells is taken and set-up with 1.5 ⁇ 10 9 DYNABEADS® M-450 CD3/CD28 T to initiate the XCELLERATETM process (approx. 3:1 beads to T-cells).
  • the mixture of cells and DYNABEADS® M-450 CD3/CD28 T are then incubated at 37° C., 5% CO 2 for approximately 8 days to generate XCELLERATED T-cells for a first infusion.
  • the remaining monocyte-depleted PBMC are cryopreserved until a second or further cell product expansion (approximately 21 days later) at which time they are thawed, washed and then a volume containing a total of 5 ⁇ 10 8 CD3 + T-cells is taken and set-up with 1.5 ⁇ 10 9 DYNABEADS® M-450 CD3/CD28 T to initiate the XCELLERATE Process for a second infusion.
  • the CD3 + T-cells activate and expand.
  • the anti-CD3 mAb used is BC3 (XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, Wash.), and the anti-CD28 mAb (B-T3, XR-CD28) is obtained from Diaclone, Besançon, France.
  • the monocyte depleted cells were mixed by rotation for 30 minutes with varying amounts of beads as summarized below in Table 1.
  • the beads used in this Example comprised the DYNABEADS® M-450 CD3CD28 T with a 1:1 CD3:CD28 antibody ratio bound on the beads.
  • Varying Bead:Cell Ratios can Selectively Expand or Delete Memory CD8 T cells Fold Increase Bead:Cell Ratio Polyclonal T cells
  • CMV Antigen-Specific T cells 10:1 149 0 5:1 294 0 3:1 346 1.4 1:1 562 20.6 1:5 113 53 1:10 79 45.8
  • stimulation with high bead:cell ratios provides a high concentration of stimulating antibody (i.e., “strong signal”), leading to over-stimulation of antigen-specific T cells, causing them to die, either by apoptosis or other mechanisms.
  • strong signal i.e., “strong signal”
  • Using lower bead:cell ratios provides a stimulation signal to antigen-specific T cells that does not over-stimulate, but rather induces rapid proliferation of these cells.
  • Cells were prepared and stimulated essentially as described in Example 3 with the following modifications: as shown in FIG. 4, panels A and B, cells were cultured either at a starting static culture with a bead:cell ratio of 1:2.5 or 1:5 OR at 1:2.5 or 1:5 starting ratio with additional beads added at day 5, 7, or 9 at 1:10, 1:25, 1:50 or 1:100 ratios as noted.
  • a comparison of total T cell expansion over 15 days shows an increase in expansion of cells when beads are added sequentially over culturing time, in cultures with both starting bead:cell ratios of 1:2.5 and 1:5.
  • Comparison of CMV-specific T cell expansion over 15 days also shows an increase in expansion of antigen-specific cells when beads are added sequentially during culture (see FIG. 4 panel A and FIG.
  • This example describes a model system for assessing CD4 T cell subsets in the Xcellerate® expansion process.
  • Toxic Shock Syndrome Toxin is a superantigen that specifically stimulates CD4+ T cells expressing TCR V ⁇ 2.
  • PBMC are composed of between 1-25% V ⁇ 2 TCR T cells.
  • a CD4 + V ⁇ 2 specific cell line is generated by stimulating PBMC with TSST for 9-14 days until T cells proliferate out of log phase.
  • These “antigen experienced” V ⁇ 2 T cells are then mixed back at varying percentages of the total culture (e.g., 1%, 2%) with a V ⁇ 2 depleted na ⁇ ve PBMC culture and stimulated with CD3/CD28 beads at varying bead:cell ratios as described herein in the Xcellerate® process.
  • T cell expansion was evaluated using varying concentrations of anti-CD3:anti-CD28 antibody ratios on the 3 ⁇ 28 DYNABEADS® M-450.
  • the process referred to as XCELLERATE IITM was used, as described in U.S. patent application Ser. No. 10/187,467. Briefly, this process is similar to XCELLERATE ITM as described in Example 3 with some modifications in which no separate monocyte depletion step was utilized and in certain processes the cells were frozen prior to initial contact with beads and further concentration and stimulation were performed.
  • FIG. 6 surprisingly, about a 68-fold expansion after 8 days of culture was observed with an anti-CD3:CD28 ratio of 1:10 antibodies on the beads.
  • This example describes the T cells expansion using essentially the Xcellerate II process as described in U.S. Patent Application Nos. 10/350,305; 10/187,467; 10/133,236; 09/960,264; 09/794,230; PCT/US01/06139; and PCT/US02/28161, followed by seeding cells into the Wave Bioreactor.
  • CD3 + cells were culture-expanded for ⁇ 2 additional days at which point the contents of the culture bags were then seeded into a 20 L Wave Bioreactor containing a 10 L volume of media. The cells were then cultured at 37° C., 5% CO 2 with the wave motion at 15 rocks/minute and with perfusion at 1 ml/minute.
  • the Wave Bioreactor provides an unexpected and dramatic improvement to the expansion process. Furthermore, hitherto unobserved cell densities and final absolute cell yields were achieved using The Wave Bioreactor.

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Cited By (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1879615A2 (fr) * 2005-05-11 2008-01-23 The Trustees Of The University Of Pennsylvania Procédés permettant l'expansion rapide de lymphocytes t spécifiques à l'antigène
US20080261307A1 (en) * 2007-03-16 2008-10-23 University Of Ulsan Foundation For Industry Cooperation Method of isolating and proliferating autologous antigen-specific CD8+ T Cell using anti-4-1BB antibodies
US20100254958A1 (en) * 2007-10-24 2010-10-07 Anne Letsch Antigen-Specific T-Cell Preparations from Bone Marrow
US20110229448A1 (en) * 2007-09-20 2011-09-22 St Vincent's Hospital Sydney Limited method for identifying antigen-specific regulatory t cells
US20130181333A1 (en) * 2012-01-17 2013-07-18 Chipmos Technologies Inc. Semiconductor package structure and manufacturing method thereof
EP2635305A1 (fr) * 2010-11-02 2013-09-11 Baylor Research Institute Augmentation des réponses des cellules mémoires th17 spécifiques d'un agent pathogène
KR101503341B1 (ko) 2014-03-12 2015-03-18 국립암센터 자가암항원 특이적 cd8+ t 세포의 분리 및 증식방법
KR20150141962A (ko) * 2013-03-14 2015-12-21 더 존스 홉킨스 유니버시티 나노스케일 인공 항원 제시 세포
WO2016100977A1 (fr) 2014-12-19 2016-06-23 The Broad Institute Inc. Procédés pour le profilage du répertoire de récepteurs de cellules t
WO2016189159A1 (fr) * 2015-05-28 2016-12-01 Ge Healthcare Bio-Sciences Ab Culture cellulaire semi-statique
WO2017069958A2 (fr) 2015-10-09 2017-04-27 The Brigham And Women's Hospital, Inc. Modulation de nouvelles cibles de points de contrôle immunitaires
WO2017075478A2 (fr) 2015-10-28 2017-05-04 The Broad Institute Inc. Compositions et méthodes d'évaluation et de modulation des réponses immunitaires à l'aide de signatures génétiques de cellules immunitaires
WO2017075451A1 (fr) 2015-10-28 2017-05-04 The Broad Institute Inc. Compositions et procédés d'évaluation et de modulation des réponses immunitaires par détection et ciblage de pou2af1
WO2017075465A1 (fr) 2015-10-28 2017-05-04 The Broad Institute Inc. Compositions et procédés d'évaluation et de modulation des réponses immunitaires par détection et ciblage de gata3
WO2017087708A1 (fr) 2015-11-19 2017-05-26 The Brigham And Women's Hospital, Inc. Hétérodimères dans l'immunité de l'interleukine 12b (p40) de type antigène lymphocytaire cd5 (cd5l)
CN107148285A (zh) * 2014-11-25 2017-09-08 Adc治疗股份有限公司 吡咯并苯并二氮杂*‑抗体缀合物
WO2018049025A2 (fr) 2016-09-07 2018-03-15 The Broad Institute Inc. Compositions et procédés pour évaluer et moduler des réponses immunitaires
WO2018067991A1 (fr) 2016-10-07 2018-04-12 The Brigham And Women's Hospital, Inc. Modulation de nouvelles cibles de points de contrôle immunitaires
WO2018080997A1 (fr) * 2016-10-24 2018-05-03 Gpb Scientific, Llc Déplacement latéral déterministe dans la préparation de cellules et de compositions à usages thérapeutiques
WO2018191553A1 (fr) 2017-04-12 2018-10-18 Massachusetts Eye And Ear Infirmary Signature tumorale pour métastase, compositions de matière et leurs procédés d'utilisation
WO2018195019A1 (fr) 2017-04-18 2018-10-25 The Broad Institute Inc. Composition permettant de détecter une sécrétion et procédé d'utilisation
US10149898B2 (en) 2017-08-03 2018-12-11 Taiga Biotechnologies, Inc. Methods and compositions for the treatment of melanoma
WO2019027465A1 (fr) 2017-08-03 2019-02-07 Taiga Biotechnologies, Inc. Méthodes et compositions pharmaceutiques destinées au traitement du mélanome
WO2019046052A1 (fr) * 2017-09-01 2019-03-07 Gpb Scientific, Llc Procédés de préparation de cellules thérapeutiquement actives au moyen de la microfluidique
WO2019094983A1 (fr) 2017-11-13 2019-05-16 The Broad Institute, Inc. Méthodes et compositions de traitement du cancer par ciblage de la voie clec2d-klrb1
US10294454B2 (en) 2016-08-24 2019-05-21 General Electric Company Methods and kits for cell activation
EP3495382A1 (fr) * 2012-10-11 2019-06-12 UTI Limited Partnership Méthodes et compositions pour le traitement de la sclérose en plaques et de troubles associés
US10324011B2 (en) 2013-03-15 2019-06-18 The Trustees Of Princeton University Methods and devices for high throughput purification
CN110241086A (zh) * 2012-06-11 2019-09-17 威尔逊沃夫制造公司 用于过继细胞疗法的改进的细胞培养方法
WO2019232542A2 (fr) 2018-06-01 2019-12-05 Massachusetts Institute Of Technology Procédés et compositions pour détecter et moduler des signatures géniques micro-environnementales à partir du lcr de patients présentant des métastases
CN110785487A (zh) * 2017-06-22 2020-02-11 Tcer公司 T细胞扩增方法及用途
WO2020072700A1 (fr) 2018-10-02 2020-04-09 Dana-Farber Cancer Institute, Inc. Lignées d'allèles uniques d'alh
WO2020081730A2 (fr) 2018-10-16 2020-04-23 Massachusetts Institute Of Technology Méthodes et compositions pour moduler un microenvironnement
CN111107871A (zh) * 2017-07-21 2020-05-05 伯克利之光生命科技公司 抗原呈递合成表面、共价官能化表面、活化t细胞及其用途
CN111182918A (zh) * 2017-08-04 2020-05-19 特鲁提诺生物科学公司 用于激活免疫细胞的方法
WO2020131586A2 (fr) 2018-12-17 2020-06-25 The Broad Institute, Inc. Méthodes d'identification de néo-antigènes
US10760055B2 (en) 2005-10-18 2020-09-01 National Jewish Health Conditionally immortalized long-term stem cells and methods of making and using such cells
US10774151B2 (en) 2017-01-06 2020-09-15 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
WO2020186101A1 (fr) 2019-03-12 2020-09-17 The Broad Institute, Inc. Procédés de détection, compositions et méthodes de modulation des cellules de sarcome synovial
WO2020191079A1 (fr) 2019-03-18 2020-09-24 The Broad Institute, Inc. Compositions et méthodes de modulation de régulateurs métaboliques de pathogénicité de lymphocytes t
US10786534B2 (en) 2013-03-11 2020-09-29 Taiga Biotechnologies, Inc. Production and use of red blood cells
WO2020236967A1 (fr) 2019-05-20 2020-11-26 The Broad Institute, Inc. Mutant de délétion de crispr-cas aléatoire
WO2020243371A1 (fr) 2019-05-28 2020-12-03 Massachusetts Institute Of Technology Procédés et compositions pour la modulation de réponses immunitaires
US10905773B2 (en) 2012-10-11 2021-02-02 Uti Limited Partnership Methods and compositions for treating multiple sclerosis and related disorders
WO2021030627A1 (fr) 2019-08-13 2021-02-18 The General Hospital Corporation Procédés de prédiction de résultats d'inhibition de point de contrôle et traitement associés
WO2021041922A1 (fr) 2019-08-30 2021-03-04 The Broad Institute, Inc. Systèmes de transposase mu associés à crispr
US10953048B2 (en) 2012-07-20 2021-03-23 Taiga Biotechnologies, Inc. Enhanced reconstitution and autoreconstitution of the hematopoietic compartment
US10976232B2 (en) 2015-08-24 2021-04-13 Gpb Scientific, Inc. Methods and devices for multi-step cell purification and concentration
US10988516B2 (en) 2012-03-26 2021-04-27 Uti Limited Partnership Methods and compositions for treating inflammation
US11000596B2 (en) 2010-11-12 2021-05-11 UTI Limited Parttiership Compositions and methods for the prevention and treatment of cancer
US11116796B2 (en) 2016-12-02 2021-09-14 Taiga Biotechnologies, Inc. Nanoparticle formulations
US11142746B2 (en) 2013-03-15 2021-10-12 University Of Maryland, Baltimore High efficiency microfluidic purification of stem cells to improve transplants
US11338024B2 (en) 2013-11-04 2022-05-24 Uti Limited Partnership Methods and compositions for sustained immunotherapy
US11369678B2 (en) 2008-08-28 2022-06-28 Taiga Biotechnologies, Inc. Compositions and methods for modulating immune cells
US11493428B2 (en) 2013-03-15 2022-11-08 Gpb Scientific, Inc. On-chip microfluidic processing of particles
US11667695B2 (en) 2008-05-16 2023-06-06 Taiga Biotechnologies, Inc. Antibodies and processes for preparing the same
US11732257B2 (en) 2017-10-23 2023-08-22 Massachusetts Institute Of Technology Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries
US11739156B2 (en) 2019-01-06 2023-08-29 The Broad Institute, Inc. Massachusetts Institute of Technology Methods and compositions for overcoming immunosuppression
US11793787B2 (en) 2019-10-07 2023-10-24 The Broad Institute, Inc. Methods and compositions for enhancing anti-tumor immunity by targeting steroidogenesis
US11865168B2 (en) 2019-12-30 2024-01-09 Massachusetts Institute Of Technology Compositions and methods for treating bacterial infections
US11897953B2 (en) 2017-06-14 2024-02-13 The Broad Institute, Inc. Compositions and methods targeting complement component 3 for inhibiting tumor growth
US11913075B2 (en) 2017-04-01 2024-02-27 The Broad Institute, Inc. Methods and compositions for detecting and modulating an immunotherapy resistance gene signature in cancer
US11957695B2 (en) 2018-04-26 2024-04-16 The Broad Institute, Inc. Methods and compositions targeting glucocorticoid signaling for modulating immune responses
US11963966B2 (en) 2017-03-31 2024-04-23 Dana-Farber Cancer Institute, Inc. Compositions and methods for treating ovarian tumors
US11981922B2 (en) 2019-10-03 2024-05-14 Dana-Farber Cancer Institute, Inc. Methods and compositions for the modulation of cell interactions and signaling in the tumor microenvironment
US11994512B2 (en) 2018-01-04 2024-05-28 Massachusetts Institute Of Technology Single-cell genomic methods to generate ex vivo cell systems that recapitulate in vivo biology with improved fidelity
WO2024124044A1 (fr) 2022-12-07 2024-06-13 The Brigham And Women’S Hospital, Inc. Compositions et procédés ciblant sat1 pour améliorer l'immunité antitumorale pendant la progression d'une tumeur
US12011480B2 (en) 2015-05-06 2024-06-18 Uti Limited Partnership Nanoparticle compositions for sustained therapy
US12036240B2 (en) 2019-06-14 2024-07-16 The Broad Institute, Inc. Compositions and methods targeting complement component 3 for inhibiting tumor growth

Families Citing this family (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE352586T2 (de) * 2000-09-29 2007-02-15 Molecular Probes Inc Modifizierte carbocyaninfarbstoffe und deren konjugate
US9726673B2 (en) 2005-11-23 2017-08-08 Genentech, Inc. Methods and compositions related to B cell assays
GB0603081D0 (en) 2006-02-15 2006-03-29 Dynal Biotech Asa Oslo Method
EP1840569A1 (fr) * 2006-03-28 2007-10-03 Deutsches Rheuma-Forschungszentrum Berlin Utilisation du récepteur 4-1BB pour l'identification et/ou la séparation de cellules Th régulatrices activées (Treg)
EP2010906B1 (fr) 2006-03-24 2014-06-11 Miltenyi Biotec GmbH Utilisation du recepteur 4-1bb pour identifier et/ou separer des lymphocytes t auxiliaires regulateurs activés (treg)
US20130330375A1 (en) 2011-02-28 2013-12-12 Cadila Pharmaceuticals Limited Therapeutic Cancer Vaccine
JP5840857B2 (ja) * 2011-04-08 2016-01-06 国立大学法人 東京大学 細胞傷害性ｔ細胞誘導用組成物
CN102816734A (zh) * 2012-05-09 2012-12-12 阮润生 一种肿瘤抗原特异性t细胞的获取方法
US8921055B2 (en) 2012-10-30 2014-12-30 Berkeley Lights, Inc. Detecting cells secreting a protein of interest
ES2771251T3 (es) 2013-03-01 2020-07-06 Us Health Métodos de producción de poblaciones enriquecidas de células T reactivas con tumores a partir de sangre periférica
CN103740643B (zh) * 2014-01-21 2015-09-30 山东省齐鲁干细胞工程有限公司 一种mhc限制性杀伤t细胞的体外诱导培养方法
MY184699A (en) 2014-04-16 2021-04-18 Juno Therapeutics Gmbh Methods, kits and apparatus for expanding a population of cells
PL3134095T3 (pl) * 2014-04-25 2020-10-19 Bluebird Bio, Inc. Ulepszone sposoby produkcji komórek do adoptywnych terapii komórkowych
EP3998278A1 (fr) 2014-04-25 2022-05-18 2seventy bio, Inc. Récepteurs d'antigènes chimériques de promoteur mnd
EP3828265A1 (fr) 2014-06-06 2021-06-02 Bluebird Bio, Inc. Compositions de lymphocytes t améliorées
WO2015191874A1 (fr) * 2014-06-12 2015-12-17 Children's National Medical Center Génération de cellules immunitaires contre le virus et généralement spécifiques ciblant plusieurs antigènes du vih pour une utilisation prophylactique et thérapeutique
JP2017520575A (ja) 2014-07-01 2017-07-27 ファイザー・インク 二重特異的ヘテロ二量体ダイアボディおよびその使用
RU2747457C2 (ru) 2014-07-24 2021-05-05 Блубёрд Био, Инк. Химерные антигенные рецепторы к bcma
PT3628687T (pt) 2014-12-12 2021-10-20 Bluebird Bio Inc Recetores do antigénio quimérico bcma
ES2954480T3 (es) 2015-08-21 2023-11-22 Mayo Found Medical Education & Res Métodos y materiales para expandir linfocitos T específicos de antígeno en cultivo
MA44909A (fr) 2015-09-15 2018-07-25 Acerta Pharma Bv Association thérapeutique d'un inhibiteur du cd19 et d'un inhibiteur de la btk
MA45489A (fr) 2015-10-22 2018-08-29 Juno Therapeutics Gmbh Procédés de culture de cellules, kits et appareil associés
JP7195141B2 (ja) 2015-10-22 2022-12-23 ジュノ セラピューティクス ゲーエムベーハー 形質導入のための方法、キット、作用物質および装置
MA45488A (fr) 2015-10-22 2018-08-29 Juno Therapeutics Gmbh Procédés, kits et appareil de culture de cellules
AU2016344745A1 (en) * 2015-10-28 2018-05-17 Life Technologies As Selective expansion of different subpopulations of T cells by the alteration of cell surfacing signals and signal ratio
EP3368051A4 (fr) 2015-10-30 2019-06-26 Children's National Medical Center Génération de lymphocytes t spécifiques contre l'antigène du vph à partir d'une population de lymphocytes t naïfs
WO2017099712A1 (fr) 2015-12-07 2017-06-15 Bluebird Bio, Inc. Compositions de lymphocytes t améliorées
EP3445399A4 (fr) * 2016-03-16 2019-10-30 NexImmune, Inc Production de cellules t spécifiques d'antigènes
WO2018009246A1 (fr) 2016-07-08 2018-01-11 American Gene Technologies International Inc. Pré-immunisation et immunothérapie du vih
KR101862502B1 (ko) * 2017-01-02 2018-05-29 성균관대학교산학협력단 재구성 인공 암세포, 이의 제조 방법, 및 이를 포함하는 항암 조성물
NZ758485A (en) 2017-04-27 2024-02-23 Juno Therapeutics Gmbh Oligomeric particle reagents and methods of use thereof
US20200239910A1 (en) 2017-08-09 2020-07-30 Juno Therapeutics, Inc. Methods and compositions for preparing genetically engineered cells
KR102296580B1 (ko) * 2017-09-01 2021-09-02 론자 워커스빌 아이엔씨. 엔드-투-엔드 세포 요법의 자동화
US20210030802A1 (en) * 2018-04-07 2021-02-04 Constant Biotechnology, Llc Glucocorticoid-resistant leukocytes and their use in the treatment of cancers and viruses
WO2020097466A1 (fr) 2018-11-08 2020-05-14 Neximmune, Inc. Compositions de lymphocytes t ayant des propriétés phénotypiques améliorées
EP4034641A4 (fr) * 2019-09-26 2024-01-03 NantBio, Inc. Multiplication primaire de lymphocytes t
WO2022187319A2 (fr) * 2021-03-02 2022-09-09 University Of Louisville Research Foundation, Inc. Séparation acoustofluidique de cellules et de particules par l'intermédiaire d'une dynamique de force de rayonnement acoustique
NL2028681B1 (en) * 2021-04-02 2022-10-17 Nutcracker Therapeutics Inc Materials and methods for generating antigen-specific t cells and treating diseases

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5595881A (en) * 1994-08-09 1997-01-21 Anergen, Inc. Method for the detection of antigen presenting cells
US5827642A (en) * 1994-08-31 1998-10-27 Fred Hutchinson Cancer Research Center Rapid expansion method ("REM") for in vitro propagation of T lymphocytes
US5858358A (en) * 1992-04-07 1999-01-12 The United States Of America As Represented By The Secretary Of The Navy Methods for selectively stimulating proliferation of T cells
US5874307A (en) * 1994-06-28 1999-02-23 The Institute Of Physical And Chemical Research Process for induction culture of cytotoxic T lymphocytes having killing activity against tumor cells
US6534055B1 (en) * 1988-11-23 2003-03-18 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US20030124122A1 (en) * 2000-02-24 2003-07-03 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US20030235908A1 (en) * 2000-02-24 2003-12-25 Xcyte Therapies, Inc. Activation and expansion of cells
US6905874B2 (en) * 2000-02-24 2005-06-14 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells

Family Cites Families (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5081029A (en) 1985-09-25 1992-01-14 Oncogen Methods of adoptive immunotherapy for treatment of aids
WO1988000970A2 (fr) 1986-08-08 1988-02-11 Regents Of The University Of Minnesota Procede de culture de leucocytes
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
US5057423A (en) 1987-12-18 1991-10-15 University Of Pittsburgh Method for the preparation of pure LAK-active lymphocytes
US6010902A (en) 1988-04-04 2000-01-04 Bristol-Meyers Squibb Company Antibody heteroconjugates and bispecific antibodies for use in regulation of lymphocyte activity
US20020076407A1 (en) 1988-11-23 2002-06-20 Carl H. June Method for selectively stimulating proliferation of t cells
US6905680B2 (en) 1988-11-23 2005-06-14 Genetics Institute, Inc. Methods of treating HIV infected subjects
US6352694B1 (en) 1994-06-03 2002-03-05 Genetics Institute, Inc. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells
US5635697A (en) 1989-03-01 1997-06-03 Symbol Technologies, Inc. Method and apparatus for decoding two-dimensional bar code
US5763266A (en) 1989-06-15 1998-06-09 The Regents Of The University Of Michigan Methods, compositions and devices for maintaining and growing human stem and/or hematopoietics cells
DE3923279A1 (de) 1989-07-14 1990-01-18 Will W Prof Dr Minuth Minusheets ist ein neues produkt, um zellen in beliebigen behaeltnissen in hochdifferenzierter form auf einer moeglichst natuerlichen unterlage zu kultivieren
CA2071478A1 (fr) 1989-10-27 1991-04-28 Jeffery A. Bluestone Methodes et compositions favorisant l'immunopotentialisation
ZA91463B (en) 1990-01-25 1992-09-30 Bristol Myers Squibb Co Method of activating cytolytic activity of lymphocytes using anti-cd28 antibody
US5470730A (en) 1990-09-28 1995-11-28 Immunex Method for producing TH -independent cytotoxic T lymphocytes
US6129916A (en) 1991-04-19 2000-10-10 Tanox, Inc. Method of Increasing activation on proliferation of T cells using antibody-microbead conjugates
US6197298B1 (en) 1991-04-19 2001-03-06 Tanox, Inc. Modified binding molecules specific for T lymphocytes and their use as in vivo immune modulators in animals
GB9120508D0 (en) 1991-09-26 1991-11-06 Nycomed As Diagnostic agents
GB9125768D0 (en) 1991-12-04 1992-02-05 Hale Geoffrey Therapeutic method
EP0563485A1 (fr) 1992-03-30 1993-10-06 Schering-Plough Génération in vitro de cellules dendritiques humaines et leurs usages
AU2593192A (en) 1992-09-14 1994-04-12 Oystein Fodstad Detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations
US5837477A (en) 1993-01-15 1998-11-17 The United States Of America As Represented By The Department Of Health And Human Services T cell receptor ligands and methods of using same
US5674704A (en) 1993-05-07 1997-10-07 Immunex Corporation Cytokine designated 4-IBB ligand
US7211259B1 (en) 1993-05-07 2007-05-01 Immunex Corporation 4-1BB polypeptides and DNA encoding 4-1BB polypeptides
ES2240962T3 (es) * 1993-06-04 2005-10-16 The United States Of America As Represented By The Secretary Of The Navy Metodo para estimular selectivamente la proliferacion de celulas t.
US5942607A (en) 1993-07-26 1999-08-24 Dana-Farber Cancer Institute B7-2: a CTLA4/CD28 ligand
WO1995004521A1 (fr) 1993-08-10 1995-02-16 W.L. Gore & Associates, Inc. Dispositif d'encapsulage de cellules
US5672505A (en) 1993-09-27 1997-09-30 Becton, Dickinson And Company Insert for a issue culture vessel
FR2717080B1 (fr) 1994-03-09 1996-12-13 Synthelabo Utilisation de l'éliprodil et de ses énantiomères pour la préparation de médicaments utiles dans le traitement des neuropathies périphériques et des maladies neurodégénératives centrales.
WO1995030412A1 (fr) 1994-05-06 1995-11-16 Kanebo Limited Potentialisateur de cytokine et remede pour des maladies dans lesquelles l'activite de la cytokine est reduite
GB9416657D0 (en) 1994-08-17 1994-10-12 Biocine Spa T cell activation
FR2729570A1 (fr) 1995-01-24 1996-07-26 Idm Immuno Designed Molecules Procede de preparation de macrophages actives, trousses et compositions pour la mise en oeuvre de ce procede
US5985653A (en) 1995-06-07 1999-11-16 Aastrom Biosciences, Inc. Incubator apparatus for use in a system for maintaining and growing biological cells
US5626561A (en) 1995-06-07 1997-05-06 Gore Hybrid Technologies, Inc. Implantable containment apparatus for a therapeutical device and method for loading and reloading the device therein
EP0773753B1 (fr) 1995-06-07 2003-10-08 Gore Hybrid Technologies, Inc. Appareil de confinement implantable destine a un dispositif therapeutque
US6096532A (en) 1995-06-07 2000-08-01 Aastrom Biosciences, Inc. Processor apparatus for use in a system for maintaining and growing biological cells
WO1997001304A1 (fr) 1995-06-29 1997-01-16 Mallinckrodt Medical, Inc. Particules d'apatite radiomarquees contenant un ion paramagnetique
WO1997005239A1 (fr) 1995-07-25 1997-02-13 Celltherapy, Inc. Immunotherapie par cellules autologues: compositions cellulaires , methodes et application au traitement d'une maladie humaine
US20020182730A1 (en) 1995-07-26 2002-12-05 Micheal L. Gruenberg Autologous immune cell therapy: cell compositions, methods and applications to treatment of human disease
US5627070A (en) 1995-07-26 1997-05-06 Celltherapy, Inc. Cell growing device for in vitro cell population expansion
AU6854696A (en) 1995-09-22 1997-04-09 Gore Hybrid Technologies, Inc. Improved cell encapsulation device
AU1152397A (en) * 1995-12-20 1997-07-14 Board Of Trustees Of The Leland Stanford Junior University Methods for in vivo t cell activation by antigen-pulsed dendritic cells
ATE244300T1 (de) 1996-01-17 2003-07-15 Imp College Innovations Ltd Immunotherapie mit verwendung von zytotoxischen t lymphozyten (ctl)
EP0904350B1 (fr) 1996-03-04 2010-08-04 Calyx Bio-Ventures Inc. Procedes modifies d'expansion rapide (rem modifie) permettant la propagation in vitro de lymphocytes t
DE19608753C1 (de) 1996-03-06 1997-06-26 Medigene Gmbh Transduktionssystem und seine Verwendung
US5972721A (en) 1996-03-14 1999-10-26 The United States Of America As Represented By The Secretary Of The Air Force Immunomagnetic assay system for clinical diagnosis and other purposes
DE69740089D1 (de) 1996-03-22 2011-02-10 Stryker Corp Verfahren zur verbesserten funktionellen Erholung der motorischen Koordiination, der Sprache oder der Sinneswahrnehmung nach Trauma oder Ischämie des ZNS
WO1997044667A2 (fr) 1996-05-21 1997-11-27 Institut Pasteur Methodes d'utilisation de complexes peptide/complexe majeur d'histocompatibilite pour obtenir ou purifier des cellules t antigene-specifiques et pour stimuler des cellules t
US5962319A (en) 1997-05-19 1999-10-05 Bml, Inc. Human-Th1-specific protein, gene encoding the protein, transformants, recombinant vectors, and antibodies related to the gene
US20010031253A1 (en) 1996-07-24 2001-10-18 Gruenberg Micheal L. Autologous immune cell therapy: cell compositions, methods and applications to treatment of human disease
US5962318A (en) * 1996-11-15 1999-10-05 St. Jude Children's Research Hospital Cytotoxic T lymphocyte-mediated immunotherapy
US5766944A (en) 1996-12-31 1998-06-16 Ruiz; Margaret Eileen T cell differentiation of CD34+ stem cells in cultured thymic epithelial fragments
JP3531712B2 (ja) 1997-06-04 2004-05-31 トヨタ自動車株式会社 車両用操舵装置
AU741130B2 (en) * 1997-09-16 2001-11-22 Oregon Health Sciences University Recombinant MHC molecules useful for manipulation of antigen-specific T-cells
US6225118B1 (en) 1997-10-01 2001-05-01 Biocure Limited Multicellular in vitro assay of angiogenesis
ATE428769T1 (de) * 1997-10-27 2009-05-15 Univ Rockefeller Methode und zusammensetzung zur herstellung von reifen dendritischen zellen
JP2001522806A (ja) * 1997-11-10 2001-11-20 アーチ・デヴェロップメント・コーポレイション エクス・ビボ活性化ｔ細胞を用いる腫瘍および腫瘍細胞の処理方法
WO1999042077A2 (fr) 1998-02-19 1999-08-26 Xcyte Therapies, Inc. Compositions et procedes de regulation de l'activation des lymphocytes
JP4601166B2 (ja) * 1998-05-11 2010-12-22 ミルテニィ バイオテック ゲーエムベーハー 抗原特異的ｔ細胞の直接的選択方法
FR2780891A1 (fr) 1998-07-10 2000-01-14 Univ Paris Curie Echange de lymphocytes t
AU774442B2 (en) * 1998-10-20 2004-06-24 Salvatore Albani Methods for isolation, quantification, characterization and modulation of antigen-specific T cells
JP4688254B2 (ja) * 1999-02-09 2011-05-25 独立行政法人理化学研究所 腫瘍ワクチン
US6797514B2 (en) 2000-02-24 2004-09-28 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US20030119185A1 (en) * 2000-02-24 2003-06-26 Xcyte Therapies, Inc. Activation and expansion of cells
ES2288952T3 (es) 2000-05-11 2008-02-01 Baylor Research Institute Composiciones y metodos para producir celulas presentadoras de antigenos.
AUPR011700A0 (en) * 2000-09-14 2000-10-05 Austin Research Institute, The Composition comprising immunogenic virus sized particles (VSP)
US6875612B2 (en) * 2001-03-30 2005-04-05 Greenville Hospital System Monocyte-specific particulate delivery vehicle
WO2002087627A1 (fr) * 2001-04-27 2002-11-07 Xcyte Therapies, Inc. Maturation des cellules presentatrices des antigenes au moyen de lymphocytes t actives
US20050130886A1 (en) * 2001-05-14 2005-06-16 Jan Holmgren Methods for promoting antigen presentation and modulating immune responses using cholera toxin and its b subunit
US20030022210A1 (en) 2001-06-01 2003-01-30 Mark Bonyhadi T cell induced tissue repair and regeneration
DE10129912A1 (de) 2001-06-21 2003-01-02 Efmt Entwicklungs Und Forschun Nadelelektrode
US20030054544A1 (en) 2001-09-14 2003-03-20 Medcell Biologics, Inc. Oxygen enriched bioreactor and method of culturing cells
US20030194395A1 (en) 2001-09-17 2003-10-16 Gruenberg Micheal L. Th1 cell adoptive immunotherapy
AU2002341717A1 (en) 2001-09-17 2003-04-01 Medcell Biologics, Llc. Cell therapy system
US20030134341A1 (en) 2001-09-19 2003-07-17 Medcell Biologics, Llc. Th1 cell adoptive immunotherapy
US20030134415A1 (en) 2001-09-19 2003-07-17 Gruenberg Micheal L. Th1 cell adoptive immunotherapy
US20030097115A1 (en) 2001-11-19 2003-05-22 Medcell Biologics, Inc. Closed sterile system devices and methods
US7638325B2 (en) 2002-01-03 2009-12-29 The Trustees Of The University Of Pennsylvania Activation and expansion of T-cells using an engineered multivalent signaling platform
US20030170238A1 (en) 2002-03-07 2003-09-11 Gruenberg Micheal L. Re-activated T-cells for adoptive immunotherapy
US20030175272A1 (en) 2002-03-07 2003-09-18 Medcell Biologics, Inc. Re-activated T-cells for adoptive immunotherapy

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6534055B1 (en) * 1988-11-23 2003-03-18 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US5858358A (en) * 1992-04-07 1999-01-12 The United States Of America As Represented By The Secretary Of The Navy Methods for selectively stimulating proliferation of T cells
US5874307A (en) * 1994-06-28 1999-02-23 The Institute Of Physical And Chemical Research Process for induction culture of cytotoxic T lymphocytes having killing activity against tumor cells
US5595881A (en) * 1994-08-09 1997-01-21 Anergen, Inc. Method for the detection of antigen presenting cells
US5827642A (en) * 1994-08-31 1998-10-27 Fred Hutchinson Cancer Research Center Rapid expansion method ("REM") for in vitro propagation of T lymphocytes
US20030124122A1 (en) * 2000-02-24 2003-07-03 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US20030235908A1 (en) * 2000-02-24 2003-12-25 Xcyte Therapies, Inc. Activation and expansion of cells
US6867041B2 (en) * 2000-02-24 2005-03-15 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US6905874B2 (en) * 2000-02-24 2005-06-14 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells

Cited By (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1879615A4 (fr) * 2005-05-11 2009-05-13 Univ Pennsylvania Procédés permettant l'expansion rapide de lymphocytes t spécifiques à l'antigène
EP1879615A2 (fr) * 2005-05-11 2008-01-23 The Trustees Of The University Of Pennsylvania Procédés permettant l'expansion rapide de lymphocytes t spécifiques à l'antigène
US10760055B2 (en) 2005-10-18 2020-09-01 National Jewish Health Conditionally immortalized long-term stem cells and methods of making and using such cells
US20080261307A1 (en) * 2007-03-16 2008-10-23 University Of Ulsan Foundation For Industry Cooperation Method of isolating and proliferating autologous antigen-specific CD8+ T Cell using anti-4-1BB antibodies
KR100882445B1 (ko) 2007-03-16 2009-02-09 울산대학교 산학협력단 항―４－１ｂｂ 항체를 이용한 항원 특이적 자가유래ｃｄ８＋ｔ 세포의 분리 및 증식 방법
US7932045B2 (en) * 2007-03-16 2011-04-26 University Of Ulsan Foundation For Industry Cooperation Method of isolating and proliferating autologous antigen-specific CD8+ T cell using anti-4-1BB antibodies
US20110229448A1 (en) * 2007-09-20 2011-09-22 St Vincent's Hospital Sydney Limited method for identifying antigen-specific regulatory t cells
US8975069B2 (en) * 2007-09-20 2015-03-10 St. Vincent's Hospital Sydney Limited Method for identifying antigen-specific regulatory T cells
US20100254958A1 (en) * 2007-10-24 2010-10-07 Anne Letsch Antigen-Specific T-Cell Preparations from Bone Marrow
US11667695B2 (en) 2008-05-16 2023-06-06 Taiga Biotechnologies, Inc. Antibodies and processes for preparing the same
US11369678B2 (en) 2008-08-28 2022-06-28 Taiga Biotechnologies, Inc. Compositions and methods for modulating immune cells
EP2635305A1 (fr) * 2010-11-02 2013-09-11 Baylor Research Institute Augmentation des réponses des cellules mémoires th17 spécifiques d'un agent pathogène
EP2635305A4 (fr) * 2010-11-02 2014-05-28 Baylor Res Inst Augmentation des réponses des cellules mémoires th17 spécifiques d'un agent pathogène
US11000596B2 (en) 2010-11-12 2021-05-11 UTI Limited Parttiership Compositions and methods for the prevention and treatment of cancer
US20130181333A1 (en) * 2012-01-17 2013-07-18 Chipmos Technologies Inc. Semiconductor package structure and manufacturing method thereof
US9053968B2 (en) * 2012-01-17 2015-06-09 Chipmos Technologies Inc. Semiconductor package structure and manufacturing method thereof
US10988516B2 (en) 2012-03-26 2021-04-27 Uti Limited Partnership Methods and compositions for treating inflammation
CN110241086A (zh) * 2012-06-11 2019-09-17 威尔逊沃夫制造公司 用于过继细胞疗法的改进的细胞培养方法
US10953048B2 (en) 2012-07-20 2021-03-23 Taiga Biotechnologies, Inc. Enhanced reconstitution and autoreconstitution of the hematopoietic compartment
EP3495382A1 (fr) * 2012-10-11 2019-06-12 UTI Limited Partnership Méthodes et compositions pour le traitement de la sclérose en plaques et de troubles associés
US10905773B2 (en) 2012-10-11 2021-02-02 Uti Limited Partnership Methods and compositions for treating multiple sclerosis and related disorders
US10786534B2 (en) 2013-03-11 2020-09-29 Taiga Biotechnologies, Inc. Production and use of red blood cells
US11939595B2 (en) 2013-03-14 2024-03-26 The Johns Hopkins University Nanoscale artificial antigen presenting cells
KR20150141962A (ko) * 2013-03-14 2015-12-21 더 존스 홉킨스 유니버시티 나노스케일 인공 항원 제시 세포
KR102164405B1 (ko) * 2013-03-14 2020-10-12 더 존스 홉킨스 유니버시티 나노스케일 인공 항원 제시 세포
US11493428B2 (en) 2013-03-15 2022-11-08 Gpb Scientific, Inc. On-chip microfluidic processing of particles
US11486802B2 (en) 2013-03-15 2022-11-01 University Of Maryland, Baltimore Methods and devices for high throughput purification
US10324011B2 (en) 2013-03-15 2019-06-18 The Trustees Of Princeton University Methods and devices for high throughput purification
US11142746B2 (en) 2013-03-15 2021-10-12 University Of Maryland, Baltimore High efficiency microfluidic purification of stem cells to improve transplants
US10852220B2 (en) 2013-03-15 2020-12-01 The Trustees Of Princeton University Methods and devices for high throughput purification
US11338024B2 (en) 2013-11-04 2022-05-24 Uti Limited Partnership Methods and compositions for sustained immunotherapy
KR101503341B1 (ko) 2014-03-12 2015-03-18 국립암센터 자가암항원 특이적 cd8+ t 세포의 분리 및 증식방법
US10570371B2 (en) 2014-03-12 2020-02-25 Eutilex Co., Ltd. Methods for isolating and proliferating autologous cancer antigen-specific CD8+T cells
US10801011B2 (en) 2014-03-12 2020-10-13 National Cancer Center Methods for isolating and proliferating autologous cancer antigen-specific CD8+ T cells
CN105473731A (zh) * 2014-03-12 2016-04-06 国立癌Center 自身癌抗原特异性cd8+t细胞的分离及增殖方法
WO2015137724A1 (fr) * 2014-03-12 2015-09-17 국립암센터 Procédé permettant d'isoler et de faire proliférer des lymphocytes t cd8+ spécifiques d'auto-antigènes tumoraux
CN107148285A (zh) * 2014-11-25 2017-09-08 Adc治疗股份有限公司 吡咯并苯并二氮杂*‑抗体缀合物
EP3757211A1 (fr) 2014-12-19 2020-12-30 The Broad Institute, Inc. Procédés pour le profilage de répertoire de récepteurs de lymphocytes t
WO2016100977A1 (fr) 2014-12-19 2016-06-23 The Broad Institute Inc. Procédés pour le profilage du répertoire de récepteurs de cellules t
US12011480B2 (en) 2015-05-06 2024-06-18 Uti Limited Partnership Nanoparticle compositions for sustained therapy
US10927344B2 (en) 2015-05-28 2021-02-23 Cytiva Sweden Ab Semi-static cell culture
WO2016189159A1 (fr) * 2015-05-28 2016-12-01 Ge Healthcare Bio-Sciences Ab Culture cellulaire semi-statique
US10976232B2 (en) 2015-08-24 2021-04-13 Gpb Scientific, Inc. Methods and devices for multi-step cell purification and concentration
WO2017069958A2 (fr) 2015-10-09 2017-04-27 The Brigham And Women's Hospital, Inc. Modulation de nouvelles cibles de points de contrôle immunitaires
US11186825B2 (en) 2015-10-28 2021-11-30 The Broad Institute, Inc. Compositions and methods for evaluating and modulating immune responses by detecting and targeting POU2AF1
WO2017075465A1 (fr) 2015-10-28 2017-05-04 The Broad Institute Inc. Compositions et procédés d'évaluation et de modulation des réponses immunitaires par détection et ciblage de gata3
WO2017075478A2 (fr) 2015-10-28 2017-05-04 The Broad Institute Inc. Compositions et méthodes d'évaluation et de modulation des réponses immunitaires à l'aide de signatures génétiques de cellules immunitaires
US11180730B2 (en) 2015-10-28 2021-11-23 The Broad Institute, Inc. Compositions and methods for evaluating and modulating immune responses by detecting and targeting GATA3
WO2017075451A1 (fr) 2015-10-28 2017-05-04 The Broad Institute Inc. Compositions et procédés d'évaluation et de modulation des réponses immunitaires par détection et ciblage de pou2af1
US11001622B2 (en) 2015-11-19 2021-05-11 The Brigham And Women's Hospital, Inc. Method of treating autoimmune disease with lymphocyte antigen CD5-like (CD5L) protein
WO2017087708A1 (fr) 2015-11-19 2017-05-26 The Brigham And Women's Hospital, Inc. Hétérodimères dans l'immunité de l'interleukine 12b (p40) de type antigène lymphocytaire cd5 (cd5l)
US11884717B2 (en) 2015-11-19 2024-01-30 The Brigham And Women's Hospital, Inc. Method of treating autoimmune disease with lymphocyte antigen CD5-like (CD5L) protein
US10294454B2 (en) 2016-08-24 2019-05-21 General Electric Company Methods and kits for cell activation
US11512288B2 (en) 2016-08-24 2022-11-29 Global Life Sciences Solutions Usa Llc Methods and kits for cell activation
WO2018049025A2 (fr) 2016-09-07 2018-03-15 The Broad Institute Inc. Compositions et procédés pour évaluer et moduler des réponses immunitaires
WO2018067991A1 (fr) 2016-10-07 2018-04-12 The Brigham And Women's Hospital, Inc. Modulation de nouvelles cibles de points de contrôle immunitaires
WO2018080997A1 (fr) * 2016-10-24 2018-05-03 Gpb Scientific, Llc Déplacement latéral déterministe dans la préparation de cellules et de compositions à usages thérapeutiques
CN110381961A (zh) * 2016-10-24 2019-10-25 Gpb科学有限责任公司 用于治疗用途的细胞和组合物的制备中的确定性侧向位移
US11116796B2 (en) 2016-12-02 2021-09-14 Taiga Biotechnologies, Inc. Nanoparticle formulations
US11859004B2 (en) 2017-01-06 2024-01-02 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
US10774151B2 (en) 2017-01-06 2020-09-15 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
US10919972B2 (en) 2017-01-06 2021-02-16 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
US11963966B2 (en) 2017-03-31 2024-04-23 Dana-Farber Cancer Institute, Inc. Compositions and methods for treating ovarian tumors
US11913075B2 (en) 2017-04-01 2024-02-27 The Broad Institute, Inc. Methods and compositions for detecting and modulating an immunotherapy resistance gene signature in cancer
WO2018191553A1 (fr) 2017-04-12 2018-10-18 Massachusetts Eye And Ear Infirmary Signature tumorale pour métastase, compositions de matière et leurs procédés d'utilisation
WO2018195019A1 (fr) 2017-04-18 2018-10-25 The Broad Institute Inc. Composition permettant de détecter une sécrétion et procédé d'utilisation
US11897953B2 (en) 2017-06-14 2024-02-13 The Broad Institute, Inc. Compositions and methods targeting complement component 3 for inhibiting tumor growth
CN110785487A (zh) * 2017-06-22 2020-02-11 Tcer公司 T细胞扩增方法及用途
CN111107871A (zh) * 2017-07-21 2020-05-05 伯克利之光生命科技公司 抗原呈递合成表面、共价官能化表面、活化t细胞及其用途
US10864259B2 (en) 2017-08-03 2020-12-15 Taiga Biotechnologies, Inc. Methods and compositions for the treatment of melanoma
EP4026554A1 (fr) 2017-08-03 2022-07-13 Taiga Biotechnologies, Inc. Procédés et compositions de traitement du mélanome
WO2019027465A1 (fr) 2017-08-03 2019-02-07 Taiga Biotechnologies, Inc. Méthodes et compositions pharmaceutiques destinées au traitement du mélanome
US10149898B2 (en) 2017-08-03 2018-12-11 Taiga Biotechnologies, Inc. Methods and compositions for the treatment of melanoma
CN111182918A (zh) * 2017-08-04 2020-05-19 特鲁提诺生物科学公司 用于激活免疫细胞的方法
US10988734B2 (en) 2017-09-01 2021-04-27 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US11306288B2 (en) 2017-09-01 2022-04-19 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
WO2019046052A1 (fr) * 2017-09-01 2019-03-07 Gpb Scientific, Llc Procédés de préparation de cellules thérapeutiquement actives au moyen de la microfluidique
US10844353B2 (en) 2017-09-01 2020-11-24 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US11149251B2 (en) 2017-09-01 2021-10-19 Gpb Scientific, Inc. Methods for preparing therapeutically active cells using microfluidics
US11732257B2 (en) 2017-10-23 2023-08-22 Massachusetts Institute Of Technology Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries
WO2019094983A1 (fr) 2017-11-13 2019-05-16 The Broad Institute, Inc. Méthodes et compositions de traitement du cancer par ciblage de la voie clec2d-klrb1
US12018080B2 (en) 2017-11-13 2024-06-25 The Broad Institute, Inc. Methods and compositions for treating cancer by targeting the CLEC2D-KLRB1 pathway
US11994512B2 (en) 2018-01-04 2024-05-28 Massachusetts Institute Of Technology Single-cell genomic methods to generate ex vivo cell systems that recapitulate in vivo biology with improved fidelity
US11957695B2 (en) 2018-04-26 2024-04-16 The Broad Institute, Inc. Methods and compositions targeting glucocorticoid signaling for modulating immune responses
WO2019232542A2 (fr) 2018-06-01 2019-12-05 Massachusetts Institute Of Technology Procédés et compositions pour détecter et moduler des signatures géniques micro-environnementales à partir du lcr de patients présentant des métastases
WO2020072700A1 (fr) 2018-10-02 2020-04-09 Dana-Farber Cancer Institute, Inc. Lignées d'allèles uniques d'alh
WO2020081730A2 (fr) 2018-10-16 2020-04-23 Massachusetts Institute Of Technology Méthodes et compositions pour moduler un microenvironnement
WO2020131586A2 (fr) 2018-12-17 2020-06-25 The Broad Institute, Inc. Méthodes d'identification de néo-antigènes
US11739156B2 (en) 2019-01-06 2023-08-29 The Broad Institute, Inc. Massachusetts Institute of Technology Methods and compositions for overcoming immunosuppression
WO2020186101A1 (fr) 2019-03-12 2020-09-17 The Broad Institute, Inc. Procédés de détection, compositions et méthodes de modulation des cellules de sarcome synovial
WO2020191079A1 (fr) 2019-03-18 2020-09-24 The Broad Institute, Inc. Compositions et méthodes de modulation de régulateurs métaboliques de pathogénicité de lymphocytes t
WO2020236967A1 (fr) 2019-05-20 2020-11-26 The Broad Institute, Inc. Mutant de délétion de crispr-cas aléatoire
WO2020243371A1 (fr) 2019-05-28 2020-12-03 Massachusetts Institute Of Technology Procédés et compositions pour la modulation de réponses immunitaires
US12036240B2 (en) 2019-06-14 2024-07-16 The Broad Institute, Inc. Compositions and methods targeting complement component 3 for inhibiting tumor growth
WO2021030627A1 (fr) 2019-08-13 2021-02-18 The General Hospital Corporation Procédés de prédiction de résultats d'inhibition de point de contrôle et traitement associés
WO2021041922A1 (fr) 2019-08-30 2021-03-04 The Broad Institute, Inc. Systèmes de transposase mu associés à crispr
US11981922B2 (en) 2019-10-03 2024-05-14 Dana-Farber Cancer Institute, Inc. Methods and compositions for the modulation of cell interactions and signaling in the tumor microenvironment
US11793787B2 (en) 2019-10-07 2023-10-24 The Broad Institute, Inc. Methods and compositions for enhancing anti-tumor immunity by targeting steroidogenesis
US11865168B2 (en) 2019-12-30 2024-01-09 Massachusetts Institute Of Technology Compositions and methods for treating bacterial infections
WO2024124044A1 (fr) 2022-12-07 2024-06-13 The Brigham And Women’S Hospital, Inc. Compositions et procédés ciblant sat1 pour améliorer l'immunité antitumorale pendant la progression d'une tumeur

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