Source: http://www.google.com/patents/US20070212767?dq=5,579,517
Timestamp: 2017-09-24 13:10:59
Document Index: 207723511

Matched Legal Cases: ['ART-1', 'ART-1', '§ 119', 'Application No. 60', 'ART-1', 'ART-1', 'ART-1', 'ART-1', 'ART-1']

Patent US20070212767 - Generation and isolation of antigen-specific t cells - Google Patents
The present invention relates generally to methods for generating, expanding, and isolating antigen-specific T cells. Compositions of antigen-specific T cells activated and expanded by the methods herein are further provided....http://www.google.com/patents/US20070212767?utm_source=gb-gplus-sharePatent US20070212767 - Generation and isolation of antigen-specific t cells
Publication number US20070212767 A1
Application number US 11/674,304
Also published as CA2525519A1, DE60334250D1, EP1623017A1, EP1623017A4, EP1623017B1, US7977095, US20040224402, US20090137017, WO2004104185A1
Publication number 11674304, 674304, US 2007/0212767 A1, US 2007/212767 A1, US 20070212767 A1, US 20070212767A1, US 2007212767 A1, US 2007212767A1, US-A1-20070212767, US-A1-2007212767, US2007/0212767A1, US2007/212767A1, US20070212767 A1, US20070212767A1, US2007212767 A1, US2007212767A1
Referenced by (20), Classifications (15), Legal Events (2)
US 20070212767 A1
(a) exposing a first population of cells wherein at least a portion thereof comprises antigen presenting cells to a surface wherein said surface has antigen attached thereto, such that said surface with antigen attached thereto is ingested by said APC;
(b) 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 and/or enriching antigen-specific T cells.
2. The method according to claim 1 wherein said APC are in direct contact with said antigen-specific T cells.
3. The method according to claim 2 wherein said APC in direct contact with said antigen-specific T cells are isolated by exposing said APC to a magnetic field.
4. The method according to claim 3 wherein said antigen-specific T cells are expanded according to the following method:
5. The method according to claim 4, further comprising exposing said T cells to IL-15.
6. The method according to claim 4, further comprising exposing said T cells to a natural ligand for CD137.
7. The method according to claim 4, further comprising exposing said T cells to an anti-CD137 antibody.
8. The method according to claim 4, further comprising exposing said T cells to an anti-NKG2D antibody or a natural ligand for NKG2D.
9. The method according to claim 3 wherein said antigen-specific T cells are expanded by exposing said antigen-specific T cells to a mitogen.
10. The method according to claim 9 wherein said mitogen is selected from the group consisting of phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), and superantigen.
11. The method according to claim 1 wherein said antigen is selected from the group consisting of 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, 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.
12. The method according to claim 1 wherein said antigen is attached to said surface by an antibody/ligand interaction.
13. The method according to claim 12 wherein said antibody/ligand interaction comprises 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-α-fetoprotein antibody/α-fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA, anti-CMV pp 65/CMV pp 65, anti-hepatitis C antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA 3B antigen, and anti-human Ig heavy and lignt chains/Ig from a myeloma cancer patient, and anti-human Ig heavy and lignt chains/Ig from a CLL cancer patient.
14. The method according to claim 1 wherein said antigen is chemically attached to said surface.
15. The method according to claim 1 wherein the attachment of said antigen to said surface comprises a biotin-avidin interaction.
16. The method according to claim 1 wherein said population of cells wherein at least a portion thereof comprises APC is derived from a source selected from the group consisting of leukapheresis product, peripheral blood, lymph node, tonsil, thymus, tissue biopsy, tumor, spleen, bone marrow, cord blood, CD34+ cells, monocytes, and adherent cells.
This application is a division of U.S. patent application Ser. No. 10/742,622, filed Dec. 19, 2003, now pending; which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/469,122 filed May 8, 2003. These applications are incorporated herein by reference in their entireties.
The various techniques available for expanding human T-cells have relied primarily on the use of accessory cells (primarily antigen presenting cells (APC)) and/or exogenous growth factors, such as interleukin-2 (IL-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. Both APC signals are thought to be required for optimal T-cell activation, expansion, and long-term survival of the T-cells upon re-infusion. The requirement for MHC-matched APCs as accessory cells presents a significant problem for long-term culture systems because APCs are relatively short-lived. Therefore, in a long-term culture system, 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. In addition, when treating viral infection, if accessory cells carry the virus, the cells may contaminate the entire T-cell population during long-term culture.
In one embodiment, the APC are in direct contact with the antigen-specific T cells. In a further embodiment, 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. In another embodiment, 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. In certain embodiments, at least one agent is an antibody or an antibody fragment. In other embodiments, the first agent is an antibody or a fragment thereof, and the second agent is an antibody or a fragment thereof. In yet another embodiment, the first and the second agents are different antibodies. In certain embodiments, 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. In another embodiment, the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody. In further embodiments, the anti-CD3 antibody and the anti-CD28 antibody are present at a ratio of about 1:1 to about 1:100. In a further embodiment, the antigen-specific T cells are expanded by exposing said antigen-specific T cells to a mitogen, such as phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), and superantigen.
In a further embodiment, 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. In certain embodiments, 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-α-fetoprotein antibody/α-fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA, anti-CMV pp 65/CMV pp 65, anti-hepatitis C antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA 3B antigen, and anti-human Ig heavy and lignt chains/Ig from a myeloma cancer patient, and anti-human Ig heavy and lignt chains/Ig from a CLL cancer patient. In certain embodiments, the antigen is chemically attached to a surface. In one embodiment, the attachment of said antigen to said surface comprises a biotin-avidin interaction. In a further embodiment, 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.
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. In certain embodiments, 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).
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 culture. Panel A shows a comparison of total T cell expansion over 15 days, comparing standard static cutter (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.
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, Ka, of at least about 106 M−1). The skilled artisan would understand that this affinity is illustrative only and that affinity constants of the ligand/anti-ligand pairs useful in the context of the present invention might be lower or in some cases higher. For example, in the case of biotin/streptavidin, 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. Exemplary ligand/anti-ligand pairs enzyme/inhibitor, hapten/antibody, lectin/carbohydrate, ligand/receptor, and biotin/avidin or streptavidin. Within the context of the present invention specification receptors and other cell surface moieties are anti-ligands, while agents (e.g., antibodies and antibody fragments) reactive therewith are considered ligands.
In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and by centrifugation through a PERCOLL™ 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. For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander). In one aspect of the present invention, 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. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
According to certain methods of the invention, antigen may comprise viral antigens such as CMV pp 65, HIV pg120, and the like. In certain embodiments, 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 idiotypes, and the like. The skilled artisan would appreciate that any tumor antigen would be useful in the context of the present invention.
In another embodiment of the present invention, APC are loaded with antigen attached to, coated on, or otherwise immobilized on particles, such as beads. In the various embodiments, commercially available beads or other particles, are useful, e.g., Miltenyi Particles, Miltenyi Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADS™, Dynal Inc., New York. In certain embodiments, 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 Dynabeads™. Exemplary Dynabeads™ in this regard are M-280, M-450, and M-500. In one embodiment, whole cells which are live, fixed, irradiated, heat-killed or otherwise manipulated, are immobilized to ingestable beads, via for example antibody/ligand specific means or chemical means. Similarly, 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). These 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.
In one particular embodiment, the particles of the present invention comprise a cell surface, such as described in U.S. patent application Ser. No. 10/336,224, PCT/U.S.03/00339. In this regard, 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. One suitable transfection technique is 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 (including voltage, resistance and pulse length) 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. Following transfection, 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-α-fetoprotein antibody/α-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-human Ig heavy and lignt chains/Ig from cancer patient, such as myeloma or CLL patient. Other protein:protein binding interactions may be suitable for attaching antigen to particles, such as beads, for example, receptor/ligand interactions may be utilized. In certain embodiments, 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. In yet further embodiments, antigen may be attached to the particles, such as beads by a biotin/avidin or streptavidin interaction. In certain embodiments, 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 (NH2) and sulfhydryl groups (SH) occurring overnight. Coupling reactions can be performed at neutral pH however high pH and incubation at 37° C. can promote covalent binding.
In one embodiment of the present invention, 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. For example, 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). In this regard, only T cells with specificity for the variety of presented antigens will optimally interact in a positive manner with the APC. 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 XCELLERATE™ 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/U.S.01/06139; and PCT/U.S.02/28161.
Generally, 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. 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. Similarly, stimulatory forms of anti-CD2 antibodies are known and available. Stimulation through CD2 with anti-CD2 antibodies is typically accomplished using a combination of at least two different anti-CD2 antibodies. Stimulatory combinations of anti-CD2 antibodies 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. 4;274(5284):94-6), 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, phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), 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. For co-stimulation of an accessory molecule on the surface of the T-cells, a ligand that binds the accessory molecule is used. For example, 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. Similarly, to stimulate proliferation of CD8+ 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).
To further re-stimulate a population of antigen-specific T cells, a co-stimulatory or accessory molecule on the surface of the T cells, such as CD28, is stimulated with a ligand that binds the accessory molecule. Accordingly, one of ordinary skill in the art will recognize that any agent, including an anti-CD28 antibody or fragment thereof capable of cross-linking the CD28 molecule, or a natural ligand for CD28 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 (IgG2a) (Bristol-Myers Squibb, Princeton, N.J.), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM), clone B-T3 (XR-CD28; Diaclone, Besançon, France) and EX5.3D10 (IgG2a) (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. 137:3260-3267, 1987; Freeman et al., J. Immunol. 143:2714-2722, 1989; Freeman et al., J. Exp. Med. 174:625-631, 1991; Freeman et al., Science 262:909-911, 1993; Azuma et al., Nature 366:76-79, 1993; Freeman et al., J. Exp. Med. 178:2185-2192, 1993).
Using certain methodologies it may be advantageous to maintain long-term stimulation of a population of T-cells following the initial activation and stimulation, by separating the T-cells from the stimulus after a period of about 12 to about 14 days. The rate of T-cell proliferation is monitored periodically (e.g., daily) by, for example, examining the size or measuring the volume of the T-cells, such as with a Coulter Counter. In this regard, a resting T-cell has a mean diameter of about 6.8 microns, and upon initial activation and stimulation, in the presence of the stimulating ligand, the T-cell mean diameter will increase to over 12 microns by day 4 and begin to decrease by about day 6. When the mean T-cell diameter decreases to approximately 8 microns, the T-cells may be reactivated and re-stimulated to induce further proliferation of the T-cells. Alternatively, the rate of T-cell proliferation and time for T-cell re-stimulation can be monitored by assaying for the presence of cell surface molecules, such as, CD154, CD54, CD25, CD137, CD134, which are induced on activated T-cells.
In another embodiment, the time of exposure to stimulatory agents such as anti-CD3/anti-CD28 (i.e., CD3×CD28)-coated particles, such as beads, may be modified or tailored to obtain a desired T-cell phenotype. One may desire a greater population of helper T-cells (TH), typically CD4+ as opposed to CD8+ cytotoxic or suppressor T-cells (TC), because an expansion of TH cells could induce desired effector function (e.g., anti-tumor, anti-viral, anti-bacterial, and the like). CD4+ T-cells, express important immune-regulatory molecules, such as GM-CSF, CD40L, and IL-2, for example. Where CD4-mediated help is preferred, a method, such as that described herein, which preserves or enhances the CD4:CD8 ratio could be of significant benefit. In one aspect of the present invention, it may be beneficial to increase the number of infused cells expressing GM-CSF, or IL-2, all of which are expressed predominantly by CD4+ T-cells. Alternatively, in situations where CD4-help is needed less and increased numbers of CD8+ T-cells are desirous, 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-γ is preferred. Further, in other applications, it may be desirable to utilize a population of TH1-type cells versus TH2-type cells (or vice versa), or supernatants therefrom. Likewise, it may be desirable in certain applications to utilize a population of regulatory T cells (e.g., Autoimmun Rev. 2002 August; 1(4):190-7; Curr Opin Immunol. 2002 December; 14(6):771-8).
In one embodiment of the present invention, 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-γ, IL-4, GM-CSF, IL-10, IL-12, TGFβ, and TNF-α. 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, α-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% C02).
In certain embodiments, it may be desirable to add some number of feeder cells to augment activation and/or expansion of antigen-specific cells. 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 myelomonocytic lineage, such as macrophages, dendritic 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 there derived antigen-specific clones.
In certain embodiments, 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. 2001, 31:1247; Salomon, et al, Immunity 2000, 12:431; and Sakaguchi, et al., Immunol. Rev. 2001, 182:18. Accordingly, 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, paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evan's syndrome, Factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever.
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 pp 65. 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.
PBMC from CMV pp 65 tetramer-positive and tetramer-negative donors were stimulated with paramagnetic Dynal M-450 beads coated with CMV lysate. As controls, CMV pp 65 tetramer-negative PBMC were cultured with CMV-lysate coated beads (FIG. 2, panel A), CMV pp 65 tetramer-positive PBMC were cultured with “naked” beads (no CMV antigen) (FIG. 2, panel B). CMV pp 65 tetramer-positive PBMC were cultured with CMV-lysate coated beads (FIG. 2, panel C). Following stimulation, activation of CMV-specific T cells was measured on Day 10 by CMV pp 65 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.
Cells were prepared and stimulated using the XCELLERATE I™ process essentially as described in U.S. patent application Ser. No. 10/187,467 filed Jun. 28, 2002. Briefly, in this process, the XCELLERATED™-cells are manufactured from a peripheral blood mononuclear cell (PBMC) apheresis product. After collection from the patient at the clinical site, the PBMC apheresis are washed and then incubated with “uncoated” DYNABEADS® M-450 Epoxy T. During this time phagocytic cells such as monocytes ingest the beads. After the incubation, 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. Following this monocyte-depletion step, a volume containing a total of 5×108 CD3+ T-cells is taken and set-up with 1.5×109 DYNABEADS® M-450 CD3/CD28 T to initiate the XCELLERATE™ 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% CO2 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 5×108 CD3+ T-cells is taken and set-up with 1.5×109 DYNABEADS® M-450 CD3/CD28 T to initiate the XCELLERATE Process for a second infusion. During the incubation period of ≈8 days at 37° C., 5% CO2, 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.
Varying Bead:Cell Ratios can Selectively Expand or
Delete Memory CD8 T 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. 4 panel B). The most dramatic increase in expansion of polyclonal cells and antigen-specific T cells over static culture was observed in those cultures where beads were added at day 0 at a ratio of 1:2.5 beads: cells and sequentially added at a 1:10 ratio at day 5.
This example describes a model system for assessing CD4 T cell subsets in the Xcellerate™ expansion process.
T cell expansion was evaluated using varying concentrations of anti-CD3:anti-CD28 antibody ratios on the 3×28 DYNABEADS® M-450. In the experiments described herein, the process referred to as XCELLERATE II™ was used, as described in U.S. patent application Ser. No. 10/187,467. Briefly, this process is similar to XCELLERATE I™ 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. As shown in 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. A 35-fold expansion of T cells was seen after 8 days of culture with a CD3:CD28 ratio of 1:3 on the beads. At a 1:1 ratio, about a 24-fold expansion was seen. As shown in FIG. 7, similar results were observed with CMVpp65-specific CD8+ T cells using anti-CD3:anti-CD28 antibody ratios as low as 1:30.
This example describes the T cells expansion using essentially the Xcellerate II process as described in U.S. patent application Ser. Nos. 10/350,305; 10/187,467; 10/133,236; 09/960,264; 09/794,230; PCT/U.S.01/06139; and PCT/U.S.02/28161, followed by seeding cells into the Wave Bioreactor.
Day 0 of the Xcellerate Process—On the first day of the Xcellerate process essentially, the required number of cryopreserved Cryocte™ containers from were removed from the storage freezer, thawed washed and filtered.
Day 0—A volume of cells containing approximately 0.5×109 CD3+ cells was then mixed with Dynabeads M-450 CD3/CD28 T at a ratio of 3:1 Dynabeads M-450 CD3/CD28 T:CD3+ T cells and incubated with rotation. After the incubation, the CD3+ T cells were magnetically concentrated and simultaneously activated. The CD3+ T cells were then resuspended in complete medium in a Lifecell Cell Culture Bag. The bag containing the cells and beads was then placed in a patient-dedicated incubator (37° C., 5% CO2).
On or around Day 3—The CD3+ cells were culture-expanded for ≈3 days at which point the contents of the single bag are split into 4 new Lifecell bags. The 4 bags were then returned to the patient-dedicated incubator (37° C., 5% CO2).
On or around Day 5—The 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% CO2 with the wave motion at 15 rocks/minute and with perfusion at 1 ml/minute.
Cell counts were determined each day and compared to cells stimulated and expanded using the static Xcellerate II process. Expansion was dramatically improved when cells were cultured in The Wave Bioreactor. Further, cell densities reached as high as 50×106 cells/ml in The Wave Bioreactor, as compared to a maximum cell density of 5×106 observed in the static Xcellerate II process. A total cell count of about 800 billion was achieved at day 12 of culture from a starting cell count of about 0.5×109 cells using The Wave Bioreactor.
Thus, 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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U.S. Classification 435/173.8, 435/372.3
International Classification C12N5/02, A61K35/12, C12N13/00, C12N5/0783
Cooperative Classification A61K2035/124, A61K2039/515, C12N2501/599, A61K2039/5158, C12N2501/58, C12N5/0636, C12N2501/515, A61K2035/122