Methods for selective expansion of different T cell subpopulations by altering cell surface signals and signal ratios

Abstract

Compositions and methods are provided for the specific expansion of subpopulations of specific T cell subtypes from the general T cell population. [Solution] A method for selectively expanding members of a T cell subpopulation, comprising exposing a mixed population of T cells to (a) a first agent that provides a primary activation signal to the members of the T cell subpopulation, thereby activating the T cells, and (b) a second agent and a third agent, each of which stimulates two or more different accessory molecules on the members of the T cell subpopulation, thereby stimulating proliferation of the activated T cells of (a), wherein the ratio of the first agent, the second agent, and the third agent is adjusted to induce selective proliferation of the members of the T cell subpopulation over members of other T cell subpopulations.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates, inter alia, to compositions of expanded T cell populations, methods for expanding T cell populations, and methods for using such cell populations. In some embodiments, the present invention relates to compositions and methods for the selective expansion of T cell subpopulations present in a mixed T cell population, as well as T cell subpopulations produced by the methods of the invention. [Background technology]

[0002] Background of the Invention For example, the ability of T cells to recognize antigenic structures associated with various cancers or infectious organisms is mediated by the T cell antigen receptor (TCR), which is composed of both α (alpha) and β (beta) chains or γ (gamma) and δ (delta) chains. The proteins that make up these chains are encoded by DNA, utilizing a unique mechanism to generate the tremendous diversity of TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides presented by major histocompatibility complex (MHC) class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of the TCR to antigenic peptides on APCs is a central event in T cell activation, occurring at the immune synapse upon contact between the T cell and APC. To maintain T cell activation, T lymphocytes typically require a second costimulatory signal. Costimulation is typically necessary for T helper cells to produce sufficient cytokine levels to induce clonal expansion. T cells can be expanded and activated ex vivo using exogenously administered cytokines and cell surface protein stimulation.

[0003] Ex vivo expanded T cells have been widely used in translational research and clinical trials for immunotherapy of cancer and opportunistic infections, as well as to prevent autoimmunity and graft-versus-host disease (GVHD) after transplantation. Although the administration of ex vivo expanded T cells has produced some favorable clinical results, the complexity of mass-producing various T cell products remains a major limitation for broader application. Early ex vivo culture conditions aimed only at high T cell numbers and resulted in cells expressing undesirable phenotypes with low in vivo proliferation. However, the focus has shifted to protocols that generate T cells with better in vivo engraftment, proliferation, and functionality.

[0004] Some of these early protocols used DYNABEADS® CD3 / CD28 CTS™ for T cell activation and expansion. This method can generate populations of T cells that exhibit a less differentiated phenotype and, in various in vivo models, can show increased and more durable antitumor activity compared to T cells expanded with soluble anti-CD3 antibodies (OKT-3) and IL-2. Similar protocols using a combination of CD3 and CD28 binding and / or signaling can be used for the isolation and activation of polyclonal T cells. However, they do not result in optimal activation of antigen-experienced memory T cells, such as tumor-infiltrating lymphocytes (TILs) and virus-specific T cells.

[0005] The quality and quantity of primary T cell activation signals, as well as the presence and type of costimulatory ligands, cytokines, and growth factors, determine the overall signal delivered to T cells and ultimately influence the fate of activated T cells. The successful expansion of such antigen-experienced T cells depends on the appropriate strength of TCR / CD3-mediated stimulation and the provision of optimal costimulatory signals and cytokines. Several reports have shown that the use of relatively strong CD3 signaling capabilities primarily expands naive T cells, which are thought to eliminate antigen-experienced T cells through activation-induced cell death (AICD) (Kalamasz et al., Immunother 27:405 (2004)). Antigen-experienced memory T cells and their various subsets have broad therapeutic implications for the treatment of cancer, autoimmune disorders, inflammatory diseases, allergic diseases, and infectious diseases. Therefore, there is a long-standing need for reliable, efficient, and rapid methods for expanding specific immune subpopulations, such as antigen-experienced memory T cells, regulatory T cells (Tregs), and Th17 cells.

[0006] The present invention addresses this need for the specific expansion of subpopulations of specific T cell subtypes from the general T cell population and also provides additional advantages. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kalamasz et al., Immunother 27:405(2004) Summary of the Invention

[0008] In particular, the present invention provides compositions and methods for the selective expansion of various T cell subpopulations, including (1) CD4 T cells, which are important for regulating immune responses; + CD4, a suppressor subset of T helper cells + CD25 + FOXP3 +These include, but are not limited to, (1) regulatory T cells (Tregs); (2) Th17 cells, a pro-inflammatory subset of CD4+ T helper cells that regulate host defense and are associated with tissue inflammation and various autoimmune diseases; and (3) memory T cells, or antigen-specific T cells, a long-lasting cell type that maintains immunity to previously exposed antigens. Disclosed herein are methods and compositions for the selective expansion of each of the above-mentioned T cell subpopulations. The present invention also provides sufficient numbers of T cell subpopulations for therapeutic use (e.g., adoptive immunotherapy, in vivo infusion, etc.). The methods and compositions described herein can be used to generate distinct T cell subpopulations for research purposes and / or clinical use. The resulting expanded T cell subpopulations have a wide range of applications in clinical settings.

[0009] In some embodiments, the present invention relates to compositions and methods for selectively expanding members of T cell subpopulations. Such methods include those that involve exposing a mixed population of T cells to (a) a first agent that provides a primary activation signal to members of the T cell subpopulation, thereby activating the T cells, and (b) a second agent and a third agent, each of which stimulates two or more different accessory molecules on members of the T cell subpopulation, thereby stimulating proliferation of the activated T cells of (a). In some embodiments, the ratio of the first agent, the second agent, and the third agent can be adjusted to induce selective expansion of the members of the T cell subpopulation over other members of the T cell subpopulation. The first agent can be an antibody (e.g., an anti-CD3 antibody). The second agent can also be an antibody. The third agent can be an antibody, a non-antibody protein, or a chemical agent (e.g., rapamycin). When a non-antibody protein is used, the protein may be a chemokine or cytokine (e.g., interleukin-1α, interleukin-2, interleukin-4, interleukin-1β, interleukin-6, interleukin-12, interleukin-15, interleukin-18, interleukin-21, transforming growth factor β1, etc.).

[0010] In some specific embodiments, the ratio of the first agent, the second agent, and the third agent may be adjusted to provide a lower concentration of the first agent relative to the second and / or third agent. Further, the lower concentration of the first agent may be about 0.06 units, about 0.1 units, about 0.2 units, about 0.3 units, about 0.34 units, or about 0.4 units. Alternatively, the first agent may be an anti-CD3 antibody at a concentration of about 0.34 units, and the second agent may be an anti-CD28 antibody at a concentration of 3.4 units.

[0011] In some embodiments, anti-CD3 may be used in an amount of 0.01 to 4.0 units (e.g., about 0.01 to about 3.5, about 0.01 to about 3.4, about 0.02 to about 3.4, about 0.05 to about 3.5, about 0.1 to about 3.4, about 0.2 to about 3.4, about 0.5 to about 3.0, about 0.1 to about 2.5, about 0.01 to about 1.0, about 0.05 to about 1.0, etc.), and anti-CD28 may be used in an amount of 1.0 to 5.0 units (e.g., about 1.0 to about 4.8, about 1.0 to about 4.5, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.9, about 1.5 to about 3.5, about 2.0 to about 3.5, about 2.5 to about 3.5, about 1.0 to about 4.8, etc.). Furthermore, the ratio of anti-CD3 to anti-CD28 may be in the range of 1:10 to 1:50 (e.g., about 1:12 to about 1:48, about 1:10 to about 1:48, about 1:12 to about 1:50, about 1:10 to about 1:40, about 1:10 to about 1:35, about 1:10 to about 1:30, etc.).

[0012] In some cases, the T cell subpopulation to be selectively expanded may be Treg cells. In such cases (and other cases), the lower concentration of the first agent may be about 0.01 units. Further, the first agent may be an anti-CD3 antibody at a concentration of about 0.01 units, about 0.06 units, about 0.1 units, about 0.2 units, about 0.3 units, about 0.34 units, or about 0.4 units, the second agent may be an anti-CD28 antibody, and the third agent may be an anti-CD137 antibody.

[0013] In some cases, the T cell subpopulation to be selectively expanded may be memory T cells. In such cases (and other cases), the lower concentration of the first agent may be about 0.005, about 0.01, or about 0.06 units. In other cases, the T cell subpopulation to be selectively expanded may be Th17 cells. Furthermore, the first agent may be an anti-CD3 antibody at a concentration of about 0.01, about 0.05, about 0.06, about 0.1, or about 0.34 units, and the second agent may be an anti-ICOS antibody.

[0014] The present invention also includes compositions and methods for selectively expanding T cell subpopulations. Such methods include (a) exposing T cells to CD3, CD28, and / or CD137 signals ex vivo and (b) culturing the T cells in a manner that permits the expansion of Th17 cells, antigen-experienced T cells, and / or regulatory T cells. In some cases, the CD3, CD28, and CD137 signals may be mediated by anti-CD3, anti-CD28, and / or anti-CD137 antibodies. In more specific cases, anti-CD3, anti-CD28, and anti-CD137 antibodies may be used at concentrations ranging from 0.01 units to 1.5 units (e.g., for expanding memory T cells). Furthermore, anti-CD3, anti-CD5, anti-ICOS, anti-CD6, anti-CD28, and anti-CD137 antibodies may be used at concentrations ranging from 0.06 units to 1.5 units (e.g., for expanding Th17 cells). Also, anti-CD3, anti-CD5, anti-ICOS, anti-CD6, anti-CD28, and anti-CD137 antibodies may be used in a range encompassing a concentration of about 0.34 units to 3.41 units (e.g., for the expansion of Treg cells). In addition, anti-CD3 antibodies may be used at lower concentrations compared to the concentrations of anti-CD5, anti-ICOS, anti-CD6, anti-CD28, and anti-CD137 antibodies. In some embodiments, T cells may be isolated using CD3 selection. Furthermore, Th17 cells expanded by the methods of the present invention may be isolated using CD3 selection. + , CD8 / CD4+ / and may produce IL-17 cytokine. Such Th17 cells may also produce IL-17, IL-21 and / or IL-22.

[0015] The memory T cells expanded by the method of the present invention include those selected from the group consisting of stem cell-like memory T cells, central memory T cells, and effector memory T cells. Stem cell-like memory cells may have one or more of the following markers: CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, IL-7Rα+, IL-2Rβ, CXCR3, and LFA-1. Central memory cells may have one or more of the following markers: CD3+, CCR7+, CD45RA-, CD45RO+, CD62L+ (L-selectin), CD27+, and CD28+. Furthermore, these central memory cells are capable of producing IL-2. Effector memory cells expanded by the methods of the present invention may have one or more of the following markers: CD28+ / -, CD27+ / -, CD3+, CD4+, CD8+, CCR7-, CD45RA-, CD45RO+. Effector memory cells may also be cells capable of producing IFNγ and IL-4. Treg cells expanded by the methods of the present invention may have one or more of the following markers: CD4 + , CD25+ 、 FOXP3 + , and CD127 負 / 低 The markers may be one or more of:

[0016] The present invention further includes compositions and methods for selectively expanding regulatory T cells. These methods include those comprising (a) exposing T cells to CD3 and CD28 signals ex vivo and (b) culturing the T cells in a manner that allows for the expansion of T regulatory cells. Such CD3 and CD28 signals may be mediated by anti-CD3 and anti-CD28 antibodies. Furthermore, anti-CD3 and anti-CD28 antibodies may each be used at a concentration range encompassing 0.34 to 3.4 units. In addition, anti-CD3 antibodies may be used at lower concentrations compared to the concentration of anti-CD28 and / or anti-CD28 antibodies.

[0017] The present invention also includes compositions and methods for selectively expanding Th17 cells. Such methods include those comprising: (a) exposing CD3+ T cells to CD3, CD28, CD5, and / or ICOS signals ex vivo; and (b) culturing the CD3+ T cells in a manner that allows for the expansion of Th17 cells, where the amounts of CD3, CD28, CD5, and / or ICOS may be the same or different. In such methods, the CD3, CD28, and ICOS signals may be mediated by anti-CD3, anti-CD28, anti-CD5, and anti-ICOS antibodies. Furthermore, anti-CD3, anti-CD28, anti-CD5, and anti-ICOS antibodies may be used in a concentration range encompassing about 0.06 to about 1.5 units for the expansion of Th17 cells. In addition, anti-CD3 antibodies may be used at lower concentrations compared to the concentrations of anti-CD28 and / or anti-ICOS antibodies.

[0018] The present invention also includes compositions and methods for selectively expanding antigen-experienced T cells. These methods include (a) exposing T cells to CD3, CD28, CD27, and / or CD137 signals ex vivo and (b) culturing the T cells in a manner that allows for the expansion of antigen-experienced T cells. The CD3, CD28, and CD27, and / or anti-CD137 signals may be provided by anti-CD3, anti-CD28, anti-CD27, and / or anti-CD137 antibodies. Anti-CD3, anti-CD28, anti-CD27, and anti-CD137 antibodies may be used at concentrations ranging from 0.01 to 1.5 units. Anti-CD3 antibodies may be used at lower concentrations compared to the concentrations of anti-CD28 and / or anti-CD137 antibodies.

[0019] The present invention also includes compositions comprising CD3+ (1) T cells and (2) beads containing (a) an anti-CD3 antibody and (b) an anti-CD28, anti-CD137, anti-ICOS, or anti-CD5 antibody capable of selectively expanding a T cell subpopulation. In some cases, the amounts of (a) the anti-CD3 antibody and (b) the anti-CD28, anti-CD137, anti-ICOS, or anti-CD5 antibody present may be the same or different. In certain cases, the T cell subpopulation may be selected from the group consisting of Th17 cells, antigen-experienced T cells, and / or regulatory T cells. Additionally, anti-CD3, anti-CD28, anti-CD5, anti-ICOS, anti-CD27, and anti-CD137 antibodies may be used at concentrations ranging from 0.01 to 1.5 units (e.g., for memory T cell expansion), 0.06 to 1.5 units (e.g., for Th17 cell expansion), or 0.34 to 3.41 units (e.g., for Treg cell expansion). In some cases, anti-CD3 antibodies may be used at lower concentrations compared to the concentrations of anti-CD28 and anti-CD137 antibodies.

[0020] The present invention further includes compositions comprising (1) T cells and (2) beads containing anti-CD3, anti-CD28, anti-ICOS, anti-CD5, and / or anti-CD137 antibodies capable of selectively expanding Th17 cells, wherein the Th17 cells are capable of producing one or more effector cytokines. In some cases, the amounts of anti-CD3 and anti-CD28 antibodies present on the beads may be the same or different. Furthermore, the one or more effector cytokines may be one or more cytokines selected from the group consisting of IL-17, IL-21, and IL-22.

[0021] The present invention includes a composition comprising (1) T cells and (2) beads containing anti-CD3, anti-CD28, and anti-CD137 antibodies that can selectively expand antigen-experienced memory T cells. In some cases, the T cells may be capable of recognizing a specific antigen, and the amounts of anti-CD3, anti-CD28, and anti-CD137 antibodies present on the beads may be the same or different. The specific antigen may be selected from the group consisting of viral antigens (e.g., CMV, EBV, influenza, HIV, etc.), bacterial (e.g., Streptococci M-protein, Neisseria fimbriae, Borrelia burgdorferi lipoprotein VisE, B. pseudomallei polysaccharide antigen, etc.), fungal or protozoan (e.g., Aspergillus fumigatus galactomannan, or F. tularensis lipopolysaccharide, etc.), and cancer antigens.

[0022] The present invention includes (1) T cells and (2) regulatory T cells (e.g., CD4+ CD25+ FOXP3+ CD127 低 / 負 and beads containing anti-CD3 and anti-CD28 antibodies capable of selectively expanding regulatory T cells (regulatory T cells that are resistant to T cell proliferation). In some cases, the amounts of anti-CD3 and anti-CD28 antibodies present on the beads may be the same or different. Regulatory T cell activity may include suppressive activity.

[0023] The present invention also includes compositions and methods for (a) treating an individual in need of treatment, (b) reconstituting the immune system of an individual in need of immune system reconstitution, and (c) administering adoptive immunotherapy to an individual in need of adoptive immunotherapy. Such methods include administering to the individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-experienced T cells, and / or regulatory T cells. The individual in need of treatment may be suffering from cancer, an inflammatory disease, an autoimmune disease, an allergic disease, an infectious disease, or a transplant-related disease. Further, exemplary cancers include lung cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, and skin cancer. The inflammatory disease may be selected from the group consisting of diabetes, rheumatoid arthritis, inflammatory bowel disease, familial Mediterranean fever, neonatal-onset multisystem inflammatory disease, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), systemic lupus erythematosus, uveitis, and Behcet's disease.

[0024] In such methods (and other methods of the invention), the T cells may be genetically modified. Furthermore, the genetic modification may result in the presence of one or more chimeric antigen receptors or genetically modified T cell receptors.

[0025] The present invention also includes compositions and methods for selectively altering the ratio of two T cell subtypes in a sample. Such methods include contacting a sample containing a mixed population of T cells with at least two stimulatory agents. In some cases, the stimulatory agents provide different amounts of signals to T cells in the mixed population. In certain cases, one T cell subtype may be selectively expanded relative to a second T cell subtype. Furthermore, the sample may include buffy coat cells from an individual and a subset of buffy coat cells (e.g., mononuclear cells). Furthermore, at least two stimulatory signals may stimulate CD3 and CD28 receptors. Also, at least one T cell subtype may be selectively removed from the mixed population. In some embodiments, Treg T cells may be increased proportionally relative to the total T cells in the mixed population. One way to do this is by contacting the cells with an amount of rapamycin suitable for eliminating non-Treg cells from the population. In additional embodiments, the total number of memory T cells may be reduced in the sample. Furthermore, the amount of the stimulatory signal CD3 may be less than half the amount of the CD28 stimulatory signal.

[0026] The present invention also includes methods for expanding Th17 cells by stimulating CD3 and CD5 cell surface receptors. In some embodiments, the present invention includes a method for expanding Th17 cells, comprising: (a) exposing a population of T cells to CD3 and CD5 signals ex vivo; and (b) culturing the population of T cells under conditions that allow for the expansion of Th17 cells. In certain embodiments of the present invention, the population of T cells may be exposed to one or more aryl hydrocarbon receptor agonists (e.g., 6-formylindolo[3,2-b]carbazole (FICZ)) and / or may not be exposed to exogenous interleukin-23. In some cases, the population of T cells is a mixed population of different T cell types. Additionally, methods of the invention include methods comprising contacting a population of T cells with one or more polarizing agents (e.g., interleukin-1β, interleukin-23, tumor growth factor-β, interleukin-6, interleukin-21, interleukin-2, anti-interleukin-4 antibody, and / or anti-interferon-γ antibody).

[0027] When used in reference to a protein, the term "exogenous" refers to a protein that is present in a composition but is not produced by cells present in the composition. For example, T cells present in a sample may produce IL-2. In such cases, exogenously added IL-2 refers to IL-2 that is placed in the sample as a non-cellular composition (e.g., IL-2 in a buffer).

[0028] In some embodiments, Th17 cells are engineered to express one or more chimeric antigen receptors, at least one of which includes a receptor with specificity for a cell surface antigen (e.g., antigen-associated) of a mammalian cell.

[0029] The present invention also includes compositions comprising a CD3 signal and a CD5 signal. In some cases, the compositions of the present invention may contain one or more aryl hydrocarbon receptor agonists (e.g., 6-formylindolo[3,2-b]carbazole (FICZ)), one or more cytokines (e.g., interleukin-1β, interleukin-23, tumor growth factor-β, interleukin-6, interleukin-21, and / or interleukin-2), and / or one or more antibodies (e.g., anti-interleukin-4 antibody and / or anti-interferon-γ antibody). In certain cases, the compositions of the present invention may contain interleukin-1β and interleukin-6. Compositions such as those described above may further comprise a population of T cells. Furthermore, the CD3 signal in the compositions of the present invention may be one or more anti-CD3 antibodies. Also, the CD5 signal in the compositions of the present invention may be one or more anti-CD5 antibodies.

[0030] The compositions of the invention may also comprise a population of T cells, a CD3 signal, a CD5 signal, an aryl hydrocarbon receptor agonist, and one or more cytokines. In certain embodiments, the compositions of the invention may comprise a population of T cells present in a mixture including: (a) a "buffy coat" sample, (b) a sample of white blood cells containing greater than 80% mixed T cells, (c) a sample containing greater than 80% CD4+ T cells, or (d) a sample containing greater than 80% Th17 cells.

[0031] The present invention further relates to methods for separating and activating T cells from a mixed population of cells (T cells and non-T cells, such as B cells). In some embodiments, such methods include: (a) contacting the mixed population of cells with one or more solid supports to which at least a first ligand is attached, the first ligand having binding affinity for a protein located on T cells present in the mixed population of cells, under conditions that allow binding of the T cells to the solid support and activation of the same T cells; and (b) separating the solid support-bound T cells from cells that are not attached to the solid support to obtain a purified T cell population. The one or more solid supports may be attached with at least a first ligand and / or at least a second ligand, each of which has binding affinity for a different protein located on individual T cells present in the mixed population of cells. Furthermore, the first ligand and the second ligand may be attached to the same solid support or different solid supports. In some cases, the first ligand may be either an anti-CD3 antibody or an anti-CD4 antibody. Furthermore, the second ligand may be any number of ligands, including a ligand selected from the group consisting of (a) an anti-CD5 antibody, (b) an anti-CD28 antibody, (c) an anti-CD137 antibody, and (d) an anti-ICOS antibody. Furthermore, the methods of the present invention include a method comprising releasing cells of the purified T cell population from a solid support, comprising releasing the T cells obtained in step (b) from the solid support. Additionally, the methods of the present invention include a method of expanding the released T cells once released from the solid support. In many cases, expansion of the released T cells occurs in culture medium (e.g., culture medium in which one or more chemokines or cytokines are present). Furthermore, one or more chemokines or cytokines may be present in step (a).Such one or more chemokines or cytokines include those selected from the group consisting of: (a) interleukin-1α, (b) interleukin-2, (c) interleukin-4, (d) interleukin-1β, (e) interleukin-6, (f) interleukin-12, (g) interleukin-15, (h) interleukin-18, (i) interleukin-21, and (j) transforming growth factor-β1.

[0032] Methods of the present invention also include methods for activating and expanding T cells. Such methods include those that involve contacting a mixed population of T cells with (a) a first agent that provides a primary activation signal to members of the T cell subpopulation (e.g., Treg cells, Th17 cells, etc.) by stimulating a molecule on the member of the T cell subpopulation, and (b) a second agent that stimulates a molecule on the member of the T cell subpopulation that is different from the molecule stimulated by the first agent. In many cases, T cells in the population (e.g., members of the T cell subpopulation) are activated and expanded. In additional cases, the first agent and the second agent may be bound to one or more solid supports. Furthermore, the T cells may be maintained under conditions that allow proliferation (e.g., proliferation of members of the T cell subpopulation). The solid support may be removed from contact with the T cells after a period of less than 120 hours. In other words, the solid support is placed in contact with the T cells for a limited period of time and then removed from contact with the T cells. This period may be from about 12 hours to about 120 hours (e.g., from about 12 hours to about 120 hours, from about 24 hours to about 120 hours, from about 36 hours to about 120 hours, from about 48 hours to about 120 hours, from about 60 hours to about 120 hours, from about 48 hours to about 96 hours, from about 60 hours to about 96 hours, from about 72 hours to about 96 hours, etc.). As discussed elsewhere herein, the first agent may be an anti-CD3 antibody. Further, the second agent may be an antibody. Additionally, the T cells may be contacted with a third agent that is an antibody or a non-antibody protein (e.g., a chemokine or cytokine).

[0033] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of the embodiment or aspect. [Brief explanation of the drawings]

[0034] [Figure 1A] Figures 1A-1B are line plots showing the fold expansion of CD4+CD25+CD127 low / - flow cytometry-sorted Tregs following activation with DYNABEADS® Treg Expander CD3 / CD28, and the retention of FOXP3+ cells (approximately 90% FOXP3+ cells). Figure 1A is a line plot showing the fold expansion following activation with DYNABEADS® Treg Expander CD3 / CD28. These data demonstrate an effective expansion of several hundred-fold. [Figure 1B] Figures 1A-1B are line plots showing the fold expansion of CD4+CD25+CD127 low / - flow cytometry-sorted Tregs and retention of FOXP3+ cells (approximately 90% FOXP3+ cells) following activation with DYNABEADS® Treg Expander CD3 / CD28. Figure 1B is a line plot showing % FOXP3+ cell retention. These data demonstrate that these cells retain high FOXP3 expression after 14 days in expansion medium. Cells are restimulated on day 9 using the same prototype beads. Increased proliferation is achieved using DYNABEADS® conjugated with lower amounts of CD3. [Figure 2] 1 is a bar graph showing the fold expansion of CMV-specific memory T cells 10 days after activation. Fold expansion negatively correlates with the increase in signal strength provided by DYNABEADS® Memory Cell Expander conjugated to higher amounts of agonistic CD3 antibody. [Figure 3]Figures 3A-3D are graphs showing IL-17 expression in T cells cultured under Th17-polarizing conditions and expanded using DYNABEADS® Th17 Expander CD3 / ICOS (anti-CD3 antibody conjugated at 1.5 and 0.06, and two different ICOS clones) or DYNABEADS® CD3 / CD28 (Cell Therapy System). Starting on day 3, IL-2 is added to the culture medium. On days 10-13, cells are stimulated with PMA-ionomycin for 4-5 hours before assessment of IL-17 expression. Histograms show intracellular expression of IL-17. Figure 3A is a graph showing IL-17 expression from cells expanded using DYNABEADS® Th17 Expander CD3 / ICOS (ISA-3), CD3 high (1.5). Figure 3B is a graph showing IL-17 expression from cells expanded with DYNABEADS® Th17 Expander CD3 / ICOS (ISA-3), CD3 low (0.06). Figure 3C is a graph showing IL-17 expression from cells expanded with DYNABEADS® Th17 Expander CD3 / ICOS (ICOS clone C398.4A purchased from eBiocienses, Affymetrix), CD3 low (0.06). Figure 3D is a graph showing IL-17 expression from cells expanded with DYNABEADS® CD3 / CD28 CTS™. The strength of the activation signal and the nature of the costimulation strongly influence the polarization and proliferation of Th17 cells. Note: Activation with DYNABEADS® Th17 Expander CD3 / ICOS (medium-0.3) results in a phenotype similar to "(high-1.5)" at a 1:1 bead:cell ratio. [Figure 4A] Figures 4A-4C are graphs showing T cells from three donors activated with DYNABEADS® Th17 Expander CD3 / ICOS. Figure 4A is a series of line graphs of fold expansion of T cells from donors A, B, and C, respectively. [Figure 4B]Figures 4A-4C are graphs showing T cells from three donors activated with DYNABEADS® Th17 Expander CD3 / ICOS. Figure 4B is a series of bar graphs showing the percentage of IL-17-producing cells obtained from selective expansion of T cells from donors A, B, and C, respectively, with DYNABEADS® Th17 Expander CD3 / ICOS at different bead:cell ratios (reported as B-C). [Figure 4C] Figures 4A-4C are graphs showing T cells from three donors activated with DYNABEADS® Th17 Expander CD3 / ICOS. Figure 4C is a series of bar graphs showing the relative numbers of IL-17-producing cells obtained from expansion of T cells from donors A, B, and C, respectively, using different bead:cell ratios (reported as B-C). [Figure 5] Effect of neutralizing antibodies. Mean % of IL-17-producing CD4 T cells stimulated with CD3 / ICOS or CD3 / CD28 beads and polarized with (+) or without (-) neutralizing antibodies (n=3 donors). P value (paired t-test) given for variations caused by blocking antibodies (NSD, not statistically different (upper panel)). Individual results; single donors (A, B, C) and stimulation with CD28 versus stimulation with ICOS (lower panel). [Figure 6] The effect of costimulation on cytokine production. Purified CD3+ T cells activated with Dynabeads prototypes CD3 / CD28, CD3 / CD5, or CD3 / ICOS expand Th17 subsets with different efficacies. Typically, CD3 / CD5 is the most efficient, followed by CD3 / ICOS, and CD3 / CD28 is the less effective stimulatory prototype. CD3 / CD5 stimulation typically induces IL-17A production in 25-50% of CD4+ T cells, with a high percentage becoming polyfunctional IL-17+IFN-γ+ upon restimulation on days 10-13. [Figure 7]Effect of costimulation on phenotype. T cells stimulated with CD3 / CD28, CD3 / CD5, or CD3 / ICOS co-express various levels of CCR4 and CCR6 after expansion, with CD3 / CD5 and CD3 / ICOS stimulation generating a higher percentage of CCR4+CCR6+ cells (77 and 73%) compared with CD3 / CD28 (46%). [Figure 8] T cells were activated by CD3 / CD5 (top) or CD3 / ICOS (bottom) and expanded in the presence of standard Th17-polarizing cytokines / antibodies (cytokine package) or 100 nM FICZ plus IL-1β and IL-6. On day 13, cultures were stimulated with PMA / ionomycin for 4–5 hours before assessment of IL-17A and IL-17F expression. Cytological findings show intracellular expression of cytokines. [Figure 9A] Early withdrawal of stimulatory DYNABEADS® enhances TH17 polarization. T cells were activated with CD3 / CD5, CD3 / ICOS, or CD3 / CD28 beads in the presence of standard Th17-polarizing cytokines / antibodies (TGF-β, IL-23, IL-6, IL-1β, and αIL-4 / α-IFN-γ neutralizing antibodies). Stimulatory DYNABEADS® were either i) left in culture throughout expansion, ii) removed 2 days (48 hours) after activation, or iii) removed 3 days (72 hours) after activation. On day 13, cultures were stimulated with PMA / ionomycin for 4–5 hours before assessment of intracellular IL-17A and IL-17F expression in CD4+ T cells. [Figure 9B] Shown are the percentage and absolute numbers of CD4+ T cells expressing the Th17-associated surface marker CD161 10 days after activation of cells prepared as described in the legend to Figure 9A. [Figure 10A]CD3 / CD5 isolated T cells can be directly polarized and expanded. T cells were isolated from PBMCs using DYNABEADS® CD3 / CD5 at bead:cell ratios of 1:3, 1:1, and 3:1. Isolation efficiency was compared using DYNABEADS® CD3 / CD28 CTS, which are commonly used for enrichment of T cells in T cell stimulation protocols. Isolation efficiency (%) is shown. [Figure 10B] Isolated T cells in Figure 10A were simultaneously expanded in the presence of standard Th17-polarizing cytokines / antibodies (TGF-β, IL-23, IL-6, IL-1β, and αIL-4 / α-IFN-γ neutralizing antibodies). Stimulatory DYNABEADS® were removed on day 3 post-activation. On day 13, cultures were stimulated with PMA / ionomycin for 4-5 hours before assessment of intracellular IL-17A and IL-17F expression in CD4+ T cells. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description In some embodiments, the present invention provides methods and compositions for, inter alia, the selective expansion of specific T cell subpopulations. These T cell subpopulations include, but are not limited to, Th17 cells, regulatory T cells (Tregs), and memory T cells. The specific T cell subpopulations disclosed herein can be used to treat various physiological conditions, disorders, and / or disease states.

[0036] Some aspects of the present invention In some embodiments, the present invention is based on the type and strength of signals for activation and / or proliferation of T cell subpopulations. Along these lines, it has been recognized that specific T cell subpopulations can be obtained and / or expanded in a mixed population by selective cell surface marker stimulation. It has further been recognized that changes in T cell receptor signal strength can also be used to obtain specific T cell subpopulations. In many cases, T cell subpopulations are obtained from a mixed T cell population (e.g., total T cells obtained from peripheral blood).

[0037] Stimulation of the T cell receptor can have several effects on specific T cells, such as (1) no effect on the T cell, (2) T cell activation, (3) T cell proliferation, (4) T cell polarization, (5) T cell differentiation (e.g., memory T cells), and (6) induction of apoptosis in T cells. The resulting effect often depends on the presence of the specific T cell, the nature of the stimulatory signal(s), if multiple signals are used, the ratio of the intensities of the multiple stimulatory signals (e.g., two, three, four, etc. signals), and the total or individual signal intensities to which the T cell is exposed.

[0038] In many cases, T cells are separated from other cell types prior to receptor stimulation. This may be done in a single step or multiple steps. Exemplary methods include: (1) buffy coat or apheresis isolation of mononuclear cells, (2) isolation of CD4+ cells using, for example, magnetic beads bearing agents bound to one or more CD4 receptors, and (3) fluorescence-activated cell sorting (see Example 1).

[0039] In some embodiments of the present invention, the ratio of two or more T cell signals is modulated in a manner that results in the selective expansion of one or more T cell subpopulations of a first set over one or more T cell subpopulations of a second set. In many cases, one or more (e.g., one, two, three, four, five, etc.) T cell subpopulations of the first set are smaller than one or more T cell subpopulations of the second set. In some cases, one or more T cell subpopulations of the first set may include a single T cell subpopulation, and one or more T cell subpopulations of the second set may include all other T cell subpopulations present. In some cases, a first T cell subpopulation (e.g., antigen-experienced (memory) T cells) is selectively expanded over a second T cell subpopulation (e.g., naive T cells). Additionally, one or more additional T cell subpopulations may be expanded along with the first T cell population.

[0040] In many cases, one signal is generated by stimulation of a first T cell receptor (e.g., a CD3 receptor) and another signal is generated by stimulation of a second, costimulatory T cell receptor (e.g., a CD28 receptor, a CD137 receptor, a CD27 receptor, a CD5 receptor, a CD6 receptor, an ICOS receptor, a CD134 receptor, etc.). The signal ratio may be altered in a manner that (a) promotes the proliferation of a particular T cell population, (b) promotes the elimination of another T cell population (e.g., via apoptosis, inhibition of cell growth, no proliferative effect, etc.), or is both (a) and (b). In some cases, one or more additional T cell receptors may also be stimulated, or other signals may be provided to the T cells.

[0041] Exemplary ratios of second T cell receptor stimulatory signals to first T cell receptor stimulatory signals vary depending on the T cell subpopulation to be obtained, and include about 50:1 to about 1:200 (e.g., about 1:5, about 1:10, about 1:15, about 1:20, about 1:40, about 50:1 to about 1:40, about 50:1 to about 1:30, about 40:1 to about 1:40, about 30:1 to about 1:40, about 40:1 to about 1:20, about 40:1 to about 1:10, about 50:1 to about 1:1, about 50:1 to about 5:1, about 40:1 to about 5:1, about 50:1 to about The ratio may be about 10:1, about 50:1 to about 15:1, about 50:1 to about 20:1, about 40:1 to about 5:1, about 30:1 to about 3:1, about 20:1 to about 3:1, about 15:1 to about 3:1, about 10:1 to about 5:1, about 1:5 to about 1:10, about 1:3 to about 1:20, about 1:8 to about 1:25, about 1:3 to about 1:40, about 1:5 to about 1:50, about 1:10 to about 1:50, about 1:10 to about 1:100, about 1:10 to about 1:150, about 1:10 to about 1:200, about 1:5 to about 1:150, about 1:5 to about 1:200, etc.

[0042] For illustrative purposes, the signals provided by anti-CD3 and anti-CD28 antibodies may be present in a ratio of 1:10. It has been found that for the proliferation of some T cell subpopulations, a lower amount of CD3 signal is more desirable than a second signal (e.g., CD28 and / or CD137 signal). In some cases, when three or more T cell receptor signals are provided, the ratio of each signal may be different, or two or more of the signal ratios (e.g., two out of three) may be the same. By way of example, CD3, CD28, and CD137 signals may be present in a ratio of 1:10:10. When each of these signals is produced by an antibody, this generally means that one part anti-CD3 antibody is present with ten parts of both anti-CD28 and anti-CD137 antibodies. This, of course, assumes that the amount of receptor stimulation for each of the three receptors is equal, depending on their cognate antibodies.

[0043] One issue to keep in mind is the characterization of a mixture containing a population of T cells generated by the methods of the present invention. In many cases, the compositions and methods of the present invention are directed to altering the ratio of a particular subpopulation of T cells in the mixture. For example, the methods of the present invention may result in a particular type of T cell being removed from the mixed population, for example, by apoptosis or dilution. Thus, one aspect of the present invention relates to the amount of expansion or depletion of a T cell population in the mixture, as well as the mixture itself. For example, if there are two T cell subpopulations in the mixture (e.g., Th17 T cells and Th1 T cells), and these subpopulations are present in a ratio of, for example, 1:1, the present invention includes a method in which one T cell subpopulation is expanded relative to the other T cell subpopulation. For illustrative purposes, the ratio may be from about 1:1.5 to about 1:100,000 (e.g., from about 1:1.5 to about 1:100,000, from about 1:1.5 to about 1:80,000, from about 1:1.5 to about 1:50,000, from about 1:1.5 to about 1:10,000, from about 1:1.5 to about 1:5,000, from about 1:2,500 to about 1:25,000, from about 1: The ratio may be changed to about 1:2,500 to about 1:60,000, about 1:2,500 to about 1:80,000, about 1:2,500 to about 1:100,000, about 1:5,000 to about 1:100,000, about 1:5,000 to about 1:80,000, about 1:5,000 to about 1:50,000, about 1:5,000 to about 1:25,000, etc.

[0044] Additionally, the present invention relates to compositions and methods for altering the proportion of specific subpopulations of T cells in a mixture, wherein the proportion of one T cell subpopulation is at least 200,000-fold greater than another T cell subpopulation (e.g., about 1,000-fold to about 200,000-fold, about 5,000-fold to about 200,000-fold, about 10,000-fold to about 200,000-fold, about 20,000-fold, etc.). The T cell population is increased by about 2-fold (about 50,000-fold to about 200,000-fold, about 75,000-fold to about 200,000-fold, about 1,000-fold to about 120,000-fold, about 5,000-fold to about 120,000-fold, about 10,000-fold to about 120,000-fold, about 1,000-fold to about 80,000-fold, about 10,000-fold to about 80,000-fold, etc.). An example of what is meant by "fold" is illustrated as follows: If two T cell subpopulations are present at an initial ratio of 1:2, changing their ratio to 1:8 is a 2-fold increase of one T cell subpopulation relative to the other T cell subpopulation.

[0045] Another factor that can lead to the selective expansion of individual T cell subpopulations is stimulatory signal strength. "Stimulatory signal strength" refers to the total signal strength per T cell. This includes the strength of various signals (e.g., a signal stimulating a first T cell surface receptor, a signal stimulating a second T cell surface receptor, a signal stimulating a third T cell surface receptor, etc.) and the combined signals to which each T cell in the population is exposed. Thus, the present invention also relates to the amount of stimulatory signal received by each cell in a mixture of various T cell subpopulations. The stimulatory signal can be modulated by changing the concentration of stimulatory agents, their ratio, or the ratio of cell numbers to surfaces containing the stimulatory agents. In embodiments in which the stimulatory agents are bead-based, the bead:cell ratio can be varied. Bead:cell ratios include about 1:5000, about 1:2500, about 1:1000, about 1:500, about 1:250, about 1:100, about 1:90, about 1:80, about 1:70, about 1:60, about 1:50, about 1:40, about 1:30, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 8:1, about 10:1, or about 15:1 beads per cell.

[0046] In some cases, one or more cytokines may be added to the T cell population. Often, these cytokines are IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IFN-gamma, and TGF-beta. If Th17 polarization is desired, one or more of the following cytokines may be used: IL-1 beta, IL-6, TGF-beta, IL-21, IL-23, and neutralizing anti-IL-4 and anti-IFN-gamma antibodies. Additionally, neutralizing antibodies against IL-1 beta, IL-6, TGF-beta, IL-23, and IL-4 and IFN-gamma signals may be used to selectively expand Th17 cells.

[0047] Table 1 shows several different T cell subtypes that can be obtained using the methods of the present invention. Table 1 shows various signals that can be used to selectively expand specific types of T cells.

[0048] (Table 1) T cell subtype stimulation TIFF0007623980000001.tif110163 * "and / or" when more than one agent is listed

[0049] Specifically, the present invention includes methods for selectively expanding one or more T cell subpopulations. Such methods result in the expansion or depletion of specific T cells in a sample. One example is stimulation of naive T cells, memory T cells, Th1 T cells, and regulatory T cells (Treg) with CD3 and CD28 receptors in combination with interleukin-2. It has been shown that modulation of total CD3 / CD28 stimulation can expand naive T cells while depleting memory T cells from a sample (U.S. Patent No. 8,617,884, the disclosure of which is incorporated herein by reference). In connection with one aspect of the present invention, it has been shown that different T cell subpopulations can be selectively expanded by adjusting the signal ratio and total signal intensity. As an example, Treg cells proliferate better when the CD3 signal is lower than the CD28 signal (see FIG. 1A). Identifying selective growth conditions can be used to increase the proportion of members of one T cell subpopulation over members of one or more other T cell subpopulations in a sample, even when various cells of the various T cell subpopulations proliferate in response to the same stimulus. For illustrative purposes, assuming that Treg T cells comprise 1% of a mixed population, and that naive T cells and memory T cells comprise 1.5% and 3%, respectively, of the same mixed population, a stimulatory signal may be adjusted to selectively expand Treg T cells while inducing the elimination of memory T cells. The end result may be a mixed population in which Treg T cells comprise 40% of the mixed population, and naive T cells, memory T cells, and Th1 T cells comprise 2%, 0.5%, and 2.5%, respectively.

[0050] An additional agent that can be used for the selective expansion or depletion of one or more T cell subtypes (e.g., CD4+CD25+FOXP3+ regulatory T cells, CD4+CD25+FOXP3- regulatory T cells, CD4+CD25- T cells, etc.) is rapamycin.

[0051] Mammalian Immune System The mammalian immune system uses two general adaptive mechanisms to protect the body against environmental pathogens: when a pathogen-derived molecule is encountered, the immune response is highly activated to ensure protection against the pathogen.

[0052] The first mechanism is the nonspecific (or innate) inflammatory response. The innate immune system can recognize specific molecules present on pathogens but not on the body itself. The second mechanism is the specific or acquired (or adaptive) immune response. The adaptive immune response is tailored to the pathogen in question. The adaptive immune system develops specific immunoglobulin (antibody) responses to many different molecules present on the pathogen, called antigens. In addition, a wide repertoire of T cell receptors is sampled for their ability to bind to processed forms of antigens bound to MHC class I and II on antigen-presenting cells (APCs), such as dendritic cells (DCs).

[0053] The immune system recognizes and responds to structural differences between self and non-self proteins. Proteins that the immune system recognizes as non-self are called antigens. Pathogens typically express numerous, highly complex antigens. Acquired immunity has specific memory for antigenic structures, and repeated exposure to the same antigen increases the response, increasing the level of induced protection against that particular pathogen.

[0054] Acquired immunity is mediated by specialized immune cells called B and T lymphocytes (or simply B and T cells). B cells generate and mediate their function through the action of antibodies. B cell-dependent immune responses are referred to as "humoral immunity" because antibodies are found in bodily fluids. T cell-dependent immune responses are referred to as "cell-mediated immunity" because effector activity is directly mediated by the local action of effector T cells. The local action of effector T cells is amplified by the synergistic interaction of T cells with secondary effector cells, such as activated macrophages. This results in the killing of pathogens and the prevention of disease.

[0055] Immune cells may require specific stimuli for activation. For example, the use of anti-CD3 / CD28 provides an activation signal for some T cell populations. Naive T cells are thought to require at least two signals for activation. Signal 1 is antigen-specific and is triggered by a peptide / major histocompatibility complex (MHC) complex presented by an antigen-presenting cell (APC) and received through the T cell receptor (TCR) / CD3 complex. For some T cell subpopulations, signal 2 is delivered by the antigen-presenting cell, and one of the candidate molecules for this receptor is the T cell antigen CD28. When both the TCR / CD3 and CD28 T cell receptors are occupied by the appropriate ligand, T cells are stimulated to proliferate and produce IL-2 (a cytokine essential for T cell proliferation), whereas occupation of the T cell receptor alone is thought to promote T cell anergy or apoptosis.

[0056] In vitro, it has been shown that T cell growth and cytokine production can be stimulated by culturing T cells with anti-CD3 antibodies immobilized on a solid phase (e.g., beads or tissue culture plates) and adding soluble CD28 antibodies (Sommer et al., Eur. J. Immunol. 23:2498-2502 (1993); Sunder-Plassmann et al., Blood 87:5179-5184 (1996)). More recently, it has been shown that immobilizing both CD3 and CD28 antibodies on the same or different solid phases can also induce T cell proliferation (Levine et al., J. of Immunol. 159:592-130 (1997); Li et al., Science 283:848-851 (1999)).

[0057] The present invention enables one of skill in the art to generate various T cell subpopulations in a reliable, effective, and efficient manner and to use the expanded T cell subpopulations for a variety of purposes, including, but not limited to, therapeutic and research / discovery purposes. As further described below, T cell subpopulations include, but are not limited to, regulatory T cells, Th17 cells, and antigen-experienced memory cells.

[0058] regulatory T cells Aspects of the present invention relate to methods for efficiently generating regulatory T cells (or "T regulatory cells" or "Tregs") and using them in generating T cell populations that have applications, for example, in immunotherapy. Treg cells are CD4+, CD25+, FOXP3+, CD127 負 / 低 In some cases, Treg cells expanded using the compositions and methods of the invention are CD4+, CD25+, FOXP3-. Compositions and methods for generating FOXP3 regulatory T cells are described in U.S. Patent No. 9,119,807 to Aarvak et al.

[0059] Naturally occurring regulatory T (Treg) cells negatively regulate the activation of other T cells, including effector T cells, and cells of the innate immune system, and can be used in immunotherapy for autoimmune diseases and provide transplant tolerance. Various populations of Treg cells have been described, including naturally occurring CD4+CD25+FOXP3+ cells and induced Tr1 and Th3 cells, which secrete IL-10 and TGF, respectively.

[0060] Treg cells are characterized by sustained suppression of effector T cell responses. Conventional or conventional Treg cells can be found in the spleen or lymph nodes, or in the circulation. Tregs have proven highly effective in preventing GVHD and autoimmunity in mouse models. Clinical trials using Treg adoptive transfer in transplantation and for the treatment of diabetes and other indications are ongoing. The relative frequency of Tregs in peripheral blood is approximately 1-2% of total lymphocytes, implying the need for ex vivo expansion of Tregs prior to adoptive transfer for most clinical applications. Generating sufficient Tregs by ex vivo expansion is a major challenge for the application of Treg therapy in humans.

[0061] Th17 cells T helper 17 cells (or "Th17 cells" or "Th17 helper cells") are an inflammatory subset of CD4+ T helper cells that regulate host defense and are involved in tissue inflammation and various autoimmune diseases. Th17 cells have been found in various human tumors, but their function in cancer immunity is unknown. When adoptively transferred into tumor-bearing mice, Th17 cells have been found to be more potent than Th1 or nonpolarized (Th0) T cells in eradicating melanoma (Muranski et al., Blood. 112:362-373 (2008)). Th17 cells are developmentally distinct from the Th1 and Th2 lineages. Th17 cells are CD4+ cells responsive to IL-1R1 and IL-23R signaling and produce the cytokines IL-17A, IL-17F, IL-17AF, IL-21, IL-22, IL-26 (human), GM-CSF, MIP-3α, and TNFα. The phenotype of Th17 cells is CD3 + , CD4 + , CD161+. One obstacle to the use of Th17 cells for adoptive cell transfer is the identification of robust culture conditions (tumor microenvironment) that can propagate Th17 cell subsets and their unstable phenotypes in vivo.

[0062] The present invention relates, in part, to compositions and methods for generating specific T cell subtypes. One specific T cell subtype that can be produced using the compositions and methods of the present invention is Th17 cells. Accordingly, in some embodiments, the present invention relates to compositions and methods for expanding (e.g., selectively expanding) Th17 cells using CD3 signaling (e.g., via an anti-CD3 antibody) and CD5 signaling (e.g., via an anti-CD5 antibody), one or more cytokines, and neutralizing antibodies (e.g., anti-IL-4 antibody and anti-IFNγ antibody). In some cases, the compositions and methods for selective expansion of Th17 cells are adjusted in a manner that results in a reduction or elimination of the amount of one or more neutralizing antibodies.

[0063] One problem with many T cells is T cell plasticity. In other words, some T cells can change to become different subtypes. This change in T cells is often mediated by T cells in the surrounding environment. For example, some T cell subtypes express IL-4 and / or IFNγ. These proteins exert physiological effects on surrounding T cells and, in some cases, mediate the differentiation of one type of T cell into another type of T cell.

[0064] The expansion (e.g., selective expansion) of T cell subsets, such as Th17 cells, may be achieved by using one or more signals, adjusting the intensity of one or more signals, adjusting the ratio of intensities between two or more signals, and removing one or more signals. Conditions may be selected based on the ratio of two parameters or three or more parameters. Two examples are as follows: (1) the ratio of CD3 and CD5 signals may be adjusted, and (2) adjusting the ratio of CD3 and CD5 signals may be combined with adjusting the sum of CD3 and CD5 signals. For example, when the expansion of Th17 cells is desired, two signals may be selected and used (e.g., CD3 and CD5 signals). These signals may be adjusted at a ratio of, for example, about 1:1 to about 1:50 (e.g., about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:5 to about 1:15, about 1:5 to about 1:20, about 1:8 to about 1:12, about 1:8 to about 1:15, about 1:8 to about 1:20, etc.).

[0065] The present invention also includes the use of aryl hydrocarbon receptor (AHR) antagonists for the proliferation of T cells (e.g., Th17 T cells). It has been found that Th17 T cells can be proliferated by exposing T cells to CD3 signals (e.g., agonistic anti-CD3 antibodies), CD5 signals (e.g., agonistic anti-CD5 antibodies), IL-1β, IL-6, and an AHR agonist (e.g., FICZ).

[0066] Exemplary AHR ligands that can be used in the practice of the present invention include, but are not limited to, FICZ (6-formylindolo[3,2-b]carbazole), dFICZ (6,12-diformylindolo[3,2-b]carbazole), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2-(1′H-indole-3′-carbyl)-thiazole-4-carboxylic acid methyl ester (ITE), quercetin, indole-3-carbinol, resveratrol, curcumin, M50367 {3-[2-(2-phenylethyl)benzimidazol-4-yl]-3-hydroxypropanoic acid, and VAF347 {[4-(3-chloro-phenyl)-pyrimidin-2-yl]}, tryptophan derivatives such as indigo dye and indirubin, tetrapyrroles such as bilirubin, and the arachidonic acid metabolites lipoxin A4 and prostaglandin G.

[0067] In some cases, the present invention includes compositions and methods for expanding Th17 cells, in which T cells are exposed to the following reagents: one or more agonistic anti-CD3 antibodies, one or more agonistic anti-CD5 antibodies, and one or more AHR agonists (e.g., FICZ). In some cases, one or more cytokines may also be used. Exemplary cytokines include IL-1β and IL-6. Thus, in some cases, T cells are exposed to one or more agonistic anti-CD5 antibodies, one or more AHR agonists (e.g., FICZ), IL-1β, and IL-6.

[0068] In some cases, the invention includes compositions and methods for expanding Th17 cells, wherein T cells are exposed to the following reagents: one or more agonistic anti-CD3 antibodies, one or more agonistic anti-CD5 and / or anti-CD28 antibodies, and one or more cytokines. Exemplary cytokines include TGF-β, IL-1β, IL-6, and IL-23. Thus, in some cases, T cells are exposed to one or more agonistic anti-CD3 antibodies, one or more agonistic anti-CD5 and / or anti-CD28 antibodies, TGF-β, IL-1β, IL-6, and IL-23.

[0069] Thus, the present invention includes compositions and methods for the expansion of Th17 cells, wherein the cells of the starting T cell population are not exposed to TGF-β and / or IL-23.

[0070] The T cell populations used in the practice of the present invention may be present in, for example, (i) a "buffy coat" sample, (ii) a sample of white blood cells depleted of non-T cells (e.g., a sample containing more than 80% mixed T cells), (iii) a sample of CD4+ T cells (e.g., a sample containing more than 80% CD4+ T cells), or a sample containing predominantly Th17 cells (e.g., a sample containing more than 80% Th17 cells). Thus, in some cases, the methods of the present invention relate to the selective expansion of one or more T cell subpopulations.

[0071] With regard to specific formulations for the expansion of Th17 T cells, and associated expansion methods, conditions are typically adjusted to achieve high levels of Th17 cell expansion.

[0072] One factor that can be adjusted is the ratio of CD5 signal (e.g., an agonist anti-CD5 antibody) to CD3 signal (e.g., an agonist anti-CD3 antibody). In many cases, the amount of CD3 signal is less than the CD5 signal. Exemplary ratios of CD5 signal to CD3 include ratios of about 1 to about 1.5, about 1 to about 2, about 1 to about 3, about 1 to about 5, about 1 to about 8, about 1 to about 10, about 1 to about 12, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 75, and about 1 to about 100.

[0073] Exemplary antibodies that can be used in the practice of the invention include the BC3 anti-CD3 antibody (a clone expressing this antibody is available from the American Type Culture Collection (HB-10166)) and the UCHT2 anti-CD5 antibody, available from several sources, including eBioscience, Inc., San Diego, CA.

[0074] In general, antibodies tend to have different specificities and affinities for their cognate ligands. Therefore, the ratio of CD3 to CD5 signals may be adjusted to be equivalent to that of BC3 to UCHT2 antibodies. In other words, the ratios shown above are partially related to data derived from BC3 and UCHT2 antibodies, and the ratio of these antibodies induces a physiological effect on T cells. Therefore, other CD3 and CD5 signals may be used, and the amounts of these other signals may be adjusted to achieve the same physiological effect. In other words, the present invention includes various CD3 and CD5 signaling agents that achieve the same physiological effect as the ratio of BC3 and UCHT2 antibodies described above. Such physiological effects may be measured by methods described elsewhere herein (e.g., Example 10 and related data).

[0075] In addition to CD3 and CD5 signaling agents, reagents may be present in the compositions and used in the methods of the invention. Some of these additional reagents include AHR agonists (e.g., FICZ), cytokines (e.g., IL-1β and IL-6), and one of more neutralizing antibodies (e.g., anti-αIL-4 and anti-α-IFN-γ neutralizing antibodies). The reagents may be adjusted to achieve the results shown in, for example, Figure 8. Specifically, the amounts of AHR agonist and cytokine used are typically in the range of about 1 nM to about 1 mM (e.g., about 1 nM to about 800 nM, about 1 nM to about 500 nM, about 1 nM to about 250 nM, about 25 nM to about 1 mM, about 25 nM to about 500 nM, about 25 nM to about 300 nM, about 50 nM to about 300 nM, about 75 nM to about 500 nM, about 75 nM to about 300 nM, etc.).

[0076] memory T cells Memory T cells, or antigen-experienced cells, are experienced prior to encountering an antigen. These T cells are long-lived and can recognize antigens and rapidly and potently influence the immune response to antigens to which they were previously exposed. Memory T cells can include: stem cell-like memory cells (T SCM ), central memory cells (T CM ), effector memory cells (T EM ). T SCM The cells have the phenotype CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but not T SCM The cells also express high amounts of IL-2Rβ, CXCR3, and LFA-1. CM The cells express L-selectin and CCR7 and are T CM The cells secrete IL-2 but not IFNγ or IL-4. EM The cells do not express L-selectin or CCR7, but produce effector cytokines such as IFNγ and IL-4.

[0077] The present invention provides methods and compositions for the selective expansion of the above-mentioned T cell subpopulations. Previous methods do not enable those skilled in the art to selectively expand specific T cell subtypes in the manner described herein. While signal strength can be adjusted to some extent by varying the ratio of T cells to standard DYNABEADS® CD3 / CD28 (Kalamazset et al., J. Immunother. 27:405-418 (2004)), the local concentration of stimulatory CD3 antibodies at the contact area between DYNABEADS® and the cell surface is unaffected by this approach and remains high. The data herein disclose amounts and ratios of primary antibodies and various costimulatory antibodies bound to the surface of DYNABEADS® that allow for fine tuning of T cell responses and result in the selective activation and expansion of various specific T cell subsets, such as Th17, antigen-experienced T cells, and Tregs.

[0078] I. Definition Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0079] Antibodies for use in the methods of the invention may be of any species, class, or subtype, provided that such antibodies are capable of reacting with a target of interest, e.g., CD3, TCR, or CD28, as appropriate.

[0080] Thus, for purposes of use in the present invention, an "antibody" includes: (a) any of the various classes or subclasses of immunoglobulins (e.g., IgG, IgA, IgM, IgD, or IgE from any animal, e.g., any of the commonly used animals, e.g., sheep, rabbit, goat, mouse, camelid, or egg yolk); (b) monoclonal or polyclonal antibodies; (c) Intact antibodies or antibody fragments, monoclonal or polyclonal, including those that contain the binding region of an antibody, e.g., fragments that do not contain the Fc portion (e.g., Fab, Fab', F(ab')2, scFv, V, H H-fragment, or other single-domain antibody), are so-called "half-molecule" fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components within an intact antibody. Fv can be defined as a fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (d) Antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, antibody fragments, "humanized antibodies," chimeric antibodies, or synthetically produced or modified antibody-like structures.

[0081] Also included are functional derivatives or "equivalents" of antibodies, such as single chain antibodies, CDR-grafted antibodies, etc. Single chain antibodies (SCAs) may be defined as genetically engineered molecules containing the variable region of a light chain and the variable region of a heavy chain joined by a suitable polypeptide linker as a fused single chain molecule. Also included are VHH antibodies, which may be monovalent or bivalent.

[0082] Methods for preparing antibody fragments and synthetic and derivatized antibodies are well known in the art and have been widely described in the literature and will not be described here.

[0083] The term "activated," as used herein, refers to the state of a cell after ligation of cell surface moieties sufficient to induce measurable morphological, phenotypic, and / or functional changes. Within the context of T cells, such activation can be the state of a T cell that has been sufficiently stimulated to induce cell proliferation. Activation of T cells can also induce cytokine production and / or secretion, as well as up- or down-regulation of expression of cell surface molecules such as receptors or adhesion molecules, or up- or down-regulation of secretion of certain molecules, and the capacity for regulatory or cytotoxic effector function. In the context of other cells, the term connotes either up- or down-regulation of specific physicochemical processes.

[0084] As used herein in reference to antibodies, the term "unit" is limited to anti-CD3-mediated physiological effects. Specifically, 0.34 units of anti-CD3 is the amount of antibody that, when contacted with a mixed T cell population in the presence of 3.4 units of anti-CD28, results in a 1000-fold expansion of Treg cells after 14 days (see Example 2 and Figure 1). A unit is defined by the number of antibodies present. Thus, in the above example, there are 10 times more anti-CD28 molecules than anti-CD3 molecules.

[0085] The term "stimulation," as used herein, refers to a primary response induced by ligation of cell surface moieties. For example, in the context of receptors, such stimulation involves receptor ligation and a subsequent signaling event. With respect to stimulation of T cells, such stimulation, in one embodiment, refers to ligation of T cell surface moieties, which subsequently induces a signaling event, such as binding of the TCR / CD3 complex. Furthermore, a stimulatory event may activate the cell and up- or down-regulate the expression of cell surface molecules, such as receptors or adhesion molecules, or up- or down-regulate the secretion of molecules, such as down-regulation of transforming growth factor beta (TGF-β). Thus, even in the absence of a direct signaling event, ligation of cell surface moieties may result in reorganization of the cytoskeletal structure or fusion of cell surface moieties, each of which may serve to enhance, modify, or alter subsequent cellular responses.

[0086] As used herein, the terms "agent," "ligand," or "stimulatory agent" refer to a molecule that binds to one or more distinct populations of cells (e.g., members of a T cell subpopulation) and induces a cellular response. An agent may bind to any cell surface moiety, such as a receptor, antigenic determinant, or other binding site present on a target cell population. Agents may be proteins, peptides, antibodies and antibody fragments thereof, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), and the like. In the description and context of T cell stimulation, antibodies are used as a prototypical example of such an agent.

[0087] As used herein in reference to T cells, the terms "selective proliferation" and "selectively expanding" refer to the ability of certain T cells to proliferate under conditions in which other T cells either do not proliferate or proliferate at a slower rate. As an example, assume that T cell subtypes 1 and 2 are present in a mixed population at percentages of 5% and 10%, respectively, of the total T cells present. If certain conditions result in T cell subtype 1 accounting for 30% of the total T cells present and T cell subtype 2 accounting for 12%, then T cell subtype 1 is selectively expanded over T cell subtype 2, even though T cell subtype 2 is now a larger portion of the total T cell population. T cell subtype 2 is selectively expanded over the general members of the mixed population of T cells, in the sense that, as a total percentage of T cells, T cell subtype 2 is now present at an increased "frequency." Thus, selective proliferation refers to the expansion of a particular T cell subtype over the general population of T cells and often also results in the expansion of the T cell subtype. The above example can be referred to as a condition for the selective proliferation of T cell subtype 1, although T cell subtype 2 also proliferates.

[0088] As used herein, the term "expose" refers to bringing into a state or condition of close or direct contact.

[0089] As used herein, the term "proliferation" means to grow or multiply by producing new cells.

[0090] A "subject" can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice, and rats. In one aspect, the subject is a human. A "subject" can be a "patient" (e.g., someone under the care of a physician), although in some cases the subject is not a patient.

[0091] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to proliferation and / or activation and / or polarization of T cells.

[0092] As used herein, "separation" includes any means of substantially purifying one component from another (e.g., by filtration, affinity, buoyant density, or magnetic attraction).

[0093] As used herein, "surface" refers to any surface capable of having an agent attached thereto, including but not limited to metals, glass, plastic copolymers, colloids, lipids, and cell surfaces. Essentially, any surface capable of having an agent bound or attached thereto.

[0094] As used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0095] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating the disorder and / or symptoms associated therewith. It is understood that treating a disorder or condition does not require, although does not preclude, the complete elimination of the disorder or condition or symptoms associated therewith.

[0096] II. Methods for generating T cell subpopulations The methods of the present invention can be utilized to selectively expand one or more substantially pure specific T cell subpopulation(s). Exemplary T cell subpopulations include, but are not limited to, Treg cells, Th17 cells, and memory T cells. Exemplary uses for expanded T cell subpopulations are disclosed herein.

[0097] Source of mixed populations of T cells The starting source of the mixed population of T cells may be blood (e.g., circulating blood), which may be isolated from a subject. Circulating blood may be obtained from one or more units of blood or from 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. T cells can be obtained from several sources, including blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gastrointestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, or any other lymphoid tissue, and tumor. T cells can be obtained from autologous or allogeneic sources, including T cell lines and umbilical cord blood. T cells can also be obtained from xenogeneic sources, such as mice, rats, non-human primates, and pigs.

[0098] In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll separation. T cells may be isolated from the subject's circulating blood. Blood may be obtained from the subject 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. Prior to exposure to the sensitizing composition and subsequent activation and / or stimulation, a source of T cells is obtained from the subject. In some embodiments, cells collected by apheresis may be washed to remove the plasma protein fraction, and the cells may be placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments of the present invention, cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution may be calcium-free, magnesium-free, or free of many, but not all, divalent cations. As one of ordinary skill in the art will readily appreciate, the washing step may be accomplished by methods known to those of ordinary skill in the art, such as by use of a semi-automated "flow-through" centrifuge (e.g., Baxter Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as calcium (Ca) and magnesium (Mg)-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0099] In other embodiments, T cells are isolated from peripheral blood lymphocytes by lysing or removing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific T cell subpopulations can be further isolated by positive or negative selection techniques.

[0100] In some embodiments, T cells can be positively selected for CD3+ cells. Any selection technique known to those of skill in the art may be used. One non-limiting example is flow cytometry sorting. In another embodiment, T cells can be isolated by incubation with a solid support bound to an anti-CD3 antibody. One non-limiting example is anti-CD3 / anti-CD28-conjugated beads, such as DYNABEADS® Human T-Expander CD3 / CD28 (Life Technologies Corp., catalog number 11141D), for a period sufficient to positively select the desired T cells. In a further embodiment, the period ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In another embodiment, the period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. Longer incubation times, such as 24 hours, may increase cell yield. In any situation where there are few T cells compared to other cell types, longer incubation times may be used to isolate T cells. In one aspect of the present invention, enrichment of T cell populations by negative selection can be achieved by a combination of antibodies directed against surface markers unique to the negatively selected cells. One possible method is cell sorting and / or selection by magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. In some embodiments, the expansion fold may vary based on the starting material due to variability in donor cells. In some embodiments, a normal starting density is approximately 0.5 x 10 6 ~1.5×10 6 It could be.

[0101] Additionally, T cell subpopulations can be generated by selection based on the presence or absence of one or more marker(s). For example, Treg cells are CD4+, CD25+, CD127 負 / 低, and optionally may be obtained from a mixed population based on selection of cells that are FOXP3+. In some cases, Treg cells may be FOXP3-. In this case, selection effectively refers to "choosing" cells based on one or more definable characteristics. Furthermore, selection can be positive or negative, in that it can be selection for cells that have one or more characteristics (positive) or cells that do not have one or more characteristics (negative).

[0102] With respect to Treg cells, by way of example, these cells may be obtained from a mixed population by binding these cells to a surface (e.g., magnetic beads) to which an antibody that binds to CD4 and / or CD25 is bound, and by binding non-Treg cells to a surface (e.g., magnetic beads) to which an antibody that binds to CD127 is bound. As a specific example, CD3+ cells may be isolated using magnetic beads to which an antibody that binds to CD3 is bound. Once released, the resulting CD3+ cells may be contacted with magnetic beads to which an antibody that binds to CD4 is bound. The resulting CD3+, CD4+ cells may then be contacted with magnetic beads to which an antibody that binds to CD25 is bound. The resulting CD3+, CD4+, CD25+ cells may then be contacted with magnetic beads to which an antibody that binds to CD127 is bound, whereby the cells that are recovered are those that do not bind to the beads.

[0103] In some cases, multiple traits may be used simultaneously to obtain T cell subpopulations (e.g., Treg cells). For example, surfaces bound with antibodies that bind to more than one cell surface marker may also be used. As a specific example, CD4+, CD25+ cells may be obtained from a mixed population by binding these cells to a surface bound with antibodies that bind to CD4 and CD25. Simultaneous selection for multiple traits may result in undesired cell types "co-purifying" with the desired cell type(s). This is because, using the specific example above, in addition to CD4+, CD25+ cells, cells that are CD4+, CD25- and CD4-, CD25+ may be obtained.

[0104] Flow cytometry is particularly useful for separating cells based on desired characteristics. Cells may be separated based on a detectable label associated with a molecule that binds to the cells of interest (e.g., a natural ligand such as IL-7 that binds CD127, an antibody specific for CD25, etc.). Thus, ligands that bind to cellular components that can be detected and / or differentiated by a flow cytometry system may be used to purify / isolate T cells with a particular characteristic. Furthermore, the presence or absence of multiple characteristics may be determined simultaneously by flow cytometry.

[0105] Thus, the invention includes methods for obtaining members of one or more T cell subpopulations, in which members of the T cell subpopulations are identified by particular characteristics and separated from cells that differ with respect to these characteristics. Exemplary characteristics that can be used in the methods of the invention include the presence or absence of the following proteins: CD3, CD4, CD5, CD8, CD11c, CD14, CD19, CD20, CD25, CD33, CD34, CD45, CD56, CD123, CD127, CD278, CD335, and FOXP3.

[0106] The selection of cell-related characteristics to be obtained will depend on the cells present in a particular sample. For example, umbilical cord blood (UCB) has been shown to contain Treg cells, which can alleviate the effects of graft-versus-host disease. However, UCB contains a proportion of cells not normally found in peripheral blood. One category of cells found in UCB is hematopoietic stem cells (HSCs). HSCs are generally CD34+, and can be separated from CD4+, CD25+ cells by binding to a surface with a suitable ligand or by a labeled molecule that binds to one or more of CD4+, CD25+, and / or CD34+.

[0107] Thus, the present invention relates to compositions and methods for purifying cells, whereby the number of cells of interest in a mixture is increased by at least 5-fold (e.g., about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 10-fold to about 40-fold, about 10-fold to about 30-fold, etc.). An example of purification is where a cell type represents 5% of the total cells in a mixed population before purification, and represents 25% of the total cells in the mixed population before purification. This is an example of a 5-fold purification.

[0108] As can be seen in Figures 10A and 10B, CD3 / CD5 isolated T cells can be directly polarized and expanded. One starting material for Th17 polarization protocols is CD3 or CD4 enriched cells (positively or negatively isolated).

[0109] Upstream isolation is difficult and costly, complicating the process for Th17 generation. We found that CD3 / CD5 beads (the same as those used in Th17 polarization) efficiently isolated CD3 cells (T cells) with efficiencies similar to those achieved using DYNABEADS® CD3 / CD28 CTS (>90% at a 3:1 bead:cell ratio and 80% at a 1:1 bead:cell ratio) (Figure 10A). As shown in Figure 10B, such positively CD3 / CD5 isolated T cells can be directly polarized to Th17 cells.

[0110] The present invention also relates to compositions and methods for simultaneous activation and purification of T cells. Examples of such compositions and methods include forming a mixture containing magnetic beads bound to anti-CD3 and anti-CD5 antibodies, and including Th17 T cells in the reaction mixture. In this case, Th17 T cells can be both activated with anti-CD3 and anti-CD5 antibodies and separated from other cells that do not contain CD3 and / or CD5 surface markers. Naturally, proteins other than CD3 and CD5, as well as various combinations of proteins (e.g., CD3 and CD28; CD3, CD28, and CD137; CD3 and CD137; CD3 and CD278; etc.), can also be used for both activation and purification of T cells.

[0111] Furthermore, when magnetic beads are used to simultaneously activate and purify T cells, it has been found that the ratio of cells to beads can be adjusted to increase purification efficiency. For example, using DYNABEADS® CD3 / CD28 CTS™ (Thermo Fisher, catalog number 40203D), it has been found that a 3:1 bead-to-cell ratio can be used to obtain a T cell population that is over 90% pure, and a 1:1 bead-to-cell ratio can be used to obtain a T cell population that is approximately 80% pure. Accordingly, the present invention provides compositions and methods for purifying T cells, in which the ratio of cells to beads is about 20:1 to about 1:5 (e.g., about 20:1 to about 1:2, about 20:1 to about 1:1, about 20:1 to about 2:1, about 20:1 to about 3:1, about 10:1 to about 1:1, about 10:1 to about 2:1, about 10:1 to about 3:1, etc.). The present invention also provides compositions and methods for purifying T cells, in which the cell to bead ratio is adjusted to yield an activated T cell population that is at least 80% (e.g., about 80% to about 99%, about 83% to about 99%, about 85% to about 99%, about 88% to about 99%, about 90% to about 99%, about 93% to about 99%, about 95% to about 99%, about 80% to about 95%, about 85% to about 95%, etc.) pure.

[0112] The purified T cells obtained by the above methods may be mixed or specific to a specific type (e.g., Treg cells). For example, CD3 and CD28 proteins are present on the surface of several different T cell subtypes. Thus, T cells purified using anti-CD3 and anti-CD28 antibodies are often mixed populations. Specific T cell subtypes may be expanded from such mixed populations using additional agents, such as chemokines, cytokines, or other agents (e.g., rapamycin, aryl hydrocarbon receptor agonists, etc.). Thus, the purified mixed T cell population may be used to generate T cells of one or more subtypes by adjusting activation and / or growth conditions.

[0113] For example, Th17 cells may be purified, activated, and expanded by processes such as the following: T cells may be purified from a mixed cell population (positive isolation) using either anti-CD3 or anti-CD4 antibodies bound to a solid support. The cells bound to the solid support are collected and exposed to a solid support containing anti-CD3 and anti-CD5 antibodies. The T cells bound to these beads are separated from unbound cells and then exposed to an agent that promotes Th17 cell polarization (e.g., IL-1β, IL-6, aryl hydrocarbon receptor antagonist molecules, etc.). These T cells are then maintained under conditions that allow for the expansion of Th17 cells. In some cases, other T cells may be expanded under the conditions used. Conditions are usually adjusted so that Th17 cells expand faster than other T cell subtypes.

[0114] Expansion and / or proliferation into various T cell subpopulations A mixed population of T cells isolated from a subject can be expanded into various T cell subpopulations by varying the exposure of these T cells to a primary activation signal using a primary agent. The primary activation signal is anti-CD3 and can be achieved using a primary agent that is anti-CD3 (e.g., an anti-CD3 antibody or other agent that has binding specificity for CD3). The primary activation signal can be used in combination with a second and / or third agent that can be directed against CD28, CTLA-4, CD137, CD27, CD5, CD6, CD134, CD2, LFA-1, CD40, SLAM, GITR, and / or ICOS.

[0115] The cells of the present invention can be expanded by incubating them in a culture medium containing the agents described above and herein. The compositions of the present invention comprise specific T cell proliferation agents bound to a surface at a predetermined ratio. The T cell proliferation agents of the present invention may be present together with one or more costimulatory signals. The ratio of costimulatory signal to T cell proliferation agent may be 1:1000, about 1:500, about 1:100, about 1:50, about 1:40, about 1:30, about 1:20, about 1:10, about 1:5, about 1:4, about 1:3, or about 1:2. In some embodiments, two or more costimulatory signals may be present at ratios of, for example, 1:1000:1000, about 1:500:500, about 1:100:100, about 1:50:50, about 1:40:40, about 1:30:30, about 1:20:20, about 1:10:10, about 1:5:5, about 1:4:4, about 1:3:3, or about 1:2:2. In some embodiments, additional costimulatory signals may be present. In some embodiments, the ratio of primary signal to individual costimulatory signals may not be the same (e.g., the ratio of costimulatory signal to primary signal is 1:4:3). Furthermore, the ratio of cells to signals can be controlled by the total amount of signal applied in selective expansion. For example, in bead-bound stimulatory signals, the bead:cell ratio may be varied.

[0116] In one embodiment, isolated T cells are expanded with beads containing a surface acting agent. Isolated T cells can be activated and expanded by incubating them in vitro with beads or other compositions of the invention at a ratio of about 1 bead per cell. In other embodiments, beads can be used in culture to activate and expand T cells at a ratio of about 100 cells / bead, about 10 cells / bead, about 5 cells / bead, about 2 cells / bead, about 2 beads / cell, about 5 beads / cell, about 10 beads / cell, or about 100 beads / cell.

[0117] The bead:cell ratio, total concentration, and / or relative proportion of costimulatory signals can each be adjusted to achieve the maximum expansion fold, the percentage of a desired cell type within the expanded cell population, or the relative number of a desired cell type. The resulting cell population can contain about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the desired cell type compared to the total cell population. The relative number of a desired cell type is defined as the percentage of desired cells multiplied by the expansion fold of the total cell population. The relative number of the desired cell type may include about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 350, about 400, or about 500.

[0118] With respect to Tregs, the stimulation method may be performed in the presence of other cells, such as other T cells, e.g., CD4+ and CD25+ cells, which themselves can be significantly expanded during the method of the present invention. Thus, in the above-described protocol, the T cell isolation step may involve the isolation of a significant portion (i.e., at least about 20, 30, 40, 50, 60, 70, 80, or 90%) or all of the CD4+ cells, i.e., both CD25+ and CD4+ cells, from the sample. Thus, in one embodiment of the present invention, the isolation step includes the isolation of CD4+ cells. Furthermore, other cells may be present before or after the isolation step, such that Tregs, Th17, or memory T cells form only a portion of the cells used in the stimulation method. Thus, in the stimulation step, CD4+ and CD25+ cells may be present as part of the cells subjected to stimulation, i.e., an enriched preparation may be used, such as a preparation comprising at least about 50, 60, 70, 80, or 90% of the total cells subjected to stimulation.

[0119] In some cases, at least some CD4, CD25 cells are absent, e.g., at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of these cells present in the starting material are absent. Alternatively, the starting cells for stimulation / expansion may be substantially all CD4, CD25 cells, such as at least about 80%, about 90%, about 95%, or about 98% CD4, CD25 cells.

[0120] Stimulation of T cells to selectively expand Treg cells can include stimulation with anti-CD3 and anti-CD28 agents. In contrast to previous anti-CD3 / CD28 costimulatory approaches, in some embodiments, the present invention utilizes a low anti-CD3 to anti-CD28 ratio, such that the anti-CD3 agent is present in lower amounts than the anti-CD28 agent. The anti-CD3 to anti-CD28 ratio can be about 1:1000, about 1:500, about 1:100, about 1:50, about 1:40, about 1:30, about 1:20, about 1:10, about 1:5, about 1:4, about 1:3, or about 1:2. In some cases, selective expansion of Treg cells can include stimulation with (1) anti-CD3 and (2) anti-CD5 and / or anti-ICOS agents.

[0121] Stimulation of T cells to selectively expand Th17 cells can include stimulatory agents that innervate CD3, CD28, CD5, and / or ICOS receptors. In some embodiments, Th17 cells can be generated by using anti-CD3 and anti-ICOS antibodies. In some embodiments of the present invention, the level of CD3 receptor stimulation can be lower than the level of ICOS stimulation. Using antibodies as an example, the ratio of anti-ICOS to anti-CD3 can be about 1:1000, about 1:500, about 1:100, about 1:50, about 1:40, about 1:30, about 1:20, about 1:10, about 1:5, about 1:4, about 1:3, or about 1:2.

[0122] Stimulation of T cells to selectively expand T memory cells can include stimulation with anti-CD3 and anti-CD27, or anti-CD28, or anti-CD137. In many cases, the anti-CD3 agent can be present at a lower concentration compared to the anti-CD28, anti-CD27, and / or anti-CD137 agents. The ratio of anti-CD3 to anti-CD28 and anti-CD27 to anti-CD137 can be about 1:1000:1000, about 1:500:500, about 1:100:100, about 1:50:50, about 1:40:40, about 1:30:30, about 1:20:20, about 1:10:10, about 1:5:5, about 1:4:4, about 1:3:3, or about 1:2:2.

[0123] Following isolation, T cells may be activated by incubating them in culture medium with a composition of the invention for a period of 1-5 days, 1-4 days, 2-3 days, about 2 days, or about 3 days without perturbation. After activation without perturbation, the isolated T cells are expanded by adding fresh culture medium and cytokines as needed. Expansion can occur for a period of 7-20 days, 8-15 days, 10-14 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. Cytokines for T cell expansion are described above and discussed below with reference to the Examples.

[0124] The expansion of T cells can be such that a specified number of T cells of a T cell subpopulation can be produced. For example, about 10 6 cells, approximately 10 7 cells, approximately 10 8 cells, or approximately 10 10 cells (e.g., about 10 6 ~about 10 10 cells, approximately 10 7 ~about 10 10 cells, approximately 10 8 ~about 10 10 cells, approximately 10 9 ~about 10 10 cells, approximately 10 6 ~about 10 9 cells, approximately 10 6 ~about 109 T cell subpopulations comprising T cells (e.g., individual T cells) can be produced by expanding T cells using the methods and compositions of the invention.

[0125] Using the methods of the present invention, activation / proliferation of T cell subpopulations can be achieved after about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days, about 30 days, etc. (e.g., about 2 hours to about 30 days, about 8 hours to about 30 days, about 20 hours to about 30 days, about 1 day to about 30 days, about 3 days to about 30 days, about 5 days to about 30 days, about 7 days to about 30 days, etc.).

[0126] In some cases, the solid phase (e.g., beads) containing one or more stimulatory signals (e.g., anti-CD3 antibodies, anti-CD28 antibodies, interleukin-2, etc.) is not removed from the T cell population after expansion. In other cases, once the T cell population has expanded to the required level described above, the expanded population may then be separated from the solid phase by an appropriate technique. For example, if one of the one or more stimulatory signals is bound to a solid phase and this solid phase is a tissue culture well, plate, or bottle, the T cell population may be obtained by removing the culture medium containing the T cells. If the solid phase comprises, for example, magnetic beads, a magnetic field may be used to attract the beads to the side of the container, and the culture medium containing the T cells may then be poured off. Other particulate solid supports (e.g., non-magnetic beads) may be centrifuged or filtered to separate them from the cells. While the majority of T cells are typically located in the culture medium, some T cells will remain bound to the solid phase after expansion. If desired, such T cells may be detached using a pipette or other suitable means, for example, by resuspension. Such resuspension is typically performed prior to separation of the T cells from the solid phase to improve yield. Once separated from the solid phase, the T cell population may then be further processed and / or manipulated in any desired manner, or may be used directly for a suitable application, such as, for example, in vitro experimentation and research, non-therapeutic applications, therapeutic applications, etc., as described below.

[0127] When a soluble activator is used, the activator may be removed by competition with an appropriate ligand, e.g., CD3 or CD28, if necessary, although in many cases the T cells are recovered from the culture medium and used for the applications described herein without further purification. Conveniently, for large-scale applications, a suitable isolation and preparation platform may be used to select T cell populations for stimulation and / or expansion protocols and / or to isolate generated T cell populations. In this regard, particular mention may be made of DYNAL's DYNAMAG™ CTS platform, which may use, for example, closed sterile disposable pouches for cell isolation prior to expansion and for the optional magnetic cell separation step performed for DYNABEAD® removal after expansion.

[0128] If necessary, T cell populations expanded / activated according to the methods of the invention may be restimulated by contacting the cells with an additional activating agent in the same manner as the initial stimulation. Generally, restimulation is only necessary or desirable if the T cells are cultured for an extended period of time, e.g., more than 10-16 days.

[0129] Following expansion, Treg cells may be restimulated by incubating with the same beads used for primary activation. Treg cells may be restimulated about 16, 14, 11, 10, 9, 8, 7, 6, or 12 hours after primary activation.

[0130] While expansion of T cell subpopulations according to the methods of the invention may result in a relatively small numerical increase, often the result is a significant increase, such as at least about a 2-fold, preferably at least about a 5-fold, about 20-fold, or about 50-fold, and more preferably at least about a 100-fold, about 500-fold, about 1,000-fold, about 2,000-fold, about 5,000-fold, about 10,000-fold, about 20,000-fold, about 30,000-fold, about 40,000-fold, about 50,000-fold, about 75,000-fold, about 100,000-fold or more increase in cell number. Such numerical increases may be measured at any appropriate time point in the cell expansion protocol.

[0131] The present invention provides methods for generating one or more substantially pure population(s) of T cell subpopulations. For purposes of the present invention, a substantially pure population of T cell subpopulations contains about 10% or less of undesired cells (e.g., not of the desired subpopulation cell type). For example, for purposes of illustration, if Treg cells are the desired T cell subpopulation, a substantially pure Treg subpopulation will contain about 10% or less of cells that are not Treg cells. Of course, this also applies to other T cell subpopulations. In some embodiments, substantially pure may contain about 25% or less, about 20% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, and / or about 1% or less of T cells that are not members of the desired subpopulation(s).

[0132] In standard Th17 generation protocols (X-VIVO™ 15 medium containing polarizing cytokines), stimulatory solid supports (e.g., DYNABEADS®) are typically maintained in the culture medium throughout the culture period. Particularly when small numbers of cells are contained and prepared for viral transduction (genetic modification), simplification of the process occurs when the solid supports (e.g., beads) can be removed before the end of the culture period (e.g., 48 or 72 hours after activation). An improved one-way process is the elimination of the need to remove large-scale solid supports (e.g., magnetic beads) near or at the end of the culture period.

[0133] We also found that early removal of the activating substrate resulted in improved polarization of Th0 cells toward a Th17 phenotype, as shown in Figures 9A and 9B. Experiments with early bead removal also showed up to 200-fold expansion, unaffected by bead removal.

[0134] It was observed that the effect of early bead removal on Th17 polarization was greater in ICOS-costimulated cells than in CD5-costimulated cells, and was almost negligible after CD28-costimulation (Figures 9A and 9B).

[0135] In some cases, it may be desirable to remove from the T cell mixture any solid supports (e.g., beads, such as magnetic beads) used to activate the T cells. Moreover, in some cases, it may be desirable to remove such solid supports after a fairly short period of time (e.g., within 5 days). Accordingly, the present invention includes compositions and methods for T cell activation in which T cells are exposed to an agent that stimulates one or more cell surface markers for less than 5 days (e.g., from about 1 to about 5 days, from about 2 to about 5 days, from about 3 to about 5 days, from about 1 to about 4 days, from about 2 to about 3 days, etc.).

[0136] In a specific embodiment, multiple samples of mixed populations of T cells are exposed to (1) a solid surface (e.g., magnetic beads) containing anti-CD3 and anti-CD5 antibodies, (2) IL-1β, and (3) IL-6, (4) IL-23, and (5) TGF-β (and, optionally, an aryl hydrocarbon receptor agonist) under conditions that allow T cell expansion. For one sample, the solid surface is not removed. For a second sample, the solid surface is removed after one day. For a third sample, the solid surface is removed after two days. For a fourth sample, the solid surface is removed after three days. Expansion of activated T cells may be performed essentially as described in Example 10. At the end of the expansion process (approximately 14 days), the Th17 phenotype of the expanded cells, the number of Th17 cells, and the ratio of Th17 cells to other cell types are determined.

[0137] III. Compositions of the Invention The compositions of the invention comprise surfaces having immobilized agents in amounts, ratios, or combinations capable of stimulating the proliferation of specific T cell subpopulations. In alternative embodiments, the agents may be present in solution.

[0138] Agents contemplated by the present invention include protein ligands and synthetic ligands. Agents that can bind to cell surface moieties and, under certain conditions, cause ligation and assembly leading to signal transduction include, but are not limited to, lectins (e.g., phytohemagglutinin (PHA), lentil lectin, concanavalin A), antibodies, antibody fragments, peptides, polypeptides, glycopeptides, receptors, B cell receptor and T cell receptor ligands, MHC-peptide dimers or tetramers, extracellular matrix components, steroids, hormones (e.g., growth hormone, corticosteroids, prostaglandins, tetra-iodothyrohormone, corticosteroids, prostaglandins, tetra-iodothyromine), bacterial moieties (e.g., lipopolysaccharides), mitogens, superantigens and their derivatives, growth factors, cytokines, adhesion molecules (e.g., L-selectin, LFA-3, CD54, LFA-1), chemokines, and small molecules. The agents may be isolated from natural sources such as cells, blood products, and tissues, or may be isolated from cells grown in vitro, recombinantly prepared by chemical synthesis, or by other methods known in the art.

[0139] In one embodiment, the agent of the present invention includes antibodies. Antibodies of the present invention include, but are not limited to, anti-CD3 (Thermo Fisher Scientific, Norway); anti-CD137 (6B4 provided by the University of Navarra, Pamplona, ​​Spain, or 4B4-1 from Affymetrix / BioScience, CA, USA); anti-CD28 (XR-CD28: Thermo Fisher Scientific, Norway); anti-ICOS (ISA-3) (Affymetrix / BioScience, CA, USA), anti-CD2, anti-CD5, anti-CD6, anti-CD134, anti-CD40L, anti-CTLA-4, anti-GITR, anti-LFA-1, anti-SLAM, anti-CD27, anti-HVEM, anti-LIGHT, anti-DR3, anti-TIM1, and anti-CD226.

[0140] Antibodies of the present invention can be obtained from public sources such as the American Type Culture Collection (ATCC), and antibodies against T cell accessory molecules and cell surface proteins can be produced by standard techniques. Methods for generating antibodies for use in the methods of the present invention are known in the art. Antibodies may also be produced as genetically engineered immunoglobulins (Igs) or Ig fragments designed to have specific desired properties. As a non-limiting example, antibodies can include recombinant IgGs, which are chimeric fusion proteins containing at least one variable (V) region domain from a first mammalian species and at least one constant region domain from a second, distinct mammalian species. Most commonly, chimeric antibodies have murine variable region sequences and human constant region sequences. Such murine / human chimeric immunoglobulins can be "humanized" by grafting complementarity-determining regions (CDRs) that confer binding specificity for antigens from the murine antibody in human-derived V region framework regions and human-derived constant regions. Antibodies containing CDRs of different specificities can also be combined to generate multispecific (e.g., bispecific or trispecific) antibodies. Fragments of these molecules may be generated by proteolytic digestion, or optionally by proteolytic digestion followed by mild reduction of disulfide bonds and alkylation, or by recombinant genetic engineering techniques.

[0141] Agents of the present invention may further include any molecule that specifically binds to the cell surface receptors CD3, CD137, CD28, CTLA-4, GITR, ICOS (ISA-3), CD2, CD5, CD6, CD134, anti-CD40L, LFA-1, LFA-2, and LFA-3. Agents of the present invention may include peptides, small organic molecules, peptidomimetics, soluble T cell receptors, or antibodies, etc. Agents of the present invention may also be naturally occurring cell surface receptor ligands. Suitable agents of the present invention may be identified by assays for determining affinity and binding specificity known in the art, including competitive and non-competitive assays. Assays of interest include ELISA, RIA, flow cytometry, etc.

[0142] Agents contemplated by the present invention may further include protein ligands, natural ligands, and synthetic ligands. Agents that can bind to cell surface moieties and, under certain conditions, cause ligation and assembly leading to signal transduction include, but are not limited to, lectins (e.g., PHA, lentil lectin, concanavalin A), antibodies, antibody fragments, peptides, polypeptides, glycopeptides, receptors, B cell receptor and T cell receptor ligands, extracellular matrix components, steroids, hormones (e.g., growth hormone, corticosteroids, prostaglandins, tetra-iodothyronine), bacterial moieties (such as lipopolysaccharides), mitogens, antigens, superantigens and their derivatives, growth factors, cytokines, viral proteins (e.g., HIV gp-120), adhesion molecules (such as L-selectin, LFA-3, CD54, LFA-1), chemokines, and small molecules. The agents may be isolated from natural sources such as cells, blood products, and tissues, or may be isolated from recombinantly prepared, in vitro grown cells, or by other methods known to those skilled in the art.

[0143] In one embodiment of the invention, where stimulation of T cells is desired, useful agents include ligands capable of binding to the CD3 / TCR complex, CD2, and / or CD28 and initiating activation or proliferation, respectively. Consequently, the term ligand includes proteins that are "natural" ligands for cell surface proteins, such as the B7 molecule for CD28, and synthetic ligands, such as antibodies directed against cell surface proteins. Such antibodies and fragments thereof can be produced according to conventional techniques, such as hybridoma methods and recombinant DNA and protein expression techniques. Useful antibodies and fragments can be derived from any species, including humans, or can be formed as chimeric proteins using sequences from more than one species.

[0144] In an alternative embodiment, the costimulatory signal also includes rapamycin. Rapamycin may be contacted with the cells before, simultaneously with, and / or after contacting the cells with an activator. Rapamycin may be present throughout the proliferation / expansion steps in methods according to the invention. Rapamycin may be added in one or more steps. Thus, for example, as described in the Examples herein, isolated CD4+ cells may be stimulated simultaneously with an activator and rapamycin. In such methods, subsequent growth and passaging may be performed in the presence of rapamycin but in the absence of an activator.

[0145] Rapamycin may be used at a concentration of about 0.01 μM to about 10 μM, such as about 0.5 μM to about 2 μM, or about 1 μM. Rapamycin is a protein kinase inhibitor with a molecular weight of 914.2, also known as sirolimus, rapamune, AY-22989, RAPA, and NSC-226080, available from Sigma, Calbio Chem, LC Labs, etc. Rapamycin is available from various commercial sources, such as AGScientific, Inc. (San Diego, Calif., USA).

[0146] Other cytokines and / or growth factors may be added to the culture medium as needed. Such cytokines and / or growth factors are added at appropriate concentrations and times. For example, IL-2 and / or IL-4 may be added to promote T cell proliferation. Other cytokines may be added, if necessary, to induce specific differentiation patterns (e.g., TGF-β and IL-10). For example, IL-4 has been shown to induce differentiation of T cell populations toward a Th2 subpopulation, while IFN-γ and IL-12 have been shown to induce differentiation toward a Th1 subpopulation (Sunder-Plassman et al., Blood 87:5179-5184 (1996)). Thus, in some embodiments of the invention, cytokines, such as IL-2, may be added in one or more steps at an exogenously added final concentration of 10 to 4,000 U / ml (e.g., about 10 U / ml to about 3,000 U / ml, about 10 U / ml to about 2,000 U / ml, about 10 U / ml to about 1,500 U / ml, about 15 U / ml to about 4,000 U / ml, about 15 U / ml to about 3,000 U / ml, about 15 U / ml to about 1,500 U / ml, about 20 U / ml to about 2,000 U / ml, etc.). In certain cases, IL-2 may be added at 20 U / ml during stimulation, periodically at 1,000 U / ml during the initial culture period, and at 20 U / ml during the pre-harvest period.

[0147] IL-4 may be added exogenously, for example, at a final concentration of about 1,000 to 5,000 U / ml or about 100 to 10,000 U / ml (e.g., about 1,000 U / ml to about 5,000 U / ml, about 5,000 U / ml to about 5,000 U / ml, about 250 U / ml to about 5,000 U / ml, about 100 U / ml to about 5,000 U / ml, about 800 U / ml to about 2,500 U / ml, etc.). In some cases, about 1,000 U / ml may be added during stimulation and culture until harvest. Suitable cytokines and growth factors, and their effects on T cells, are well known and described in the art. Once the T cell population has been contacted with the activator and, optionally, rapamycin under conditions appropriate for T cell growth, it may be grown for a period of time selected according to the desired final number of T cells and rate of cell proliferation. Passaging of the cells may be performed during this period, which is typically about 3 to about 10 days, but may be as long as about 14 to about 20 days or even longer, provided that viability and continued proliferation of the T cells is maintained.

[0148] The compositions of the present invention may include a surface on which the aforementioned agents are immobilized. The surfaces of the present invention may be any surface to which a ligand can be attached or incorporated and which is biocompatible, i.e., substantially non-toxic to the target cells being stimulated. Biocompatible surfaces may be biodegradable or non-biodegradable. The surfaces may be natural or synthetic. Synthetic surfaces may be polymeric. The surfaces may include collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, extracellular matrix compositions, liposomes, or cell surfaces. Polysaccharides may include, by way of example, cellulose, agarose, dextran, chitosan, hyaluronic acid, or alginic acid. Other polymers may include polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinyl acetate, block copolymers, polypropylene, polytetrafluoroethylene (PTFE), or polyurethanes. The polymer may be lactic acid or copolymers. Copolymers may include lactic acid and glycolic acid (PLGA). Non-biodegradable surfaces may include polymers such as poly(dimethylsiloxane) and poly(ethylene-vinyl acetate). Biocompatible surfaces include, by way of example, glass (e.g., bioglass), collagen, chitin, metals, hydroxyapatite, aluminates, bioceramic materials, hyaluronic acid polymers, alginates, acrylate polymers, lactic acid polymers, glycolic acid polymers, lactic acid / glycolic acid polymers, purified proteins, purified peptides, or extracellular matrix compositions. Other polymers include surfaces that may include glass, silica, silicon, hydroxyapatite, hydrogels, collagen, acrolein, polyacrylamide, polypropylene, polystyrene, nylon, or any number of synthetic organic polymer plastics. The surface may include biological structures such as liposomes or cell surfaces such as red blood cells (RBCs). The surface may be in the form of a lipid, plate, bag, pellet, fiber, mesh, or particle. Particles can include colloidal particles, microparticles, non-particles, beads, and the like.In various embodiments, commercially available surfaces such as beads or other particles are useful (e.g., Miltenyi Particles, Miltenyi Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADS®, Dynal Inc., New York; PURABEADS®, Prometric Biosciences).

[0149] Proteins, such as antibodies, may be attached to a solid support (e.g., beads) in any number of ways. One method for attaching proteins to a support is via biotin and avidin or streptavidin. One system that has been developed is called Strep-tag® (IBA GmbH, Göttingen, Germany). This system allows for the use of an eight amino acid sequence tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1)) that binds to streptavidin. Furthermore, such a system allows for the release of bound cells and proteins via competitive binding with biotin. Thus, the present invention includes compositions and methods for attaching proteins (e.g., antibodies) to a solid support via streptavidin interaction and the purification of cells bound to such a solid support. Cell purification methods include the following: An antibody interacts with a solid support via avidin or streptavidin, where the antibody binds to a cell surface marker (e.g., CD4). The solid support is separated from unbound cells, and then the bound cells are released. Release can occur by methods such as proteolytic cleavage of the antibody or by competition with added biotin. Such supports can be used for cell purification, T cell activation.

[0150] A similar product is available from Miltenyi Biotec (Bergisch Gladbach, Germany, catalog number 130-090-485). This product contains anti-biotin-conjugated beads that can be used to bind biotinylated antibodies. Such beads can also be used for cell purification and T cell activation.

[0151] When beads are used, they may be of any size that results in target cell stimulation. In some embodiments, beads of about 5 nm to about 500 μm in size are used. The choice of bead size often depends on the particular application for which the beads will function. For example, when using paramagnetic beads, the beads typically range in size from less than 1 μm, to about 2.8 μm to about 500 μm, and generally from about 2.8 μm to about 50 μm. Finally, beads of about 10 -5 It is possible to select superparamagnetic non-particles as small as 4.5 μm. An exemplary bead is DYNABEADS® M-450, which is 4.5 μm in diameter. Therefore, beads used in the practice of the present invention generally have a diameter of about 0.00001 nm to about 500 μm (e.g., about 0.01 nm to about 500 μm, about 1 nm to about 500 μm, about 5 nm to about 500 μm, about 0.00001 nm to about 50 μm, about 0.1 nm to about 50 μm, about 10 nm to about 50 μm, about 50 nm to about 50 μm, about 50 nm to about 25 μm, about 50 nm to about 1 μm, about 10 μm to about 500 μm, about 1 μm to about 10 μm, etc.).

[0152] In some embodiments, DYNABEADS® may be used. DYNABEADS® has the advantage of allowing parameters such as ligand composition and stoichiometry to be varied for systematic examination of their effects on T cell activation and differentiation. DYNOSPHERES® (Thermo Fisher Scientific, Lillestrom, Norway) can be coupled with agents at different stoichiometries for the expansion of specific T cell subpopulations. DYNOSPHERES® binds to bare beads coated with epoxy groups, allowing antibodies or other agents of the invention to be coupled to the beads without a binding group.

[0153] The agent may be conjugated, attached, incorporated, coupled to, or integrated into the surface by a variety of methods known and available in the art. The agent may be a natural ligand, a protein ligand, or a synthetic ligand. Binding may be covalent or non-covalent, electrostatic, or hydrophobic, and may be achieved by a variety of binding means, including, for example, chemical, mechanical, enzymatic, electrostatic, or other means by which the ligand can stimulate the cell. Binding of the agent may be direct or indirect (e.g., tethered). For example, an antibody may be directly bound (i.e., directly attached) to the surface or may be via indirect binding (e.g., avidin or streptavidin / biotin).

[0154] With respect to cell surfaces, the binding may be via genetic expression of the agent using any number of techniques known in the art, such as transfection or transduction of an expression vector containing the coding region of the agent of interest. Antibodies against the ligand may be bound to the surface via anti-idiotypic antibodies. Another example includes the use of protein A or protein G or other nonspecific antibody-binding molecules bound to any suitable surface, including DYNABEADS®, as a method for binding antibodies to any suitable surface. Alternatively, the ligand may be bound to the surface by chemical means, such as cross-linking to the surface using commercially available cross-linking reagents (e.g., those available from Pierce, Rockford, Ill.), or other means. In certain embodiments, the ligand may be covalently bound to the surface. Furthermore, in one embodiment, commercially available tosyl-activated DYNABEADS® or DYNABEADS® with epoxy surface reactive groups are incubated with the ligand of interest according to the manufacturer's instructions. Briefly, such conditions typically involve incubation in a phosphate or borate buffer at about pH 4 to pH 9.5 at a temperature ranging from about 4° to 37°C.

[0155] The activators are generally mixed together in an appropriate ratio before contacting with the beads. While "appropriate reaction conditions" can vary according to the particular activator used, exemplary conditions include, for example, a phosphate buffer solution (e.g., 0.5 M phosphate) at about pH 7.4 and about 4×10 8 This may involve incubation of beads and an activating agent, e.g., an antibody, at a particle concentration of about beads / ml. Human serum albumin (e.g., recombinant HSA) or other serum albumin, such as bovine serum albumin, may optionally be present (e.g., at a concentration of about 0.05% w / v) to stabilize the activating agent and block any remaining hydrophobic patches on the surface of the beads.

[0156] Once the appropriate reaction mixture is prepared, the reaction is allowed to proceed for a suitable time and under suitable conditions to promote absorption of the activator onto the surface in the required ratio. Again, the specific conditions may vary depending on the components of the reaction mixture, but exemplary conditions include incubation at about 37°C for about 6 to about 24 hours with gentle tilting and rotation.

[0157] Whatever conditions are selected, once the immobilization reaction is complete, the beads, with the appropriate ratio of activators immobilized on their surfaces, can be removed from the remaining aqueous medium by placing a magnet on the side of the reaction vessel and discarding the supernatant. The beads are generally washed to remove any excess unbound antibody and are then ready for use. Once prepared, such beads may be used immediately or stored for later use.

[0158] In some embodiments, DYNABEADS® M-450 may be used for binding to surface-acting agents. According to the manufacturer, DYNABEADS® M-450 are 4.5 micrometer epoxy beads. These are non-porous, monodisperse superparamagnetic beads. High solution mobility allows DYNABEADS® to constantly interact with the solution, facilitating antibody binding. The beads are also available with surface tosyl groups.

[0159] Agents can be immobilized on beads either on the same bead, ie, "cis," or on separate beads, ie, "trans."

[0160] In an alternative embodiment, the activator of the present invention may be in solution.

[0161] Activation and proliferation of specific T cell subpopulations can be targeted by specific surface acting agents and specific ratios of surface acting agents. In some embodiments, compositions of the invention for T cell (e.g., Treg cell) proliferation comprise an anti-CD3 agent and an anti-CD28 agent. T cell (e.g., Treg cell) expansion compositions of the invention can comprise low, intermediate, or high relative levels of surface-bound CD3. By way of example, a low anti-CD3 agent may be present on a surface at a concentration of about 0.01 to about 0.04, about 0.05 to about 0.4, about 0.06 to about 0.4, about 0.07 to about 0.4, about 0.08 to about 0.4, about 0.09 to about 0.4, about 0.1 to about 0.4, about 0.2 to about 0.4, about 0.3, or about 0.34 units; a medium anti-CD3 agent may be present on a surface of the invention at a concentration of about 0.5 to about 1.5, about 0.6 to about 0.8, or about 0.75 units; and a high anti-CD3 agent may be present on a surface of the invention at a concentration of about 2 to about 4, about 3 to about 4, about 3.4, or about 3.41 units. In some embodiments, beads for the expansion of T cells (e.g., Treg cells) are used that comprise low relative levels of anti-CD3 antibody in conjunction with anti-CD28 costimulation.

[0162] In some embodiments, compositions of the invention for activating and expanding T cells (e.g., memory T cells) comprise an anti-CD3 agent, an anti-CD137 agent, and an anti-CD28 agent. T cell (e.g., memory T cell) expansion compositions of the invention may comprise low, moderate-low, moderate-high, or high relative levels of surface-bound CD3. By way of example, a low anti-CD3 agent may be present on a surface of the invention at a concentration of about 0.005 to about 0.02, about 0.008 to about 0.015, or about 0.01 units; a medium to low anti-CD3 agent may be present on a surface of the invention at a concentration of about 0.02 to about 0.08, about 0.03 to about 0.07, or about 0.05 units; a medium to high anti-CD3 agent may be present on a surface of the invention at a concentration of about 0.09 to about 0.16, about 0.1 to about 0.15, about 0.14, or about 0.142 units; and a high anti-CD3 agent may be present on a surface of the invention at a concentration of about 1 to about 2, about 1.3 to about 1.7, about 1.4 to about 1.6, or about 1.5 units. In some embodiments for the expansion of T cells (eg, memory T cells), beads containing low relative levels of anti-CD3 antibodies in conjunction with anti-CD28 and anti-CD137 costimulation are used.

[0163] In some embodiments, compositions of the invention for activating and expanding T cells (e.g., Th17 cells) comprise an anti-CD3 antibody and an anti-ICOS antibody, an anti-CD5 antibody, or an anti-CD28 antibody. T cell (e.g., Th17 cell) expansion compositions of the invention can contain low, medium, or high relative levels of CD3 bound to the surface. By way of example, a low anti-CD3 agent may be present on a surface of the invention at a concentration of about 0.02 to about 0.1, about 0.03 to about 0.08, about 0.04 to about 0.07, about 0.05 to about 0.07, or about 0.06 units; a medium anti-CD3 agent may be present on a surface of the invention at a concentration of about 0.1 to about 0.5, about 0.2 to about 0.5, or about 0.3 units; and a high anti-CD3 agent may be present on a surface of the invention at a concentration of about 1 to 2, about 1.3 to about 1.7, or about 1.5 units. In some embodiments for the expansion of T cells (eg, Th17 cells), beads containing low relative levels of anti-CD3 antibodies in conjunction with anti-CD5, anti-CD28, and / or anti-ICOS costimulation are used.

[0164] The antibody may be bound to a solid support (e.g., a bead) by any number of means. Some methods of binding the antibody involve contacting the antibody with a solid support under conditions that allow the antibody to be covalently bound to the solid support. A commercially available product that can be used to prepare antibody-bound beads is DYNABEADS® Antibody Coupling Kit (Thermo Fisher Scientific, Cat. No. 14311D).

[0165] The DYNABEADS® Antibody Coupling Kit allows for the covalent attachment of antibodies and other proteins, such as lectins and functional enzymes, to the surface of DYNABEADS® M-270 Epoxy. The surface epoxy groups allow for the attachment of proteins through primary amino and sulfhydryl groups. The beads can be coupled until "saturated," resulting in low background binding when exposed to post-coupling treatments.

[0166] The amount of protein used in the reaction mixture to saturate the beads varies depending on the "loading" of the beads used. As an example, if DYNABEADS® M-270 Epoxy beads are used, it is recommended that at least 10 μg of protein be used per mg of beads to ensure saturation. This is true because the loading of these beads typically varies from about 7 to 9 μg of protein per mg of beads. Furthermore, antibody loading may be adjusted per bead by varying the ratio of antibodies contacted with the beads. Thus, antibody / bead binding may be adjusted to change the ratio of ligands present on the beads relative to each other and / or the average total amount of ligand on each bead.

[0167] DYNABEADS® M-450 Epoxy (Thermo Fisher Scientific, Catalog No. 14011) may also be used in the practice of the present invention. In the process herein, 1 ml (4 x 10 8 1 ml (4 × 10 8 Approximately 200 μg of antibody (Ab) should be used per 100 beads (1000 μg). 1 ml of the washed and resuspended beads is placed in a tube. The tube is then placed in a magnet for 1 minute and the supernatant is discarded. The tube is then removed from the magnet and the beads are resuspended in Buffer A (0.1 M sodium phosphate buffer, pH 7.4-8.0 or 0.1 M sodium borate buffer, pH 7.6-9.5). 200 μg of antibody(ies) is then added to bring the volume up to 1 ml. The mixture is then incubated for 15 minutes, and then BSA is added to 0.01-0.1% w / v. The mixture is then incubated at room temperature for 16-24 hours with gentle tilting and rotation. The tube is then placed in a magnet for 1 minute and the supernatant is discarded. 1 ml of Buffer B (Ca 2+ and Mg 2+Add 1 ml of phosphate-buffered saline (PBS), 0.1% bovine serum albumin (BSA), and 2 mM EDTA, pH 7.4. The mixture is then mixed and incubated for 5 minutes with gentle tilting and rotation. This washing procedure is repeated twice. Remove the tube and beads from the magnet and add 1 ml of Buffer B (4 x 10 8 Resuspend in 0.5 mL of PBS (0.5 mL of beads / ml).

[0168] Another product that can be used in the practice of the present invention is PIERCE™ NHS-activated magnetic beads (Thermo Fisher Scientific, catalog number 88827). These beads have N-hydroxysuccinimide (NHS) functional groups on the block-shaped magnetic bead surface. They are superparamagnetic (no magnetic memory) and have a nominal average diameter of 1 μm. These beads have a density of 2.0 g / cm and a binding capacity of ≥26 μg of beads of rabbit IgG / mg.

[0169] First, the protein solution and magnetic beads are brought to room temperature, then 300 μl of magnetic beads are placed in a 1.5 ml microcentrifuge tube. The tube is placed in a magnetic stand, the beads are collected, and the supernatant is discarded. 1 ml of ice-cold wash buffer A (ice-cold 1 mM hydrochloric acid) is introduced into the tube and gently vortexed for 15 seconds. The tube is placed in a magnetic stand, the beads are collected, and the supernatant is discarded. Next, 300 μl of protein solution is added to the tube, and the tube is then vortexed for 30 seconds. The mixture is incubated on a rotator at room temperature for 1 to 2 hours. During the first 30 minutes of incubation, the tube is vortexed for 15 seconds every 5 minutes. For the remaining time, the tube is vortexed for 15 seconds every 15 minutes until the incubation is complete. The beads are collected on the magnetic stand, and the flow-through is set aside. 1 ml of wash buffer B (0.1 M glycine, pH 2.0) is added to the beads and the tube is vortexed for 15 seconds. The tube is placed in a magnetic stand, the beads are collected, and the supernatant is discarded. The wash and collection are repeated once. 1 ml of ultrapure water is then added to the beads and the tube is vortexed for 15 seconds. The tube is placed in a magnetic stand, the beads are collected, and the supernatant is discarded. 1 ml of quenching buffer (3 M ethanolamine, pH 9.0) is added to the beads and the tube is vortexed for 30 seconds. The tube is then placed on a rotator for 2 hours at room temperature. The tube is placed in a magnetic stand, the beads are collected, and the supernatant is discarded. 1 ml of water is added to the tube, mixed well, the beads are collected, and the supernatant is discarded. Add 1 ml of storage buffer (50 mM boric acid, 0.05% sodium azide, pH 8.5) to the tube, mix well, collect the beads, and discard the supernatant. Repeat this wash step two additional times. Add 300 μl of storage buffer to the beads, mix well, and store at 4°C until ready to use. The final concentration of protein-coupled magnetic beads should be approximately 10 mg / ml.

[0170] Other beads that can be used in the practice of the present invention are DYNABEADS® M-270 Carboxylic Acid (Thermo Fisher Scientific, catalog number 14306D), DYNABEADS® M-450 Tosylactivated (Thermo Fisher Scientific, catalog number 14013), DYNABEADS® M-280 Tosylactivated (Thermo Fisher Scientific, catalog number 14204), and DYNABEADS® MyOne™ Tosylactivated (Thermo Fisher Scientific, catalog number 65502).

[0171] In many cases, the solid support (e.g., beads) used in the practice of the present invention is saturated with protein. The ratio of proteins used can vary. For example, 100% of the total load can be ligand (e.g., antibody). In such cases, one ligand can be present, or multiple ligands can be present. When multiple ligands are present, they can be present in the same ratio (1:1) or different ratios (e.g., 1:10). Exemplary ratios of ligands that can be used in the practice are described elsewhere herein.

[0172] Table 2: Exemplary antibody mixtures TIFF0007623980000002.tif35161

[0173] Total ligand loading can also be adjusted. As shown in Table 2 using antibody ligands, the solid support may be loaded at less than 100%. In many cases where this is done, it is desirable to block the ligand binding sites from further reaction. One way to do this is by contacting the solid support with an agent (e.g., a protein) that does not have ligand binding activity suitable for the particular application (e.g., human serum albumin (HSA), bovine serum albumin (BSA), one or more antibodies that do not bind to T cell receptors, etc.) (non-ligand agent) under conditions that allow both the ligand(s) and the non-ligand agent to bind to the solid support. This allows adjustment of the ratios of different ligands and different total signals. Some examples along these lines are listed in Table 2.

[0174] The total signal to T cells can be adjusted in any number of ways. For example, if the signal per solid support (e.g., bead) is low, each T cell can be contacted with more solid support surfaces. When beads are used, this means that the ratio of T cells to beads can be adjusted. For example, when using beads that are 50% loaded with antibody, two times as many beads can be used to obtain the same signal as beads that are 100% loaded.

[0175] IV. Methods of Use of the Invention The T cell subpopulations of the present invention can be used in any number of physiological conditions, disorders, and / or disease states for therapeutic and / or research / discovery purposes. Conditions or diseases characterized by an abnormal immune response can be autoimmune diseases, such as diabetes, multiple sclerosis, myasthenia gravis, neuritis, lupus, rheumatoid arthritis, psoriasis, or inflammatory bowel disease. Conditions in which immunosuppression is beneficial include conditions in which a normal or activated immune response would be detrimental to the mammal, e.g., in allogeneic transplants of body fluids or regions, or in infertility treatments where an adequate immune response contributes to pregnancy failure and miscarriage, to avoid rejection. The use of such cells before, during, or after transplantation avoids widespread chronic graft-versus-host disease, which can occur in treated patients (e.g., cancer patients). These cells can be expanded immediately after collection or storage (e.g., by freezing), before or after expansion, and before using them in therapy. Such therapy can be performed in conjunction with known immunosuppressive therapies.

[0176] Once a suitable T cell population or subpopulation has been isolated from a patient or animal, genetic or any other suitable modification or manipulation may optionally be performed prior to expanding the resulting T cell population using the methods and supports of the present invention, which may take the form of, for example, stimulating / restimulating the T cells with anti-CD3 and anti-CD28 antibodies to activate / reactivate the T cells.

[0177] In certain embodiments, it may be desirable to administer activated T cells to a subject, then subsequently draw blood (or perform apheresis), activate T cells from the blood according to the present invention, and reinfuse these activated and expanded T cells back into the patient. This process may be performed several times every few weeks. In certain embodiments, T cells may be activated from a blood draw of 10 ml to 400 ml. In certain embodiments, T cells are activated from a blood draw of about 20 ml, about 30 ml, about 40 ml, about 50 ml, about 60 ml, about 70 ml, about 80 ml, about 90 ml, or about 100 ml. Administration of the subject compositions may be performed in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implant, or transplant. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention may be administered by iv injection. The T cell compositions may be injected directly into a tumor, lymph node, or site of infection / inflammation (autoimmunity).

[0178] Compositions of T cell subpopulations generated according to the present invention have many potential applications, including experimental and therapeutic uses. Specifically, it is anticipated that such T cell populations will be extremely useful in suppressing unwanted or inappropriate immune responses. In such methods, a small number of T cells are removed from a patient, then manipulated and expanded ex vivo before reinfusing these T cells into the patient. Examples of diseases that can be treated in this manner include autoimmune diseases and conditions in which suppressed immune activity is desirable (e.g., for allograft tolerance). A treatment method can include providing a mammal, obtaining a biological sample from the mammal containing T cells, expanding / activating the T cells ex vivo according to the methods of the present invention described above, and administering the expanded / activated T cells to the mammal to be treated. The initial mammal and the treated mammal can be the same or different. The mammal can generally be any mammal, such as a cat, dog, rabbit, horse, pig, cow, goat, sheep, monkey, or human. The initial mammal (the "donor") can be syngeneic, allogeneic, or xenogeneic. The therapy may be used in mammals with an abnormal immune response (e.g., autoimmune diseases including diabetes, multiple sclerosis, myasthenia gravis, neuritis, lupus, rheumatoid arthritis, psoriasis, and inflammatory bowel disease), or undergoing tissue transplantation, or infertility treatment.

[0179] The main technical hurdles associated with such therapies include the purification of the cells of interest from the patient and the propagation and / or manipulation of those cells in vitro. Such therapies generally require large numbers of cells, and therefore, it can be considered essential to optimize methods for inducing T cell proliferation in vitro to maximize the number of T cells produced and minimize the time required to produce sufficient numbers of T cells. The compositions and methods of the present invention result in large numbers of T cells of specific subpopulations.

[0180] The T cell subpopulations of the present invention can be used in a variety of applications and therapeutic modalities. The T cell subpopulations of the present invention can be used in the treatment of disease states, including, but not limited to, cancer, autoimmune diseases, allergic diseases, inflammatory diseases, infectious diseases, and graft-versus-host disease (GVHD). Broad exemplary T cell therapies include infusion into a subject of T cell subpopulations selectively expanded ex vivo by the methods of the present invention, with or without immunodepletion, or infusion into a subject of xenogeneic ex vivo expanded T cells isolated from a donor subject (e.g., adoptive cell transfer).

[0181] Autoimmune disorders Autoimmune diseases or disorders are diseases resulting from inappropriate and excessive responses to self-antigens. Research has implicated defective Treg cells in autoimmune disorders. Examples of autoimmune diseases include, but are not limited to, diabetes mellitus, uveitis and multiple sclerosis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, hemolytic anemia, pemphigus vulgaris, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthropathy, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, Crohn's disease, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus. In autoimmune disease states, CD4 + CD25 +Tregs may be reduced in number or functionally defective. Peripheral nerve-derived Tregs with reduced capacity to suppress T cell proliferation have been observed in patients with multiple sclerosis (Viglietta et al., J. Exp. Med. 199:971-979 (2004)), polyglandular autoimmune syndrome type 2 (Kriegel et al., J. Exp. Med. 199:1285-1291 (2004)), type 1 diabetes (Lindley et al., Diabetes 54:92-929 (2005)), psoriasis (Sugiyama et al., J. Immunol. 174:164-173 (2005)), and myasthenia gravis (Balandina et al., Blood 105:735-741 (2005)).

[0182] Treatment of autoimmune disorders using T cell therapy can involve different mechanisms. In one embodiment, blood or another source of immune cells may be removed from a subject suffering from an autoimmune disorder. The methods of the present invention described herein can be used to selectively expand T cell types other than memory T cells from a patient sample. After removal and expansion of autologous cells, inappropriate memory T cells can be depleted in the subject requiring removal of inappropriate memory T cells by known methods, including administration of low-dose total body irradiation, thymic irradiation, antithymocyte globulin, and chemotherapy. Exemplary chemotherapeutic agents of the present invention include, but are not limited to, campath, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation therapy. After depletion of inappropriate memory T cells capable of recognizing autoantigens, ex vivo expanded autologous T cells can be readministered to the subject to reconstitute or restimulate the subject's immune system.

[0183] Alternatively, or in addition to the above-mentioned therapeutic modalities, Treg cells can be isolated from sources including peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gastrointestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, or any other lymphoid tissue and tumor. These T cells can be selectively expanded using the methods of the present invention. These expanded Treg cells can be re-administered to patients to suppress inappropriate immune responses. This Treg therapy can be administered to subjects who have not undergone immunodepletion, or to suppress minimal remaining immune responses after immunodepletion.

[0184] Graft-versus-host disease A major problem in hematopoietic stem cell transplantation is graft-versus-host disease (GVHD), which is caused by alloreactive T cells present in the infused hematopoietic stem cell preparation. In organ transplantation, graft rejection mediated by alloreactive host T cells is a major problem and is usually overcome by long-term immunosuppression of the transplant recipient.

[0185] In a manner similar to that described above, treating or reducing the risk or severity of adverse GVHD events associated with T cell therapy may involve different mechanisms. In one embodiment, blood or another source of immune cells may be removed from a subject suffering from GVHD. The methods of the invention described herein may be used to selectively expand T cell types other than memory T cells, selectively expanding cell types that do not involve persistent recognition of antigens derived from exogenous tissues. Following autologous cell removal and ex vivo expansion, inappropriate memory T cells may be depleted in a subject requiring removal of inappropriate memory T cells by known methods, including administration of low-dose total body irradiation, thymic irradiation, antithymocyte globulin, and chemotherapy. Exemplary chemotherapeutic agents of the invention include, but are not limited to, campath, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation therapy. After depletion of inappropriate memory T cells capable of recognizing antigens on exogenous tissue, ex vivo expanded autologous T cells can be readministered to the subject to reconstitute or restimulate the subject's immune system.

[0186] Alternatively, or in addition to the above-mentioned therapeutic modalities, Treg cells removed from the patient's blood may be selectively expanded, and these expanded Treg cells may be readministered to the patient to suppress minimal residual immune responses after immunodepletion or to suppress inappropriate immune responses in subjects who have not undergone immunodepletion.

[0187] Allergic diseases Allergic diseases can also be affected by T cell dysfunction. Studies have shown that impaired CD4 T cell function in allergen-specific T helper type 2 (Th2) cells + CD25 +Treg-mediated inhibition has been shown to be present in patients with seasonal allergies (Ling EM, et al., Lancet 2004;363:608-15; Grindebacke H, et al., Clin Exp Allergy 2004;34:1364-72). Furthermore, altered proportions of T cell populations have been associated with allergic and asthmatic individuals compared with healthy subjects (Akdis M, et al., J Exp Med 2004;199:1567-75; Tiemessen MM, et al., J Allergy Clin Immunol 2004;113:932-9).

[0188] In a manner similar to that described above, treatment, prevention, or alleviation of allergic disorders using T cell therapy may involve different mechanisms. Like autoimmune disorders and GVHD, allergic disorders are caused by an inappropriate immune response. Suppression of the response or depletion of cells capable of recognizing inappropriate antigens can alleviate allergic symptoms. In a T cell therapy method for the treatment of allergies, blood can be withdrawn from a subject suffering from an allergic disorder. The methods of the invention described herein can be used to selectively expand non-T memory T cell types, which selectively expand cell types that do not involve persistent recognition of antigens derived from inappropriate antigens (e.g., legume proteins). After withdrawal and expansion of autologous cells, inappropriate memory T cells can be depleted in subjects requiring removal of inappropriate memory T cells by known methods, including administration of low-dose total body irradiation, thymic irradiation, antithymocyte globulin, and chemotherapy. Exemplary chemotherapeutic agents of the present invention include, but are not limited to, campath, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation therapy. Following depletion of inappropriate memory T cells capable of recognizing antigens on exogenous tissue, ex vivo expanded autologous T cells can be readministered to the subject to reconstitute or restimulate the subject's immune system.

[0189] Alternatively, or in addition to the above-mentioned therapeutic modalities, Treg cells removed from the patient's blood may be selectively expanded, and these expanded Treg cells may be readministered to the patient to suppress minimal residual immune responses after immunodepletion or to suppress inappropriate immune responses in subjects who have not undergone immunodepletion.

[0190] inflammatory diseases T cell therapy is implicated in the treatment of inflammatory diseases and inflammation-related disorders, many of which can also be classified as autoimmune disorders. Non-limiting examples of inflammatory diseases and inflammation-related disorders include diabetes mellitus, rheumatoid arthritis, inflammatory bowel disease, familial Mediterranean fever, neonatal-onset multisystem inflammatory disease, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), and Behcet's disease.

[0191] Due to the role of Treg cells in suppressing inappropriate immune responses to non-pathogenic antigens, a decrease in the number or impaired function of these T cell subpopulations can contribute to inflammatory diseases, such as inflammatory bowel disease (M Himmell, et al., Immunology 2012 Jun;136(2):115-122) and rheumatoid arthritis (M Noack, et al., Autoimmunity Reviews 2014 Jun;13(6):668-677).

[0192] Inflammatory diseases are mechanistically similar to autoimmune disorders. Thus, inflammatory diseases may be caused in part by an inappropriate immune response. Suppression of the response or depletion of cells capable of recognizing inappropriate antigens can alleviate inflammatory symptoms. In a method of T cell therapy for the treatment of inflammatory diseases, blood can be withdrawn from a subject suffering from an inflammatory disorder. The methods of the invention described herein can be used to selectively expand non-T memory T cell types, selectively expanding cell types that do not involve persistent recognition of inappropriate antigens (e.g., carbamylated proteins in rheumatoid arthritis mediated by anti-carbamylated protein (anti-CarP) antibodies). After autologous cell withdrawal and expansion, inappropriate memory T cells can be depleted in subjects requiring removal of inappropriate memory T cells by known methods, including administration of low-dose total body irradiation, thymic irradiation, antithymocyte globulin, and chemotherapy. Exemplary chemotherapeutic agents of the present invention include, but are not limited to, campath, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation therapy. Following depletion of inappropriate memory T cells capable of recognizing autoantigens and initiating the resulting inflammatory response, ex vivo expanded autologous T cells can be readministered to the subject to reconstitute the subject's immune system.

[0193] Alternatively, or in addition to the above-mentioned therapeutic modalities, Treg cells removed from the patient's blood may be selectively expanded, and these expanded Treg cells may be readministered to the patient to suppress minimal residual immune responses after immunodepletion or to suppress inappropriate immune responses in subjects who have not undergone immunodepletion.

[0194] Hyperproliferative disorders Evidence from cancer patients further suggests T cell dysfunction associated with hyperproliferative disorders, including cancer. For example, increased Treg activity may lead to poor immune responses to tumor antigens and contribute to immune dysfunction. Increased CD4+CD25+ populations have been observed in either the blood or tumors of patients with lung, pancreatic, breast, liver, and skin cancer (Woo EY, et al.; J Immunol 2002;168:4272-6; Wolf AM, et al. Clin Cancer Res 2003;9:606-12; Liyanage UK, et al. J Immunol 2002;169:2756-61; Viguier M, et al. J Immunol 2004;173:1444-53; Ormandy LA, et al. Cancer Res 2005;65:2457-64).

[0195] The methods of the present invention can be used to expand T cells specific for tumor antigens or hyperproliferative disorder antigens, or antigens associated with hyperproliferative disorders. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response.

[0196] Cancers that can be treated include non-vascularized or not yet substantially vascularized tumors, and vascularized tumors. Cancers can include non-solid tumors (e.g., hematological tumors, such as leukemias and lymphomas) or solid tumors. Cancer types that can be treated with the products of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, certain leukemias or lymphatic tumors, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also included are adult and pediatric tumors / cancers. These include skin cancer, brain cancer and other central nervous system cancers, head cancer, neck cancer, muscle / sarcoma cancer, bone cancer, lung cancer, esophageal cancer, stomach cancer, pancreatic cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, breast cancer, kidney cancer, prostate cancer, bladder cancer, or thyroid cancer, or cancers including glioblastoma.

[0197] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (hematogenous) cancers include leukemias, including acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0198] A solid tumor is an abnormal volume that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form the tumor (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcoma and carcinoma, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, pulmonary fibroblasts, and pulmonary fibroblasts. These include: alveolar carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0199] One approach to treating subjects or patients in need of treatment is to use the expanded T cells of the present invention and genetically engineer the T cells to target antigens expressed on tumor cells by expressing a chimeric antigen receptor (CAR). CARs are antigen receptors designed to recognize cell surface antigens in a manner independent of human leukocyte antigens. In treatments utilizing CARs, immune cells may be collected from the patient's blood or other tissues. T cells are engineered as described below to express a CAR on their surface, allowing the T cells to recognize specific antigens (e.g., tumor antigens). These CAR T cells can then be expanded by the methods of the present invention and infused into the patient. In certain embodiments, T cells are expressed at a density of 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , or 1 × 10 12 Once infused into the patient, the T cells continue to proliferate, express the CAR, and mount an immune response against T cells bearing the specific antigen that the CAR was engineered to recognize.

[0200] In one embodiment, the present invention provides cells (e.g., T cells) engineered to express a CAR, wherein the CAR T cells exhibit anti-tumor properties. The CAR of the present invention can be engineered to include an extracellular domain with an antigen-binding domain fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). When expressed in T cells, the CAR can redirect antigen recognition based on antigen-binding specificity.

[0201] The antigen-binding portion of the CAR comprises a target-specific binding agent called an antigen-binding portion. The selection of the portion depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular disease state. Thus, antigen-binding domains in the CAR of the present invention include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0202] Expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion of a CAR polypeptide to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0203] The T cells of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. In another embodiment, the present invention provides a gene therapy vector.

[0204] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0205] Furthermore, the expression vector can be introduced into cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication function within at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (For example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0206] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and introduced into target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0207] Additional promoter regions (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have recently been shown to contain functional regions downstream of the start site as well. The spacing between promoter regions is often flexible, so that promoter function is preserved when regions are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter regions can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual regions appear to be able to function cooperatively or independently to activate transcription. Methods for generating CAR T cells are known in the art (see, e.g., U.S. Patent No. 8,906,682).

[0208] In embodiments in which the T cells are CAR T cells, the choice of antigen-binding moiety of the invention will depend on the particular type of cancer being treated. Tumor antigens are known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chronic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RUL RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, surviving and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF-1), IGF-II, IGF-I receptor, and mesothelin.

[0209] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CDd20; ROR1, CD22, CD23, and lambda / kappa light chains, are other candidate target antigens in B-cell lymphomas.

[0210] The type of tumor antigen referred to can also be a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells, but instead are also expressed on normal cells under conditions that do not allow for immune tolerance to the antigen to be induced. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or TAAs can be antigens that are normally present at very low levels on normal cells but occur at significantly higher levels on tumor cells.

[0211] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA224, BTAA, CA 125, CA 15-3 / CA 27 / 29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, C250, GA733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, MAC-2 binding protein / cyclophilin C-related protein, TAAl6, TAG72, TLP, and TPS, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-met, PSMA, glycolipid F77, EGRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and others.

[0212] infectious disease The immune response to infectious diseases involves a balance between anti-pathogen and anti-inflammatory responses. T cells play a major role in this complex balance. Infectious pathogens that can elicit a T cell response can be bacterial, viral, parasitic, or fungal. Treg cells have been implicated in contributing to the chronicity of infections caused by Helicobacter pylori (Lundgren A, et al. Infect Immun 2003;71:1755-62), hepatitis B virus (HBV), and hepatitis C virus (HCV) (Cabrera R, et al., Hepatology 2004;40:1062-71; Stoop JN, et al., Hepatology 2005;41:771-8; Sugimoto K, et al., Hepatology 2003;38:1437-48). This expansion of specific T cell subpopulations may contribute to the protracted nature of these infections by inappropriately suppressing memory T cell responses. The compositions of the present invention can be used to specifically expand specific T cell subpopulations and can be used in the treatment of such infectious diseases.

[0213] Infectious diseases can be caused by direct contact with pathogens and transmission from person to person, animal to person, or mother to fetus. Alternatively, infectious diseases can be transmitted by indirect contact, for example, from contact with infected surfaces such as door handles, tables, counters, or faucet handles. Infectious diseases can also be transmitted through insect bites or food contamination. Certain autoimmune disorders, such as HIV or AIDS, and some cancers can increase susceptibility to infectious diseases. Certain treatment regimens that suppress the immune system can also promote susceptibility to infectious diseases. Exemplary infectious diseases include smallpox, malaria, tuberculosis, typhus, plague, diphtheria, typhoid, cholera, dysentery, and pneumonia.

[0214] As discussed above, there are several mechanisms by which selective expansion of T cells can be used in the treatment of disease states. In infectious disease states, patients suffering from an infectious disease lack sufficient immunity to the infectious agent. The methods of the present invention can be used to selectively expand xenogeneic T memory cells from donors immune to a particular infectious agent, which can be utilized in adoptive T cell transfer. The ex vivo expanded T cells from the infectious agent-experienced donor can then be infused into the patient infected with the infectious disease. In certain embodiments, T cells are in the range of 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , or 1 × 10 12 The infectious antigen-competent donor memory T cells are then able to help mount an autologous immune response in the patient.

[0215] Alternative methods utilizing the present invention in the treatment of infectious diseases include the selective expansion of autologous or heterologous Th17 cells for reinfusion or adoptive cell transfer, respectively. Using the methods described herein, blood- or tissue-derived T cells isolated from a patient can be expanded ex vivo. These expanded T cells can then be infused into the patient to help induce B cells secreting antibodies against specific infectious antigens (e.g., Streptococcus M-protein, Neisseria fimbriae, Borrelia burgdorferi lipoprotein VisE, Burkholderia pseudomallei polysaccharide antigen, Aspergillus fumigatus galactomannan, or Francisella tularensis lipopolysaccharide). In certain embodiments, T cells are infused at a concentration of 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9, 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , or 1 × 10 12 The cells are administered to the subject.

[0216] Combination therapy For many of the disease states listed above, existing treatments may be recommended. The T cell subpopulation expanded by the present invention may be used as the only alternative therapy or in conjunction with other known therapies in some cases. T cell therapy may be administered before, simultaneously with, or subsequent to the administration of other therapies.

[0217] The methods of the present invention can also be used in conjunction with vaccines to promote antigen reactivity and in vivo efficacy. In one embodiment, the compositions of the present invention are administered to a patient in conjunction with a composition that promotes in vivo T cell proliferation, e.g., IL-2, IL-4, IL-7, IL-10, IL-12, and / or IL-15. Furthermore, given the relatively long half-life of T cells expanded according to the present invention in the body, these cells may carry a desired nucleic acid sequence of interest and potentially home to the site of cancer, disease, or infection, thereby serving as an ideal vehicle for gene therapy, as described above. Thus, cells expanded according to the present invention may be delivered to a patient in conjunction with a vaccine, one or more cytokines, one or more therapeutic antibodies, etc. Virtually any therapy that would benefit from a more robust T cell population can be used in conjunction with the compositions of the present invention.

[0218] V. Kits of the Invention Also provided herein are kits comprising (i) compositions for isolating T cells from a subject, (ii) compositions for ex vivo culture of T cells, and (iii) compositions for selective expansion of one or more T cell subpopulations (e.g., Th17, Treg, memory T cells, etc.). Kits of the invention may optionally include compositions for reactivating Treg cells.

[0219] The kit may also include written instructions for using the kit, such as instructions for washing steps, culture conditions, and duration of incubation of isolated T cells with the compositions of the invention for selective expansion of specific T cell subpopulations.

[0220] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. [Example]

[0221] The present invention can be further understood by reference to the following examples, which are provided for purposes of illustration and are not meant to be limiting.

[0222] Example 1. Primary T cells and culture conditions Peripheral blood mononuclear cells (PBMCs) are isolated from healthy donor buffy coats or apheresis under informed consent (HemaCare Corp., CA USA and Astarte Biologics, LLC, WA, USA). Antigen-specific memory T cells are expanded in PBMCs from cytomegalovirus (CMV)-positive donors after stimulation with the CMV pp65 peptide NLVPMVATV (Astarte Biologics LLC). CD4 + CD25 + CD127 低 / -Regulatory T cells (Tregs) are obtained from a lymphocyte fraction that has been purified and CD4-enriched (DYNABEADS® UNTOUCHED™ Human CD4 T cells, Cat. No. 11346D, Thermo Fisher Scientific, CA USA) by fluorescence-activated cell sorting based on antibody labeling of CD4, CD25, and CD127, resulting in CD4-expressing Treg cells (Oslo University Hospital, Ulleval Blood Bank, Oslo, Norway).

[0223] T cells are activated either directly from PBMCs using DYNABEADS® with various ligand compositions and stoichiometries as described, or from isolated or enriched T cell subsets. Activated T cells are incubated at 37°C and 5% CO in X-VIVO™ 15 (Lonza Biologics, MD, USA, Cat. No. BE04-418F) with 2-5% CTS™ Immune Cell SR and 0.25 μg / ml gentamicin (both Thermo Fisher Scientific, NY, USA, Cat. No. 15710-049). 2 and cultured at 0.5–2 × 10 6 Expansion is achieved by adding cytokines and fresh medium every 1-3 days to maintain a cell concentration of 100 cells / ml.

[0224] Flow-sorted T regulatory cells (Tregs) are grown in medium containing 100 ng / ml rapamycin (PHZ1235, Thermo Fisher Scientific, NY, USA) and 300 IU IL-2 / ml (Thermo Fisher Scientific, NY, USA), while CMV-stimulated T cells are grown in 100 IU IL-2 / ml. Th17 / Tc17 cells are grown in medium containing polarizing cytokines (IL-6, IL-1β, IL-23, and TGF-β, all Thermo Fisher Scientific, CA, USA) in the presence of anti-IL-4 and anti-IFN-γ neutralizing antibodies (Thermo Fisher Scientific, CA, USA) as described by Paulos et al. (Paulos et al., Science Transl. Med 55:55ra78 (2010)). 100 IU IL-2 / ml is added on day 3 after activation.

[0225] Example 2. Generation of Dynabeads®-based proliferation platforms with various ligand compositions and ratios DYNOSPHERES® MS-4.5-REK particles (Thermo Fisher Scientific, Lillestrom, Norway) are coupled with different amounts and ratios of stimulatory and costimulatory ligands optimized for activation and polarization of various T cell subsets: i) Tregs, ii) antigen-experienced memory T cells, and iii) Th17 / Tc17 cells, respectively. The relative amounts of stimulatory anti-CD3 antibodies and their co-bound costimulatory ligands are summarized in Table 3.

[0226] (Table 3) TIFF0007623980000003.tif101161

[0227] Example 3. T cell stimulation and proliferation T cells were activated either directly from PBMCs or from isolated or enriched T cell subsets by adding one DYNABEADS® per T cell unless other ratios were noted. In most experiments, DYNABEADS® CD3 / CD28 CTS™ (CD3 high) were included as a baseline stimulator. Additionally, an alternative Treg activation reagent (MACS GMP ExpAct Treg Kit; Miltenyi catalog number 170-076-119) was included to assess Treg proliferation levels. Activated T cells were left undisturbed for 2-3 days and then expanded for 10-14 days by adding fresh media and cytokines as needed. Tregs were restimulated on day 9 using the same stimulatory beads as used for primary activation (day 0). Cells were analyzed using a Coulter Counter (Beckman Coulter, CA USA) to measure absolute cell proliferation.

[0228] Example 4. Antibodies and flow cytometry analysis The following cells were used for Treg analysis: CD4 PerCP (Invitrogen, MHCD0431), CD25 APC (Invitrogen, MHCD2505), and CD127 PE (Invitrogen, A18684). Intracellular FOXP3 expression was analyzed using Alexa Fluor 488 (BD560047) after fixation / permeabilization (eBioscience). Flow cytometry data were collected on a BD LSRII (BD Biosciences) and analyzed with FACS Diva software (BD Biosciences). Lymphocytes were gated according to their forward and size distribution characteristics. Appropriate irrelevant isotype controls were used to set the negative gate (IgG2a PerCP (Thermo Fisher Scientific, Catalog No. MA1-10426), IgG1 APC (Thermo Fisher Scientific, Catalog No. MG105), IgG1 PE (Thermo Fisher Scientific, Catalog No. MG104), and IgG1 Alexa Fluor 488 (BD Biosciences, Catalog No. 557702)).

[0229] CMV-specific CD8+ T cells were purified by Pro5 Pentamer APC (NLVPMVATV / A * 02:01) (ProImmune, Oxford UK) and CD8-PE (Thermo Fisher Scientific, Catalogue No. MHCD0804) are used and identified with appropriate isotype controls (IgG1 PE, Thermo Fisher Scientific, Catalogue No. MG104).

[0230] Intracellular IL-17 expression in PMA / ionomycin-activated Th17 cells was detected after fixation / permeabilization (eBioscience) using IL-17A-PE (catalog no. A18695, clone: ​​4H1524, Molecular Probes) and appropriate isotype control (IgG2b PE, catalog no. 400313 BioLegend). Flow cytometry data were collected on a BD LSRII (BD Biosciences) and analyzed with FACS Diva software (BD Biosciences).

[0231] Example 5. Functionality by flow cytometry Th17-polarized and proliferating T cells were characterized for intracellular IL-17 expression 4-5 hours after PMA / ionomycin stimulation (day 13 post-DYNABEADS® activation) using a Cell Stimulation Cocktail Kit (eBioscience, catalog no. 00-4971) containing the protein transport inhibitors brefeldin and monensin. This stimulation was performed according to the protocol provided by eBioscience. IL-17 expression was assessed by intracellular IL-17 staining by flow cytometry as described.

[0232] Example 6. Treg activation and proliferation As shown in Figure 1, CD4 + CD25 + CD127 低 / -Flow cytometry-sorted Tregs (approximately 90% FOXP3+ cells) were efficiently activated with DYNABEADS® Treg prototypes, expanded hundreds of times (top), and retained high FOXP3 expression after 14 days in expansion culture (bottom). Cells were restimulated on day 9 using the same prototype beads. Increased proliferation was achieved with DYNABEADS®, which bind lower amounts of CD3. As seen in Figure 1, the stimulation protocol utilizing DYNABEADS® CD3 / CD28 (low CD3 - approximately 0.34) resulted in the highest Treg proliferation. DYNABEADS® CD3 / CD28 (high CD3 - approximately 3.4) did not generate any efficient Treg proliferation. DYNABEADS® CD3 / CD28 low (CD3 -approximately 0.34) and DYNABEADS® CD3 / CD28 medium (CD3 -approximately 0.75) perform similarly or better than alternative Treg stimulating reagents. DYNABEADS® CD3 / CD28 CTS™ (CD3 -approximately 1.5) results in suboptimal proliferation.

[0233] Example 7. Activation and expansion of antigen-experienced (CMV-specific memory) T cells As seen in Figure 2, the fold expansion of CMV-specific memory T cells 10 days after activation negatively correlates with the increased signal strength provided by DYNABEADS® prototypes conjugated with more agonistic CD3 antibodies. Using cytomegalovirus (CMV)-specific T cells as a model, we compare the proliferation kinetics and phenotype of T cells expanded from peptide-stimulated PBMCs from CMV+ donors using different DYNABEADS® prototypes conjugated with CD3, CD28, and / or CD137 antibodies, and DYNABEADS® CD3 / CD28 CTS™. The superiority of beads conjugated with low amounts of CD3 (which provide a weaker CD3 activation signal) is demonstrated here. DYNABEADS® CD3 / CD28 CTS™ is suboptimal and does not expand CMV-specific T cells. DYNABEADS® CD3 / CD137 / CD28 results in the highest memory T cell expansion.

[0234] Example 8. Activation, polarization and proliferation of Th17 cells T cells are cultured using Th17-polarizing conditions and expanded using DYNABEADS® (CTS beads) conjugated with antibodies against CD3 / ICOS (various amounts of anti-CD3 antibody conjugated; 1.5 and 0.06, and two different ICOS clones) or CD3 / CD28 (CTS beads). From day 3, IL-2 is added to the culture medium. On day 13, the cultures are stimulated with PMA-ionomycin for 4-5 hours before assessing IL-17 expression. The histograms show the intracellular expression of IL-17; DYNABEADS® CD3 / ICOS (ISA-3), CD3 high and low (1.5 and 0.06), DYNABEADS® CD3 / ICOS (different ICOS C398.4A, eBiocienses, purchased from Affymetrix), CD3 low (0.06), and DYNABEADS® CD3 / CD28 CTS™. The strength of the activation signal and the nature of the costimulation significantly influence the polarization and proliferation of Th17 cells. Activation with DYNABEADS® CD3 / ICOS (medium -0.3) results in a phenotype similar to that of "(high -1.5)" (not shown).

[0235] Successful polarization and expansion of Th17 cells was only achieved by activating T cells with DYNABEADS® CD3 / ICOS (ISA-3 clone), which was designed to provide a weak CD3 signal (CD3 -0.06). Only the ISA-3 ICOS clone was functional in this study.

[0236] DYNABEADS® CD3 / CD28 CTS™ (CD3-1.5) results in low / no Th17 polarization / expansion.

[0237] Example 9: Effect of bead:cell ratio on selective expansion of IL-17-producing cells T cells were collected from three donors (A, B, and C). PBMCs were collected from healthy donors by leukapheresis and further purified on a Ficoll-sodium metrizoate density gradient. Astarte Biologics (Normal PBMCs). CD3+ T cell enrichment was performed using the Dynabeads® Untouched™ Human T Cells Kit (Thermo Fisher Scientific catalog number 11344D) according to the provided manual.

[0238] Negatively isolated CD3+ T cells were stimulated with DYNABEADS® CD3 / ICOS (low-medium-high) at a given bead:cell ratio (BC 1:1, 1:3, 1:5) (Table 4) and cultured for 3 days with Th17-polarizing cytokines: TGF-β1 (Thermo Fisher Scientific, PHG9214; 10 ng / ml, IL-1β: PHC0816 10 ng / ml, IL-23: PHC9324, 20 ng / ml, rIL-6: PHC0066 10 ng / ml, and IL-2 (100 μl, from day 3). Anti-IFNγ antibody AHC4032 Anti-human IL-17, E.Bioscience (Cat. No. BMS129) 10 μg / ml. Split as needed. On day 10, total proliferation (T cell proliferation) is measured, and cells are restimulated and the fraction of IL-17-producing cells is assessed by intracellular flow cytometry staining for IL-17 (% IL-17 producing cells). Data shown in Figures 4A-4C demonstrate the effect of bead:cell ratio on the selective expansion of Th17 cells. The relative number of IL-17-producing cells is reported as the percentage of IL-17-producing cells multiplied by the total T cell proliferation fold.

[0239] (Table 4) TIFF0007623980000004.tif91136AB=CD3 / ICOS antibody ratio BC = cell to bead ratio

[0240] Example 10: Protocol for generating Th17 cells preface IL-17A-secreting T cells (Th17 and Tc17 cells) induce tumor regression to a greater extent than nonpolarized T cells or IFN-γ-secreting Th1 or Tc1 cells after adoptive transfer into tumor-bearing mice (Muranski et al., Blood, 112:362-73, (2008), Nelson et al., J Immunol, 194:1737-47 (2015), Guedan et al., Blood, 124:1070-80 (2014)). The factors regulating the generation of human Th17 cells are still under debate, and Th17 cell differentiation has been reported to be controlled by various transcription factors, including RORγt, IRF4, RUNX1, BATF, and STAT3 (Nalbant and Eskier, Front. Biosci. (Elite Ed.), 8:427-35 (2016)). Identifying robust culture conditions capable of expanding Th17 cells for adoptive cell transfer is desirable. DYNABEADS® CD3 / CD28 CTS™ activation results in a T cell population characterized by a Th1-polarized phenotype, while a weaker CD3 signal provided to T cells under certain polarization conditions induces Th17 development (Purvis et al., Blood, 11:4829-37 (2010)). It has been found that optimized CD3 signal strength can be achieved by using a low bead-to-cell ratio of 1:5 and reducing the amount of agonistic CD3 antibody bound to the beads. A weak CD3 signal combined with ICOS costimulation was found to generate a larger fraction of IL-17A-producing T cells compared to beads providing CD28 costimulation.

[0241] The following alternative pathways and protocols have been shown to generate Th17 cells: i) CD5 or CD6 costimulation in the presence of IL-1β, IL-6, IL-23, and TGF-β (de Wit et al., Blood, 118:6107-14 (2011)); ii) association of SLAMF3 and SLAMF6 with Ag-mediated CD3 / TCR stimulation (Chatterjee et al., J Immunol, 188:1206-12 (2012)); iii) agonists acting on the aryl hydrocarbon receptor (ARH) (Veldhoen et al., J Exp Med, 206:43-9 (2009)); and iv) a cholesterol precursor that functions as a potent retinoic acid receptor-related orphan receptor (ROR) agonist (Hu et al., Nat Chem Biol, 11:141-147 (2015)). However, the commonly used medium, RPMI, supports only low levels of Th17 polarization. Other media richer in aromatic amino acids, precursors of aryl hydrocarbon receptor (AHR) agonists, consistently result in higher Th17 proliferation, indicating that AHR activation is essential for Th17 polarization. RORγt is the master transcription factor for Th17 cells. When injected into mice, RORγt agonists induce high levels of Th17 cytokines, increase the levels of costimulatory receptors such as CD137, and decrease the expression of the co-inhibitory receptor PD-1 (Hu et al., “RORγt agonist regulates immune checkpoint receptors to enhance anti-tumor immunity,” AACR abstract #565, New Orleans 2016). Furthermore, T cells treated with RORγt agonists in vitro maintained low PD-1 expression after adoptive transfer.

[0242] A significant Th17 polarization of T cells was found following low intensity CD3 activation and CD5 co-stimulation under polarizing conditions, including the use of an AHR agonist.

[0243] Materials and Methods Primary T cells and culture conditions: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats or apheresis (Astarte Biologics, WA, USA) of healthy donors with informed consent. Negatively isolated T cells (DYNABEADS® Untouched Human T Cells, catalog number 11344D, Thermo Fisher Scientific, Waltham, MA) were activated using DYNABEADS® with various ligand compositions and stoichiometries as described. Polarization conditions are described in the text. Activated T cells were cultured in X-Vivo15™ (Cat. No. BE02-060F, Lonza Biologics, MD, USA) plus 2–5% CTS™ Immune Cell SR (Cat. No. A25961-02, Thermo Fisher Scientific, Waltham, MA) and 0.25 μg / mL gentamicin (Cat. No. 15750060, Thermo Fisher Scientific) at 37°C and 5% CO2, as indicated, at 0.5–2 × 10 6 Expansion is achieved by adding cytokines and fresh medium every 1-3 days to maintain a cell concentration of 100 cells / ml.

[0244] Protocol Summary: Th17 / Tc17 cells were grown in medium containing polarizing cytokines (IL-6, IL-1β, IL-23, and TGF-β (catalog numbers IL6-PHC066, IL-1β-PHC0816, IL23-PHC9324, and TGF-β-PHG9214, Thermo Fisher Scientific, Waltham, MA) in the presence of anti-IL-4 and anti-IFN-γ neutralizing antibodies (catalog numbers AC0642 and ACH4032, Thermo Fisher Scientific, Waltham, MA) as described by Paulos et al., Sci Transl Med, 2:55ra78, (2010). Polarizing cytokines and antibodies were added only for the initial activation (days 0–3). Cells were split and maintained in medium containing 100 IU IL-2 / mL and IL-23 from day 3 postactivation.

[0245] Stimulating Dynabeads: The amounts of stimulating anti-CD3 antibodies and their co-bound costimulatory ligands bound to Dynospheres are summarized in Table 5.

[0246] Table 5. Dynabeads Th17 Expander prototype and control TIFF0007623980000005.tif28159 Anti-CD3 and anti-CD28 antibodies XR-CD3 and XR-CD28 (Thermo Fisher Scientific, Norway), anti-ICOS antibody ISA-3 (Affymetrix / eBioScience CA USA), anti-CD5 antibody clone UCHT2 (Affymetric / eBioScience CA USA).

[0247] Th17 cell polarization and expansion: T cells isolated from healthy donor PBMCs by negative isolation (DYNABEADS® Untouched Human T Cells, catalog no. 11344D, Thermo Fisher Scientific, Waltham, MA) were activated using DYNABEADS®-Th17 prototypes (1:5 cell-to-bead ratio) designed to deliver different costimulatory signals in the presence of polarizing cytokines (IL-1β, IL-21, IL-23, IL-6, TGF-β) and neutralizing IL-4 and IFNγ antibodies (Table 5). Activated T cells were left undisturbed for 2–3 days and then expanded for 10–14 days by adding fresh medium and cytokines, along with IL-2 and IL-23, as needed. T cell proliferation was assessed by cell counting (Coulter Counter, Beckman Coulter, CA USA). On days 10-14 of growth, cells were stimulated with PMA / ionomycin for 5 hours in medium containing monensin (BD GOLGISTOP™, Catalog No. 554724, BD Biosciences) and brefeldin A (BD GOLGIPLUG™, Catalog No. 555029, BD Biosciences) before being analyzed by flow cytometry for phenotypic markers and intracellular expression of IL-17A and IFNγ, and after fixation and permeabilization.

[0248] Table 6: Polarizing agents and their functions TIFF0007623980000006.tif147165

[0249] Intracellular cytokine expression in Th17 cells activated with PMA / ionomycin (Cell Stimulation Cocktail Kit containing the protein transport inhibitors brefeldin and monensin, catalog no. 00-4971, eBioscience) was detected using IL-17A-PE (catalog no. A18695, clone: ​​4H1524, Molecular Probes) along with IL17-F, IFN-γ, and appropriate isotype controls (IgG2b PE, catalog no. 400313, BioLegend). Flow cytometry data were collected using a BD LSRII (BD Biosciences) and analyzed with FACS Diva software (BD Biosciences).

[0250] Results and Discussion i) Effect of neutralizing antibodies The αIL-4 / α-IFNγ antibody was utilized in a Th17 cell polarization protocol to disrupt Th1 / Th2 polarization.

[0251] To improve Th17 generation after CD3 / CD28 activation, blocking antibodies were introduced. Clinical-grade blocking antibodies are expensive and further complicate Th17 cell generation protocols.

[0252] Protocol modification: T cells are activated using optimized conditions with either DYNABEADS® CD3 / ICOS or DYNABEADS® CD3 / CD28. Th17 polarization is promoted with cytokines TGF-β, IL-23, IL-6, and IL-1β. The effect of αIL-4 / α-IFN-γ neutralizing antibodies on the fraction of IL-17-producing CD4+ cells after expansion is compared in Figure 5.

[0253] Conclusion: Stimulation with CD3 / ICOS results in a higher percentage fraction of IL-17-producing T cells compared to CD3 / CD28 (42.5% vs. 16.5% of CD4 cells). The generation of Th17 cells after stimulation of CD4+ T cells with DYNABEADS® CD3 / ICOS (plus Th17-polarizing cytokines) is less critically dependent on neutralizing IFN-γ and IL-4 antibodies. Stimulation with DYNABEADS® CD3 / CD28 requires the addition of Th1 / Th2-blocking antibodies.

[0254] ii) Effect of CD5 costimulation It has been reported that CD5 promotes Th17 development by increasing IL-23R expression, resulting in prolonged STAT3 activation and increased levels of ROR-γt compared to CD28 costimulation (de Wit et al., Blood, 118:6107-14 (2011)).

[0255] Protocol modification: T cells were activated with DYNABEADS® CD3 / CD5, DYNABEADS® CD3 / ICOS, or DYNABEADS® CD3 / CD28 using equivalent signal strength and stoichiometry. Th17 polarization was promoted by the addition of cytokines (TGF-β, IL-23, IL-6, IL-1β) and an αIL-4 / α-IFN-γ neutralizing antibody. The fraction of IL-17- and IFN-γ-producing CD4+ T cells (intracellular staining) and the fraction of CCR4+CCR6+ expressing cells (a Th17-associated phenotype) were compared (Figures 6 and 7).

[0256] Conclusions: Stimulation with CD3 / CD5 results in a comparable or slightly higher Th17 profile compared with CD3 / ICOS and is superior to CD3 / CD28 stimulation as judged by IL-17 production and CCR4 / CCR6 co-expression.

[0257] iii) Effect of AHR agonists The AHR ligand FICZ (5,11-dihydroindolo(3,2-b)carbazole-6-carboxaldehyde) has been shown to upregulate the Th17 program (Veldhoen et al., J. Exp. Med., 206:43-9 (2009)).

[0258] Protocol modification: T cells were activated with DYNABEADS® CD3 / CD5, DYNABEADS® CD3 / ICOS, or DYNABEADS® CD3 / CD28 at comparable signal strength and stoichiometry. Th17 polarization was promoted by i) cytokines (TGF-β, IL-23, IL-6, IL-1β) and αIL-4 / α-IFN-γ neutralizing antibody, or ii) the AHR agonist FICZ plus IL-6 and IL-1βα. Th17 polarization was assessed by intracellular staining for IL-17 and IFN-γ following PMA / ionomycin activation of polarized T cells (Figure 8).

[0259] Conclusions: FICZ had promising effects on T cells costimulated with CD5 but not ICOS. The FICZ, IL-1β, and IL-6 protocol generated fewer IL-17-producing cells compared to standard polarization conditions and requires optimization.

[0260] Example 11: Expansion of Treg cells using DYNABEADS™ Treg beads (>80% FOXP3) Table 7: Materials used in this example and their sources TIFF0007623980000007.tif40160

[0261] Sorted Treg cells (>80% FOXP3) were diluted in X-VIVO15 + 5% CTS Immune Cell Serum Replacement (X-VIVO™ 15 / CTS) to 2 million cells / mL. DYNABEADS™ were washed once by magnetic isolation, and the beads were diluted to 4 million cells / mL in X-VIVO™ 15 / CTS. Control beads were washed once, and the beads were diluted to 4 million cells / mL in X-VIVO™ 15 / CTS with 300 U / mL rIL2. Equal volumes of cells, X-VIVO™ 15 / CTS, and beads were then added to wells of a 48-well plate to a total volume of 400 μl. The procedure was completed in triplicate for each sample.

[0262] Table 8: Reagent mixture TIFF0007623980000008.tif45160

[0263] Day 0: Treg expansion mix was prepared in 48-well plates with 300 U / mL rIL2 using X-VIVO™ 15 / CTS as described above.

[0264] Day 2: 200 μL of X-VIVO™ 15 / CTS and 300 U / mL rIL2 were added to each well and the resulting cell suspension was mixed.

[0265] Days 3-12: Cell concentration was estimated daily by microscopy to be approximately 1.5-2 million cells / cm. 2 When the cells reached a density of 100 μg / ml, they were transferred to larger wells or flasks. Fresh X-VIVO™ 15 / CTS was added along with 300 U / mL rIL2 as needed.

[0266] Day 5: 250 μL of X-VIVO™ 15 / CTS was removed from each sample and 250 μL of fresh X-VIVO™ 15 / CTS was added along with 300 U / mL rIL2. The cell suspension was then mixed.

[0267] Day 7: Approximately 1.5-2 million cells / cm 2 Upon reaching a cell density of 100 μg / ml, the cells were transferred to larger wells / flasks and fresh X-VIVO™ 15 / CTS was added along with 300 U / ml rIL2.

[0268] Day 9: Fresh X-VIVO™ 15 / CTS was added again along with 300 U / mL rIL2 and the cell suspension was mixed.

[0269] Day 12: The volume was determined and cells were counted in each well. 100,000-200,000 cells were used for FoxP3 staining using the reagents listed in Table 9.

[0270] (Table 9) TIFF0007623980000009.tif46159

[0271] Fixation / permeabilization solution was prepared by mixing Fixation / Permeabilization (1 part) with Fixation / Permeabilization Diluent (3 parts). Fixation / Permeabilization Buffer / Wash Solution was prepared by mixing Permeabilization Concentrate (1 part) with Water (9 parts). Fresh solutions were prepared daily. Typically, 100,000-200,000 cells were stained.

[0272] The cells were washed once with 1 mL of DPBS / 0.1% HAS, centrifuged at 350×g for 8 minutes, and the supernatant was removed.

[0273] The cells were pulse-vortexed and then vortexed to mix. 1 mL of fixation / permeabilization (1x) solution was added, and the cells were vortexed again. The cell suspension was then incubated at 4°C for 60 minutes. 2 mL of permeabilization buffer / wash solution (1x) was then added, and the suspension was centrifuged at 400 x g for 8 minutes at 4°C, and the supernatant was removed. The addition of permeabilization buffer / wash solution and subsequent centrifugation was repeated once.

[0274] 50 μL of staining mixture (2.5 μL CD4 PerCP, 2.5 μL CD25 APC, 5 μL FOXP3 AF488, 2 μL CD127PE + 38 μL permeabilization buffer / wash solution (1x)) was then added to the samples, followed by incubation at 4° C. for 30 minutes. Samples were then analyzed by flow cytometry.

[0275] Other embodiments While the present invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0276] This patent and the scientific literature referenced herein establish information that is available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference.

[0277] While the present invention has been particularly shown and described with reference to certain embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0278] Exemplary subject matter of the present invention is represented by the following clauses:

[0279] Clause 1 1. A method for selectively expanding members of a T cell subpopulation, comprising: A mixed population of T cells (a) a first agent that provides a primary activation signal to said member of said T cell subpopulation, thereby activating said T cell; (b) a second agent and a third agent, each of which stimulates two or more different accessory molecules on the members of the T cell subpopulation, thereby stimulating proliferation of the activated T cells of (a); and including exposure to the ratio of the first agent, the second agent, and the third agent is adjusted to induce selective proliferation of the members of the T cell subpopulation over members of other T cell subpopulations; method.

[0280] Clause 2 The method of clause 1, wherein said first agent is an anti-CD3 antibody.

[0281] Clause 3 The method of clause 1, wherein said second agent is an antibody.

[0282] Clause 4 The method of clause 1, wherein said third agent is an antibody or a non-antibody protein.

[0283] Clause 5 The method of clause 4, wherein said non-antibody protein is a chemokine or cytokine.

[0284] Clause 6 The chemokine or cytokine is (a) interleukin-1α, (b) interleukin-2, (c) interleukin-4, (d) interleukin-1β, (e) interleukin-6, (f) interleukin-12, (g) interleukin-15, (h) interleukin-18, (i) interleukin-21, and (j) Transforming growth factor-β1 and wherein the protein is one or more proteins selected from the group consisting of: Clause 4 Method.

[0285] Clause 7 10. The method of claim 1, wherein the ratio of the first agent, the second agent, and the third agent is adjusted so that the concentration of the first agent is lower compared to the second or third agent.

[0286] Article 8 The method of clause 7, wherein the lower concentration of the first agent is about 0.34 units.

[0287] Article 9 The method of clause 8, wherein said first agent is an anti-CD3 antibody at a concentration of about 0.34 units and said second agent is an anti-CD28 antibody at a concentration of 3.4 units.

[0288] Article 10 The method of clause 8, wherein said selectively expanded T cell subpopulation is Treg cells.

[0289] Article 11 The method of clause 7, wherein the lower concentration of the first agent is about 0.01 units.

[0290] Article 12 12. The method of clause 11, wherein the first agent is an anti-CD3 antibody at a concentration of about 0.01 units, the second agent is an anti-CD28 antibody, and the third agent is an anti-CD137 antibody.

[0291] Article 13 13. The method of clause 12, wherein said selectively expanded T cell subpopulation is memory T cells.

[0292] Article 14 The method of clause 7, wherein the lower concentration of the first agent is about 0.06 units.

[0293] Article 15 15. The method of clause 14, wherein said first agent is an anti-CD3 antibody at a concentration of about 0.34 units and said second agent is an anti-ICOS antibody.

[0294] Article 16 16. The method of clause 15, wherein said selectively expanded T cell subpopulation is Th17 cells.

[0295] Article 17 1. A method for selectively expanding a T cell subpopulation, comprising: (a) exposing T cells to CD3, CD28, and / or CD137 signals ex vivo; (b) culturing the T cells in a manner that allows for the expansion of Th17 cells, antigen-experienced T cells, and / or regulatory T cells; A method comprising:

[0296] Article 18 18. The method of clause 17, wherein said CD3, CD28, and CD137 signals are mediated by anti-CD3, anti-CD28, and / or anti-CD137 antibodies.

[0297] Article 19 18. The method of clause 17, wherein said anti-CD3, anti-CD28, and anti-CD137 antibodies are used in a range encompassing concentrations of 0.01 units to 1.5 units for expansion of memory T cells.

[0298] Article 20 18. The method of clause 17, wherein said anti-CD3, anti-CD28, and anti-CD137 antibodies are used in a range encompassing a concentration of 0.06 units to 1.5 units for the expansion of Th17 cells.

[0299] Article 21 18. The method of clause 17, wherein said anti-CD3, anti-CD28, and anti-CD137 antibodies are used in a range encompassing a concentration of about 0.34 units to 3.41 units for the expansion of Treg cells.

[0300] Article 22 18. The method of clause 17, wherein said anti-CD3 antibody is used at a lower concentration compared to the concentrations of said anti-CD28 and anti-CD137 antibodies.

[0301] Article 23 The method of clause 17, wherein said T cells are isolated using CD3 selection.

[0302] Article 24 The Th17 cells are CD3 + , CD8 / CD4 + / and producing IL-17 cytokine.

[0303] Article 25 18. The method of clause 17, wherein said Th17 cells are capable of producing IL-17, IL-21, and / or IL-22.

[0304] Article 26 18. The method of clause 17, wherein said memory T cells are selected from the group consisting of stem cell-like memory T cells, central memory T cells, and effector memory T cells.

[0305] Article 27 27. The method of clause 26, wherein said stem cell-like memory cells have one or more of the following markers: CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, IL-7Rα+, IL-2Rβ, CXCR3, and LFA-1.

[0306] Article 28 27. The method of clause 26, wherein said central memory cells have one or more of the following markers: CD3+, CCR7+, CD45RA-, CD45RO+, CD62L+ (L-selectin), CD27+, and CD28+.

[0307] Article 29 29. The method of Clause 28, wherein said central memory cells are capable of producing IL-2.

[0308] Article 30 27. The method of clause 26, wherein said effector memory cells have one or more of the following markers: CD28+ / -, CD27+ / -, CD3+, CD4+, CD8+, CCR7-, CD45RA-, CD45RO+.

[0309] Article 31 27. The method of clause 26, wherein said effector memory cells are capable of producing IFNγ and IL-4.

[0310] Article 32 The Treg cells express the following markers: CD4 + , CD25+, FOXP3 + , and CD127 負 / 低 17. The method of claim 17, comprising one or more of:

[0311] Article 33 1. A method for selectively expanding T regulatory cells, comprising: (a) exposing T cells to CD3 and CD28 signals in vitro; (b) culturing said T cells in a manner that allows for the expansion of T regulatory cells; A method comprising:

[0312] Article 34 34. The method of clause 33, wherein said CD3 and CD28 signals are mediated by anti-CD3 and anti-CD28 antibodies.

[0313] Article 35 35. The method of clause 34, wherein said anti-CD3 and anti-CD28 antibodies are used in a concentration range encompassing 0.34 to 3.4 units.

[0314] Article 36 35. The method of clause 34, wherein said anti-CD3 antibody is used at a lower concentration compared to the concentration of anti-CD28 and / or antibody.

[0315] Article 37 A method for selectively expanding Th17 cells, comprising: (a) exposing CD3+ T cells to CD3, CD28 and / or ICOS signals ex vivo; (b) culturing said CD3+ T cells in a manner that allows for the expansion of Th17 cells; Including, The amount of CD3, CD28, and / or ICOS is not the same.

[0316] Article 38 38. The method of clause 37, wherein said CD3, CD28, and ICOS signals are mediated by anti-CD3, anti-CD28, and anti-ICOS antibodies.

[0317] Article 39 38. The method of clause 37, wherein said anti-CD3, anti-CD28 and anti-ICOS antibodies are used in a concentration range encompassing 0.06 to 1.5 units for the expansion of Th17 cells.

[0318] Article 40 38. The method of clause 37, wherein said anti-CD3 antibody is used at a lower concentration compared to the concentration of anti-CD28 and / or anti-ICOS antibodies.

[0319] Article 41 1. A method for selectively expanding antigen-experienced T cells, comprising: (a) exposing T cells to CD3, CD28, CD27, and / or CD137 signals ex vivo; (b) culturing said T cells in a manner that allows for the expansion of antigen-experienced T cells; A method comprising:

[0320] Article 42 42. The method of clause 41, wherein said CD3, CD28, and CD27, and / or anti-CD137 signals are provided by anti-CD3, anti-CD28, anti-CD27, and / or anti-CD137 antibodies.

[0321] Article 43 42. The method of clause 41, wherein said anti-CD3, anti-CD28, anti-CD27, and anti-CD137 antibodies are used in a concentration range encompassing 0.01 to 1.5 units.

[0322] Article 44 42. The method of clause 41, wherein said anti-CD3 antibody is used at a lower concentration compared to the concentration of anti-CD28 and / or anti-CD137 antibodies.

[0323] Article 45 CD3+ (1) T cells, and (2) beads containing (a) anti-CD3 antibodies and (b) anti-CD28, anti-CD137, or ICOS antibodies capable of selectively expanding T cell subpopulations. A composition comprising: the amount of (a) anti-CD3 antibody and (b) anti-CD28, anti-CD137, or ICOS antibody present are not the same; composition.

[0324] Article 46 46. ​​The composition of clause 45, wherein said T cell subpopulation is selected from the group consisting of Th17 cells, antigen-experienced T cells and / or regulatory T cells.

[0325] Article 47 46. ​​The composition of clause 45, wherein the anti-CD3, anti-CD28, anti-CD27 and anti-CD137 antibodies are used in a concentration range encompassing 0.01 to 1.5 units for expansion of memory T cells.

[0326] Article 48 46. ​​The composition of clause 45, wherein said anti-CD3, anti-CD28, and anti-CD137 antibodies are used in a concentration range encompassing 0.06 to 1.5 units for the expansion of Th17 cells.

[0327] Article 49 46. ​​The composition of clause 45, wherein said anti-CD3 and anti-CD28 antibodies are used in a concentration range encompassing 0.34 to 3.41 units for the expansion of Treg cells.

[0328] Article 50 46. ​​The composition of clause 45, wherein said anti-CD3 antibody is used at a lower concentration compared to the concentrations of anti-CD28 and anti-CD137 antibodies.

[0329] Article 51 (1) Beads containing anti-CD3, anti-CD28, and anti-CD137 antibodies capable of selectively expanding T cells and (2) Th17 cells. A composition comprising: the Th17 cells are capable of producing one or more effector cytokines, and the amount of anti-CD3 antibody and the amount of anti-CD28 antibody present on the beads are not the same; composition.

[0330] Article 52 The composition of clause 51, wherein said one or more effector cytokines are selected from the group consisting of IL-17, IL-21, and IL-22.

[0331] Article 53 (1) T cells, and (2) beads containing anti-CD3, anti-CD28, and anti-CD137 antibodies that can selectively expand antigen-experienced memory T cells. A composition comprising: the T cells can recognize a specific antigen, and the amount of anti-CD3 antibody and the amount of anti-CD28 antibody present on the beads are not the same; composition.

[0332] Article 54 54. The composition of clause 53, wherein said specific antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, and a cancer antigen.

[0333] Article 55 55. The composition of clause 54, wherein said viral antigen is selected from the group consisting of CMV, EBV, influenza, and HIV.

[0334] Article 56 54. The composition of clause 53, wherein said antigen is selected from the group consisting of Streptococci M-protein, Neisseria fimbriae, Borrelia burgdorferi lipoprotein VisE, B. pseudomallei polysaccharide antigen, Aspergillus fumigatus galactomannan, and F. tularensis lipopolysaccharide.

[0335] Article 57 (1) Beads containing anti-CD3 and anti-CD28 antibodies that can selectively expand T cells and (2) regulatory T cells A composition comprising: the amount of anti-CD3 antibody and the amount of anti-CD28 antibody present on the beads are not the same; composition.

[0336] Article 58 The regulatory T cells are CD4+CD25+FOXP3+CD127 低 / 負 The composition of Article 57.

[0337] Article 59 58. The composition of clause 57, wherein said regulatory T cell activity comprises suppressive activity.

[0338] Article 60 A method of treating an individual in need thereof, comprising administering to said individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-experienced T cells, and / or regulatory T cells.

[0339] Article 61 61. The method of clause 60, wherein said individual in need of treatment is suffering from cancer, an inflammatory disease, an autoimmune disease, an allergic disease, or an infectious disease.

[0340] Article 62 62. The method of clause 61, wherein said cancer is lung cancer, pancreatic cancer, breast cancer, liver cancer, and skin cancer.

[0341] Article 63 62. The method of clause 61, wherein said inflammatory disease is selected from the group consisting of diabetes mellitus, rheumatoid arthritis, inflammatory bowel disease, familial Mediterranean fever, neonatal-onset multisystem inflammatory disease, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), systemic erythroplasia, uveitis, and Behcet's disease.

[0342] Article 64 A method for reconstituting the immune system of an individual in need thereof, comprising administering to the individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-experienced T cells, and / or regulatory T cells.

[0343] Article 65 A method of administering adoptive immunotherapy to an individual in need thereof, comprising administering to the individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-experienced T cells, and / or regulatory T cells.

[0344] Article 66 66. The method of any of clauses 60 to 65, wherein said T cells are genetically modified.

[0345] Article 67 67. The method of clause 66, wherein said genetic modification is a chimeric antigen receptor.

[0346] Article 68 67. The method of Clause 66, wherein said genetic modification is a genetically modified T cell receptor.

[0347] Article 69 1. A method for selectively altering the ratio of two T cell subtypes in a sample, comprising: contacting a sample containing a mixed population of T cells with at least two stimulatory agents; the stimulatory agents provide different amounts of signals to the T cells in the mixed population, such that one T cell subtype is selectively expanded relative to a second T cell subtype; method.

[0348] Article 70 70. The method of clause 69, wherein said sample comprises buffy coat cells from the individual.

[0349] Article 71 70. The method of clause 69, wherein said at least two stimulatory signals stimulate CD3 and CD28 receptors.

[0350] Article 72 70. The method of Clause 69, wherein at least one T cell subtype is selectively deleted from said mixed population.

[0351] Article 73 70. The method of clause 69, wherein Treg T cells are expanded proportionally relative to total T cells in said mixed population.

[0352] Article 74 70. The method of clause 69, wherein the total number of memory T cells is reduced in said sample.

[0353] Article 75 70. The method of clause 69, wherein the amount of stimulatory signal CD3 is less than half the amount of CD28 stimulatory signal.

[0354] Article 76 A method for expanding Th17 cells, comprising: (a) exposing a population of T cells to CD3 and CD5 signals ex vivo; (b) culturing the population of T cells under conditions that allow for the expansion of Th17 cells; Including, the population of T cells is exposed to an aryl hydrocarbon receptor agonist or is not exposed to exogenous interleukin-23; method.

[0355] Article 77 77. The method of clause 76, wherein said population of T cells is a mixed population of different T cell types.

[0356] Article 78 77. The method of clause 76, further comprising contacting said population of T cells with one or more polarizing agents.

[0357] Article 79 77. The method of Clause 76, further comprising contacting, wherein said one or more polarizing agents are one or more agents selected from the group consisting of interleukin-1 beta, interleukin-23, tumor growth factor-beta, interleukin-6, interleukin-21, interleukin-2, an anti-interleukin-4 antibody, and an anti-interferon gamma antibody.

[0358] Article 80 77. The method of Clause 76, wherein said population of T cells is further exposed to an aryl hydrocarbon receptor agonist.

[0359] Article 81 81. The method of clause 80, wherein said aryl hydrocarbon receptor agonist is 6-formylindolo[3,2-b]carbazole (FICZ).

[0360] Article 82 77. The method of Clause 76, wherein said population of T cells is further exposed to interleukin-1β and interleukin-6.

[0361] Article 83 77. The method of clause 76, wherein said Th17 cells are engineered to express one or more chimeric antigen receptors.

[0362] Article 84 77. The method of clause 76, wherein at least one of said one or more chimeric antigen receptors has specificity for a cell surface antigen of a mammalian cell.

[0363] Article 85 77. The method of Clause 76, wherein said cell surface antigen of said mammalian cell is an antigen associated with a tumor cell.

[0364] Article 86 A composition comprising a CD3 signal, a CD5 signal, an aryl hydrocarbon receptor agonist, and one or more cytokines.

[0365] Article 87 87. The composition of clause 86, wherein said one or more cytokines comprises both interleukin-1β and interleukin-6.

[0366] Article 88 The composition of clause 86, further comprising a population of T cells.

[0367] Article 89 The composition of clause 86, wherein said CD3 signal is an anti-CD3 antibody.

[0368] Article 90 The composition of clause 86, wherein said CD5 signal is an anti-CD5 antibody.

[0369] Article 91 A composition comprising a population of T cells, a CD3 signal, a CD5 signal, an aryl hydrocarbon receptor agonist, and one or more cytokines.

[0370] Article 92 the population of T cells (a) "Buffy coat" sample, (b) a sample of white blood cells containing more than 80% mixed T cells; (c) a sample containing more than 80% CD4+ T cells; or (d) Samples containing more than 80% Th17 cells present in a mixture comprising Article 91 Composition.

[0371] Article 93 1. A method for the separation and activation of T cells from a mixed population of cells, comprising: (a) contacting the mixed population of cells with the solid support having attached thereto at least a first ligand that has binding affinity for a protein located on T cells present in the mixed population of cells under conditions that allow binding of the T cells to the solid support and activation of the same; (b) separating the T cells bound to the solid support from cells not bound to the solid support to obtain a purified population of T cells. A method comprising:

[0372] Article 94 94. The method of Clause 93, wherein said solid support has attached thereto at least a first ligand and a second ligand, each of said first ligand and said second ligand having binding affinity for a different protein located on an individual T cell present in said mixed population of cells.

[0373] Article 95 the first ligand is either an anti-CD3 antibody or an anti-CD4 antibody; The second ligand is (a) Anti-CD5 antibody, (b) anti-CD28 antibody, (c) anti-CD137 antibody, and (d) Anti-ICOS antibody is a ligand selected from the group consisting of Article 94 Method.

[0374] Article 96 94. The method of clause 93, further comprising releasing cells of the purified T cell population obtained in step (b) from said solid support.

[0375] Article 97 97. The method of clause 96, further comprising expanding said released T cells.

[0376] Article 98 98. The method of Clause 97, wherein expansion of said released T cells occurs in culture medium.

[0377] Article 99 99. The method of Clause 98, wherein one or more chemokines or cytokines are present in said culture medium.

[0378] Article 100 94. The method of clause 93, wherein said one or more chemokines or cytokines are present in step (a).

[0379] Article 101 the one or more chemokines or cytokines (a) interleukin-1α, (b) interleukin-2, (c) interleukin-4, (d) interleukin-1β, (e) interleukin-6, (f) interleukin-12, (g) interleukin-15, (h) interleukin-18, (i) interleukin-21, and (j) Transforming growth factor-β1 selected from the group consisting of Article 100 Method.

[0380] Article 102 1. A method for the activation and expansion of T cells, comprising: A mixed population of T cells (a) a first agent that provides a primary activation signal to a member of a T cell subpopulation by stimulating a molecule on said member of said T cell subpopulation; and (b) a second agent that stimulates a molecule on a member of said T cell subpopulation that is different from the molecule stimulated by said first agent. contacting the thereby activating and expanding T cells in the population; the first agent and the second agent are bound to one or more solid supports; the T cells are maintained under conditions that allow their proliferation; the solid support is removed from contact with the T cells after a period of less than 120 hours. method.

[0381] Article 103 The method of Clause 102, wherein said first agent is an anti-CD3 antibody.

[0382] Article 104 The method of Clause 102, wherein said second agent is an antibody.

[0383] Article 105 The method of Clause 102, wherein said T cell is contacted with a third agent that is an antibody or a non-antibody protein.

[0384] Article 106 The method of Clause 105, wherein said non-antibody protein is a chemokine or cytokine.

Claims

1. 1. A method for expanding antigen-experienced T cells in vitro, the method comprising: (a) exposing antigen-experienced T cells to (i) an anti-CD3 antibody and (ii) an anti-CD137 antibody ex vivo; (b) culturing the antigen-experienced T cells under conditions that permit proliferation of the antigen-experienced T cells; Including, The anti-CD3 antibody and the anti-CD137 antibody are present in a ratio of 1:10 to 1:150; method.

2. The method of claim 1 , wherein the antigen-experienced T cells are contacted with one or more cytokines.

3. 3. The method of claim 2, wherein at least one of the one or more cytokines is interleukin-2, interleukin-7, or interleukin-15.

4. The method of claim 1 , wherein the antigen-experienced T cells are present in a mixed population of T cells.

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