Selective expansion of different T cell subsets by changing cell surface signals and signal ratios

Abstract

The present invention relates particularly to compositions of expanded T cell populations, methods for expanding T cell populations, and methods of using such cell populations. In some aspects, the present invention relates to compositions and methods for selectively expanding a subpopulation of T cells present in a mixed T cell population, as well as T cell subpopulations produced by the methods of the present invention.

Description

[0001] This application is a divisional application of CN201680076706.3. Technical Field

[0002] This invention particularly relates to compositions for amplifying T cell populations, methods for amplifying T cell populations, and methods for using such cell populations. In some aspects, this invention relates to compositions and methods for selectively amplifying T cell subsets present in a mixed T cell population, and to T cell subsets generated by the methods of this invention. Background Technology

[0003] The ability of T cells to recognize antigenic environments associated with organisms such as various cancers or infections is conferred by T-cell antigen receptors (TCRs), which are composed of α (alpha) and β (beta) chains, or γ (gamma) and δ (delta) chains. The proteins that make up these chains are encoded by DNA, which employs unique mechanisms to generate the vast diversity of TCRs. This multi-subunit immune recognition receptor is associated with the CD3 complex and binds to peptides presented by major histocompatibility complex (MHC) classes I and II proteins on the surface of antigen-presenting cells (APCs). The binding of the TCR to antigenic peptides on APCs is a major event in T-cell activation, occurring at the immune synapse at the contact point between the T cell and the APC. To maintain T-cell activation, T lymphocytes typically require a second co-stimulatory signal. Co-stimulation is usually necessary for helper T cells to produce sufficient levels of cytokines to induce clonal expansion. T cells can be expanded and activated in vitro using exogenously administered cytokines and cell surface protein stimulants.

[0004] In vitro expanded T cells are widely used in translational research and clinical trials for immunotherapy of cancer and incidental infections, and for the prevention of post-transplant autoimmune diseases and graft-versus-host disease (GVHD). While the application of in vitro expanded T cells has yielded some positive clinical results, the complexity of producing large quantities of various T cell products remains a major limitation to wider application. Although early in vitro culture conditions aimed only to produce high T cell numbers and express unfavorable phenotypes with low in vivo proliferative capacity, the focus has shifted to protocols for generating T cells that are more readily implanted, proliferate, and function in vivo.

[0005] Some of these early protocols utilized Dynabeads in the activation and expansion of T cells. ® CD3 / CD28CTS TMThis method can generate a population of T cells that exhibit a less differentiated phenotype and demonstrate increased antitumor activity and better persistence compared to T cells expanded with soluble anti-CD3 antibodies (OKT-3) and IL-2 in various in vivo models. Similar protocols using combinations of CD3 and CD28 binding and / or signal transduction can be used for the isolation and activation of polyclonal T cells. However, they do not provide optimal activation of antigen-stimulated memory T cells such as tumor-infiltrating lymphocytes (TILs) and virus-specific T cells.

[0006] It is believed that the quality and quantity of primary T cell activation signals, as well as the presence and type of co-stimulatory ligands, cytokines, and growth factors, determine the overall signal delivered to T cells and ultimately their impact on the fate of activated T cells. Successful expansion of antigen-stimulated T cells depends on sufficient intensity of stimulation via TCR / CD3, as well as the provision of optimal co-stimulatory signals and cytokines. Some reports suggest that using strong CD3 signaling transduction primarily expands naïve T cells and is thought to result in the deletion of antigen-stimulated T cells due to activation-induced cell death (AICD) (Kalamasz et al., Immunotherapy). Immunother ) 27 :405 (2004)). Antigen-stimulated memory T cells and their various subtypes have broad therapeutic significance in the treatment of cancer, autoimmune disorders, inflammatory diseases, allergic diseases, and infectious diseases. Therefore, there is a long-standing and urgent need for reliable, effective, and rapid methods to expand specific immune subsets, such as antigen-stimulated memory T cells, regulatory T cells (Tregs), and Th17 cells.

[0007] This invention addresses the need for subpopulation-specific expansion of specific T cell subtypes from the general T cell population, and also provides additional benefits. Summary of the Invention

[0008] This invention (particularly) provides compositions and methods for selectively amplifying various T cell subsets. These T cell subsets include, but are not limited to: (1) CD4 + CD25 + FOXP3 + Regulatory T cells (Tregs), a type of CD4 cell that plays an important role in regulating the immune response. + (2) Th17 cells, a type of CD4 cell that regulates host defense and is involved in tissue inflammation and various autoimmune diseases. +Inflammatory subtypes of helper T cells; and (3) memory T cells or antigen-specific T cells, a long-lived cell type that retains immunity to previously exposed antigens. Methods and compositions for selectively expanding the various T cell subsets described above are disclosed herein. This invention provides sufficient quantities of T cell subsets for therapeutic uses (e.g., adoptive immunotherapy, in vivo infusion, etc.). The methods and compositions described herein can be used to generate different T cell subsets for research purposes and / or for clinical use. The resulting expanded T cell subsets have wide applicability in clinical settings.

[0009] In some aspects, the present invention relates to compositions and methods for selectively expanding members of a T cell subset. This method includes: exposing a mixed T cell population to (a) a first reagent that provides a primary activation signal to members of the T cell subset, thereby activating the T cells; and exposing the mixed T cell population to (b) a second and a third reagent, each of the second and third reagents stimulating two or more different accessory molecules on members of the T cell subset, thereby stimulating the proliferation of the activated T cells of (a). In some aspects, the ratio of the first, second, and third reagents can be adjusted to induce selective expansion of said T cell subset members relative to other T cell subset members. The first reagent may be an antibody (e.g., an anti-CD3 antibody). The second reagent may also be an antibody. The third reagent may be an antibody, a non-antibody protein, or a chemical agent (e.g., rapamycin). When using non-antibody proteins, the protein can be a chemokine or a 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 certain aspects, the ratio of the first, second, and third reagents can be adjusted such that the concentration of the first reagent is lower than that of the second and / or third reagents. Furthermore, the lower concentration of the first reagent can be approximately 0.06 units, approximately 0.1 units, approximately 0.2 units, approximately 0.3 units, approximately 0.34 units, or approximately 0.4 units. Additionally, the first reagent can be an anti-CD3 antibody at a concentration of approximately 0.34 units, and the second reagent can be an anti-CD28 antibody at a concentration of 3.4 units.

[0011] In some respects, antiCD3 can be used in amounts from 0.01 to 4.0 (e.g., from about 0.01 to about 3.5, from about 0.01 to about 3.4, from about 0.02 to about 3.4, from about 0.05 to about 3.5, from about 0.1 to about 3.4, from about 0.2 to about 3.4, from about 0.5 to about 3.0, from about 0.1 to about 2.5, from about 0.01 to about 1.0, from about 0.05 to about 1.0, etc.), and antiCD28 can be used in amounts from 1.0 to 5.0 (e.g., from about 1.0 to about 4.8, from about 1.0 to about 4.5, from about 1.0 to about 3.5, from about 1.0 to about 3.0, from about 1.0 to about 2.9, from about 1.5 to about 3.5, from about 2.0 to about 3.5, from about 2.5 to about 3.5, from about 1.0 to about 4.8, etc.). In addition, the ratio of anti-CD3 to anti-CD28 can be in the range of 1:10 to 1:50 (e.g., from about 1:12 to about 1:48, from about 1:10 to about 1:48, from about 1:12 to about 1:50, from about 1:10 to about 1:40, from about 1:10 to about 1:35, from about 1:10 to about 1:30, etc.).

[0012] In some cases, the selectively expanded T cell subset may be Treg cells. In this case (and others), the lower concentration of the first reagent may be about 0.01 units. Furthermore, the first reagent may be an anti-CD3 antibody at concentrations 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 reagent may be an anti-CD28 antibody; and the third reagent may be an anti-CD137 antibody.

[0013] In some cases, the selectively expanded T cell subset may be memory T cells. In this case (and others), the lower concentration of the first reagent may be about 0.005, about 0.01, or about 0.06 units. In other cases, the selectively expanded T cell subset may be Th17 cells. Furthermore, the first reagent 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 reagent may be an anti-ICOS antibody.

[0014] The present invention also includes compositions and methods for selectively expanding T cell subsets. Such methods include those comprising (a) and (b): (a) exposing T cells in vitro to CD3, CD28, and / or CD137 signaling, and (b) culturing the T cells in a manner that allows for the expansion of Th17 cells, antigen-stimulated T cells, and / or regulatory T cells. In some cases, CD3, CD28, and CD137 signaling 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 in concentrations ranging from 0.01 units to 1.5 units (e.g., for the expansion of memory T cells). Furthermore, anti-CD3, anti-CD5, anti-ICOS, anti-CD6, anti-CD28, and anti-CD137 antibodies may be used in concentrations ranging from 0.06 units to 1.5 units (e.g., for the expansion of Th17 cells). Additionally, anti-CD3, anti-CD5, anti-ICOS, anti-CD6, anti-CD28, and anti-CD137 antibodies can be used in concentrations ranging from approximately 0.34 units to 3.41 units (e.g., for Treg cell amplification). Furthermore, anti-CD3 antibodies can be used at lower concentrations compared to the concentrations of anti-CD5, anti-ICOS, anti-CD6, anti-CD28, and anti-CD137 antibodies. In some aspects, T cells can be isolated using CD3 selection. Moreover, Th17 cells amplified by the method of the present invention can be CD3-selective. + CD8 / CD4 + / These Th17 cells can also produce the IL-17 cytokine. They may also be able to 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 may be able to produce IL-2. Effector memory cells expanded by the method 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 method of the present invention may have one or more of the following markers: CD4+ + CD25+, FOXP3 + and CD127 阴性 / 低 .

[0016] The present invention further includes compositions and methods for selectively expanding T regulatory cells. These methods include those comprising (a) and (b): (a) exposing T cells in vitro to CD3 and CD28 signaling, and (b) culturing T cells in a manner that allows for the expansion of T regulatory cells. This CD3 and CD28 signaling can be mediated by anti-CD3 and anti-CD28 antibodies. Furthermore, the anti-CD3 and anti-CD28 antibodies can each be used in a concentration range including 0.34 to 3.4 units. Additionally, the anti-CD3 antibody can be used at a lower concentration compared to the concentration of the anti-CD28 and / or antibody.

[0017] The present invention also includes compositions and methods for selectively expanding Th17 cells. This method includes those comprising (a) and (b): (a) exposing CD3+ T cells in vitro to CD3, CD28, CD5, and / or ICOS signaling, and (b) culturing the CD3+ T cells in a manner that allows for the expansion of Th17 cells, wherein the amounts of CD3, CD28, CD5, and / or ICOS may be the same or different. In this method, CD3, CD28, and ICOS signaling may be mediated by anti-CD3, anti-CD28, anti-CD5, and anti-ICOS antibodies. Furthermore, the expansion of Th17 cells using anti-CD3, anti-CD28, anti-CD5, and anti-ICOS antibodies may be used in a concentration range from about 0.06 to about 1.5 units. Additionally, anti-CD3 antibodies may be used at concentrations lower than those of anti-CD28 and / or anti-ICOS antibodies.

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

[0019] The present invention also includes a composition comprising CD3+ (1) T cells and (2) granules, the granules containing (a) anti-CD3 antibodies and (b) anti-CD28, anti-CD137, anti-ICOS, or anti-CD5 antibodies capable of selectively amplifying T cell subsets. In some cases, the amounts of (a) anti-CD3 antibodies and (b) anti-CD28, anti-CD137, anti-ICOS, or anti-CD5 antibodies present may be the same or different. In certain cases, the T cell subsets may be selected from a group consisting of Th17 cells, antigen-stimulated T cells, and / or regulatory T cells. In addition, anti-CD3, anti-CD28, anti-CD5, anti-ICOS, anti-CD27, and anti-CD137 antibodies can be used in concentration ranges from 0.01 to 1.5 units (e.g., for the expansion of memory T cells), from 0.06 to 1.5 units (e.g., for the expansion of Th17 cells), or from 0.34 to 3.41 units (e.g., for the expansion of Treg cells). In some cases, anti-CD3 antibodies can be used at lower concentrations compared to anti-CD28 and anti-CD137 antibodies.

[0020] The present invention further includes a composition comprising (1) T cells and (2) granules, the granules containing anti-CD3, anti-CD28, anti-ICOS, anti-CD5, and / or anti-CD137 antibodies capable of selectively amplifying Th17 cells, wherein the Th17 cells may be capable of producing one or more effector cytokines. In some cases, the amounts of anti-CD3 and anti-CD28 antibodies present on the granules 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] This invention includes a composition comprising (1) T cells and (2) beads, the beads containing anti-CD3, anti-CD28, and anti-CD137 antibodies capable of selectively amplifying antigen-stimulated memory T cells. In some cases, the T cells may be able to recognize specific antigens, and the amounts of anti-CD3, anti-CD28, and anti-CD137 antibodies present on the beads may be the same or different. Specific antigens may be selected from the group consisting of: viral antigens (e.g., CMV, EBV, influenza, HIV, etc.), bacterial antigens (e.g., streptococcal M-protein, Neisseria fimbriae, Borrelia burgdorferi lipoprotein VisE, Burkholderia melioides polysaccharide antigen, etc.), fungal or protozoan antigens (e.g., Aspergillus fumigatus galactomannan or Francisella tularensis lipopolysaccharide, etc.), and cancer antigens.

[0022] The present invention also includes a composition comprising (1) T cells and (2) granules, the granules containing the ability to selectively amplify regulatory T cells (e.g., regulatory T cells CD4+CD25+FOXP3+CD127). 低 / 阴性 Anti-CD3 and anti-CD28 antibodies are present on the beads. 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 inhibitory activity.

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

[0024] In this method (and other methods of the present invention), T cells may be genetically modified. Furthermore, the genetic modification may induce 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 the sample with at least two stimulants, the sample comprising a mixed T cell population. In some cases, the stimulants provide different amounts of signaling to the T cells in the mixed population. In certain cases, one T cell subtype may be selectively amplified relative to a second T cell subtype. Furthermore, the sample may contain erythrocyte sedimentation rate (ESR) tandem cells from an individual, and a subtype of ESR tandem cells (e.g., monocytes). Additionally, the at least two stimuli may stimulate CD3 and CD28 receptors. Additionally, at least one T cell subtype may be selectively removed from the mixed population. In some aspects, the proportion of TregT cells relative to all T cells in the mixed population may be increased. One way to do this is by contacting the cells with rapamycin in an amount suitable for removing non-Treg cells from the population. In other aspects, the total number of memory T cells in the sample may be reduced. Furthermore, the amount of the CD3 stimuli may be less than half the amount of the CD28 stimuli.

[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 methods for expanding Th17 cells, which may include: (a) exposing a T cell population in vitro to CD3 and CD5 signaling, and (b) culturing the T cell population under conditions that allow for the expansion of Th17 cells. In specific embodiments of the present invention, the T cell population 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 T cell population is a mixed population of different T cell types. Furthermore, the methods of the present invention include those comprising contacting the T cell population 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 referring to the use of proteins, the term "exogenous" refers to proteins present in the composition rather than those produced by cells present in the composition. For example, T cells present in a sample may produce IL-2. In this case, exogenously added IL-2 refers to IL-2 added to the sample as a non-cellular component (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 these chimeric antigen receptors includes those that are specific to cell surface antigens of mammalian cells (e.g., antigen-associated).

[0029] The present invention also includes compositions comprising CD3 and CD5 signaling. 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. The compositions (e.g., those described above) may further contain a T cell population. Furthermore, the CD3 signaling in the compositions of the present invention may be one or more anti-CD3 antibodies. Additionally, the CD5 signaling in the compositions of the present invention may be one or more anti-CD5 antibodies.

[0030] The compositions of the present invention may further comprise T cell populations, CD3 signaling, CD5 signaling, aryl hydrocarbon receptor agonists, and one or more cytokines. In a particular embodiment, the compositions of the present invention may comprise T cell populations present in a mixture comprising: (a) an erythrocyte sedimentation rate (ESR) brown-yellow layer sample, (b) a leukocyte sample containing more than 80% mixed T cells, (c) a sample containing more than 80% CD4+ T cells, or (d) a sample containing more than 80% Th17 cells.

[0031] The present invention further relates to a method for isolating and activating T cells from a mixed cell population (T cells and non-T cells, such as B cells). In some embodiments, the method comprises: (a) contacting the mixed cell population with one or more solid carriers having at least a first ligand thereto bound to it, the first ligand having binding affinity to a protein located on a T cell present in the mixed cell population, under conditions allowing T cells to bind to a solid carrier and activate the same T cell; and (b) isolating the T cells bound to the solid carrier from the T cells not bound to the solid carrier, to obtain a purified T cell population. The one or more solid carriers may have at least a first ligand and / or at least a second ligand thereto bound to them, wherein each of the first and second ligands has binding affinity to a different protein located on a separate T cell present in the mixed cell population. Furthermore, the first and second ligands may bind to the same solid carrier or different solid carriers. In some cases, the first ligand may be an anti-CD3 antibody or an anti-CD4 antibody. Furthermore, the second ligand can be any number of ligands, including ligands selected from the group consisting of: (a) anti-CD5 antibody, (b) anti-CD28 antibody, (c) anti-CD137 antibody, and (d) anti-ICOS antibody. Additionally, the method of the present invention includes those methods comprising releasing the purified T cell population from a solid carrier (including releasing the T cells obtained in step (b) from the solid carrier). Furthermore, the method of the present invention includes methods comprising amplifying the released T cells once released from the solid carrier. In many cases, the amplification of the released T cells occurs in a culture medium (e.g., a 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] The method of the present invention also includes methods for activating and expanding T cells. This method includes contacting a mixed population of T cells with (a) and (b): (a) a first reagent that provides a primary activation signal to the T cell subset members (e.g., Treg cells, Th17 cells, etc.) by stimulating molecules on the T cell subset members, said molecules being different from those stimulated by the first reagent. In many cases, T cells in the population (e.g., T cell subset members) are activated and expanded. In other cases, the first and second reagents may be bound to one or more solid carriers. Furthermore, the T cells may be maintained under conditions that allow for expansion (e.g., expansion of T cell subset members). Additionally, the solid carrier may be disconnected from the T cells after a period of less than 120 hours. That is, the solid carrier is in contact with the T cells for a limited period of time, and then the contact with the T cells is disconnected. This time period can 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 reagent can be an anti-CD3 antibody. Furthermore, the second reagent can also be an antibody. Additionally, T cells can be contacted with a third reagent, which is an antibody or a non-antibody protein (e.g., a chemokine or cytokine).

[0033] Each aspect and embodiment described herein can be used together unless explicitly or clearly excluded from the context of the embodiment or aspect. Attached Figure Description

[0034] Figure 1A and 1B Is it displaying the use of Dynabeads? ® Line graph showing the fold increase of Tregs after activation with the Treg amplifying agent CD3 / CD28 and the retention of FOXP3+ cells (approximately 90% FOXP3+ cells) by flow cytometry after sorting by CD4+CD25+CD127 low / -. Figure 1A Is it displaying the use of Dynabeads? ® A line graph showing the amplification fold after activation with the Treg amplifying agents CD3 / CD28. The data indicate several hundredfold effective amplification. Figure 1B This is a line graph showing the retention of %FOXP3+ cells. The data indicate that cells maintained high FOXP3 expression in expanded cultures after 14 days. Cells were restimulated on day 9 using the same prototype beads. Increased expansion was achieved using Dynabeads conjugated with lower CD3 levels. ® accomplish.

[0035] Figure 2 This is a bar graph showing the fold increase in CMV-specific memory T cells 10 days after activation. The fold increase is negatively correlated with the increased signal intensity, which is generated by Dynabeads conjugated with an increased amount of agonist CD3 antibody. ® Memory cell expansion agents are provided.

[0036] Figures 3A to 3D show the cultured using Th17-polarization conditions and Dynabeads. ® Th17 amplification agents CD3 / ICOS (anti-CD3 antibodies conjugated at 1.5 and 0.06; and two different ICOS clones) or Dynabeads ® A graph showing IL-17 expression in T cells expanded using the CD3 / CD28 (cell therapy system). IL-2 was added to the culture starting on day 3. From days 10 to 13, the culture was stimulated with PMA-inomycin for 4–5 hours before assessing IL-17 expression. The histogram shows intracellular IL-17 expression. Figure 3A shows the expression of IL-17 in cells using Dynabeads. ® Figure 3B shows IL-17 expression in cells amplified with the Th17 amplifying agent CD3 / ICOS (ISA-3) and high CD3 (1.5) amplification. Figure 3B also shows IL-17 expression in cells amplified using Dynabeads. ® Figure 3C shows IL-17 expression in cells amplified with the Th17 amplifying agent CD3 / ICOS (ISA-3) and low CD3 (0.06) amplification. Figure 3C also shows IL-17 expression in cells amplified using Dynabeads. ® Figure 3D shows IL-17 expression in cells amplified with the Th17 amplification agent CD3 / ICOS (ICOS clone C398.4A, purchased from eBiocienses, Affymetrix, USA) and low CD3 (0.06) amplification. Figure 3D shows the expression of IL-17 in cells amplified using Dynabeads. ® CD3 / CD28CTS TM A graph showing IL-17 expression in expanded cells. The intensity of activation signals and the nature of co-stimulation highly influence Th17 cell polarization and expansion. Note: Dynabeads was used. ® Th17 amplifying agent CD3 / ICOS (medium-0.3) activation at a 1:1 bead:cell ratio produced a phenotype similar to "(high-1.5)".

[0037] Figures 4A to 4C Is it displaying the use of Dynabeads? ® A diagram of T cells from three donors activated by the Th17 amplifying agent CD3 / ICOS. Figure 4A These are a series of line graphs showing the fold expansion of T cells from donors A, B, and C, respectively. Figure 4B It is a series of bar charts that show the effects of using Dynabeads ® The percentage of IL-17-producing cells generated by selectively amplifying T cells from donors A, B, and C with the Th17 amplifying agent CD3 / ICOS at different bead-to-cell ratios (recorded as BC). Figure 4C It is a series of bar graphs showing the relative number of IL-17-producing cells produced by expanding T cells from donors A, B, and C at different pearl-to-cell ratios (recorded as BC).

[0038] Figure 5 The effect of neutralizing antibodies. Mean % of IL-17-producing CD4 T cells (n=3 donors) stimulated with CD3 / ICOS beads or CD3 / CD28 beads and polarized with (+) or without (-) neutralizing antibodies. P-values ​​given for variation caused by blocking antibodies (paired T test) (NSD, not statistically significant (top block). Individual results; individual donors (A, B, C) and ICOS versus CD28 stimulation (bottom block).

[0039] Figure 6 The effect of co-stimulation on cytokine production. Purified CD3+ T cells activated using Dynabeads prototype CD3 / CD28, CD3 / CD5, or CD3 / ICOS expand Th17 isoforms with varying degrees of efficiency. CD3 / CD5 is generally the most efficient, followed by CD3 / ICOS, while CD3 / CD28 is the least efficient stimulatory prototype. CD3 / CD5 stimulation typically induces IL-17A production in 25% to 50% of CD4+ T cells, with a higher proportion of multifunctional IL-17+INF-γ+ observed during restimulation at days 10 to 13.

[0040] Figure 7 Effects of co-stimulation on phenotype. T cells stimulated by CD3 / CD28, CD3 / CD5, or CD3 / ICOS co-expressed various levels of CCR4 and CCR6 after expansion. CD3 / CD5 and CD3 / ICOS stimulation produced fractions of CCR4+CCR6+ cells (77% and 73%, respectively) compared to CD3 / CD28 stimulation (46%).

[0041] Figure 8T cells were activated using CD3 / CD5 (upper) or CD3 / ICOS (lower) plots and expanded in the presence of standard Th17-polarizing cytokines / antibodies (cytokine-encapsulated), or activated using 100 nm FICZ and IL-1β and IL-6. On day 13, cultures were stimulated with PMA / inomycin for 4–5 hours before assessment of IL-17A and IL-17F expression. Flow cytometry was used to visualize intracellular cytokine expression.

[0042] Figure 9A Irritating Dynabeads ® Early removal of these substances improves TH17 polarization. T cells are activated using 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 neutralizing antibodies against αIL-4 / α-IFN-γ). Stimulated Dynabeads ® i) Keep in the culture throughout the expansion, ii) Remove after 2 days (48 hours) of activation, or iii) Remove after 3 days (72 hours) of activation. On day 13, stimulate the culture with PMA / inomycin for 4–5 hours before assessing intracellular IL-17A and IL-17F expression in CD4+ T cells.

[0043] Figure 9B Display as shown Figure 9A The illustration shows the fraction and absolute number of CD4+ T cells expressing the Th17-related surface marker CD161 10 days after cell activation.

[0044] Figure 10A CD3 / CD5 isolated T cells can be directly polarized and expanded. T cells are produced using Dynabeads. ® CD3 / CD5 was separated from PBMCs at microsphere:cell ratios of 1:3, 1:1, and 3:1. Separation efficiency was determined using Dynabeads. ® CD3 / CD28CTS were compared, and these are commonly used in T cell stimulation protocols to enrich T cells. Separation efficiency (%) is shown.

[0045] Figure 10B . Figure 10A Isolated T cells 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 antibody). Stimulating Dynabeads were removed after 3 days of activation. ®On day 13, the culture was stimulated with PMA / inomycin for 4–5 hours before assessing intracellular IL-17A and IL-17F expression in CD4+ T cells. Detailed Implementation

[0046] In some aspects, the present invention provides methods and compositions for selectively expanding specific T cell subsets. These T cell subsets include, but are not limited to, Th17 cells, regulatory T cells (Tregs), and memory T cells. The specific T cell subsets disclosed herein can be used to treat various physiological conditions, diseases, and / or disease states.

[0047] Some aspects of the present invention

[0048] In some aspects, the present invention is based on the type and intensity of signals used to activate and / or amplify T cell subsets. According to these aspects, it has been observed that specific T cell subsets within a mixed population can be obtained and / or increased by selective stimulation with cell surface markers. It has also been observed that changes in the intensity of T cell receptor signals can be used to obtain specific T cell subsets. In many cases, T cell subsets are obtained from a mixed T cell population (e.g., total T cells obtained from peripheral blood).

[0049] Stimulation of T cell receptors can have a number of effects on specific T cells, such as (1) no effect on T cells, (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 effects are often a function of various factors, such as the specific T cells present, the nature of the stimulating signal, the intensity ratio of multiple stimulating signals (e.g., two, three, four, etc.) when multiple signals are used, and the total or individual signal intensity to which the T cell is exposed.

[0050] In many cases, T cells will be separated from other cell types before receptor stimulation. This can be done in a single step or in multiple steps. Exemplary methods include: (1) separating monocytes by erythrocyte sedimentation rate (ESR) amber or blood component apheresis, (2) separating CD4+ cells using, for example, magnetic beads with one or more CD4 receptor binding agents, and (3) sorting fluorescently activated cells (see Example 1).

[0051] In some aspects of the invention, the ratio of two or more T cell signals is modulated in a manner that results in the selective amplification of a first group of one or more T cell subsets rather than a second group of one or more T cell subsets. In many cases, the first group of one or more (e.g., one, two, three, four, five, etc.) T cell subsets will be smaller than the second group of one or more T cell subsets. In some cases, the first group of one or more T cell subsets may comprise a single T cell subset, while the second group of one or more T cell subsets may comprise all other T cell subsets present. In some cases, the first T cell subset (e.g., antigen-stimulated (memory) T cells) will be selectively amplified rather than the second T cell subset (e.g., naive T cells). Furthermore, one or more additional T cell subsets may be amplified together with the first T cell population.

[0052] In many cases, stimulation of a first T cell receptor (e.g., the CD3 receptor) produces one signal, while stimulation of a second co-stimulatory T cell receptor (e.g., CD28, CD137, CD27, CD5, CD6, ICOS, CD134, etc.) produces another signal. The signal ratio can be altered in such a way that it (a) enhances the expansion of a specific T cell population, (b) enhances the elimination of another T cell population (e.g., through apoptosis, inhibition of cell growth, or by a lack of expansion), or both (a) and (b). In some cases, one or more additional T cell receptors may be stimulated, or other signals may be delivered to the T cells.

[0053] The exemplary ratio of the stimulation signal of the first T cell receptor to the stimulation signal of the second T cell receptor will vary depending on the T cell subset sought to be obtained, and can range from about 50:1 to about 1:200 (e.g., about 1:5, about 1:10, about 1:15, about 1:20, about 1:40, from about 50:1 to about 1:40, from about 50:1 to about 1:30, from about 40:1 to about 1:40, from about 30:1 to about 1:40, from about 40:1 to about 1:20, from about 40:1 to about 1:10, from about 50:1 to about 1:1, from about 50:1 to about 5:1, from about 40:1 to about 5:1, from about 50:1 to about 10:1, from... From about 50:1 to about 15:1, from about 50:1 to about 20:1, from about 40:1 to about 5:1, from about 30:1 to about 3:1, from about 20:1 to about 3:1, from about 15:1 to about 3:1, from about 10:1 to about 5:1, from about 1:5 to about 1:10, from about 1:3 to about 1:20, from about 1:8 to about 1:25, from about 1:3 to about 1:40, from about 1:5 to about 1:50, from about 1:10 to about 1:50, from about 1:10 to about 1:100, from about 1:10 to about 1:150, from about 1:10 to about 1:200, from about 1:5 to about 1:150, from about 1:5 to about 1:200, etc.).

[0054] To illustrate, the signals provided by anti-CD3 and anti-CD28 antibodies can exist in a 1:10 ratio. It has been found that for the amplification of some T cell subsets, a lower amount of CD3 signal is expected than the second signal (e.g., CD28 and / or CD137 signal). In some cases, when more than two T cell receptor signals are provided, the proportion of each signal can be different, or the proportions of two or more signals can be equal (e.g., two out of three). As an example, CD3, CD28, and CD137 signals can exist in a 1:10:10 ratio. When each of these signals is produced by an antibody, this typically means that for every 10 units of anti-CD28 and anti-CD137 antibodies, there is one unit of anti-CD3 antibody. This, of course, assumes that the amount of receptor stimulation for each of the three receptors by their homologous antibodies is equal.

[0055] One issue to consider is the characterization of mixtures containing T cell populations produced by the methods of the present invention. In many cases, the compositions and methods of the present invention will involve altering the proportion of specific subsets of T cells in the mixture. For example, the methods of the present invention may result in the removal of certain types of T cells from the mixture through, for example, apoptosis or dilution. Therefore, one aspect of the present invention relates to the amount of T cell populations added or removed in the mixture, as well as the mixture itself. For example, if two T cell subsets (e.g., Th17 T cells and Th1 T cells) are present in the mixture and these subsets are present in, for example, a 1:1 ratio, then the present invention includes a method for increasing the proportion of one T cell subset relative to the other T cell subsets. For illustration, the ratio can be changed from approximately 1:1.5 to approximately 1:100,000 (e.g., from approximately 1:1.5 to approximately 1:100,000, from approximately 1:1.5 to approximately 1:80,000, from approximately 1:1.5 to approximately 1:50,000, from approximately 1:1.5 to approximately 1:10,000, from approximately 1:1.5 to approximately 1:5,000, from approximately 1:2,500 to approximately 1:25,000, from...). (Changed from approximately 1:2,500 to approximately 1:60,000, from approximately 1:2,500 to approximately 1:80,000, from approximately 1:2,500 to approximately 1:100,000, from approximately 1:5,000 to approximately 1:100,000, from approximately 1:5,000 to approximately 1:80,000, from approximately 1:5,000 to approximately 1:50,000, from approximately 1:5,000 to approximately 1:25,000, etc.).

[0056] Furthermore, the present invention relates to compositions and methods for altering the proportion of specific T cell subsets in a mixture, wherein the proportion of one T cell subset relative to another T cell subset is increased by at least 200,000 times (e.g., from about 1,000 times to about 200,000 times, from about 5,000 times to about 200,000 times, from about 10,000 times to about 200,000 times, from about 20,000 times to about 2...). (From approximately 50,000 to approximately 200,000 times, from approximately 75,000 to approximately 200,000 times, from approximately 1,000 to approximately 120,000 times, from approximately 5,000 to approximately 120,000 times, from approximately 10,000 to approximately 120,000 times, from approximately 1,000 to approximately 80,000 times, from approximately 10,000 to approximately 80,000 times, etc.). An example illustrating the meaning of "times" is as follows: If two T cell subsets exist in an initial ratio of 1:2, then changing the ratio to 1:8 represents a two-fold increase in one T cell subset relative to the other.

[0057] Another factor that can lead to the selective amplification of individual T cell subsets is the intensity of the stimulus signal. By "stimulus signal intensity," we mean the total signal intensity on a per-T-cell basis. This includes the intensity of various signals (e.g., signals stimulating first T-cell surface receptors, signals stimulating second T-cell surface receptors, signals stimulating third T-cell surface receptors, etc.) and combinations thereof that each T cell in the population is exposed to. Therefore, the present invention also relates to the amount of stimulus signal received by each cell in a mixture of various T-cell subsets. The stimulus signal can be modulated by changing the concentration of the stimulant, its proportion, or the ratio of the surface containing said stimulant to the cell count. In embodiments where the stimulant is bead-based, the bead-to-cell ratio can be varied. The pearl-to-cell ratio may include approximately 1:5,000, 1:2,500, 1:1,000, 1:500, 1:250, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 8:1, 10:1, or 15:1 pearls / cells.

[0058] In some cases, one or more cytokines can be added to the T cell population. In many cases, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IFN-γ, and TGF-β are used. When Th17 polarization is desired, one or more of the following cytokines can be used: IL-1β antibodies, IL-6 antibodies, TGF-β antibodies, IL-21 antibodies, IL-23 antibodies, and neutralizing antibodies against IL-4 and IFN-γ. Furthermore, signaling from IL-1β antibodies, IL-6 antibodies, TGF-β antibodies, IL-23 antibodies, and neutralizing antibodies against IL-4 and IFN-γ can be used to selectively expand Th17 cells.

[0059] Table 1 shows many different T cell subtypes that can be obtained using the methods of the present invention. Table 1 also shows various signals that can be used to selectively amplify specific types of T cells.

[0060]

[0061] Specifically, the present invention includes a method for selectively amplifying one or more T cell subsets. This method results in the increase or removal of specific T cells in a sample. As an example, CD3 and CD28 receptors, along with interleukin-2, stimulate naive T cells, memory T cells, Th1 T cells, and regulatory T cells (Tregs). It has been shown that naive T cells can be amplified, while memory T cells can be removed from the sample by modulating total CD3 / CD28 stimulation (see U.S. Patent No. 8,617,884, the disclosure of which is incorporated herein by reference). Regarding one aspect of the invention, it has now been shown that different T cell subsets can be selectively amplified by modulating the signal ratio and total signal intensity. As an example, Treg cells amplify well when CD3 signal is lower than CD28 signal (see...). Figure 1A The confirmation of selective amplification conditions can be used to increase the proportion of one T cell subset member in a sample compared to one or more other T cell subset members, even when various cells from different T cell subsets amplify in response to the same stimulus. For illustration, assuming TregT cells comprise 1% of the mixed population and naive T cells and memory T cells comprise 1.5% and 3% of the same mixed population, respectively, the stimulus signal can be modulated to induce the elimination of memory T cells while selectively amplifying TregT cells. The final result could be where TregT cells comprise 40% and naive T cells, memory T cells, and Th1T cells comprise 2%, 0.5%, and 2.5% of the mixed population, respectively.

[0062] Another factor that can be used to selectively increase or decrease one or more T cell subtypes (such as CD4+CD25+FOXP3+ regulatory T cells, CD4+CD25+FOXP3- regulatory T cells, CD4+CD25- T cells, etc.) is rapamycin.

[0063] mammalian immune system

[0064] The mammalian immune system uses two general adaptive mechanisms to protect the body from environmental pathogens. When encountering molecules from a pathogen, the immune response is highly activated to ensure protection against the organism's pathogenic effects.

[0065] The first mechanism is a nonspecific (or innate) inflammatory response. The innate immune system recognizes specific molecules present on pathogens but not in the body itself. The second mechanism is a specific or acquired (or adaptive) immune response. The adaptive immune response is routinely regulated in response to the pathogen in question. The adaptive immune system produces specific immunoglobulins (antibodies) in response to the many different molecules (called antigens) present in pathogens. Additionally, a wide variety of T-cell receptors are sampled to test their ability to bind processed forms of antigens, which bind to class I and class II MHCs on antigen-presenting cells (APCs) such as dendritic cells (DCs).

[0066] The immune system recognizes and responds to structural differences between its own proteins and non-own proteins. Proteins that the immune system recognizes as non-own proteins are called antigens. Pathogens typically express large numbers of highly complex antigens. Acquired immunity has a specific memory for antigenic structure; repeated exposure to the same antigen increases the response, which increases the level of protection induced against that specific pathogen.

[0067] Acquired immunity is mediated by specialized immune cells called B lymphocytes and T lymphocytes (or simply B cells and T cells). B cells are produced and function through antibody action. B cell-dependent immune responses are called "humoral immunity," due to the detection of antibodies in body fluids. T cell-dependent immune responses are called "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 through synergistic effects between T cells and secondary effector cells such as activated macrophages. The result is that pathogens are killed and prevented from causing disease.

[0068] Immune cells can acquire specific stimuli for activation. For example, the use of anti-CD3 / CD28 provides activation signals for some T cell populations. It is believed that naïve T cells require at least two signals for activation. The first signal is antigen-specific and is induced by a peptide / major histocompatibility complex (MHC) presented by antigen-presenting cells (APCs) and received via the T cell receptor (TCR) / CD3 complex. For some T cell subsets, a second signal can be delivered by antigen-presenting cells, and one of the candidate molecules for its receptor is the T cell antigen CD28. It is thought that when both the TCR / CD3 and CD28 T cell receptors are occupied by appropriate ligands, T cells proliferate and produce IL-2 (a cytokine essential for T cell proliferation) upon stimulation, while occupation of only the T cell receptor favors T cell unresponsiveness or apoptosis.

[0069] In vitro studies have 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., *European Journal of Immunology*). Eur. J. Immunol. ) 23 :2498-2502 (1993)), Sunder-Plassmann et al., Blood ( blood ) 87 :5179-5184 (1996)). It has recently been shown that co-immobilizing CD3 and CD28 antibodies onto the same or different solid phases can also induce T cell proliferation (Levine et al., *Journal of Immunology*). J. of Immunol. ) 159 :592130(1997); Li et al., Science ( Science ) 283 :848-851 (1999)).

[0070] This invention allows those skilled in the art to reliably, effectively, and efficiently generate various T cell subsets and use the expanded T cell subsets for different purposes, including, but not limited to, therapeutic and research / discovery purposes. As further discussed below, T cell subsets include, but are not limited to, regulatory T cells, Th17 cells, and antigen-stimulated memory cells.

[0071] Regulatory T cells

[0072] This invention relates to methods for efficiently generating regulatory T cells (or "regulatory T cells" or "Tregs"), and the use of these methods in the generation of T cell populations applicable, for example, immunotherapy. Treg cells can be generated via CD4+, CD25+, FOXP3+, and CD127+. 阴性 / 低 Such markers are used for characterization. In some cases, the Treg cells expanded using the compositions and methods of the present invention will be CD4+, CD25+, and FOXP3-. Compositions and methods for generating FOXP3- regulatory T cells are disclosed in U.S. Patent No. 9,119,807 to Aarvak et al.

[0073] 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 to provide transplant tolerance. Various Treg cell populations have been described, including naturally occurring CD4+CD25+FOXP3+ cells, as well as induced Tr1 and Th3 cells that secrete IL-10 and TGF, respectively.

[0074] Treg cells are characterized by sustained suppression of effector T cell responses. Conventional or routine Treg cells can be found, for example, in the spleen or lymph nodes or in circulation. Tregs have demonstrated high efficacy in preventing GVHD and autoimmune diseases in mouse models. Clinical trials of adoptive transfer of Tregs for the treatment of transplantation, diabetes, and other indications are underway. The relative frequency of Tregs in peripheral blood is approximately 1 to 2% of total lymphocytes, highlighting the necessity of in vitro expansion of Tregs prior to adoptive transfer for most clinical applications. Generating sufficient Tregs during in vitro expansion is a major challenge for the application of human Treg therapies.

[0075] Th17 cells

[0076] T helper 17 cells (or “Th17 cells” or “Th17 helper cells”) are an inflammatory subtype of CD4+ helper T 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 remains unclear. When adopted into mice with tumors, Th17 cells were found to be more effective than Th1 or nonpolarized (Th0) T cells in eradicating melanoma (Muranski et al., 《 blood( blood ) .112 :362-373 (2008)). Th17 cells develop differently from Th1 and Th2 cell lines. Th17 cells are CD4+ cells that respond to IL-1R1 and IL-23R signal transduction and produce 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 using Th17 cells for adoptive cell transfer is determining robust culture conditions (tumor microenvironment) for expanding Th17 cell subtypes and their unstable phenotypes in vivo.

[0077] This invention relates in part to compositions and methods for generating specific T cell subtypes. One specific T cell subtype that can be generated using the compositions and methods of this invention is Th17 cells. Therefore, in some aspects, this invention relates to compositions and methods for amplifying (e.g., selectively amplifying) Th17 cells using CD3 signaling (e.g., via anti-CD3 antibodies) and CD5 signaling (e.g., via anti-CD5 antibodies), one or more cytokines, and neutralizing antibodies (e.g., anti-IL-4 antibodies and anti-IFNγ antibodies). In some cases, the compositions and methods used for selectively amplifying Th17 cells are modulated in a manner that reduces or eliminates one or more neutralizing antibodies.

[0078] One problem with many T cells is their plasticity. That is, some T cells can change into different subtypes. Furthermore, this T cell change is often mediated by T cells in the surrounding environment. For example, many T cell subtypes express IL-4 and / or IFNγ. These proteins have physiological effects on surrounding T cells and, in some cases, mediate the differentiation of one type of T cell into another.

[0079] The expansion (e.g., selective expansion) of T cell subtypes such as Th17 cells can be achieved by using one or more signals, adjusting the intensity of one or more signals, adjusting the intensity ratio between two or more signals, and eliminating one or more signals. Each condition can be selected based on the ratio of two or more parameters. Two examples are as follows: (1) the ratio of CD3 to CD5 signals can be adjusted, and (2) the ratio of CD3 to CD5 signals can be adjusted in conjunction with the regulation of total CD3 and CD5 signals. For example, when it is desired to expand Th17 cells, two signals can be selected for use (e.g., CD3 and CD5 signals). These signals can be adjusted in ratio, for example, from about 1:1 to about 1:50 (e.g., from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:1 to about 1:20, from about 1:1 to about 1:15, from about 1:5 to about 1:15, from about 1:5 to about 1:20, from about 1:8 to about 1:12, from about 1:8 to about 1:15, from about 1:8 to about 1:20, etc.).

[0080] The present invention also includes the use of aryl hydrocarbon receptor (AHR) antagonists for the expansion of T cells (e.g., Th17T cells). Th17T cells have been found to be expanded by exposing T cells to CD3 signaling (e.g., agonist anti-CD3 antibodies), CD5 signaling (e.g., agonist anti-CD5 antibodies), IL-1β, IL-6, and AHR agonists (e.g., FICZ).

[0081] Exemplary AHR ligands that can be used to practice the present invention include, but are not limited to, FICZ (6-formylindo[3,2-b]carbazole), dFICZ (6,12-diformylindo[3,2-b]carbazole), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), methyl 2-(1'H-indo-3'-carbonyl)-thiazolyl-4-carboxylate (ITE), quercetin, indole-3-methanol, resveratrol, curcumin, M50367{3-[2-(2-phenylethyl)benzimidazol-4-yl]-3-hydroxypropionic acid and VAF347{[4-(3-chloro-phenyl)-pyrimidin-2-yl]}, tryptophan derivatives such as indigo dye and indirubin, tetrapyrroles such as bilirubin, and arachidonic acid metabolites lipoxygen A4 and prostaglandin G.

[0082] In some cases, the present invention includes compositions and methods for expanding Th17 cells, wherein T cells are exposed to one or more agonist anti-CD3 antibodies, one or more agonist 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 agonist anti-CD5 antibodies, one or more AHR agonists (e.g., FICZ), IL-1β, and IL-6.

[0083] In some embodiments, the present invention includes compositions and methods for expanding Th17 cells, wherein T cells are exposed to one or more agonist anti-CD3 antibodies, one or more agonist anti-CD5 and / or anti-CD28 antibodies, and one or more cytokines. Exemplary cytokines include TGF-β, IL-1β, IL-6, and IL-23. Therefore, in some embodiments, T cells are exposed to one or more agonist anti-CD3 antibodies, one or more agonist anti-CD5 and / or anti-CD28 antibodies, TGF-β, IL-1β, IL-6, and IL-23.

[0084] Therefore, the present invention includes compositions and methods for expanding Th17 cells, wherein the cells of the initiating T cell population are not exposed to TGF-β and / or IL-23.

[0085] The T cell populations used to implement the present invention may be present, for example, in: (i) "erythrocyte sedimentation rate (ESR) brown-yellow layer" samples, (ii) leukocyte samples from which non-T cells have been removed (e.g., samples containing more than 80% mixed T cells), (iii) CD4+ T cell samples (e.g., samples containing more than 80% CD4+ T cells), or samples primarily containing Th17 cells (e.g., samples containing more than 80% Th17 cells). Therefore, in some cases, the method of the present invention relates to the selective expansion of one or more T cell subsets.

[0086] Regarding the specific formulations used to expand Th17T cells, and the associated expansion methods, conditions are typically adjusted to achieve high levels of Th17 cell expansion.

[0087] One modifiable factor is the ratio of CD3 signaling (e.g., agonist anti-CD3 antibody) to CD5 signaling (e.g., agonist anti-CD5 antibody). In many cases, the amount of CD3 signaling will be lower than that of CD5 signaling. Exemplary CD3 to CD5 signaling ratios include those ranging from about 1 to about 1.5, from about 1 to about 2, from about 1 to about 3, from about 1 to about 5, from about 1 to about 8, from about 1 to about 10, from about 1 to about 12, from about 1 to about 15, from about 1 to about 20, from about 1 to about 25, from about 1 to about 30, from about 1 to about 40, from about 1 to about 50, from about 1 to about 75, from about 1 to about 100, etc.

[0088] Exemplary antibodies that can be used to practice this invention include BC3 anti-CD3 antibody and UCHT2 anti-CD5 antibody. BC3 anti-CD3 antibody is a clone expressing the antibody and is available from the American Type Culture Collection (HB-10166). UCHT2 anti-CD5 antibody is available from many sources, including eBioscience, Inc., San Diego, California.

[0089] Antibodies generally vary in their specificity and affinity for associated ligands. Therefore, the ratio of CD3 and CD5 signaling can be adjusted to equal the ratio of BC3 and UCHT2 antibodies. That is, the ratios mentioned above relate in part to data obtained from BC3 and UCHT2 antibodies, whose ratios produce physiological effects on T cells. Therefore, other CD3 and CD5 signaling agents can be used, and the amounts of these other signals can be adjusted to obtain the same physiological effects. In other words, the present invention includes various CD3 and CD5 signaling transducers that achieve the same physiological effects as the BC3 and UCHT2 antibody ratios given above. Such physiological effects can be measured by methods described elsewhere herein (e.g., Example 10, and related data).

[0090] The compositions of the present invention may contain reagents other than CD3 and CD5 signaling transducers and may be used in the methods of the present invention. Some of these additional reagents include AHR agonists (e.g., FICZ), cytokines (e.g., IL-1β and IL-6), and one or more neutralizing antibodies (e.g., anti-αIL-4 and anti-α-IFN-γ neutralizing antibodies). The reagents can be adjusted to obtain, for example... Figure 8 The results are shown. In particular, the amounts of AHR agonists and cytokines used are typically in the range of about 1 nm to about 1 mM (e.g., from about 1 nm to about 800 nm, from about 1 nm to about 500 nm, from about 1 nm to about 250 nm, from about 25 nm to about 1 mM, from about 25 nm to about 500 nm, from about 25 nm to about 300 nm, from about 50 nm to about 300 nm, from about 75 nm to about 500 nm, from about 75 nm to about 300 nm, etc.).

[0091] Memory T cells

[0092] Memory T cells, or antigen-stimulated cells, are cells that have been exposed to an antigen before. These T cells are long-lived and can recognize antigens and rapidly and strongly influence the immune response to antigens to which they have been previously exposed. Memory T cells may include: stem cell-like memory cells (T cells). SCM ), central memory cells (T) CM ), effector memory cells (T) EM T SCM The cells exhibit the phenotypes CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express high levels of IL-2Rβ, CXCR3, and LFA-1. CM Cells express L-selectin and CCR7; they secrete IL-2 but not IFNγ or IL-4. T EM The cells do not express L-selectin or CCR7, but they do produce effector cytokines such as IFNγ and IL-4.

[0093] This invention provides methods and compositions for selectively amplifying the above-mentioned T cell subsets. Existing methods do not allow those skilled in the art to selectively amplify specific T cell subtypes in the manner described herein. Although standard Dynabeads can be modified... ® The CD3 / CD28 ratio to T cells regulates signal intensity to some extent (Kalamazs et al., J. Immunotherapy Immunother ).27 :405–418 (2004)), but in Dynabeads ® The local concentration of irritating CD3-antibody at the contact area between the antibody and the cell surface remained unaffected and remained high in this method. The data disclosed in this paper are consistent with those of Dynabeads. ® The amount and ratio of the surface-conjugated primary antibody and various co-stimulatory antibodies allow for fine-tuning of the T cell response, resulting in selective activation and amplification of various specific T cell subtypes, such as Th17, antigen-stimulated T cells, and Treg.

[0094] I. Definition

[0095] 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 invention pertains.

[0096] The antibody used in the method of the present invention can be any species, class, or subtype, as long as the antibody can react with the target site (e.g., CD3, TCR, or CD28, if appropriate).

[0097] Therefore, the "antibody" used in this invention includes:

[0098] (a) Any of the classes or subclasses of immunoglobulins (e.g., IgG, IgA, IgM, IgD or IgE obtained from any animal, such as any animal commonly used, such as sheep, rabbit, goat, mouse, camel or egg yolk).

[0099] (b) Monoclonal or polyclonal antibodies,

[0100] (c) Monoclonal or polyclonal complete antibodies or antibody fragments, said fragments being those containing the antibody-binding region, such as fragments lacking the Fc portion (e.g., Fab, Fab', F(ab')2, scFv, V). H H fragments (or other single-domain antibodies) are so-called "half-molecule" fragments obtained by reducing and cleaving the disulfide bonds linking the heavy chain component in the intact antibody. Fv can be defined as a fragment containing a light chain variable region and a heavy chain variable region expressed as two chains.

[0101] (d) Antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, antibody fragments, “humanized antibodies”, chimeric antibodies or synthetically manufactured or modified antibody-like structures.

[0102] This also includes functional derivatives or antibody "equivalents," such as single-chain antibodies and CDR-transplanted antibodies. Single-chain antibodies (SCAs) can be defined as genetically engineered molecules containing a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a fused single-chain molecule. It also includes VHH antibodies, which can be monovalent or bivalent.

[0103] Methods for preparing antibody fragments, as well as synthetic and derivatized antibodies, are well known in the art and are extensively described in the literature, and are not described herein.

[0104] As used herein, the term "activation" refers to a cell state following sufficient T cell surface group attachment to induce measurable morphological, phenotypic, and / or functional changes. In the case of T cells, this activation can be a T cell state following adequate stimulation to induce cell proliferation. T cell activation can also induce the production and / or secretion of cytokines, and the upregulation or downregulation of the expression of cell surface molecules such as receptors or adhesion molecules, or the upregulation or downregulation of the secretion of certain molecules and the upregulation or downregulation of the function of regulatory effectors or cytolytic effectors. In the case of other cells, the term refers to the upregulation or downregulation of specific physicochemical processes.

[0105] As used in this article regarding antibodies, the term "unit" is associated with anti-CD3-mediated physiological effects. Specifically, 0.34 units of anti-CD3 is the amount of antibody that, upon exposure to a mixed T cell population in the presence of 3.4 units of anti-CD28, would result in a 1000-fold amplification of Treg cells after 14 days (see Example 2 and Figure 1). Units are defined by the number of antibody molecules present. Therefore, in the above case, there are 10 times more anti-CD28 molecules than anti-CD3 molecules.

[0106] As used herein, the term "stimulation" refers to an initial response induced by the binding of cell surface groups. For example, in the case of receptors, such stimulation requires receptor binding and subsequent signal transduction events. In the case of T cells, stimulation refers to the binding of T cell surface groups, which in one embodiment subsequently induces signal transduction events, such as binding of the TCR / CD3 complex. Furthermore, said stimulatory events can activate cells and upregulate or downregulate the expression of cell surface molecules such as receptors or adhesion molecules, or upregulate or downregulate the secretion of molecules, such as downregulating tumor growth factor β (TGF-β). Therefore, the binding of cell surface groups, even in the absence of direct signal transduction events, can lead to cytoskeleton reorganization or to the binding of cell surface groups, each of which can be used to enhance, modify, or alter subsequent cellular responses.

[0107] As used herein, the terms "factor," "ligand," or "stimulator" refer to a molecule that binds to one or more defined cell populations (e.g., members of T cell subsets) and induces a cellular response. The factor may bind to any cell surface group, such as receptors, antigenic determinants, or other binding sites present on the target cell population. The factor may be a protein, peptide, antibody and its fragments, fusion protein, synthetic molecule, organic molecule (e.g., small molecule), etc. In this specification and in the case of T cell stimulation, antibodies are used as a prototype example of such a factor.

[0108] As used in this article regarding T cells, the terms "selective amplification" and "selective expansion" refer to the ability of certain T cells to expand under conditions where other T cells do not expand or expand at a lower rate. As an example, suppose T cell isotypes 1 and 2 exist in a mixed population as 5% and 10% of the total number of T cells, respectively. If certain conditions result in T cell isotype 1 comprising 30% of the total number of T cells and T cell isotype 2 comprising 12%, then T cell isotype 1 is selectively expanded relative to T cell isotype 2, even though T cell isotype 2 now constitutes a larger proportion of the total T cell population. T cell isotype 2 is selectively expanded relative to the general members of the mixed T cell population; in a sense, it exists at an increased "frequency" according to the total percentage of T cells. Therefore, selective amplification involves the expansion of a specific T cell isotype relative to the general T cell population and usually results in the expansion of the T cell isotype as well. The above example can be termed the conditions for selective expansion of T cell isotype 1, even though T cell isotype 2 is also expanded.

[0109] As used in this article, the term “exposure” refers to being brought into a state or condition that is very close to or directly exposed to.

[0110] As used in this article, the term "proliferation" means growth or reproduction by producing new cells.

[0111] The “object” can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, livestock, poultry, pets, primates, mice, and rats. In one respect, the object is a human. The “object” can be a “patient” (e.g., under the care of a physician), but in some cases the object is not a patient.

[0112] As used in this article, a “co-stimulatory signal” refers to a signal that, when combined with a primary signal such as the TCR / CD3 linker, leads to T cell proliferation and / or activation and / or polarization.

[0113] As used herein, “separation” includes any means of purifying one component from another (e.g., by filtration, affinity, buoyancy density, or magnetic attraction).

[0114] As used herein, "surface" means any surface capable of having elements attached to it, and includes, without limitation, metals, glass, plastics, copolymers, colloids, lipids, cell surfaces, etc. Essentially, it refers to any surface capable of retaining elements bound to or connected to it.

[0115] As used herein, the singular terms “a (or an)”, “a (or an)”, and “the” include plural objects unless the context clearly indicates otherwise.

[0116] As used herein, the terms “treat,” “treating,” “treatment,” etc., refer to the reduction or relief of disorders and / or associated symptoms. It should be understood that, although not excluded, treating a disorder or condition does not require the complete elimination of the disorder, condition, or associated symptoms.

[0117] II. Methods for generating T cell subsets

[0118] The method of this invention can be used to selectively amplify one or more substantially pure specific T cell subsets. Exemplary T cell subsets include, but are not limited to, Treg cells, Th17 cells, and memory T cells. Examples of uses of the amplified T cell subsets are disclosed herein.

[0119] Sources of mixed T cell populations

[0120] The initial source of a mixed T-cell population can be blood separable from the subject (e.g., circulating blood). Circulating blood can be obtained from one or more blood units, or from apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. T cells can be obtained from many sources, including blood monocytes, bone marrow, thymus, tissue biopsies, tumors, lymph node tissue, gastrointestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, or any other lymphoid tissue and tumor. T cells can be obtained from T-cell lines, or from autologous or allogeneic sources, including umbilical cord blood. T cells can also be obtained from xenogeneic sources, such as mice, rats, non-human primates, and pigs.

[0121] In some embodiments of the invention, T cells may be obtained from blood units collected from the subject using any number of techniques known to those skilled in the art (e.g., Ficol separation). T cells may be separated from the subject's circulating blood. Blood may be obtained from the subject via apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. The source of T cells is obtained from the subject prior to exposure to the sensitizing composition and subsequent activation and / or stimulation. In some embodiments, cells collected via apheresis may be washed to remove plasma components and placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments of the invention, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. Those skilled in the art will readily understand that the washing step can be performed using methods known to them, such as using a semi-automated "continuous flow" centrifuge (e.g., the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as calcium (Ca)-free and magnesium (Mg)-free PBS. Alternatively, unwanted components in apheresis samples can be removed, and the cells can be directly resuspended in a culture medium.

[0122] In other embodiments, red blood cells are lysed or removed and monocytes are removed (e.g., by PERCOLL). TM Gradient centrifugation separates T cells from peripheral blood lymphocytes. A specific subset of T cells can be further separated using positive or negative selection techniques.

[0123] In some embodiments, T cells can be positively selected against CD3+ cells. Any selection technique known to those skilled in the art can be used. One non-limiting example is flow cytometry sorting. In another embodiment, T cells can be isolated by incubating with a solid carrier 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 (LifeTechnologies Corp., catalog number 11141D), for a period of time sufficient to positively select the desired T cells. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values ​​in between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. Longer incubation times, such as 24 hours, can increase cell yield. In any case where fewer T cells are present compared to other cell types, a longer incubation time can be used to isolate T cells. In one aspect of the invention, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies targeting unique surface markers on the negatively selected cells. One possible method is cell sorting and / or selection via magnetic immunoadsorption or flow cytometry (which uses a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells). In some embodiments, the amplification fold may vary depending on the starting material due to the variability of donor cells. In some embodiments, a normal starting density may be around 0.5 × 10⁻⁶. 6 –1.5×10 6 between.

[0124] Furthermore, T cell subsets can be generated through selection based on the presence or absence of one or more markers. For example, Treg cells can be selected based on CD4+. + CD25+, CD127 阴性 / 低 And optional FOXP3 + Cell selection is derived from a mixed population. In some cases, Treg cells may be FOXP3-. In this case, selection actually refers to "selecting" cells based on one or more definable characteristics. Furthermore, selection can be positive or negative, meaning that cells may possess one or more characteristics (positive), or cells may not possess one or more characteristics (negative).

[0125] Regarding Treg cells, for illustration, these cells can be obtained from a mixed population by binding these cells to a surface (e.g., magnetic beads) with antibodies bound to CD4 and / or CD25 and non-Treg cells to a surface (e.g., magnetic beads) with antibodies bound to CD127. As a specific example, magnetic beads with antibodies bound to CD3 can be used to separate CD3+ cells. Once released, the obtained CD3+ cells can be contacted with magnetic beads with antibodies bound to CD4. The resulting CD3+, CD4+ cells can then bind to magnetic beads with antibodies bound to CD25. The resulting CD3+, CD4+, CD25+ cells can then bind to magnetic beads with antibodies bound to CD127, wherein the collected cells are those that do not bind to the beads.

[0126] In some cases, multiple features can be used simultaneously to obtain T cell subsets (e.g., Treg cells). For example, a surface containing antibodies that bind to two or more cell surface markers can also be used. As a specific example, CD4+, CD25+ cells can be obtained from a mixed population by binding these cells to a surface containing antibodies that bind to CD4 and CD25. Simultaneously selecting multiple features may result in many undesirable cell types being “co-purified” with the target cell type. This is because, using the specific example above, CD4+, CD25-, and CD4-, CD25+ cells can be obtained in addition to CD4+, CD25+ cells.

[0127] Flow cytometry is particularly useful for isolating cells based on desired characteristics. Cells can be isolated based on detectable markers associated with molecules binding to target cells, such as natural ligands, like IL-7 binding to CD127, or an antibody specific to CD25. Therefore, the detection and / or differentiation of ligands bound to cellular components by a flow cytometry system can be used to purify / isolate T cells with specific characteristics. Furthermore, the presence or absence of multiple characteristics can be simultaneously determined by flow cytometry.

[0128] Therefore, the present invention includes a method for obtaining members of one or more T cell subsets, wherein T cell subset members are identified by specific characteristics and isolated from cells by differences in these characteristics. Examples of characteristics that can be used in the methods of the present 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.

[0129] The selection of characteristics associated with the cells sought to be obtained varies depending on the cells present in a particular sample. For example, umbilical cord blood (UCB) has been shown to contain Treg cells, which can mitigate the effects of graft-versus-host disease. However, UCB contains a proportion of cells not typically found in peripheral blood. One class of cells present in UCB is hematopoietic stem cells (HSCs). HSCs are typically CD34+, and can be isolated from CD4+ and CD25+ cells by binding to a surface with a suitable ligand to which they bind, or by a marker molecule that binds to one or more of CD4+, CD25+, and / or CD34+.

[0130] Therefore, the present invention relates to compositions and methods for purifying cells, wherein the number of target cells in the mixture is increased at least 5-fold (e.g., from about 5-fold to about 50-fold, from about 10-fold to about 50-fold, from about 15-fold to about 50-fold, from about 20-fold to about 50-fold, from about 25-fold to about 50-fold, from about 10-fold to about 40-fold, from about 10-fold to about 30-fold, etc.). One example of purification is a cell type that constitutes 5% of the total cells in the mixture before purification and 25% of the total cells in the mixture before purification. This is an example of 5-fold purification.

[0131] from Figure 10A and Figure 10B It can be seen that CD3 / CD5 isolated T cells can be directly polarized and expanded. One starting material for the Th17 polarization protocol is CD3 or CD4 enriched cells (positive or negative isolation).

[0132] Upstream separation is laborious and expensive, and complicates the process of generating Th17. It has been found that CD3 / CD5 beads (the same ones used for Th17 polarization) are compatible with the use of Dynabeads. ® CD3 / CD28CTS has similar efficiency (>90% at a 3:1 microsphere:cell ratio and 80% at a 1:1 microsphere:cell ratio) for effectively isolating CD3 cells (T cells). Figure 10A ).like Figure 10B As shown, these positively CD3 / CD5 separated T cells can be directly polarized into Th17 cells.

[0133] The present invention also relates to compositions and methods for simultaneously activating and purifying T cells. One example of such compositions and methods involves forming a mixture containing magnetic beads having anti-CD3 and anti-CD5 antibodies bound thereto, wherein the reaction mixture comprises Th17T cells. In this case, Th17T cells can be activated by the anti-CD3 and anti-CD5 antibodies and isolated from other cells that do not contain CD3 and / or CD5 surface markers. Of course, proteins other than CD3 and CD5, and their respective combinations (e.g., CD3 and CD28; CD3, CD28, and CD137; CD3 and CD137; CD3 and CD278, etc.) can be used to activate and purify T cells.

[0134] Furthermore, it has been found that when using magnetic beads to simultaneously activate and purify T cells, the bead-to-cell ratio can be adjusted to improve purification efficiency. For example, it has been found that using Dynabeads® CD3 / CD28CTS™ (Thermo Fisher Scientific, catalog number 40203D), a bead-to-cell ratio of 3:1 can yield a T cell population with >90% purity, while a 1:1 bead-to-cell ratio can yield a T cell population with approximately 80% purity. Therefore, the present invention provides compositions and methods for purifying T cells, wherein the bead-to-cell ratio is from about 20:1 to about 1:5 (e.g., from about 20:1 to about 1:2, from about 20:1 to about 1:1, from about 20:1 to about 2:1, from about 20:1 to about 3:1, from about 10:1 to about 1:1, from about 10:1 to about 2:1, from about 10:1 to about 3:1, etc.). The present invention also provides compositions and methods for purifying T cells, wherein the mitochondrial cell ratio is adjusted to produce a population of activated T cells with a purity of at least 80% (e.g., from about 80% to about 99%, from about 83% to about 99%, from about 85% to about 99%, from about 88% to about 99%, from about 90% to about 99%, from about 93% to about 99%, from about 95% to about 99%, from about 80% to about 95%, from about 85% to about 95%, etc.).

[0135] The purified T cells obtained through the above methods can be of mixed types or of a specific type (e.g., Treg cells). For example, CD3 and CD28 proteins are present on the surface of many different T cell subtypes. Therefore, T cells purified using anti-CD3 and anti-CD28 antibodies are typically a mixed population. Specific T cell subtypes can be expanded from this mixed population using additional factors (e.g., chemokines, cytokines, or other factors such as rapamycin, aryl hydrocarbon receptor agonists, etc.). Therefore, one or more T cell subtypes can be generated using purified mixed T cell populations by adjusting activation and / or expansion conditions.

[0136] For example, Th17 cells can be purified, activated, and expanded through the following process. T cells can be purified from a mixed cell population using anti-CD3 or anti-CD4 antibodies bound to a solid carrier (positive separation). Cells bound to the solid carrier are collected and exposed to a solid carrier containing anti-CD3 and anti-CD5 antibodies. T cells bound to these beads are separated from unbound cells and then exposed to factors that promote Th17 cell polarization (e.g., IL-1β, IL-6, aryl hydrocarbon receptor antagonists, etc.). The T cells are then maintained under conditions that allow Th17 cell expansion. In some cases, other T cells can be expanded under the conditions used. Conditions are typically adjusted so that Th17 cells expand faster than other T cell subtypes.

[0137] Expanding and / or proliferating into various T cell subsets

[0138] The mixed T cell population isolated from the subject can be expanded into various T cell subsets by altering its exposure to a primary activation signal using primary factors. The primary activation signal is anti-CD3 and can be obtained using anti-CD3 primary factors (e.g., anti-CD3 antibodies or other factors with specific binding to CD3). The primary activation signal can be used in combination with a second and / or third reagent, which can target CD28, CTLA-4, CD137, CD27, CD5, CD6, CD134, CD2, LFA-1, CD40, SLAM, GITR, and / or ICOS.

[0139] Cells of the present invention can be expanded by culturing in a culture using factors as described above and herein. The compositions of the present invention comprise specific T-cell expansion factors conjugated to a surface in a defined ratio. The T-cell expansion factors of the present invention may be present with one or more co-stimulatory signals. The ratio of T-cell expansion factors to co-stimulatory signals may be 1:1,000, 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 co-stimulatory signals may be present in ratios such as 1:1,000:1,000, approximately 1:500:500, approximately 1:100:100, approximately 1:50:50, approximately 1:40:40, approximately 1:30:30, approximately 1:20:20, approximately 1:10:10, approximately 1:5:5, approximately 1:4:4, approximately 1:3:3, or approximately 1:2:2. In some embodiments, additional co-stimulatory signals may be present. In some embodiments, the ratio of individual co-stimulatory signals to the primary signal may be different (e.g., the ratio of the primary signal to the co-stimulatory signal is 1:4:3). Furthermore, the signal-to-cell ratio can be controlled by selectively amplifying the total amount of signal applied. For example, in stimulation signals that bind to beads, the bead-to-cell ratio can be varied.

[0140] In one embodiment, isolated T cells are expanded using beads containing surface-active factors. Isolated T cells can be activated and expanded in vitro by culturing with the beads of the present invention or other compositions at a ratio of about one bead per cell. In other embodiments, the beads can be used in cultures to activate and expand T cells at ratios of about 100 cells per bead, about 10 cells per bead, about 5 cells per bead, about 2 cells per bead, about 2 beads per cell, about 5 beads per cell, about 10 beads per cell, or about 100 beads per cell.

[0141] Beads: The relative proportions of cell ratio, total concentration, and / or co-stimulatory signals can be individually adjusted to obtain the maximum fold increase, the percentage of the target cell type in the expanded cell population, or the relative number of the target cell type. Compared to the total cell population, the resulting cell population may contain approximately 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the target cell type. The relative number of the target cell type is defined as the fraction of target cells multiplied by the fold increase of the total cell population. The relative number of the target cell type may contain approximately 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, or 500.

[0142] For Tregs, the stimulation method can be performed in the presence of other cells (e.g., other T cells such as CD4+ and CD25+ cells, which themselves can proliferate to a greater extent during the method of the present invention). Therefore, in the above-described embodiments, the step of isolating T cells may include isolating a large portion (i.e., at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90%) or all of the CD4+ cells, i.e., including both CD25+ cells from the sample. Therefore, in one embodiment of the invention, the isolation step includes isolating CD4+ cells. Furthermore, other cells may be present so that Tregs, Th17s, or memory T cells form only a portion of the cells used for the stimulation method before or after the isolation step. Therefore, in the stimulation step, CD4+ and CD25+ cells may be present as part of the stimulated cells, i.e., enrichment preparations may be used, such as preparations containing at least about 50, about 60, about 70, about 80, or about 90% of the total stimulated cells.

[0143] In some cases, at least a portion of CD4- and CD25- cells are absent, for example, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the cells present in the starting material are absent. Alternatively, the starting cells used for stimulation / expansion may be substantially entirely CD4+ and CD25+ cells, for example, at least about 80%, about 90%, about 95%, or about 98% of CD4+ and CD25+ cells.

[0144] Stimulating T cells to selectively expand Treg cells may include stimulation with anti-CD3 and anti-CD28 factors. In some aspects, compared to previous anti-CD3 / CD28 co-stimulation methods, the present invention utilizes a low anti-CD3 to anti-CD28 ratio so that the anti-CD3 factor is present at a lower level than the anti-CD28 factor. The anti-CD3 to anti-CD28 ratio may be about 1:1,000, 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 may include stimulation with (1) anti-CD3 and (2) anti-CD5 and / or anti-ICOS factors.

[0145] To selectively amplify Th17 cells, stimulation of T cells may include stimulants that stimulate CD3, CD28, CD5, and / or ICOS receptors. In some embodiments, Th17 cells may be generated using anti-CD3 and anti-ICOS antibodies. In some embodiments of the invention, the level of CD3 receptor stimulation may be at a lower concentration than the level of ICOS stimulation. Using antibodies as an example, the ratio of anti-CD3 to anti-ICOS may be about 1:1,000, 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.

[0146] To selectively expand T memory cells, stimulation of T cells may include anti-CD3 and anti-CD27, or anti-CD28, or anti-CD137 stimulation. In many cases, anti-CD3 will be present at a lower concentration than anti-CD28, anti-CD27, and / or anti-CD137. The ratio of anti-CD3 to anti-CD28 and anti-CD27 or anti-CD137 may be approximately 1:1,000:1,000, approximately 1:500:500, approximately 1:100:100, approximately 1:50:50, approximately 1:40:40, approximately 1:30:30, approximately 1:20:20, approximately 1:10:10, approximately 1:5:5, approximately 1:4:4, approximately 1:3:3, or approximately 1:2:2.

[0147] After isolation, T cells can be activated by undisturbed culture in a culture medium using the composition of the present invention for 1 to 5 days, 1 to 4 days, 2 to 3 days, about 2 days, or about 3 days. Following undisturbed activation, the isolated T cells can be expanded as needed by adding freshly prepared culture medium and cytokines. Expansion can occur for 7 to 20 days, 8 to 15 days, 10 to 14 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. The cytokines used for T cell expansion have been described above and discussed in the following examples.

[0148] T cell amplification can result in a predetermined number of cells capable of producing a T cell subset. For example, amplifying T cells using the method and composition of the present invention can produce cells containing approximately 10... 6 10 cells, approximately 10 7 10 cells, approximately 10 8 One cell, or about 10 10 Cells (e.g. from about 10) 6 To about 10 10 Cells, from about 10 7 To about 10 10 Cells, from about 10 8 To about 10 10 Cells, from about 10 9 To about 10 10 Cells, from about 10 6 To about 10 9 Cells, from about 10 6 To about 10 9 T cell subsets (e.g., individual cells, etc.).

[0149] Using the method of the present invention, activation / expansion in T cell subsets can be achieved within approximately 2 hours, approximately 4 hours, approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 20 days, approximately 30 days, etc. (e.g., from approximately 2 hours to approximately 30 days, from approximately 8 hours to approximately 30 days, from approximately 20 hours to approximately 30 days, from approximately 1 day to approximately 30 days, from approximately 3 days to approximately 30 days, from approximately 5 days to approximately 30 days, from approximately 7 days to approximately 30 days, etc.).

[0150] In some cases, a solid phase (e.g., beads) containing one or more stimulating signals (e.g., anti-CD3 antibody, anti-CD28 antibody, interleukin-2, etc.) is not removed from the T cell population after amplification. In other cases, once the T cell population has amplified to the required level as described above, the amplified population can be separated from the solid phase in an appropriate manner. For example, if one or more stimulating signals are attached to a solid phase and that solid phase is a tissue culture well, plate, or flask, the T cell population can be obtained by removing the culture medium containing the T cells. If the solid phase contains, for example, magnetic beads, a magnetic field is used to attract the beads to the sidewall of the container, and then the culture medium containing the T cells can be, for example, discarded. Other specific solid carriers (e.g., non-magnetic beads) can be removed from the cells by centrifugation or filtration. Although most T cells are usually present in the culture medium, some T cells may attach to the solid phase after amplification. If necessary, these T cells can be separated by resuspension, for example, using a pipette or other suitable tool. This resuspension is usually performed before the T cells are separated from the solid phase to improve yield. Once separated from the solid phase, the T cell population can be further processed and / or manipulated in any desired manner, or used directly for suitable applications such as in vitro experiments and research, non-therapeutic applications, therapeutic applications, etc., as discussed below.

[0151] When soluble activators are used, these can be removed (if desired) by competitive removal with appropriate ligands, such as CD3 or CD28; however, in many cases, T cells are collected from the culture medium and used for applications as described herein without further purification. Conveniently, for large-scale applications, suitable isolation and preparation platforms can be used to select T cell populations for stimulation and / or expansion protocols and / or isolation of the resulting T cell populations. In this regard, DYNAL'S Dynamag™ CTS platform deserves special mention, in which a closed, sterile, disposable bag can be used for any magnetic cell isolation step performed, such as for cell isolation prior to expansion and for removal of Dynabead® after expansion.

[0152] If necessary, the expanded / activated T cell population according to the method of the invention can be restimulated by exposing the cells to more activators in a manner similar to the initial stimulation. Generally, restimulation is only necessary or desirable if the T cells will be cultured for a longer period of time (e.g., more than 10 to 16 days).

[0153] Treg cells can be restimulated after expansion using the same bead culture method used for primary activation. Treg cells can be restimulated at approximately 16 days, 14 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, or 12 hours after primary activation.

[0154] Expanding T cell subsets according to the method of the present invention can result in a small increase in number, but in many cases, the result is a significant increase, for example, an increase in cell number of at least about 2 times, preferably at least about 5 times, about 20 times, or about 50 times, more preferably at least about 100 times, about 500 times, about 1,000 times, about 2,000 times, about 5,000 times, about 10,000 times, about 20,000 times, about 30,000 times, about 40,000 times, about 50,000 times, about 75,000 times, about 100,000 times, or greater. This increase in number can be measured at any appropriate time point in the cell expansion protocol.

[0155] This invention provides a method for generating a population of one or more substantially pure T cell subsets. For the purposes of this invention, a population of substantially pure T cell subsets contains about 10% or less of undesirable cells (e.g., cells that are not the desired subset cell type). For example, to illustrate, if Treg cells are the desired T cell subset, then a substantially pure Treg subset would contain about 10% or less of non-Treg cells. Of course, this also applies to other T cell subsets. In some embodiments, substantially pure may include 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 subset.

[0156] In standard Th17 production protocols (X-Vivo™ 15 medium with polarizing cytokines), stimulating solid carriers (e.g., Dynabeads) are used. ® Solid carriers (e.g., magnetic beads) are typically retained in the culture throughout the entire culture phase. The process is simplified if the solid carriers can be removed before the end of the culture phase (e.g., 48 or 72 hours after activation), especially when cells are contained in small volumes and are prepared for viral transduction (genetic modification). One way to improve the process is to eliminate the need to remove large-scale solid carriers (e.g., magnetic beads) at or near the end of the culture.

[0157] like Figure 9A and Figure 9B As shown, early removal of the activation vector has been found to improve the polarization of Th0 cells towards the Th17 phenotype. Experiments with early bead removal have also shown up to 200-fold expansion, which was unaffected by bead removal.

[0158] Early microbeta removal was observed to have a greater effect on Th17 polarization in cells co-stimulated with ICOS than in cells co-stimulated with CD5, and this effect was almost negligible after CD28 co-stimulation. Figure 9A and Figure 9B ).

[0159] In some cases, it may be desirable to remove the solid carriers (e.g., beads such as magnetic beads) used to activate T cells from a T cell mixture. Furthermore, in some cases, it may be desirable to remove such solid carriers after a short period of time (e.g., within 5 days). Therefore, the present invention includes compositions and methods for T cell activation, wherein T cells are exposed to factors that stimulate one or more cell surface markers for an exposure time of less than 5 days (e.g., from about 1 day to about 5 days, from about 2 days to about 5 days, from about 3 days to about 5 days, from about 1 day to about 4 days, from about 2 days to about 3 days, etc.).

[0160] In one particular embodiment, multiple mixed T cell population samples were exposed to: (1) a solid surface (e.g., magnetic beads) containing anti-CD3 and anti-CD5 antibodies, (2) IL-1β, (3) IL-6, (4) IL-23, and (5) TGF-β (and optionally an aryl hydrocarbon receptor agonist) under conditions permissible for T cell expansion. For one sample, the solid surface was not removed. For the second sample, the solid surface was removed after 1 day. For the third sample, the solid surface was removed after 2 days. For the fourth sample, the solid surface was removed after 3 days. Expansion of activated T cells could be performed substantially 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 were determined.

[0161] III. The compositions of the present invention

[0162] The compositions of the present invention comprise a surface having factors fixed in number, proportion, or combination to stimulate the expansion of specific T cell subsets. In alternative embodiments, the factors may be in solution.

[0163] The factors considered in this invention include protein ligands and synthetic ligands. Factors that can bind to cell surface groups and, under certain conditions, lead to linkages and aggregations that induce 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, tetraiodothyronine, corticosteroids, prostaglandins, tetraiodothyronine), bacterial groups (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. These factors can be isolated from natural sources such as cells, blood products, and tissues, or isolated from cells propagated in vitro, recombinantly prepared, chemically synthesized, or prepared by other methods known in the art.

[0164] In one embodiment, the factors of the present invention include antibodies. The 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 Navarre, Pamplona, ​​Spain, or 4B4-1 from Affymetrix / BioScience, California, USA), anti-CD28 (XR-CD28: Thermo Fisher Scientific, Norway), anti-ICOS (ISA-3) (Affymetrix / BioScience, California, 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.

[0165] The antibodies of this invention are available from public sources such as the United States Type Culture Collection (ATCC), and antibodies, T-cell helper molecules, and cell surface proteins can be produced using standard techniques. Methods for producing antibodies used in the methods of this invention are known in the art. Antibodies can also be produced as genetically engineered immunoglobulins (Ig) or Ig fragments designed to have specific target properties. As a non-limiting example, antibodies may include recombinant IgG, which is a chimeric fusion protein having at least one variable (V) region domain from a first mammalian species and at least one constant region domain from a second, different mammalian species. Most commonly, chimeric antibodies have a murine variable region sequence and a human constant region sequence. Such murine / human chimeric immunoglobulins can be "humanized" by transplanting a complementarity-determining region (CDR) derived from a murine antibody (which confers antigen-binding specificity) into a human V region framework region and a human constant region. Antibodies containing CDRs with different specificities can also be combined to produce multispecific (bispecific or trispecific, etc.) antibodies. These molecular fragments can be produced by proteolytic digestion, or optionally by proteolytic digestion followed by mild disulfide bond reduction and alkylation, or by recombinant genetic engineering techniques.

[0166] The factors of this invention may further comprise any molecule that specifically binds to 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. The factors of this invention may comprise peptides, small organic molecules, peptide mimics, soluble T-cell receptors, antibodies, etc. The factors of this invention may be naturally occurring cell surface receptor ligands. Suitable factors of this invention can be identified by assays known in the art for determining binding affinity and specificity, including competitive and non-competitive assays. Assays of interest include ELISA, RIA, flow cytometry, etc.

[0167] The factors considered in this invention may further include protein ligands, natural ligands, and synthetic ligands. Factors that can bind to cell surface groups and, under certain conditions, cause linkages and aggregations that induce 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, tetraiodothyronine), bacterial groups (e.g., lipopolysaccharides), mitogens, antigens, superantigens and their derivatives, growth factors, cytokines, viral proteins (e.g., HIV gp-120), adhesion molecules (e.g., L-selectin, LFA-3, CD54, LFA-1), chemokines, and small molecules. These factors may be isolated from natural sources such as cells, blood products, and tissues, or from cells propagated in vitro, or prepared by recombinant methods, or by other methods known to those skilled in the art.

[0168] In one aspect of the invention, when it is desired to stimulate T-cells, available factors include ligands capable of binding to the CD3 / TCR complex, CD2, and / or CD28 and initiating activation or proliferation, respectively. Thus, the term ligand includes those proteins that are “natural” ligands for cell surface proteins (e.g., the B7 molecule targeting CD28) and those that are artificial ligands (e.g., antibodies targeting cell surface proteins). Such antibodies and fragments can be produced according to conventional techniques, such as hybridoma methods and recombinant DNA and protein expression technologies. Available antibodies and fragments can be obtained from any species (including humans) or can be formulated as chimeric proteins employing sequences from more than one species.

[0169] In alternative embodiments, the co-stimulatory signal may also include rapamycin. Rapamycin may be introduced into the cells before, simultaneously with, and / or after contact with the activator. Rapamycin may be present throughout the proliferation / expansion steps of the method 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 the activator and rapamycin. In this method, subsequent growth and passage may be performed in the presence of rapamycin, but not in the presence of the activator.

[0170] Rapamycin can be used at concentrations from about 0.01 µM to about 10 µM (e.g., 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, rapamycin, AY-22989, RAPA, and NSC-226080, and is available from Sigma, CalbioChem, LCLabs, etc. Rapamycin is available from various commercial sources, such as AGScientific, Inc. (San Diego, California, USA).

[0171] Other cytokines and / or growth factors may be added to the culture as needed. These cytokines and / or growth factors are added at appropriate concentrations and at appropriate time points. For example, IL-2 and / or IL-4 may be added to enhance T cell proliferation. Other cytokines may be added if necessary to induce specific differentiation patterns (e.g., TGF-β and IL-10). For example, it has been shown that IL-4 triggers T cell differentiation into the Th2 subset, and IFN-γ and IL-12 trigger differentiation into the Th1 subset (Sunder-Plassmann et al., Blood (…). blood ) 87 ;5179-5184 (1996)). Therefore, in some embodiments of the invention, cytokines (e.g., IL-2) may be added in one or more steps to achieve a final exogenously added concentration of 10 to 4,000 U / ml (e.g., from about 10 U / ml to about 3,000 U / ml, from about 10 U / ml to about 2,000 U / ml, from about 10 U / ml to about 1,500 U / ml, from about 15 U / ml to about 4,000 U / ml, from about 15 U / ml to about 3,000 U / ml, from about 15 U / ml to about 1,500 U / ml, from about 20 U / ml to about 2,000 U / ml, etc.). In some specific cases, IL-2 may be added at 20 U / ml during stimulation, periodically at 1,000 U / ml during the initial culture phase, and at 20 U / ml during the period prior to collection.

[0172] For example, IL-4 can be exogenously added at a final concentration, such as about 1,000 to 5,000 U / ml or about 100 to 10,000 U / ml (e.g., from about 1,000 U / ml to about 5,000 U / ml, from about 500 U / ml to about 5,000 U / ml, from about 250 U / ml to about 5,000 U / ml, from about 100 U / ml to about 5,000 U / ml, from about 800 U / ml to about 2,500 U / ml, etc.). In some cases, about 1,000 U / ml can be added during stimulation and culture until collection. Appropriate cytokines and growth factors and their effects on T cells are well known and described in the art. Once the T cell population is contacted with an activator and optionally rapamycin under appropriate conditions for T cell growth, growth can be allowed to proceed for a period of time, the duration of which is selected based on the desired final T cell number and cell expansion rate. Cell passage can be performed during this period. This period typically ranges from about 3 to about 10 days, but can last from about 14 to about 20 days or even longer, as long as the survival rate and continued proliferation of T cells are maintained.

[0173] The compositions of the present invention may be contained on a surface on which the factors described above are immobilized. The surface of the present invention may be any surface capable of having ligands bound to or integrated therein, and is biocompatible, i.e., substantially non-toxic to the target cells to be stimulated. The biocompatible surface may be biodegradable or non-biodegradable. The surface may be natural or synthetic. Synthetic surfaces may be polymers. The surface may comprise collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan, extracellular matrix composition, liposomes, or cell surfaces. Polysaccharides may include, for example, cellulose, agarose, dextran, chitosan, hyaluronic acid, or alginate. Other polymers may include polyesters, polyethers, polyanhydrides, polyalkyl cyanoacrylates, polyacrylamide, polyorthoesters, polyphosphazenes, polyvinyl acetate, block copolymers, polypropylene, polytetrafluoroethylene (PTFE), or polyurethane. The polymer may be lactic acid or a copolymer. Copolymers may comprise lactic acid and glycolic acid (PLGA). Non-biodegradable surfaces may include polymers such as poly(dimethylsiloxane) and poly(ethylene-vinyl acetate). Biocompatible surfaces include, for example, glass (e.g., bioglass), collagen, chitosan, metals, hydroxyapatite, aluminates, bioceramic materials, hyaluronic acid polymers, alginates, acrylate polymers, lactic acid polymers, glycolic acid polymers, lactic / glycolic acid polymers, purified proteins, purified peptides, or extracellular matrix compositions. Other polymeric surfaces may include glass, silica, silicon, hydroxyapatite, hydrogels, collagen, acrolein, polyacrylamide, polypropylene, polystyrene, nylon, or any number of plastic synthetic organic polymers, etc. Surfaces may contain biological structures such as liposomes or cell surfaces, such as erythrocytes (RBCs). Surfaces may be in the form of lipids, plates, bags, pellets, fibers, sieves, or particles. Particles may include colloidal particles, microspheres, non-particles, beads, etc. In various embodiments, commercially available surfaces (e.g., beads or other particles) may be used (e.g., Miltenyi Particles, Miltenyi Biotec, Germany; Sepharosebeads, Pharmacia Fine Chemicals, Sweden; Dynabeads). ® Dynal Inc., New York; PuraBeads ® (PrometricBiosciences).

[0174] Proteins like antibodies can be conjugated to solid carriers (such as beads) in any number of ways. One way to link proteins to carriers is via biotin and avidin or avidin-streptolysin. One system that has been developed is called... Strep-tag® (IBAGMBH, Göttingen, Germany). This system allows the use of an eight-amino acid sequence tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1)) that binds to avidin. Furthermore, this system allows the release of bound cells and proteins through competitive binding to biotin. Therefore, the present invention includes compositions and methods for linking proteins (e.g., antibodies) to a solid carrier via avidin-streptomycin interaction, and for purifying cells bound to such a solid carrier. The cell purification method includes the following: The interaction between the solid carrier and the antibody is formed via avidin or avidin-streptomycin, wherein the antibody binds to a cell surface marker (e.g., CD4). The solid carrier is separated from unbound cells, and then the bound cells are released. Release can be carried out by methods such as proteolytic cleavage of the antibody or by competition with added biotin. This carrier can be used for cell purification, as well as T cell activation.

[0175] Similar products are available from Miltenyi Biotec (Bergüschgradbach, Germany, catalog number 130-090-485). These products contain beads with an anti-biotin bound to them, which can be used to bind to biotinylated antibodies. These beads can also be used for cell purification and T-cell activation.

[0176] When beads are used, they can be of any size capable of achieving target cell stimulation. In some embodiments, beads with sizes ranging from about 5 nm to about 500 µM are used. The choice of bead size typically depends on the specific application of the beads. For example, when paramagnetic beads are used, the bead size is typically from less than 1 µM to about 2.8 µM to about 500 µM, typically from about 2.8 µM to about 50 µM. Finally, superparamagnetic non-particles, which can be as small as about 10 nm, may be used. -5 nm. An exemplary bead, Dynabeads ® M-450 is 4.5 µM. Therefore, the beads used to practice this invention typically have a diameter from about 0.00001 nm to about 500 µM (e.g., from about 0.01 nm to about 500 µM, from about 1 nm to about 500 µM, from about 5 nm to about 500 µM, from about 0.00001 nm to about 50 µM, from about 0.1 nm to about 50 µM, from about 10 nm to about 50 µM, from about 50 nm to about 50 µM, from about 50 nm to about 25 µM, from about 50 nm to about 1 µM, from about 10 µM to about 500 µM, from about 1 µM to about 10 µM, etc.).

[0177] In some embodiments, Dynabeads can be used® Dynabeads ® It has the advantage that it allows parameters such as ligand composition and stoichiometry to be altered for systematic examination of their effects on T cell activation and differentiation. (Dynospheres) ® (Thermo Fisher Scientific, Lillestrøm, Norway) Dynospheres can conjugate specific T cell subsets with different stoichiometry to various factors. ® The antibodies or other factors of the present invention can be conjugated to beads without linker groups by conjugating them to bare beads coated with epoxy groups.

[0178] Factors can be conjugated to, attached to, bound to, coupled to, or integrated into surfaces using various methods known and available in the art. Factors can be natural ligands, protein ligands, or synthetic ligands. The connection can be covalent or non-covalent, electrostatic or hydrophobic, and can be achieved through various connection means, including (e.g.) chemical, mechanical, enzymatic, electrostatic, or other means, while the ligand is capable of stimulating cells. Factor connection can be direct or indirect (e.g., tethering). For example, antibodies can be directly attached to the surface (i.e., direct attachment) or indirectly (e.g., avidin or avidin-streptin / biotin).

[0179] Regarding cell surfaces, binding can be achieved via gene expression of the factor using any number of techniques known in the art (e.g., transfection or transduction of expression vectors containing the coding region of the target factor). Antibodies to the ligand can be bound to the surface using anti-individual genotype antibodies. Another example includes the use of protein A or protein G, or other non-specific antibody-binding molecules (including Dynabeads) bound to any suitable surface. ® As a method of conjugation with antibodies, the ligand can alternatively be attached to the surface using commercially available cross-linking agents (e.g., those available from Pierce, Rockford, Ill.) via chemical methods (e.g., surface cross-linking) or other methods. In some embodiments, the ligand is covalently bound to the surface. Furthermore, in one embodiment, commercially available p-toluenesulfonyl-activated Dynabeads... ® Or Dynabeads with epoxy-surface reactive groups ® Use the target ligand for incubation according to the manufacturer's instructions. In short, these conditions typically involve incubation at a temperature of approximately 4°C to 37°C in a phosphate or borate buffer at a pH of approximately 4 to 9.5.

[0180] Appropriate proportions of activators are typically mixed together before they come into contact with the beads. "Appropriate reaction conditions" can vary depending on the specific activator used, but exemplary conditions may include, for example, a particle concentration of approximately 4 × 10⁻⁶.8 Beads and activators, such as antibodies, are cultured in phosphate buffer (e.g., 0.5 M phosphate) at approximately pH 7.4 per bead / 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 approximately 0.05% w / v) to stabilize the activator and block any remaining hydrophobic flakes on the bead surface.

[0181] Once the appropriate reaction mixture is set up, allow the reaction to proceed for the appropriate time and under suitable conditions to promote the adsorption of the activator to the surface in the desired proportion. Again, specific conditions may vary depending on the composition of the reaction mixture, but exemplary conditions include incubation at approximately 37°C for approximately 6 to approximately 24 hours while slowly tilting and rotating.

[0182] Regardless of the conditions chosen, once the fixation reaction is complete, the beads, with the activator immobilized on their surface in an appropriate proportion, can be removed from the remaining aqueous culture medium by placing a magnet on the side of the reaction vessel, and the supernatant is discarded. The beads are typically washed to remove any excess unbound antibody before use. Once prepared, these beads can be used immediately or stored for future use.

[0183] In some embodiments, Dynabeads ® M-450 can be used in conjunction with surface interaction factors. According to the manufacturer, Dynabeads... ® M-450 consists of 4.5-micron epoxy beads. They are non-porous, monodisperse, superparamagnetic beads. High solution flowability allows Dynabeads ® It continuously interacts with the solution to facilitate conjugation with antibodies. The beads can also have a surface p-toluenesulfonyl group.

[0184] Factors can be fixed to the same bead, i.e., "cis", or fixed to different beads, i.e., "trans".

[0185] In alternative embodiments, the activating factor of the present invention may be in solution.

[0186] Activation and expansion of specific T cell subsets can be achieved through specific surface factors and their ratios. In some embodiments, the compositions of the present invention used to expand T cells (e.g., Treg cells) contain anti-CD3 and anti-CD28 factors. The T cell (e.g., Treg cell) expansion compositions of the present invention may contain low, moderate, or high relative levels of CD3 adhering to the surface. For example, low concentrations of anti-CD3 factor may be present on the surface at concentrations 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; moderate concentrations of anti-CD3 factor may be present on the surface of the invention at concentrations of about 0.5 to about 1.5, about 0.6 to about 0.8, or about 0.75 units; and high concentrations of anti-CD3 factor may be present on the surface of the invention at concentrations of about 2 to about 4, about 3 to about 4, about 3.4, or about 3.41 units. In some embodiments, beads for amplifying T cells (e.g., Treg cells) are used, which contain low relative levels of anti-CD3 antibody and anti-CD28 co-stimulation.

[0187] In some embodiments, the compositions of the present invention used to activate and expand T cells (e.g., memory T cells) comprise anti-CD3 factor, anti-CD137 factor, and anti-CD28 factor. The T cell (e.g., memory T cell) expansion compositions of the present invention may contain low, low-medium, medium-high, or high relative levels of CD3 adhering to the surface. For example, low levels of anti-CD3 factor may be present on the surface of the invention at concentrations of about 0.005 to about 0.02, about 0.008 to about 0.015, or about 0.01 units; moderately low levels of anti-CD3 antibody may be present on the surface of the invention at concentrations of about 0.02 to about 0.08, about 0.03 to about 0.07, or about 0.05 units; moderately high levels of anti-CD3 factor may be present on the surface of the invention at concentrations of about 0.09 to about 0.16, about 0.1 to about 0.15, about 0.14, or about 0.142 units; and high levels of anti-CD3 factor may be present on the surface of the invention at concentrations 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 used to expand T cells (e.g., memory T cells), beads containing low relative levels of anti-CD3 antibody and co-stimulation with anti-CD28 and anti-CD137 are used.

[0188] In some embodiments, the compositions of the present invention used to activate and expand T cells (e.g., Th17 cells) comprise an anti-CD3 antibody, as well as an anti-ICOS antibody, an anti-CD5 antibody, or an anti-CD28 antibody. The T cell (e.g., Th17 cell) expansion compositions of the present invention may contain low, moderate, or high relative levels of CD3 adhering to a surface. For example, low anti-CD3 factors may be present on the surface of the present invention at concentrations 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; moderate anti-CD3 factors may be present on the surface of the present invention at concentrations of about 0.1 to about 0.5, about 0.2 to about 0.5, or about 0.3 units; and high anti-CD3 factors may be present on the surface of the present invention at concentrations of about 1 to 2, about 1.3 to about 1.7, or about 1.5 units. In some embodiments used to expand T cells (e.g., Th17 cells), beads containing low relative levels of anti-CD3 antibody and co-stimulation with anti-CD5, anti-CD28, and / or anti-ICOS are used.

[0189] Antibodies can be conjugated to solid carriers (e.g., beads) in any number of ways. Some methods for conjugating antibodies involve contacting the antibody with the solid carrier under conditions that allow for covalent coupling. One commercially available product for preparing antibody-conjugated beads is the Dynabeads® Antibody Coupling Kit (Thermo Fisher Scientific, catalog number 14311D).

[0190] The Dynabeads® Antibody Coupling Kit enables the covalent coupling of antibodies and other proteins, such as lectins and functional enzymes, to the surface of Dynabeads® M-270 Epoxy. Surface epoxy groups allow protein coupling via primary amino and thiol groups. Beads can be coupled to “saturation,” resulting in low background binding upon exposure to post-coupling treatment.

[0191] The amount of protein used in the reaction mixture to saturate the beads varies depending on the “load” of the beads used. As an example, when using Dynabeads® M-270Epoxy beads, it is recommended to use at least 10 µg of protein per mg of beads to ensure saturation. This is because the load of these beads typically varies between approximately 7 µg and 9 µg of protein per mg of beads. Furthermore, the antibody loading can be adjusted on a per-bead basis by changing the proportion of antibody in contact with the beads. Therefore, antibody / bead coupling can be adjusted to change the ratio between the ligands present on the beads and / or the average total amount of ligands on each bead.

[0192] In practice, Dynabeads® M-450Epoxy (Thermo Fisher Scientific, catalog number 14011) can also be used. The procedure here is used to couple 1 ml (4 × 10⁻⁶) of the solution. 8 ) beads. Per 1 ml (4×10) 8 The beads should be prepared using approximately 200 μg of antibody (Ab). Place 1 ml of washed and resuspended beads in a tube. Place the tube in a magnet for 1 minute and discard the supernatant. Remove the tube from the magnet and resuspend the beads in buffer A (0.1 M sodium phosphate buffer, pH 7.4 to 8.0, or 0.1 M sodium borate buffer, pH 7.6 to 9.5). Then add 200 μg of antibody to bring the volume to 1 ml. Incubate the mixture for 15 minutes, then add BSA to 0.01 to 0.1% w / v. Incubate the mixture at room temperature for 16 to 24 hours while gently tilting and rotating. Place the tube in a magnet for 1 minute and discard the supernatant. Then add 1 ml of buffer B (Ca-free). 2+ and Mg 2+ The mixture was prepared with phosphate-buffered saline (PBS), 0.1% bovine serum albumin (BSA), and 2 mM EDTA (pH 7.4). The mixture was then gently tilted and rotated while incubating for 5 minutes. This washing step was repeated twice. The tube was then removed from the magnet, and the beads were resuspended in 1 ml of buffer B (4 × 10⁻⁶). 8 (1 bead / ml)

[0193] Another product that can be used in the practice of this invention is Pierce™ NHS-Activated Magnetic Beads (Thermo Fisher Scientific, catalog number 88827). These beads have N-hydroxysuccinimide (NHS) functional groups on the surface of a closed magnetic bead. They are superparamagnetic (without magnetic memory) and have a nominal average diameter of 1 µM (nominal). These beads have a density of 2.0 g / cm³ and a binding capacity of ≥26 µg rabbit IgG / mg bead.

[0194] First, allow the protein solution and magnetic beads to reach room temperature, then place 300 µl of magnetic beads into a 1.5 ml microcentrifuge tube. Place the tube in a magnetic holder, collect the beads, and discard the supernatant. Introduce 1 ml of ice-cold wash buffer A (ice-cold 1 mM hydrochloric acid) into the tube and gently vortex for 15 seconds. Place the tube in a magnetic holder, collect the beads, and discard the supernatant. Then add 300 µl of protein solution to the tube and vortex for 30 seconds. Incubate the mixture on a vortex mixer at room temperature for 1 to 2 hours. During the first 30 minutes of incubation, vortex the tube for 15 seconds every 5 minutes. For the remaining time, vortex the tube for 15 seconds every 15 minutes until incubation is complete. Collect the beads using a magnetic holder and preserve the throughflow. Add 1 ml of wash buffer B (0.1 M glycine, pH 2.0) to the beads and vortex the tube for 15 seconds. Place the tube in a magnetic holder, collect the beads, and discard the supernatant. Repeat the washing and collection process once. Add 1 ml of ultrapure water to the beads and vortex the tube for 15 seconds. Place the tube in a magnetic holder, collect the beads, and discard the supernatant. Add 1 ml of quenching buffer (3M ethanolamine, pH 9.0) to the beads and vortex the tube for 30 seconds. Then place the tube on a vortex mixer at room temperature for 2 hours. Place the tube in a magnetic holder, collect the beads, and discard the supernatant. Add 1 ml of water to the tube, mix thoroughly, collect the beads, and discard the supernatant. Add 1 ml of storage buffer (50 mM borate, 0.05% sodium azide, pH 8.5) to the tube, mix thoroughly, collect the beads, and discard the supernatant. Repeat this washing step twice more. Add 300 µl of storage buffer to the beads, mix thoroughly, and store at 4°C until ready for use. The final concentration of the protein-coupled magnetic beads should be approximately 10 mg / ml.

[0195] Other beads that can be used to practice this invention are Dynabeads® M-270 CarboxylicAcid (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).

[0196] In many cases, the solid carriers (e.g., beads) used to practice this invention are saturated with protein. The proportion of protein used can vary. For example, 100% of the total load can be a ligand (e.g., an antibody). In this case, one ligand may be present, or multiple ligands may be present. When multiple ligands are present, they may be present in the same proportion (1:1) or in different proportions (e.g., 1:10). Exemplary proportions of ligands that can be used for practice are described elsewhere herein.

[0197]

[0198] The total ligand load can also be adjusted. As shown in Table 2 using antibody ligands, solid carriers can be loaded to less than 100% capacity. In many cases, when this is done, it will be desirable to block further reactions at the ligand-linking sites. One way to do this is by contacting the solid carrier with a factor (e.g., a protein) that does not have ligand-binding activity associated with a specific application (e.g., human serum albumin (HSA), bovine serum albumin (BSA), one or more antibodies that do not bind to T-cell receptors, etc.), while allowing ligand and non-ligand factors to bind to the solid carrier. This allows for the adjustment of different ligand ratios and different total signal-to-weight ratios. Many examples of these aspects are listed in Table 2.

[0199] The total signaling of T cells can be modulated in any number of ways. For example, if the signal / solid carrier (e.g., beads) ratio is low, then each T cell can come into contact with more solid carrier surface. When using beads, this means the ratio of beads to T cells can be adjusted. For example, if beads with a 50% antibody load are used, then twice as many beads can be used to obtain the same total signal as 100% loaded beads.

[0200] IV. Using the method of the present invention

[0201] The T-cell subsets of this invention can be used in any number of physiological conditions, diseases, and / or disease states for therapeutic and / or research / discovery purposes. Conditions or diseases characterized by abnormal immune responses can be autoimmune diseases such as diabetes, multiple sclerosis, myasthenia gravis, neuritis, lupus, rheumatoid arthritis, psoriasis, or inflammatory bowel disease. Conditions where immunosuppression would be advantageous include those where a normal or activated immune response is detrimental to mammals (e.g., humoral or partial allogeneic transplantation) to avoid rejection, or fertility treatments involving inappropriate immune responses to pregnancy failure and miscarriage. Using these cells before, during, or after transplantation avoids widespread chronic graft-versus-host disease, which can occur in patients undergoing treatment (e.g., cancer patients). Cells can be expanded immediately after collection or stored (e.g., by freezing) before or after expansion and before their therapeutic use. This treatment can be combined with known immunosuppressive therapies.

[0202] Once a suitable population or subset of T cells is isolated from a patient or animal, it may optionally be genetically modified or manipulated, or any other suitable modification or manipulation, prior to amplification of the resulting T cell population using the methods and vectors of the present invention. Manipulation may, for example, take the form of stimulating / restimulating T cells with anti-CD3 and anti-CD28 antibodies to activate / reactivate them.

[0203] In some embodiments, it is desirable to administer activated T cells to a subject, followed by a blood draw (or apheresis) to activate the T cells therein according to the invention, and then re-infuse these activated and expanded T cells to the patient. This process can be performed multiple times every few weeks. In some embodiments, T cells can be activated from blood draws ranging from 10 ml to 400 ml. In some embodiments, T cells are activated from blood draws 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 composition to the subject can be performed in any convenient manner, including by aerosol inhalation, injection, oral administration, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention can be administered by intravenous injection. T-cell compositions can be directly injected into tumors, lymph nodes, or sites of infection / inflammation (autoimmunity).

[0204] The T-cell subset composition produced according to the present invention has numerous potential uses, including experimental and therapeutic uses. In particular, it is conceivable that such T-cell populations would be very useful in suppressing unwanted or inappropriate immune responses. In this method, a small number of T cells are removed from a patient and then manipulated and expanded in vitro before being re-infused into the patient. Examples of diseases that can be treated in this manner are autoimmune diseases and conditions in which the suppression of immune activity is desired (e.g., allogeneic transplant resistance). A treatment method may comprise: providing a mammal; obtaining a biological sample from a mammal containing T cells; expanding / activating T cells in vitro according to the method of the present invention as described above; and administering the expanded / activated T cells to the mammal to be treated. The first mammal and the mammal to be treated may 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 first mammal (“donor”) can be isomorphic, allogeneic, or xenomorphic. Therapies can be administered to mammals with abnormal immune responses (e.g., autoimmune diseases, including, for example, diabetes, multiple sclerosis, myasthenia gravis, neuritis, lupus, rheumatoid arthritis, psoriasis, and inflammatory bowel disease), tissue transplantation, or fertility treatments.

[0205] Key technical hurdles associated with this therapy include purifying target cells from the patient, and the in vitro expansion and / or manipulation of these cells. This therapy typically requires large numbers of cells; therefore, optimizing methods for in vitro induction of T cell proliferation is crucial to maximizing the number of T cells generated and minimizing the time required to generate a sufficient number of T cells. The compositions and methods of the present invention provide a large number of specific subsets of T cells.

[0206] The T-cell subsets of this invention can be used in a variety of applications and therapeutic modalities. These T-cell subsets 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). Broadly defined examples of T-cell therapy include infusing a subject with a T-cell subset selectively expanded externally by the methods of this invention, either after or not after immune elimination, or infusing a subject with allogeneic, externally expanded T-cells isolated from a donor subject (e.g., adoptive cell transfer).

[0207] Autoimmune disorders

[0208] Autoimmune diseases or disorders are those illnesses resulting from inappropriate and excessive responses to autoantigens. Research has linked defective Treg cells to autoimmune disorders. Autoimmune diseases include: diabetes mellitus, uveitis and multiple sclerosis, Addison's disease, steatorrhea, dermatomyositis, Graf's disease, Hashimoto's thyroiditis, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Abulemi's disease, pemphigus vulgaris, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthritis, vasculitic lentigines, leukoplakia, myxedema, pernicious anemia, ulcerative colitis, Crohn's disease, bullous epidermolysis bullosa, epididymitis, glomerulonephritis, diffuse toxic goiter, infectious polyneuritis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus erythematosus as non-limiting examples. In autoimmune disease states, CD4... + CD25 + Tregs can be present in reduced quantities or have functional impairments. This has already been observed in patients with multiple sclerosis (Viglietta et al.). 《 Journal of Experimental Medicine ( J. Exp. Med. ) 199 :971–979 (2004)), type II autoimmune polyendocrine syndrome (Kriegel et al., 《 Journal of Experimental Medicine ( J. Exp. Med. ) 199 :1285–1291 (2004)), Type I diabetes (Lindley et al., Diabetes ( Diabetes ) 54 :92–929 (2005)), psoriasis (Sugiyama et al., Immunology) miscellaneous Zhi ( J. Immunol. ) 174 :164–173 (2005)) and myasthenia gravis (Balandina et al., Blood ( blood ) 105 In patients with Treg cells from peripheral blood (735–741 (2005)), the ability of Treg cells to suppress T cell proliferation was reduced.

[0209] The use of T-cell therapy to treat autoimmune disorders can involve different mechanisms. In one embodiment, another source of blood or immune cells can be removed from a subject suffering from an autoimmune disorder. The method of the present invention discussed 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 removed from the subject in need by known methods, including low-dose whole-body radiation, thymic radiation, anti-thymocyte globulin, and chemotherapy. Illustrative chemotherapeutic agents of the present invention include, but are not limited to, alemtuzumab, anti-CD3 antibodies, cytoxin, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation. After removal of inappropriate memory T cells capable of recognizing autoantigens, externally expanded autologous T cells can be administered to the subject to rebuild or restimulate their immune system.

[0210] As an alternative to, or in addition to, the treatments described above, Treg cells can be isolated from sources including peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsies, tumors, lymph node tissue, gastrointestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, or any other lymphoid tissue and tumor origin. These T cells can be selectively expanded using the methods of this invention. These expanded Treg cells can be re-administered to a patient to suppress inappropriate immune responses. This Treg therapy can be administered to suppress minimal residual immune responses after immune depletion, or to suppress immune responses in subjects that have not undergone immune depletion.

[0211] graft-versus-host disease

[0212] A major problem in hematopoietic stem cell transplantation is graft-versus-host disease, which is caused by alloreactive T cells present in the infused hematopoietic stem cell preparation. In organ transplantation, transplant rejection mediated by alloreactive host T cells is a major problem, usually overcome through long-term immunosuppression in the transplant recipient.

[0213] In methods similar to those described above, the use of T-cell therapy to treat adverse graft-versus-host disease events, reduce their risk or severity, may involve different mechanisms. In one embodiment, another source of blood or immune cells may be removed from a subject suffering from graft-versus-host disease. The methods of the present invention discussed herein can be used to selectively expand T cell types other than memory T cells, thereby selectively expanding those cell types that do not contain durable recognition of antigens from exogenous tissues. After removal and external expansion of autologous cells, inappropriate memory T cells can be removed from the subject in need by known methods, including low-dose whole-body radiation, thymic radiation, anti-thymocyte globulin, and chemotherapy. Illustrative chemotherapeutic agents of the present invention include (but are not limited to) alemtuzumab, anti-CD3 antibodies, cytoxin, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation. After removal of inappropriate memory T cells capable of recognizing antigens on exogenous tissues, the subject can be re-administered with externally expanded autologous T cells to rebuild or restimulate their immune system.

[0214] As an alternative to, or in addition to, the treatments described above, Treg cells removed from the patient's blood can be selectively expanded. These expanded Treg cells can then be re-administered to the patient to suppress inappropriate immune responses, to minimize residual immune responses after immune depletion, or to suppress immune responses in subjects that have not undergone immune depletion.

[0215] Allergic diseases

[0216] Allergic diseases can also be affected by T cell dysfunction. Studies have shown that impaired CD4+ cells are present in patients with seasonal allergies. + CD25 + Treg-mediated suppression of allergen-specific T helper type 2 (Th2) cells (Ling EM et al., *The Lancet*, 2004; 363:608–15.; Grindebacke H et al., *Clinical Exp Allergy*, 2004; 34:1364–72.). Furthermore, compared to healthy individuals, individuals with allergies and asthma showed a higher proportion of altered T cell populations (Akdis M et al., *Journal of Experimental Medicine*, 2004; 199:1567–75.; Tiemessenm M et al., *Journal of Allergy Clin Immunol*, 2004; 113:932–9.).

[0217] In methods similar to those described above, the use of T-cell therapy to treat, prevent, or alleviate allergic diseases may involve different mechanisms. Similar to autoimmune disorders and graft-versus-host disease, allergic diseases are caused by inappropriate immune responses. Suppressing this response, or removing cells capable of recognizing inappropriate antigens, can alleviate allergic symptoms. In methods of T-cell therapy used to treat allergies, blood may be removed from the subject suffering from the allergic disorder. The methods of the present invention discussed herein can be used to selectively expand non-T memory cell T-cell types, thereby selectively expanding those cell types that do not contain durable recognition of antigens (e.g., legume proteins) from inappropriate antigens. After removing and expanding autologous cells, inappropriate memory T cells can be removed from the subject in need by known methods, including low-dose whole-body radiation, thymic radiation, anti-thymocyte globulin, and chemotherapy. Illustrative chemotherapeutic agents of the present invention include, but are not limited to, alemtuzumab, anti-CD3 antibodies, cytoxin, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation. After removing inappropriate memory T cells that can recognize antigens on exogenous tissues, the subject can be given externally expanded autologous T cells to rebuild or restimulate their immune system.

[0218] As an alternative to, or in addition to, the treatments described above, Treg cells removed from the patient's blood can be selectively expanded. These expanded Treg cells can then be re-administered to the patient to suppress inappropriate immune responses, to minimize residual immune responses after immune depletion, or to suppress immune responses in subjects that have not undergone immune depletion.

[0219] Inflammatory diseases

[0220] T-cell therapy is involved in the treatment of inflammatory diseases and inflammation-related disorders. Many of these diseases 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 of multisystem inflammatory disease; tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS); interleukin-1 receptor antagonist deficiency (DIRA); and Behcet's disease.

[0221] Because of the role of Treg cells in suppressing inappropriate immune responses to non-pathogen antigens, a decrease in the number or impairment of these T cell subsets can lead to inflammatory diseases. This is certainly true for, for example, inflammatory bowel disease (MHimmell et al., Immunology, June 2012; 136(2):115-122) and rheumatoid arthritis (MNoack et al., Autoimmunity Reviews, June 2014; 13(6):668-677).

[0222] Inflammatory diseases are mechanistically similar to autoimmune disorders. Accordingly, inflammatory diseases can be partly caused by inappropriate immune responses. Suppressing this response, or removing cells that recognize inappropriate antigens, can alleviate inflammatory symptoms. In methods of T-cell therapy used to treat inflammatory diseases, blood can be removed from the subject suffering from the inflammatory disorder. The method of the present invention discussed herein can be used to selectively expand non-T memory cell T cell types, thereby selectively expanding those cell types that do not contain persistent recognition of inappropriate antigens, such as carbamylated proteins in rheumatoid arthritis mediated by anti-carbamylated protein (anti-CarP) antibodies. After removal and expansion of autologous cells, inappropriate memory T cells can be removed from the subject in need by known methods, including low-dose whole-body radiation, thymic radiation, anti-thymocyte globulin, and chemotherapy. Illustrative chemotherapeutic agents of the present invention include (but are not limited to) alemtuzumab, anti-CD3 antibodies, cytoxin, fludarabine, cyclosporine, FK506, mycophenolic acid, steroids, FR901228, and radiation. After removing inappropriate memory T cells that can recognize autoantigens and enhance the resulting inflammatory response, the subject can be given externally expanded autologous T cells to rebuild their immune system.

[0223] As an alternative to, or in addition to, the treatments described above, Treg cells removed from the patient's blood can be selectively expanded. These expanded Treg cells can then be re-administered to the patient to suppress inappropriate immune responses, to minimize residual immune responses after immune depletion, or to suppress immune responses in subjects that have not undergone immune depletion.

[0224] Hyperproliferative disorders

[0225] Evidence from cancer patients further links T-cell dysfunction to hyperproliferative diseases, including cancer. For example, increased Treg activity can lead to a poor immune response to tumor antigens and result in immune dysfunction. Increased CD4+CD25+ clusters have been found in the blood or tumors themselves of patients with lung cancer, pancreatic cancer, breast cancer, liver cancer, and skin cancer (WooEY et al.; J Immunol 2002; 168:4272–6.; WolfAM et al. Clin Cancer Res 2003; 9:606–12.; LiyanageUK et al. J Immunol 2002; 169:2756–61.; ViguierM et al. J Immunol 2004; 173:1444–53.; OrmandyLA et al. Cancer Res 2005; 65:2457–64.).

[0226] The methods of this invention can be used to amplify T cells that are specific to tumor antigens, hyperproliferative disease antigens, or antigens associated with hyperproliferative diseases. Tumor antigens are proteins produced by tumor cells that trigger an immune response, particularly a T cell-mediated immune response.

[0227] Treatable cancers include non-vascularized or not yet significantly vascularized tumors, as well as vascularized tumors. Cancers may include non-solid tumors (e.g., hematologic malignancies such as leukemia and lymphoma) or may include solid tumors. Types of cancers treated with the products of this invention include, but are not limited to, epithelial carcinomas, germ cell tumors, and sarcomas, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, epithelial carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included. This includes the following cancers: 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 malignant glioma.

[0228] Blood cancers are cancers of the blood or bone marrow. Examples of blood (or blood-derived) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, as well as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (such as chronic myeloid leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (slowly progressive and high-grade forms), multiple myeloma, Woldanström macroglobulinemia, heavy chain disease, spinal dysplasia syndrome, hairy cell leukemia, and spinal dysplasia.

[0229] Solid tumors are abnormal masses that typically do not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (e.g., sarcoma, epithelial carcinoma, and lymphoma). Examples of solid tumors like sarcoma and epithelial carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, benign synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, 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, pheochromocytic sebaceous gland carcinoma of the adrenal gland, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, and liver cancer. Cholangioma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (e.g., brainstem glioma and mixed glioma), glioblastoma (also known as multimorphic glioblastoma), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0230] One approach to treating patients or individuals in need of this treatment is to use T cells expanded and genetically modified according to the present invention to target antigens expressed on tumor cells via chimeric antigen receptor (CAR) expression. CARs are antigen receptors designed to recognize cell surface antigens in a manner independent of human leukocyte antigens. In CAR-based therapy, immune cells can be collected from a patient's blood or other tissues. T cells are engineered, as described below, to express CARs on their surface, thereby allowing them to recognize specific antigens (e.g., tumor antigens). These CAR T cells can then be expanded and infused into the patient using the methods of the present invention. In some embodiments, T cells are produced at a rate of 1 × 10⁻⁶. 5 1×10 6 1×107 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 recipient. After infusion, the T cells continue to expand and express CAR, thereby allowing for an increased immune response against the T cells, which contain CARs engineered to recognize specific antigens.

[0231] 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 having an antigen-binding domain fused to an intracellular signaling domain of the T cell antigen receptor complex ζ chain (e.g., CD3ζ). When expressed in T cells, the CAR is able to redirect antigen recognition based on antigen-binding specificity.

[0232] The antigen-binding domain of a CAR contains a target-specific binding element, or antigen-binding group. The choice of domain depends on the type and number of ligands defining the surface of the target cell. For example, antigen-binding domains can be selected to recognize ligands that act as cell surface markers on target cells associated with a specific disease state. Therefore, the antigen-binding domains in the CAR of this invention include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0233] Expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operatively binding a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the target nucleic acid sequence.

[0234] The T cells of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patents 5,399,346, 5,580,859, and 5,589,466. In another embodiment, the present invention provides a gene therapy vector.

[0235] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0236] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2001) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain a replication origin function (in at least one organism), a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0237] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retrovirus particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or in vitro to the target cells. Many retrovirus systems are known in the art. In some embodiments, adenovirus vectors are used. Many adenovirus vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0238] Additional promoter elements (such as enhancers) regulate the frequency of transcription initiation. These are typically located 30 to 110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually variable to maintain promoter function when the elements invert or move relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, the elements appear to act synergistically or independently to activate transcription. Methods for manufacturing CAR T cells are known in the art (see, for example, U.S. Patent 8,906,682).

[0239] In embodiments where the T cells are CAR T cells, the selection of the antigen-binding groups of the present invention will depend on the specific type of tumor antigen of the cancer to be treated, which is known in the art and includes, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RULRU2 (AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, susceptin and telomerase, prostate-epithelial tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin-like growth factor (IGF-1), IGF-II, IGF-I receptor, and mesothelin antibody.

[0240] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignancy. Malignant tumors express a number of proteins that can act 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-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is tumor-fetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific immunoglobulins constitute truly tumor-specific immunoglobulin antigens, which are unique to the individual tumor. B-cell differentiation antigens such as CD19, CDd20, ROR1, CD22, CD23, and the λ / κ light chain are other candidates for target antigens in B-cell lymphoma.

[0241] The types of tumor antigens mentioned can also be tumor-specific antigens (TSA) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells and are also expressed on normal cells, provided that an immune resistance to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0242] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2; tumor-specific multipotent antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens arising from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other major 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, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA224, BTAA, CA125, CA15-3, CA27 / 29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, C250, GA733, and EpCA. M, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, MAC-2 binding protein, cyclic protein C-related protein, TAAl6, TAG72, TLP and TPS, CD19, CD20, CD22, ROR1, mesothelin antibody, CD33 / IL3Ra, c-met, PSMA, glycolipid F77, EGRvIII, GD-2, MY-ESO-1TCR, MAGEA3TCR and others.

[0243] Infectious diseases

[0244] The immune response to infectious diseases involves a balance between antipathogenic and anti-inflammatory responses. T cells are particularly involved in this complex balance. Infectious pathogens capable of inducing T cell responses can be bacteria, viruses, parasites, or fungi. Treg cells have been associated with chronicity of Helicobacter pylori infection (Lundgren A et al., *Infect Immunology*, 2003; 71:1755–62.), hepatitis B virus (HBV) infection, and hepatitis C virus (HCV) infection (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 increase in specific T cell subsets can lead to the persistent nature of these infections by inappropriately suppressing memory T cell responses. The compositions of the present invention can be used to specifically amplify specific T cell subsets and can be used to treat such infectious diseases.

[0245] Infectious diseases can be caused by direct contact with pathogens and spread from person to person, from animals to humans, or from mother to unborn child. Infectious diseases can also be transmitted through indirect contact, such as contact with contaminated surfaces like doorknobs, tables, counters, or faucet handles. They can further spread through insect bites or food contamination. Certain autoimmune disorders, such as HIV or AIDS, and some cancers can increase susceptibility to infectious diseases. Some treatments that suppress the immune system can also increase susceptibility to infectious diseases. Examples of infectious diseases include: smallpox, malaria, tuberculosis, typhus, plague, diphtheria, typhoid fever, cholera, dysentery, and pneumonia.

[0246] As described above, several mechanisms exist for the selective expansion of T cells to treat disease states. In infectious disease states, the infected patient lacks sufficient immunity to the infectious agent. The method of the present invention can be used to selectively expand allogeneic T memory cells from donors immunized against a specific infectious agent and for adoptive T cell transfer. Externally expanded T cells from donors immunized against the infectious agent can then be infused into the infected patient. In some embodiments, the T cells are distributed at a rate of 1 × 10⁻⁶. 5 1×10 6 1×10 7 1×10 8 5×10 8 1×10 9 5×10 9 1×10 105×10 10 1×10 11 5×10 11 Or 1×10 12 Individual cells are administered to the recipient. Then, donor memory T cells adapted to infectious antigens can help enhance the patient's autoimmune response.

[0247] Alternative methods for treating infectious diseases according to this invention include selectively expanding autologous or allogeneic Th17 cells for reinfusion or adoptive cell transfer, respectively. Using the methods described herein, T cells can be expanded externally from blood or tissue isolated from a patient. These expanded T cells can then be infused into the patient to help induce B cells to secrete antibodies against specific infectious antigens, such as Streptococcal M-protein, Neisseria fimbriae, Borrelia burgdorferi lipoprotein VisE, Burkholderia melioides polysaccharide antigen, Aspergillus fumigatus galactomannan, or Francisella tularensis lipopolysaccharide. In some embodiments, T cells are used at a rate of 1 × 10⁻⁶. 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 Apply to the object using individual cells.

[0248] adjunctive therapy

[0249] Existing therapies can be suggested for many of the disease states listed above. The T-cell subsets expanded by this invention can be used as a standalone alternative therapy in some cases, or in combination with other known therapies. T-cell therapy can be administered before, simultaneously with, or after other therapies.

[0250] The methods of the present invention can also be used with vaccines to enhance antigen reactivity and improve in vivo efficacy. In one embodiment, the compositions of the present invention are administered to a patient together with compositions that increase the number of T cells in the body (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, and / or IL-15). Furthermore, considering the long half-life of T cells expanded by the present invention in vivo, these cells can serve as perfect carriers for gene therapies as described above, by carrying desired target nucleic acid sequences and potentially being placed at sites of cancer, disease, or infection. Therefore, cells expanded by the present invention can be delivered to patients together with vaccines, one or more cytokines, one or more therapeutic antibodies, etc. In fact, any therapy that benefits from a more robust T cell population can be used with the compositions of the present invention.

[0251] V. The reagent kit of the present invention

[0252] This document also provides a kit comprising (i) a composition for isolating T cells from a subject; (ii) a composition for culturing T cells in vitro; and (iii) a composition for selectively expanding one or more T cell subsets (e.g., Th17, Treg, memory T cells, etc.). The kit of the present invention may optionally include a composition for reactivating Treg cells.

[0253] The kit may also include written instructions for using the kit, such as instructions on washing steps, culture conditions, and the duration for which isolated T cells are cultured using the compositions of the present invention for selective amplification of specific T cell subsets.

[0254] The intention is that each maximum numerical limit given throughout this specification includes each lower numerical limit, as if such a lower numerical limit were explicitly stated herein. Each minimum numerical limit given throughout this specification will include each upper numerical limit, as if such an upper numerical limit were explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as if all such narrower numerical ranges were explicitly stated herein.

[0255] Example

[0256] The invention can be further understood by referring to the following examples, which are provided by way of example and are not intended to be limiting.

[0257] Example 1. Initial T cells and culture conditions.

[0258] With informed consent, peripheral blood mononuclear cells (PBMCs) were isolated from the erythrocyte sedimentation rate (ESR) amber layer or apheresis components from healthy donors (HemaCareCorp., California, USA, and AstarteBiologics, LLC, Washington, USA). Following stimulation with the CMV pp65 peptide NLVPMVATV (AstarteBiologics LLC), antigen-specific memory T cells were enriched in PBMCs from CMV-positive donors. Regulatory T cells (Tregs) were separated from Dynabeads and CD4-enriched cells using fluorescently activated cell sorting based on CD4, CD25, and CD127 antibody labeling. ® Untouched™ human CD4 T cells (catalog number 11346D, Thermo Fisher Scientific, California, USA) were obtained from the lymphocyte fraction and produced cells expressing CD4. + CD25 + CD127 低 / - Treg cells (Oslo University Hospital, Oslo Ulvall Blood Bank, Norway).

[0259] T cells are activated directly from PBMCs, or using Dynabeads with the various ligand compositions and stoichiometry described above. ® Activation from isolated or enriched T cell subtypes. Activated T cells were incubated at 37°C and 5% CO2. 2 Cells were cultured in X-Vivo™ 15 (Lonza Biologics, Maryland, USA, catalog number BE04-418F) with 2 to 5% CTS™ immune cells (SR) and 0.25 µg / ml gentamicin (both from Thermo Fisher Scientific, New York, USA, catalog number 15710-049), and maintained at 0.5 to 2 × 10⁻⁶ cells per 1 to 3 days by adding cytokines and freshly prepared medium. 6 Amplification was performed using a cell concentration of 1 cell / ml.

[0260] Flow cytometry-sorted regulatory T cells (Tregs) were expanded in media 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 were expanded in 100 IU IL-2 / ml. Th17 / Tc17 cells, such as those from Paulos et al. (Paulos et al., Science Translational Medicine. Med) > 55As described in :55ra78 (2010), the amplification was performed in a medium containing polarizing cytokines (IL-6, IL-1β, IL-23, and TGF-β, all from Thermo Fisher Scientific, California, USA) in the presence of anti-IL-4 and anti-IFN-γ neutralizing antibodies (Thermo Fisher Scientific, California, USA). 100 IU IL-2 / ml was added on day 3 after activation.

[0261] Example 2. Dynabeads-based methods using various ligand compositions and ratios ® The generation of the amplification platform.

[0262] Dynospheres ® MS-4.5-REK particles (Thermo Fisher Scientific, Lillestrøm, Norway) are conjugated with different amounts and ratios of stimulating and co-stimulating ligands optimized for the activation and polarization of various T cell subtypes (i) Treg, ii) antigen-stimulated memory T cells, and iii) Th17 / Tc17 cells. The relative amounts of the stimulating anti-CD3 antibody and its co-conjugated co-stimulating ligands are summarized in Table 3.

[0263]

[0264] Example 3. Stimulation and expansion of T cells.

[0265] T cells are activated directly from PBMCs, or by adding one Dynabead per cell. ® Activation from isolated or enriched T cell subtypes, unless otherwise specified. In most experiments, Dynabeads ® CD3 / CD28CTS TM (High CD3) was included as a reference stimulus. An alternative Treg activation reagent (MACSGMPExpActTregKit; Miltenyi Catalog No. 170-076-119) was also included in the experiment to assess Treg amplification levels. Activated T cells were kept undisturbed for 2 to 3 days, and then amplified for 10 to 14 days as needed by adding freshly prepared culture medium and cytokines. On day 9, Tregs were restimulated using the same stimulating beads as those used for primary activation (day 0). Cells were analyzed on a Coulter Counter (Beckman Coulter, California, USA) to measure absolute cell amplification.

[0266] Example 4. Antibody and flow cytometry analysis.

[0267] The following antibodies were used for Treg analysis: CD4PerCP (Invitrogen, MHCD0431), CD25APC (Invitrogen, MHCD2505), and CD127PE (Invitrogen, A18684). Intracellular FOXP3 expression was analyzed using Alexa Fluor488 (BD560047) after fixation / permeabilization (eBioscience). Flow cytometry data were collected on BDLSRII (BDBiosciences) and analyzed using FACSDiva software (BDBiosciences). Lymphocytes were gated based on their forward and size scattering characteristics. Appropriate unrelated isotype controls were used to set the negative gate (IgG2aPerCP (Thermo Fisher Scientific, catalog number MA1-10426), IgG1APC (Thermo Fisher Scientific, catalog number MG105), IgG1PE (Thermo Fisher Scientific, catalog number MG104), and IgG1AlexaFluor488 (BDBiosciences, catalog number 557702)).

[0268] CMV-specific CD8+ T cells were used with Pro5 Pentamer APC (NLVPMVATV / A) 02:01 (ProImmune, Oxford, UK) and CD8-PE (Thermo Fisher Scientific, catalog number MHCD0804) and appropriate isotype control (IgG1PE, Thermo Fisher Scientific, catalog number MG104) were used for confirmation.

[0269] Intracellular IL-17 expression in PMA / inotropic mycoside-activated Th17 cells was detected after fixation / permeabilization (eBioscience) using IL-17A-PE (catalog number A18695, clone: ​​4H1524, MolecularProbes) and an appropriate isotype control (IgG2bPE, catalog number 400313BioLegend). Flow cytometry data were collected on BDLSRII (BDBiosciences) and analyzed using FACSDiva software (BDBiosciences).

[0270] Example 5. Functions of flow cytometry.

[0271] Following stimulation with PMA / inomycin ([1]Day 13 after Dynabeads® activation) for 4 to 5 hours using the Cell Stimulation Cocktail Kit (eBioscience, catalog number 00-4971) containing the protein transport inhibitors brevidin and monensin, Th17-polarized and expanded T cells were characterized by their intracellular IL-17 expression. Stimulation was performed according to the protocol provided by eBioscience. IL-17 expression was assessed by intracellular IL-17 staining using the flow cytometry method described above.

[0272] Example 6. Activation and amplification of Tregs

[0273] As shown in Figure 1, CD4 + CD25 + CD127 低 / - Treg cells (approximately 90% FOXP3+ cells) sorted by flow cytometry were processed using Dynabeads. ® The Treg prototype was effectively activated and expanded several hundredfold in the amplified culture after 14 days (top) while still maintaining high FOXP3 expression (bottom). Cells were restimulated on day 9 using the same prototype beads. Dynabeads conjugated with lower amounts of CD3 were used. ® Amplification is achieved. As shown in Figure 1, Dynabeads is used to increase the number of amplifications. ® A CD3 / CD28 stimulation protocol (low CD3 – approximately 0.34) elicited the highest Treg amplification. Dynabeads ® CD3 / CD28 (high CD3 – approximately 3.4) does not produce any effective Treg amplification. Dynabeads ® CD3 / CD28 low (CD3 – approximately 0.34) and Dynabeads ® A moderate CD3 / CD28 ratio (CD3–approximately 0.75) is comparable to or better than alternative Treg stimulators. Dynabeads ® CD3 / CD28CTS TM (CD3–approximately 1.5) induces suboptimal amplification.

[0274] Example 7. Activation and expansion of antigen-stimulated (CMV-specific memory) T cells

[0275] like Figure 2 As observed, the fold increase in CMV-specific memory T cells on day 10 post-activation was related to the increased amount of Dynabeads conjugated with agonistic CD3 antibodies. ®The increased signal strength provided by the prototype was negatively correlated. Using cytomegalovirus (CMV)-specific T cells as a model, different Dynabeads were used. ® Prototype (which is conjugated with CD3, CD28 and / or CD137 antibodies) and Dynabeads ® CD3 / CD28CTS TM The growth kinetics and phenotypes of T cells expanded from peptide-stimulated PBMCs (derived from CMV+ donors) were compared. The superiority of Dynabeads conjugated with low CD3 levels (providing weaker CD3 activation signals) is demonstrated in this paper. ® CD3 / CD28CTS TM It is suboptimal and does not amplify CMV-specific T cells. Dynabeads ® CD3 / CD137 / CD28 showed the highest expansion of memory T cells.

[0276] Example 8. Activation, polarization, and expansion of Th17 cells

[0277] T cells were cultured under Th17-polarized conditions and used with Dynabeads conjugated with antibodies against CD3 / ICOS (conjugated with various amounts of anti-CD3 antibodies; 1.5 and 0.06, and two different ICOS clones) or CD3 / CD28 (CTS beads). ® Amplification. Starting on day 3, IL-2 was added to the culture. On day 13, the culture was stimulated with PMA-inomycin for 4 to 5 hours before assessing IL-17 expression. Histograms show intracellular IL-17 expression; Dynabeads ® CD3 / ICOS (ISA-3), CD3 High and Low (1.5 and 0.06), Dynabeads ® CD3 / ICOS (different ICOSC398.4A, purchased from eBiocienses, Ambow, Inc.), low CD3 (0.06) and Dynabeads ® CD3 / CD28CTS TM The intensity of activation signals and the nature of co-stimulatory responses significantly influence the polarization and proliferation of Th17 cells. Note: Use Dynabeads. ® Activation of CD3 / ICOS (medium-0.3) produces a phenotype similar to "(high-1.5)", not shown.

[0278] Only when using Dynabeads ®CD3 / ICOS (ISA-3 clone) (designed to provide weak CD3 signaling (CD3-0.06)) is required to activate T cells for successful Th17 cell polarization and expansion. In this study, only the ISA-3ICOS clone was effective.

[0279] Dynabeads ® CD3 / CD28CTS TM (CD3-1.5) induces low Th17 polarization / amplification, or does not induce Th17 polarization / amplification.

[0280] Example 9: The effect of the bead-cell ratio on the selective expansion of IL-17-producing cells.

[0281] T cells were collected from three donors (A, B, and C). PBMCs were collected from healthy donors via leukocyte removal and further purified using a Ficol-methyl diatrizoate density gradient (AstarteBiologics, NormalPBMCs). CD3+ T cells were enriched using the Dynabeads® Untouched™ HumanTCells Kit (Thermo Fisher Scientific catalog number #11344D) according to the provided instructions.

[0282] Negatively isolated CD3+ T cells were stimulated with Dynabeads® CD3 / ICOS (low to medium-high) at given bead:cell ratios (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 UL ml, from day 3)) was isolated. Anti-IFNγ antibody AHC4032 10 µg / ml and anti-human IL4, from E. Bioscience (catalog number BMS129), were also isolated as needed. On day 10, total expansion (T cell expansion) was measured, cells were restimulated, and the IL-17-producing cell fraction was assessed by intracellular flow cytometry staining for IL-17 (% of IL-17-producing cells). Figures 4A to 4C The data shown illustrates the effect of the bead-to-cell ratio on the selective expansion of Th17 cells. The relative number of IL-17-producing cells is recorded as the fraction of IL-17-producing cells multiplied by the total T cell expansion fold.

[0283]

[0284] Example 10: A protocol for generating Th17 cells.

[0285] introduction

[0286] Following adoptive transfer to mice with tumors, T cells secreting IL-17A (Th17 and Tc17 cells) caused greater tumor regression than non-polarized T cells secreting Th1 or Tc1 cells or IFN-γ (Muranski et al.). Blood , 112 :362-73, (2008), Nelson et al. Journal of Immunology (J Immunol) , 194 :1737-47 (2015), Guedan et al., Blood 124 :1070-80 (2014)). The factors regulating the production of human Th17 cells remain a subject of 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, Preface to Biological Sciences (Front) (Biosci.) (Elite editor) , 8 :427-35 (2016)). The aim is to confirm robust culture conditions for the expansion of Th17 cells via adoptive cell transfer. Dynabeads® CD3 / CD28CTS TM Activation produces T cell products characterized by the Th1 polarization phenotype, while weak CD3- signaling provided to T cells under certain polarization conditions can induce the production of Th17 (Purvis et al.). Blood , 11 :4829-37 (2010)). It has been found that optimized CD3 signaling intensity can be achieved by reducing the amount of agonistic CD3 antibodies conjugated to mitochondria along with a low mitochondrial-to-cell ratio of 1:5. It has been found that weak CD3 signaling along with ICOS co-stimulation produces a higher fraction of IL-17A-producing T cells compared to mitochondria providing CD28 co-stimulation.

[0287] Alternative pathways and protocols that have been reported to generate Th17 cells have been demonstrated: i) co-stimulation with CD5 or CD6 in the presence of IL-1β, IL-6, IL-23, and TGF-β (deWit et al., Blood , 118 :6107-14(2011)),ii) to perform SLAMF3 and SLAMF6 and Ag-mediated CD3 / TCR stimulation (Chatterjee et al., Immunology Journal (J Immunol) , 188 :1206-12 (2012)), iii) the effect of agonists on aryl hydrocarbon receptors (ARH) (Veldhoen et al., Journal of Experimental Medicine (J Exp Med) , 206 :43-9 (2009), and iv) cholesterol precursors, which act as potent retinoic acid receptor-associated orphan receptor (ROR) agonists (Hu et al., National Chemical Biology (Nat Chem) Biol) , 11 :141-147 (2015). However, the commonly used medium RPMI only supports low levels of Th17 polarization. Other media rich in aromatic amino acids (precursors to aryl hydrocarbon receptor (AHR) agonists) have consistently induced higher Th17 amplification and demonstrated that AHR activation is necessary for Th17 polarization. RORγt is the most important transcription factor for Th17 cells. When injected into mice, RORγt agonists produce elevated levels of Th17 cytokines, increase the level of co-stimulatory receptors such as CD137, and decrease the expression of the co-inhibitory receptor PD-1 (Hu et al., "RORg agonist regulate immune checkpoint receptors to enhance anti-tumor immunity," AACR abstract). #565 (New Orleans, 2016). Furthermore, T cells treated in vitro with RORγt agonists maintained low PD-1 expression after adoptive transfer.

[0288] Significant Th17 polarization of T cells was observed after low CD3 intensity activation and CD5 co-stimulation under polarized conditions (including the use of AHR agonists).

[0289] Materials and methods

[0290] Initial T cells and culture conditions: Peripheral blood mononuclear cells (PBMCs) were isolated from the erythrocyte sedimentation rate (ESR) amber layer or single blood component from healthy donors (Astarte Biologics, Washington, USA). Negatively isolated T cells (Dynabeads® Untouched Human T Cells, catalog number 11344D, Thermo Fisher Scientific, Waltham, MA) were activated using Dynabeads® with the various ligand compositions and stoichiometry described above. Polarization conditions are described in the text. Activated T cells were cultured at 37°C and 5% CO2 in X-Vivo15™ (catalog number BE02-060F, Lonza Biologics, MD, USA) with 2 to 5% CTS™ immune cell SR (catalog number A25961-02, Thermo Fisher Scientific, Waltham, MA) and 0.25 µg / ml gentamicin (catalog number 15750060, Thermo Fisher Scientific), and maintained at the specified 0.5 to 2 × 10⁻⁶ cells per 1 to 3 days by adding cytokines and freshly prepared medium. 6 Amplification was performed using a cell concentration of 1 cell / ml.

[0291] General solutions: such as those proposed by Paulos et al. Science Translational Medicine , 2 As described in :55ra78, (2010), Th17 / Tc17 cells were expanded in the presence of anti-IL-4 and anti-IFN-γ neutralizing antibodies (catalog numbers AC0642 and ACH4032, Thermo Fisher Scientific, Waltham, MA) in a 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)). The addition of polarizing cytokines and antibodies was only for initial activation (days 0 to 3). Cells were separated and maintained in a medium containing 100 IU IL-2 / ml and IL-23 starting from day 3 of activation.

[0292] Irritating Dynabeads: The relative amounts of irritating anti-CD3 antibodies and their co-conjugated costimulatory ligands to Dynospheres are summarized in Table 5.

[0293] Table 5. Dynabeads Th17 amplifier prototype and control

[0294]

[0295] Th17 cell polarization and expansion. T cells isolated from healthy donor PBMCs via negative isolation (Dynabeads® Untouched Human T Cells, catalog 11344D, Thermo Fisher Scientific, Waltham, MA) were activated using the Dynabeads®-Th17 prototype (microglobulin-to-cell ratio 1:5) 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 kept undisturbed for 2 to 3 days, followed by expansion for 10 to 14 days by the addition of freshly prepared culture medium and IL-2 and IL-23 cytokines. T cell expansion was assessed by cell counting (Coulter Counter, Beckman Coulter, California, USA). On days 10 to 14 after expansion, following fixation and permeabilization, cells were stimulated for 5 hours with PMA / inomycin in a medium containing monensin (BDGolgiStop™, catalog number 554724, BDBiosciences) and brefedipine A (BDGolgiPlug™, catalog number 555029, BDBiosciences) before analysis of phenotypic markers and intracellular expression of IL-17A and IFNγ by flow cytometry.

[0296] Table 6. Polarizing agents and their functions

[0297] Polarizing agent activity on T cells

[0298]

[0299] Flow cytometry, phenotype, and function: Intracellular cytokine expression in PMA / inomycin-activated Th17 cells (CellStimulationCocktail Kit containing the protein transport inhibitors brefidobacterium and monensin, catalog 00-4971, eBioscience) was detected using IL-17A-PE (catalog 00-4971, clone: ​​4H1524, MolecularProbes) and IL-17-F, IFN-γ, and an appropriate isotype control (IgG2bPE, catalog 00313 BioLegend). Flow cytometry data were collected on BDLSRII (BDBiosciences) and analyzed using FACSDiva software (BDBiosciences).

[0300] Results and discussion

[0301] i) The role of neutralizing antibodies

[0302] αIL-4 / α-IFNγ antibody was used in the Th17 cell polarization protocol to block Th1 / Th2 polarization.

[0303] Blocking antibodies are introduced after CD3 / CD28 activation to improve Th17 cell production. Clinical-grade blocking antibodies are expensive and add complexity to Th17 cell production protocols.

[0304] Protocol variation: T cells were activated using optimized conditions with Dynabeads® CD3 / ICOS or Dynabeads® CD3 / CD28. Th17 polarization was promoted via cytokines TGF-β, IL-23, IL-6, and IL-1β. The effect of αIL-4 / α-IFN-γ neutralizing antibody on the fraction of IL-17-producing CD4+ cells after amplification was investigated. Figure 5 A comparison was made.

[0305] Conclusion: Compared to CD3 / CD28, stimulation with CD3 / ICOS resulted in a higher fraction of IL-17-producing T cells (42.5% of CD4 cells versus 16.5% of CD4+ cells). Stimulation of CD4+ T cells with Dynabeads® CD3 / ICOS (+Th17 polarizing cytokines) reduced the dependence of Th17 cell production on neutralizing IFN-γ and IL-4 antibodies. Stimulation with Dynabeads® sCD3 / CD28 requires the addition of Th1 / Th2 blocking antibodies.

[0306] ii) Effects of CD5 co-stimulation

[0307] It has been reported that by increasing IL-23R expression, CD5 promotes Th17 production, resulting in sustained STAT3 activation and enhanced levels of ROR-γt relative to CD28 co-stimulation (deWit et al.). Blood , 118 :6107-14 (2011)).

[0308] Scheme Transformation: T cells were activated using Dynabeads® CD3 / CD5, Dynabeads® CD3 / ICOS, or Dynabeads® CD3 / CD28 at equal signal intensities and chemotactic values. Th17 polarization was promoted by adding cytokines (TGF-β, IL-23, IL-6, IL-1β) and a neutralizing antibody against αIL-4 / α-IFN-γ. The fractions of CD4+ T cells producing IL-17 and IFN-γ (intracellular staining) were compared with the fractions of cells expressing CCR4+CCR6+ (a Th17-associated phenotype). Figure 6 and Figure 7 ).

[0309] Conclusion: As determined by IL-17 production and CCR4 / CCR6 co-expression, stimulation by CD3 / CD5 produces a Th17 profile similar to or slightly higher than that of CD3 / ICOS, and is superior to CD3 / CD28 stimulation.

[0310] iii) Effects of AHR agonists

[0311] It has been shown that the AHR ligand FICZ (5,11-dihydroindolo(3,2-b)carbazole-6-carbaldehyde) upregulates the Th17 program (Veldhoen et al., *Journal of Experimental Medicine*). J.Exp.Med. ）》 , 206 :43-9 (2009).

[0312] Scheme Transformation: T cells were activated using Dynabeads® CD3 / CD5, Dynabeads® CD3 / ICOS, or Dynabeads® CD3 / CD28 at equal signal intensities and chemotactic values. Th17 polarization was promoted by adding i) cytokines (TGF-β, IL-23, IL-6, IL-1β) and αIL-4 / α-IFN-γ neutralizing antibodies or ii) the AHR agonist FICZ plus IL-6 and IL-1βa. Th17 polarization was assessed by intracellular staining for IL-17 and IFN-γ after activation of polarized T cells using PMA / inomycin. Figure 8 ).

[0313] Conclusion: FICZ exhibits good activity on T cells co-stimulated via CD5 rather than ICOS. Compared to standard polarization conditions, the FICZ, IL-1β, and IL-6 regimen produces fewer IL-17-producing cells and requires optimization.

[0314] Example 11: Expanding Treg cells (>80% FOXP3) using Dynabeads™ Treg beads

[0315]

[0316] Sorted Treg cells (>80% FOXP3) were diluted to 2 million cells / ml in X-Vivo™ 15 + 5% CTS (X-Vivo™ 15 / CTS). Dynabeads™ were washed once by magnetic separation and the beads were diluted to 4 million cells / ml in X-Vivo™ 15 / CTS. Control beads were washed once and 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 the wells of a 48-well plate until the total volume was 400 µl. Each sample was prepared in triplicate.

[0317]

[0318] Day 0: As above, prepare the Treg amplification mixture in a 48-well plate using X-Vivo™15 / CTS with 300 U / ml rIL2.

[0319] Day 2: Add 200 µLX-Vivo™15 / CTS and 300 U / ml rIL2 to each well and mix the resulting cell suspension.

[0320] Days 3-12: Estimate cell density daily using a microscope. When it reaches approximately 1.5 to 2 million cells / cm³... 2 When the cell density is reached, transfer the cells to larger wells or flasks. Add freshly prepared X-Vivo™ 15 / CTS at 300 U / ml rIL2 as needed.

[0321] Day 5: Remove 250 µL of X-Vivo™15 / CTS from each sample and add 250 µL of freshly prepared X-Vivo™15 / CTS with 300 U / ml rIL2. Then mix the cell suspensions.

[0322] Day 7: Determine the well volume and cell count. When approximately 1.5 to 2 million cells / cm³ is reached... 2 When the cell density is reached, the cells are transferred to larger wells / flasks and freshly prepared X-Vivo™ 15 / CTS with 300 U / ml rIL2 is added.

[0323] Day 9: Add freshly prepared X-Vivo™15 / CTS with 300 U / ml rIL2 and mix the cell suspension.

[0324] Day 12: Determine the volume in each well and count the cells. Perform FoxP3 staining using the reagents listed in Table 9 and 100,000 to 200,000 cells.

[0325]

[0326] Prepare a fixation / permeabilization solution by mixing fixation / permeabilization (1 part) with fixation / permeabilization diluent (3 parts). Prepare a fixation / permeabilization buffer / wash solution by mixing permeabilization concentrate (1 part) with water (9 parts). Prepare fresh samples daily. Typically, staining is performed on 100,000 to 200,000 cells.

[0327] Cells were washed once with 1 ml DPBS / 0.1% HAS and centrifuged at 350 x g for 8 minutes to remove the supernatant.

[0328] Vortex the cells and then mix them by vortexing. Add 1 ml of fixation / permeabilization (1x) solution and vortex the cells again. Incubate the cell suspension at 4°C for 60 minutes. Add 2 ml of permeabilization buffer / wash buffer (1x) and centrifuge the suspension at 400xg for 8 minutes at 4°C to remove the supernatant. Repeat the process of adding permeabilization buffer / wash buffer and centrifuging once.

[0329] Then, 50 µL of the staining mixture (2.5 µL CD4PerCP, 2.5 µL CD25APC, 5 µL FOXP3AF488, 2 µL CD127PE + 38 µL permeation buffer / wash buffer (1x)) was added to the sample, followed by incubation at 4 °C for 30 min. The sample was then analyzed by flow cytometry.

[0330] Other embodiments

[0331] Although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate the scope of the invention and not to limit it, the scope of which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0332] The patents and scientific literature mentioned herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference.

[0333] Although the invention has been specifically shown and described with reference to particular embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as defined in the appended claims.

[0334] The following clauses represent exemplary subject matter of the present invention:

[0335] Clause 1. A method for selectively expanding members of a T cell subset, the method comprising exposing a mixed T cell population to:

[0336] (a) The first reagent, which provides a primary activation signal to members of the T cell subset, thereby activating the T cells, and

[0337] (b) The second and third reagents, each of which stimulates two or more different helper molecules on T cell subset members, thereby stimulating the proliferation of the T cells activated in (a), wherein the ratio of the first, second and third reagents is regulated to selectively induce the expansion of T cell subset members relative to other T cell subset members.

[0338] Clause 2. The method according to Clause 1, wherein the first reagent is an anti-CD3 antibody.

[0339] Clause 3. The method according to Clause 1 or 2, wherein the second reagent is an antibody.

[0340] Clause 4. The method according to any one of the preceding clauses, wherein the third reagent is an antibody or a non-antibody protein.

[0341] Clause 5. The method according to any one of the preceding clauses, wherein the non-antibody protein is a chemokine or cytokine.

[0342] Clause 6. The method according to Clause 4, wherein the chemokine or cytokine is one or more proteins selected from the group consisting of:

[0343] (a) Interleukin-1α,

[0344] (b) Interleukin-2,

[0345] (c) Interleukin-4,

[0346] (d) Interleukin-1β,

[0347] (e) Interleukin-6,

[0348] (f) Interleukin-12,

[0349] (g) Interleukin-15,

[0350] (h) Interleukin-18,

[0351] (i) Interleukin-21,

[0352] (j) Interleukin-7,

[0353] (k) Interleukin-23 and

[0354] (l) Transforming growth factor β1.

[0355] Clause 7. The method according to any one of the preceding clauses, wherein the ratio of the first reagent, the second reagent, and the third reagent is adjusted such that the concentration of the first reagent is lower than that of the second or third reagent.

[0356] Clause 8. The method according to Clause 7, wherein the lower concentration of the first reagent is about 0.34 units.

[0357] Clause 9. The method according to Clause 8, wherein the first reagent is an anti-CD3 antibody at a concentration of about 0.34 units and the second reagent is an anti-CD28 antibody at a concentration of 3.4 units.

[0358] Clause 10. The method according to any one of the preceding clauses, wherein the selectively expanded T cell subset is a Treg cell.

[0359] Clause 11. The method according to any one of the preceding clauses, wherein the lower concentration of the first reagent is about 0.01 units.

[0360] Clause 12. The method according to any one of the preceding clauses, wherein the first reagent is an anti-CD3 antibody at a concentration of about 0.01 units, the second reagent is an anti-CD28 antibody, and the third reagent is an anti-CD137 antibody.

[0361] Clause 13. The method according to any one of the preceding clauses, wherein the selectively expanded T cell subset is memory T cells.

[0362] Clause 14. The method according to any one of the preceding clauses, wherein the lower concentration of the first reagent is about 0.06 units.

[0363] Clause 15. The method according to any one of the preceding clauses, wherein the first reagent is an anti-CD3 antibody at a concentration of about 0.34 units, and the second reagent is an anti-ICOS or anti-CD5 antibody.

[0364] Clause 16. The method according to any one of the preceding clauses, wherein the selectively amplified T cell subset is Th17 cells.

[0365] Clause 17. A method for selectively expanding T cell subsets, said method comprising...

[0366] (a) Exposing T cells in vitro to CD3, CD28, anti-CD5, anti-ICOS, and / or CD137 signaling, and

[0367] (b) The T cells are cultured in a manner that allows for the expansion of Th17 cells, antigen-stimulated T cells, and / or regulatory T cells.

[0368] Clause 18. The method according to Clause 17, wherein CD3, CD28, and CD137 signaling are mediated by anti-CD3, anti-CD28, and / or anti-CD137 antibodies.

[0369] Clause 19. The method according to Clause 17 or 18, wherein the anti-CD3, anti-CD28, and anti-CD137 antibodies are used for the expansion of memory T cells in a range of concentrations from 0.01 units to 1.5 units.

[0370] Clause 20. The method according to Clause 17 or 18, wherein the anti-CD3, anti-CD28, anti-CD5, anti-ICOS, and anti-CD137 antibodies are used for the amplification of Th17 cells in a range of concentrations from 0.06 units to 1.5 units.

[0371] Clause 21. The method according to Clause 17 or 18, wherein the anti-CD3, anti-CD28, and anti-CD137 antibodies are used for the expansion of Treg cells in a range of concentrations from about 0.34 units to 3.41 units.

[0372] Clause 22. The method according to any one of Clauses 17 to 21, wherein the anti-CD3 antibody is used at a lower concentration compared to the concentrations of the anti-CD28 and anti-CD137 antibodies.

[0373] Clause 23. The method according to any one of Clauses 17 to 22, wherein T cells are isolated using CD3 selection.

[0374] Clause 24. The method according to any one of Clauses 17 to 23, wherein the Th17 cells are CD3+, CD8 / CD4+, and produce the IL-17 cytokine.

[0375] Clause 25. The method according to any one of Clauses 17 to 24, wherein the Th17 cells are capable of producing IL-17, IL-21 and / or IL-22.

[0376] Clause 26. The method according to any one of Clauses 17 to 25, wherein the memory T cell is selected from the group consisting of stem cell-like memory T cells, central memory T cells, and effector memory T cells.

[0377] Clause 27. The method according to Clause 26, wherein the 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.

[0378] Clause 28. The method according to Clause 26, wherein the central memory cells have one or more of the following markers: CD3+, CCR7+, CD45RA-, CD45RO+, CD62L+ (L-selectin), CD27+, and CD28+.

[0379] Clause 29. The method according to Clause 28, wherein the central memory cells are capable of producing IL-2.

[0380] Clause 30. The method according to Clause 26, wherein the effector memory cells have one or more of the following markers: CD28+ / -, CD27+ / -, CD3+, CD4+, CD8+, CCR7-, CD45RA-, CD45RO+.

[0381] Clause 31. The method of Clause 26 or 30, wherein the effector memory cell is a cell capable of producing IFNγ and IL-4.

[0382] Clause 32. The method according to any one of Clauses 17 to 24, wherein the Treg cells have one or more of the following markers: CD4+, CD25+, FOXP3+, and CD127. 阴性 / 低 .

[0383] Clause 33. A method for selectively expanding T regulatory cells, the method comprising:

[0384] (a) Exposing T cells to CD3 and CD28 signaling in vitro, and

[0385] (b) The T cells are cultured in a manner that allows for the expansion of T regulatory cells.

[0386] Clause 34. The method according to Clause 33, wherein CD3 and CD28 signaling is mediated by anti-CD3 and anti-CD28 antibodies.

[0387] Clause 35. The method according to Clause 34, wherein the anti-CD3 and anti-CD28 antibodies are used in a concentration range including 0.34 to 3.4 units.

[0388] Clause 36. The method described in accordance with Clause 34 or 35, wherein the anti-CD3 antibody is used at a lower concentration compared to the concentration of the anti-CD28 and / or antibody.

[0389] Clause 37. A method for selectively expanding Th17 cells, the method comprising:

[0390] (a) Exposing CD3+ T cells in vitro to CD3, CD28, anti-CD5, and / or ICOS signaling, and

[0391] (b) The CD3+ T cells are cultured in a manner that allows for the expansion of Th17 cells.

[0392] The amounts of CD3, CD28, CD5 and / or ICOS differ.

[0393] Clause 38. The method according to Clause 37, wherein CD3, CD28, CD5, and ICOS signaling are mediated by anti-CD3, anti-CD28, anti-CD5, and anti-ICOS antibodies.

[0394] Clause 39. The method according to Clause 37 or 38, wherein the anti-CD3, anti-CD28, anti-CD5, and anti-ICOS antibodies are used for the amplification of Th17 cells in a concentration range including 0.06 to 1.5 units.

[0395] Clause 40. The method according to any one of Clauses 37, 28 or 39, wherein the anti-CD3 antibody is used at a lower concentration compared to the concentrations of the anti-CD28, anti-CD5 and / or anti-ICOS antibodies.

[0396] Clause 41. A method for selectively expanding antigen-stimulated T cells, the method comprising:

[0397] (a) Exposing T cells to CD3, CD28, CD27 and / or CD137 signaling in vitro, and

[0398] (b) The T cells are cultured in a manner that allows for the expansion of antigen-stimulated T cells.

[0399] Clause 42. The method according to Clause 41, wherein CD3, CD28 and CD27 and / or anti-CD137 signaling is provided by anti-CD3, anti-CD28, anti-CD27 and / or anti-CD137 antibodies.

[0400] Clause 43. The method according to Clause 41 or 42, wherein the anti-CD3, anti-CD28, anti-CD27 and anti-CD137 antibodies are used in a concentration range including 0.01-1.5 units.

[0401] Clause 44. The method according to any one of Clauses 41, 42 or 43, wherein the anti-CD3 antibody is used at a lower concentration compared to the concentration of the anti-CD28 and / or anti-CD137 antibody.

[0402] Clause 45. A composition comprising CD3+ (1) T cells and (2) beads, said beads containing (a) an anti-CD3 antibody and (b) an anti-CD28, anti-CD137 or ICOS antibody capable of selectively amplifying T cell subsets, wherein the amount of (a) anti-CD3 antibody present differs from that of (b) anti-CD28, anti-CD137 or ICOS antibody.

[0403] Clause 46. The composition according to Clause 45, wherein the T cell subset is selected from the group consisting of: Th17 cells, antigen-stimulated T cells, and / or regulatory T cells.

[0404] Clause 47. The composition according to Clause 45 or 46, wherein the anti-CD3, anti-CD28, anti-CD27, and anti-CD137 antibodies are used against the expansion of memory T cells in a concentration range including 0.01 to 1.5 units.

[0405] Clause 48. The composition according to any one of Clauses 45, 46 or 47, wherein the anti-CD3, anti-CD28 and anti-CD137 antibodies are used for the amplification of Th17 cells in a concentration range including 0.06 to 1.5 units.

[0406] Clause 49. The composition according to any one of Clauses 45 to 48, wherein the anti-CD3 and anti-CD28 antibodies are used against the expansion of Treg cells in a concentration range including 0.34 to 3.41 units.

[0407] Clause 50. The composition according to any one of Clauses 45 to 49, wherein the anti-CD3 antibody is used at a lower concentration compared to the concentrations of the anti-CD28 and anti-CD137 antibodies.

[0408] Clause 51. A composition comprising (1) T cells and (2) beads, said beads containing anti-CD3, anti-CD5, anti-CD28, anti-ICOS and anti-CD137 antibodies capable of selectively amplifying Th17 cells, said Th17 cells being capable of producing one or more effector cytokines, and wherein the amount of anti-CD3 present on the beads differs from that of the anti-CD5 / ICOS / CD28 / CD137 antibodies.

[0409] Clause 52. The composition according to Clause 51, wherein the one or more effector cytokines are selected from the group consisting of IL-17, IL-21, and IL-22.

[0410] Clause 53. A composition comprising (1) T cells and (2) beads, said beads containing anti-CD3, anti-CD28 and anti-CD137 antibodies capable of selectively amplifying antigen-stimulated memory T cells, wherein the T cells are capable of recognizing specific antigens, and wherein the amounts of anti-CD3 and anti-CD28 antibodies present on the beads are different.

[0411] Clause 54. The composition according to Clause 53, wherein the specific antigen is selected from the group consisting of: viral antigens, bacteria, fungi, protozoa, and cancer antigens.

[0412] Clause 55. The composition according to Clause 54, wherein the viral antigen is selected from the group consisting of: CMV, EBV, influenza, and HIV.

[0413] Clause 56. The composition according to Clause 53, wherein the antigen is selected from the group consisting of: Streptococcal M-protein, Neisseria fimbriae, Borrelia burgdorferi lipoprotein VisE, Burkholderia melioides polysaccharide antigen, Aspergillus fumigatus galactomannan, and Francisella tularensis lipopolysaccharide.

[0414] Clause 57. A composition comprising (1) T cells and (2) beads, said beads containing anti-CD3 and anti-CD28 antibodies capable of selectively amplifying regulatory T cells, wherein the amounts of anti-CD3 and anti-CD28 antibodies present on the beads are different.

[0415] Clause 58. The composition according to Clause 57, wherein the regulatory T cells are CD4+CD25+FOXP3+CD127 low / negative.

[0416] Clause 59. The composition according to Clause 57 or 58, wherein the activity of the regulatory T cells includes inhibitory activity.

[0417] Clause 60. A method of treating an individual in need, the method comprising administering to the individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-stimulated T cells, and / or regulatory T cells.

[0418] Clause 61. The method described in Clause 60, wherein the individual in need is affected by cancer, inflammatory disease, autoimmune disease, allergic disease, or infectious disease.

[0419] Clause 62. The method described in Clause 61, wherein the cancer is lung cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, and skin cancer.

[0420] Clause 63. According to the method of Clause 61, the inflammatory disease is selected from the group consisting of: diabetes mellitus; rheumatoid arthritis; inflammatory bowel disease; familial Mediterranean fever; neonatal onset of 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.

[0421] Clause 64. A method for rebuilding the immune system of an individual in need, the method comprising administering to the individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-stimulated T cells, and / or regulatory T cells.

[0422] Clause 65. A method of providing adoptive immunotherapy to an individual in need, the method comprising administering to the individual a pharmaceutically acceptable composition comprising Th17 cells, antigen-stimulated T cells, and / or regulatory T cells.

[0423] Clause 66. The method according to any one of Clauses 60 to 65, wherein the T cell is genetically modified.

[0424] Clause 67. The method according to Clause 66, wherein the genetic modification is a chimeric antigen receptor.

[0425] Clause 68. The method according to Clause 66, wherein the genetic modification is a genetically modified T-cell receptor.

[0426] Clause 69. A method for selectively altering the ratio of two T cell subtypes in a sample, the method comprising contacting a sample containing a mixed T cell population with at least two stimulants, wherein the stimulants provide different amounts of signaling to the T cells in the mixed population, wherein one T cell subtype selectively amplifies compared to a second T cell subtype.

[0427] Clause 70. The method according to Clause 69, wherein the sample comprises erythrocyte sedimentation rate (ESR) brown-yellow layer cells obtained from an individual.

[0428] Clause 71. The method according to Clause 69 or 70, wherein at least two stimulating signals stimulate CD3 and CD28 receptors.

[0429] Clause 72. The method described in Clauses 69, 70 or 71, wherein at least one T cell subtype is selectively removed from the mixed population.

[0430] Clause 73. The method according to any one of Clauses 69 to 72, wherein the proportion of Treg T cells relative to all T cells in the mixed population is increased.

[0431] Clause 74. The method according to any one of Clauses 69 to 73, wherein the total number of memory T cells in the sample is reduced.

[0432] Clause 75. The method of any one of Clauses 69 to 74, wherein the amount of the CD3 stimulus signal is less than half that of the CD28 stimulus signal.

[0433] Clause 76. A method for expanding Th17 cells, the method comprising:

[0434] (a) Exposing T cell populations to CD3 and CD5 signaling in vitro, and

[0435] (b) Culture T cell populations under conditions that allow for the expansion of Th17 cells.

[0436] The T cell populations mentioned above are either exposed to aryl hydrocarbon receptor agonists or not exposed to exogenous interleukin-23.

[0437] Clause 77. The method according to Clause 76, wherein the T cell population is a mixed population of different T cell types.

[0438] Clause 78. The method described in accordance with Clause 76 or 77 further comprises contacting a population of T cells with one or more polarizing agents.

[0439] Clause 79. The method according to Clause 76, 77 or 78 further comprises contact, wherein one or more polarizing agents are one or more factors selected from the group consisting of: interleukin-1β, interleukin-23, tumor growth factor-β, interleukin-6, interleukin-21, interleukin-2, anti-interleukin-4 antibody and anti-interferon-γ antibody.

[0440] Clause 80. The method according to any one of Clauses 76 to 79, wherein the T cell population is further exposed to an aryl hydrocarbon receptor agonist.

[0441] Clause 81. The method according to Clause 80, wherein the aryl hydrocarbon acceptor agonist is 6-formylindolo[3,2-b]carbazole (FICZ).

[0442] Clause 82. The method according to any one of Clauses 69 to 81, wherein the T cell population is further exposed to interleukin-1β and interleukin-6.

[0443] Clause 83. The method according to any one of Clauses 69 to 82, wherein the Th17 cells are engineered to express one or more chimeric antigen receptors.

[0444] Clause 84. The method according to Clause 83, wherein at least one of the one or more chimeric antigen receptors is specific for cell surface antigens of mammalian cells.

[0445] Clause 85. The method according to any one of Clauses 69 to 84, wherein the cell surface antigen of the mammalian cell is an antigen associated with tumor cells.

[0446] Clause 86. A composition comprising CD3 signaling, CD5 signaling, an aryl hydrocarbon receptor agonist, and one or more cytokines.

[0447] Clause 87. The composition according to Clause 86, wherein the one or more cytokines comprise interleukin-1β and interleukin-6.

[0448] Clause 88. The composition according to Clause 86 or 87 further comprises a population of T cells.

[0449] Clause 89. The composition according to Clause 86, 87 or 89, wherein the CD3 signal is an anti-CD3 antibody.

[0450] Clause 90. The composition according to any one of Clauses 86 to 89, wherein the CD5 signal is an anti-CD5 antibody.

[0451] Clause 91. A composition comprising a population of T cells, CD3 signaling, CD5 signaling, an aryl hydrocarbon receptor agonist, and one or more cytokines.

[0452] Clause 92. The composition according to Clause 91, wherein said T cell population is present in a mixture comprising:

[0453] (a) Sample with "brownish-yellow erythrocyte sedimentation rate layer",

[0454] (b) Leukocyte samples containing more than 80% mixed T cells,

[0455] (c) Samples containing more than 80% CD4+ T cells, or

[0456] (d) Samples containing more than 80% Th17 cells.

[0457] Clause 93. A method for isolating and activating T cells from a mixed cell population, the method comprising:

[0458] (a) Under conditions that allow T cells to bind to a solid carrier and activate the same T cells, a mixed cell population is brought into contact with a solid carrier having at least a first ligand thereto bind, the first ligand having binding affinity to a protein located on a T cell present in the mixed cell population, and

[0459] (b) T cells bound to a solid carrier are separated from T cells never bound to a solid carrier to obtain a purified T cell population.

[0460] Clause 94. The method according to Clause 93, wherein the solid carrier has at least a first ligand and a second ligand bound thereto, wherein each of the first ligand and the second ligand has binding affinity for different proteins located on individual T cells present in a mixed cell population.

[0461] Clause 95. The method according to Clause 94, wherein the first ligand is an anti-CD3 antibody or an anti-CD4 antibody, and wherein the second ligand is a ligand selected from the group consisting of:

[0462] (a) Anti-CD5 antibody,

[0463] (b) Anti-CD28 antibody,

[0464] (c) Anti-CD137 antibody and

[0465] (d) Anti-ICOS antibody.

[0466] Clause 96. The method according to Clauses 93, 94 or 95 further comprises releasing the purified T cell population obtained in step (b) from the solid carrier.

[0467] Clause 97. The method described in Clause 96 further comprises amplifying the released T cells.

[0468] Clause 98. The method according to Clause 97, wherein the expansion of the released T cells occurs in a culture medium.

[0469] Clause 99. The method according to Clause 98, wherein one or more chemokines or cytokines are present in the culture medium.

[0470] Clause 100. The method according to any one of Clauses 93 to 99, wherein one or more of the chemokines or cytokines are present in step (a).

[0471] Clause 101. The method according to Clause 100, wherein the one or more chemokines or cytokines are selected from the group consisting of:

[0472] (a) Interleukin-1α,

[0473] (b) Interleukin-2,

[0474] (c) Interleukin-4,

[0475] (d) Interleukin-1β,

[0476] (e) Interleukin-6,

[0477] (f) Interleukin-12,

[0478] (g) Interleukin-15,

[0479] (h) Interleukin-18,

[0480] (i) Interleukin-21 and

[0481] (j) Transforming growth factor β1.

[0482] Clause 102. A method for activating and expanding T cells, said method comprising contacting a mixed population of T cells with:

[0483] (a) The first reagent provides primary activation signals to T cell subset members by stimulating molecules on T cell subset members, and

[0484] (b) A second reagent that stimulates molecules on a subset of T cells that are different from those stimulated by the first reagent.

[0485] As a result, the T cells in the population are activated and proliferated.

[0486] The first and second reagents are combined with one or more solid carriers.

[0487] T cells are maintained under conditions that allow for proliferation, and

[0488] The solid carrier was removed from contact with T cells after a period of less than 120 hours.

[0489] Clause 103. The method according to Clause 102, wherein the first reagent is an anti-CD3 antibody.

[0490] Clause 104. The method described in accordance with Clause 102 or 103, wherein the second reagent is an antibody.

[0491] Clause 105. The method described in accordance with Clauses 102, 103 or 104, wherein the T cells are contacted with a third reagent, which is an antibody or a non-antibody protein.

[0492] Clause 106. The non-antibody protein described in accordance with Clause 105 is a chemokine or cytokine.

Claims

1. A method for selectively expanding T cell subsets, the method comprising: (a) Exposing T cells to CD3, CD28, and / or CD137 signaling in vitro, and (b) The T cells are cultured in a manner that allows for the expansion of Th17 cells, memory T cells, antigen-stimulated T cells and / or regulatory T cells.

2. The method according to claim 1, wherein the CD3, CD28, and CD137 signals are mediated by anti-CD3, anti-CD28, and / or anti-CD137 antibodies.

3. The method of claim 1, wherein the anti-CD3, the anti-CD28, and the anti-CD137 antibodies are used in a concentration range of 0.01 units to 1.5 units to induce the expansion of memory T cells.

4. The method of claim 1, wherein the anti-CD3, the anti-CD28, and the anti-CD137 antibodies are used in a concentration range of 0.06 units to 1.5 units to induce Th17 cell expansion.

5. The method of claim 1, wherein the anti-CD3, the anti-CD28, and the anti-CD137 antibodies are used in a concentration range of about 0.34 units to 3.41 units to induce Treg cell expansion.

6. The method of claim 1, wherein the anti-CD3 antibody is used at a lower concentration compared to the concentrations of the anti-CD28 and anti-CD137 antibodies.

7. The method of claim 1, wherein the T cells are isolated using CD3 selection.

8. The method according to claim 1, wherein the Th17 cells are CD3 cells. + CD8 / CD4 + / It also produces the IL-17 cytokine.

9. The method of claim 1, wherein the Th17 cells are capable of producing IL-17, IL-21 and / or IL-22.

10. The method of claim 1, wherein the memory T cell is selected from the group consisting of stem cell-like memory T cells, central memory T cells, and effector memory T cells.

11. The method of claim 10, wherein the memory stem 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.

12. The method of claim 10, wherein the central memory cells have one or more of the following markers: CD3+, CCR7+, CD45RA-, CD45RO+, CD62L+ (L-selectin), CD27+, and CD28+.

13. The method of claim 12, wherein the central memory cells are capable of producing IL-2.

14. The method of claim 10, wherein the effector memory cells have one or more of the following markers: CD28+ / -, CD27+ / -, CD3+, CD4+, CD8+, CCR7-, CD45RA-, CD45RO+.

15. The method of claim 10, wherein the effector memory cell is a cell capable of producing IFNγ and IL-4.

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