KR20050023347A - Compositions and Methods for Restoring Immune Repertoire in Patients with Immunological Defects Related to Autoimmunity and Organ or Hematopoietic Stem Cell Transplantation - Google Patents

Abstract

The present invention generally relates to methods for stimulating T cells, and more particularly to normal immune of T cells by removing unwanted (eg autoreactive, allergic, pathogenic) subpopulations of T cells from a mixed population of T cells. It is about how to restore the repertoire. The invention also relates to compositions of cells, including stimulated T cells with a repaired immune repertoire, and uses thereof.

Description

Compositions and Methods for Restoring Immune Repertoire in Patients with Immunological Defects Related to Autoimmunity and Organ or Hematopoietic Stem Cells Transplantation}

The present invention generally relates to methods for stimulating T cells, and more particularly to normal immune of T cells by removing unwanted (eg autoreactive, allergic, pathogenic) subpopulations of T cells from a mixed population of T cells. It is about how to restore the repertoire. The invention also relates to compositions of cells, including stimulated T cells with a repaired immune repertoire, and uses thereof.

Disclosure of Related Prior Art

The ability of T cells to recognize universal antigens associated with various cancers or infectious organisms is provided by T cell antigen receptors consisting of α (alpha) chains and β (beta) chains or γ (gamma) and δ (delta) chains. . The proteins that make up these chains are encoded by DNA using a unique mechanism that produces a wide variety of TCRs. These multisubunit immune recognition receptors are associated with the CD3 complex and bind to peptides presented by major histocompatibility complex (MHC) class I and II proteins on the antigen-presenting cell (APC) surface. Binding of antigenic peptides on TCR and APC is a central response of T cell activation that occurs at the immunological synapse of the point of contact between T cells and APC.

To maintain T cell activation, T lymphocytes typically require a second costimulatory signal. Costimulation is typically required for T helper cells that produce sufficient levels of cytokines to induce clonal expansion [Bretscher, Immunol. Today 13:74, 1992; June et al., Inamuzol. Today 15: 321, 1994]. Primary costimulatory signals occur when members of the B7 family ligand (CD80 (B7.1) or CD86 (B7.2)) on activated antigen-presenting cells (APC) bind to CD28 on T cells.

Methods of stimulating expansion of any subset of T cells have the potential to produce a variety of T cell compositions useful for immunotherapy. Effective stimulation can aid successful immunotherapy by increasing the reactivity and amount of T cells. In addition, in the context of autoimmunity or transplantation, successful immunotherapy can be assisted by removing unwanted autoreactive or alloreactive cells.

The various techniques available for expanding human T cells rely primarily on the use of accessory cells and / or exogenous growth factors such as interleukin-2 (IL-2). IL-2 is used in combination with anti-CD3 antibodies to stimulate T cell proliferation and mainly expand the CD8 ⁺ subpopulation of T cells. All APC signals directed against TCR / CD3 complexes and CD28 on T cell surfaces are believed to be required for optimal T cell activation, expansion, and long-term survival of T cells upon re-infusion. Because APCs survive relatively short, the requirement for MHC-matched APCs as accessory cells represents a significant problem for long term culture systems. Therefore, in long term culture systems, APC must be continuously obtained and supplemented from the source. The need for a renewable supply of accessory cells is a problem in the treatment of immunodeficiency disease in which accessory cells become diseased. In addition, when treating viral infections, if accessory cells carry the virus, the cells may contaminate the entire T cell population during long term culture.

In the absence of exogenous growth factors or accessory cells, costimulatory signals can be delivered to the T cell population, for example, by exposing T cells to CD3 ligands and CD28 ligands attached to solid surface surfaces such as beads. C. June, et al. (US Pat. No. 5,858,358); C. June et al. See WO 99/953823. Although therapeutically useful T cell populations can be obtained by these methods, the difficulty and delay in T cell preparation are not ideal.

Conventional methods available in the art use anti-CD3 and anti-CD28 for expansion of T cells. In addition, current methods available in the art did not focus on expanding T cells in a short time or on obtaining a stronger T cell population and its beneficial results. None of these methods described a method or similar method for removing an undesirable clonal or oligoclonal T cell population from the T cell population, nor did they describe the beneficial results of such a method. In addition, existing available methods tend to distort the clonality of the T cell population rather than removing undesirable reactive clones from the T cell population and restoring normal immune repertoire. For maximum in vivo effects, theoretically, ex vivo- or in vivo-generated activated T cell populations must be in a state capable of maximizing the immune response to cancer, infectious disease or other disease symptoms. In the context of autoimmunity or transplantation, the activated T cell population must be in a state of reconstructing normal T cell repertoires with little or no autoreactive or potentially pathogenic alloreactive T cells. Currently, patients with autoimmune disease are treated with long-term immunosuppression, which inhibits autoreactive T cells causing the disease. When the immunosuppressant is discontinued, the disease often recurs with the reappearance of disease-causing T cells that reappear in the patient. The main problem in hematopoietic stem cell transplantation is graft-versus-host disease (GVHD) caused by alloreactive T cells present in the injected hematopoietic stem cell sample. In organ transplantation, transplant rejection mediated by alloreactive host T cells is a major problem, which is usually overcome by prolonged immunosuppression of transplant recipients.

The present invention provides a method for producing a large number of more highly active and purer T cells that have surface receptor and cytokine production properties and appear healthier and more natural than other expansion methods, and are characterized by the undesirable autoreactivity of T cells or Additional methods are provided for reducing or eliminating alloreactive populations. The present invention provides a method of using said T cell population in situations of autoimmune disease, hematopoietic stem cells, and organ transplantation, and in other situations requiring reconstruction of the T cell immune system that has been eliminated, discarded, or dysfunctional. . In addition, the present invention provides compositions of cell populations of any target cells, including T cell populations, and parameters for their preparation, and other related advantages.

In addition, it is well recognized that the aging immune system is characterized by a contradictory increase in autoimmunity combined with a gradual decrease in responsiveness to exogenous antigens and tumors [C. Weyand et al. Mechanisms of Ageing and Development 102: 131-147, 1998; D. Schmidt et al. Molecular Medicine 2: 608-618, 1996; G. Liuzzo et al. Circulation 100: 2135-2139, 1999]. This study described that aging is associated with the development of a T helper cell subset characterized by reduced CD28 expression. CD4 ⁺ CD28 ^- T cells survive long, typically perform clonal expansion in vivo, and react with self-antigens in vitro. Reduced CD28 expression correlates with a lack of CD40 ligand expression that renders the CD4 ⁺ T cells unable to perform B cell differentiation and immunoglobulin secretion. CD4 ⁺ CD28 ^- T cell accumulation associated with aging results in immune compartments that are distorted in the opposite direction to the development of a high-affinity B cell response to the self-reactive direction and to exogenous antigens.

<Overview of invention>

One aspect of the invention provides a cell population comprising at least a portion of T cells and exposing the cell population to one or more apoptotic pro-apoptotic compositions in vitro to induce apoptosis in at least a portion of the T cells. Thereby providing a method of removing at least a substantial portion of a clonal T cell population from a mixed population of T cells derived from an individual, thereby removing at least a substantial portion of the clonal T cells from a mixed population of T cells derived from the individual. .

The present invention discloses a cell population comprising at least a portion of T cells to one or more apoptosis-inducing or growth inhibitory compositions to induce apoptosis or to present one or more clonal T cell populations present in a mixed population of T cells from an individual. At least a substantial portion of the clonal T cell subpopulation from the mixed population of T cells from the individual, comprising removing at least a substantial portion of the clonal T cell population from the mixed population of T cells by inhibiting at least a substantial portion of growth Provides a way to remove it. In one embodiment, the methods of the invention further comprise expanding the mixed population of T cells by exposing the remaining mixed population of T cells to the apoptosis-inducing composition to induce proliferation of the T cell mixed population. In one specific embodiment, said apoptosis-inducing composition comprises anti-CD3 and anti-CD28 antibodies co-fixed onto beads. In some embodiments, the apoptosis-inducing composition used to remove at least a substantial portion of the clonal T cell population from the mixed population of T cells is the same composition as the composition used to expand the remaining mixed population of T cells.

In one embodiment, the methods of the present invention further comprise expanding the remaining population of cells. In another embodiment, the methods of the present invention further comprise expanding the remaining population of cells by exposing the remaining population of cells to a surface, wherein said surface has at least a portion of the cell surface residues of said remaining T cells. One or more substances that ligation are attached to stimulate the remaining T cells. In a related embodiment, a first substance ligating with said first T cell surface residue of a T cell is attached to said surface, and a second substance ligating with said second residue of said T cell is attached to said same or second surface. Is attached, wherein said ligation by a first substance and a second substance induces proliferation of said T cells.

In one embodiment, the substance attached to the surface is an antibody or antibody fragment. In other embodiments, the first substance is an antibody or fragment thereof, and the second substance is an antibody or fragment thereof. In one embodiment, the first substance and the second substance are different antibodies. In one particular embodiment, the first agent is an antibody fragment of an anti-CD3 antibody, an anti-CD2 antibody, or an anti-CD3 or anti-CD2 antibody. In other embodiments, the second agent is an anti-CD28 antibody or antibody fragment thereof. In further embodiments, the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody.

In another embodiment, the cells are exposed to the surface of the invention for a time sufficient to increase polyclonality. In certain embodiments, such polyclonal increases occur in the monoclonality of the T cell population as determined by the Vβ, Vα, Vγ, or Vδ spectratype profiles of one or more Vβ, Vα, Vγ, or Vδ family genes. Migration to clonal or polyclonal.

Exemplary apoptosis-inducing compositions of the invention include anti-CD3 antibodies, anti-CD2 antibodies, anti-CD28 antibodies, anti-CD20 antibodies, target antigens, MHC-peptide tetramers, Fas ligands, anti-Fas antibodies, IL-2, IL-4, TRAIL, rolipram, doxorubicin, chlorambucil, fludarabine, cyclophosphamide, azathioprine, methotrexate, cyclosporin, mycophenolate, FK506, bcl-2 inhibitor, topo isomerase inhibitors, interleukin -1β-converting enzyme (ICE) - binding agents, Shigella (Shigella) IpaB protein, star right cephalosporin, ultraviolet irradiation, gamma irradiation, tumor necrosis factor, a target antigen nucleic acid molecules, proteins or peptides, and non- Protein or non-polynucleotide compound, but is not limited thereto. In some embodiments, one or more of these compositions are used simultaneously.

In certain embodiments of the invention, the apoptosis-inducing composition comprises an autoantigen. Exemplary autoantigens of the invention include myelin based protein (MBP), MBP 84-102, MBP 143-168, pancreatic islet cell antigen, collagen, CLIP-170, thyroid antigen, nucleic acid, acetylcholine receptor, S antigen, and Type II collagen is mentioned, but it is not limited to this.

The invention further provides T cell populations produced according to any of the methods described above.

The present invention exposes, at least in part, a cell population comprising T cells to one or more growth inhibitory compositions, thereby inhibiting the growth of at least a substantial portion of the one or more clonal T cell populations present in the mixed population of T cells from the individual. Providing a method of removing at least a substantial portion of a clonal T cell subpopulation from a mixed population of T cells from an individual, including; The method further includes expanding the mixed population of T cells by exposing the non-growth inhibited population of cells, ie, the remaining mixed population of T cells, to a surface to which one or more substances that bind cell surface molecules are attached. In one embodiment, said surface comprises anti-CD3 and anti-CD28 antibodies co-fixed onto beads.

One aspect of the invention provides a method of treating an autoimmune disease in a patient, comprising administering to the patient a T cell population of the invention. In one embodiment, the patient is treated with a chemotherapeutic agent prior to administering the T cell population. Exemplary chemotherapeutic agents of the invention include, but are not limited to, camppath, anti-CD3 antibodies, cytotoxin, fludarabine, cyclosporin, FK506, mycophenolic acid, steroids, FR901228, and irradiation. It is not limited to this. In some embodiments, the patient is treated with T cell ablation therapy prior to administering the T cell population of the invention.

One aspect of the invention provides a cell population comprising at least a portion of T cells, exposing the cell population to one or more substances that sensitize at least some of the T cells with further activation and stimulation, and For a time sufficient to induce apoptosis of the sensitized T cells on a surface to which one or more substances attached to the population are ligated with cell surface residues of at least a portion of the sensitized T cells, thereby stimulating the sensitized T cells A method of removing at least a substantial portion of a clonal T cell population from an individual derived T cell population, comprising exposing to remove said sensitized T cells from said population. In one embodiment, the method further comprises stimulating at least a portion of the remaining T cells to expose the cell population to the surface for a time sufficient for at least some of the remaining cells to proliferate. In a further embodiment, the method comprises attaching a first substance ligating a first T cell surface residue of a T cell to the surface and lysing a second residue of the T cell on the same or second surface thereof. A second substance to gating is attached, and the ligation by the first substance and the second substance induces proliferation of the T cells. In one embodiment, the one or more agents are antibodies or antibody fragments. In other embodiments, the first substance is an antibody or fragment thereof, and the second substance is an antibody or fragment thereof. In another embodiment, the first substance and the second substance are different antibodies. In related embodiments, the first agent is an antibody fragment of an anti-CD3 antibody, an anti-CD2 antibody, or an anti-CD3 or anti-CD2 antibody. In another embodiment, the second agent is an anti-CD28 antibody or antibody fragment thereof. In other embodiments, the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody.

In related embodiments, cells are exposed to said surface for a time sufficient to increase polyclonality. In other embodiments, the polyclonal increase is determined by the Vβ, Vα, Vγ, or Vδ spectratype profile of one or more Vβ, Vα, Vγ, or Vδ family genes, as determined by the monoclonal to oligoclonal or poly Migration to clonality.

In some embodiments, the patient requires hematopoietic stem cell transplantation. In related embodiments, the composition for sensitizing treated PBMCs and cell populations that is unable to sustain division includes donor T cells. The present invention also provides a T cell population generated according to the above method. The present invention also provides a method for reducing the risk or severity of GVHD adverse effects in a patient transplanting hematopoietic stem cells according to the methods described herein.

In certain embodiments, patients receiving cells of the invention require organ transplantation. In related embodiments, the sensitizing composition comprises irradiated donor cells and the cell population comprises recipient T cells. The present invention also provides a cell population produced according to the above method. In one embodiment, the cells are administered to a patient who has undergone an organ transplant to reduce the risk of organ rejection. In related embodiments, organ transplant patients are treated with T cell ablation therapy prior to administering the T cell population.

In one aspect of the invention, the sensitizing composition comprises an autoantigen. Exemplary autoantigens of the present invention include, but are not limited to, myelin based protein (MBP), MBP 84-102, MBP 143-168, pancreatic islet cell antigens, S antigens and type II collagen. In one embodiment of the invention, the T cell populations produced according to the invention are administered to treat patients suffering from autoimmune diseases. In related embodiments, the patient is treated with a T cell clearance therapy prior to administering the T cell population.

The invention also provides a cell population comprising B cells in at least a portion and exposing the cell population to one or more apoptosis-inducing compositions, thereby inducing apoptosis in at least a portion of the B cells, thereby removing the B cell portion from the population. Provided is a method for removing a clonal B cell population from a population of B cells from an individual comprising removing. In one embodiment, the method further comprises stimulating the remaining B cells by exposing the remaining population of cells to a surface to which at least one substance to which at least a portion of the cell surface residues of the remaining B cells is attached. . In certain embodiments, the apoptosis-inducing composition comprises an autoantigen.

The present invention also provides a composition of B-cells produced according to the above method.

In one embodiment of the present invention, a composition comprising a population of B-cells generated using the method of the present invention is used to treat a patient suffering from an autoimmune disease. In related embodiments, the patient is treated with a B cell clearance therapy prior to administering the B cell population.

One aspect of the invention provides a cell population comprising at least a portion of T cells, and exposing the surface T cell population to a composition that selectively selects and / or stimulates CD3 ⁺ and CD28 ⁺ molecules ex vivo. A method is provided for generating a substantially pure T cell population from an individual derived T cell population, comprising generating a substantially pure population of CD3 ⁺ / CD28 ⁺ T cells. In related embodiments, the resulting pure T cell population is a substantially pure CD4 ⁺ / CD3 ⁺ / CD28 ⁺ T cell population. In related embodiments, the pure T cell population is a substantially pure CD8 ⁺ / CD3 ⁺ / CD28 ⁺ T cell population.

In one aspect of the invention, the purity of CD3 ⁺ / CD28 ⁺ T cells is at least 90% pure. In further embodiments, the purity of CD3 ⁺ / CD28 ⁺ T cells is 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%. In other embodiments, the purity of CD3 ⁺ / CD28 ⁺ T cells is at least 99% pure. In related embodiments, the purity of CD3 ⁺ / CD28 ⁺ T cells is at least 99.9% pure. Thus, one aspect of the invention relates to a CD3 ⁺ / CD28 ⁺ T cell population comprising less than 10% CD28 ⁻ cells. In certain embodiments, the CD3 ⁺ / CD28 ⁺ T cell population comprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% contaminating CD28 ^-Comprises T cells.

In one embodiment, anti-CD3 antibodies are used to stimulate CD3 ⁺ surface molecules and anti-CD28 antibodies are used to stimulate CD28 ⁺ surface molecules.

Accordingly, the present invention also provides a method of generating substantially pure CD3 ⁺ CD28 ⁺ T cell populations, including CD4 ⁺ CD3 ⁺ CD28 ⁺ T cells and CD8 ⁺ CD3 ⁺ CD28 ⁺ T cells. The T cell population is then used for autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitus, Addison's disease, chronic digestive disorders, chronic fatigue syndrome, inflammatory bowel disease, ulcerative colitis, Crohn ( Crohn's disease, myofascial pain, systemic lupus erythematosus, psoriasis, Sjogren's syndrome, hyperthyroidism / Grave's disease, hypothyroidism / Hashimoto's disease, insulin-dependent diabetes (type 1), Myasthenia gravis, endometriosis, scleroderma, pernicious anemia, Goodpasture syndrome, Wegener's disease, glomerulonephritis, aplastic anemia, paroxysmal nocturnal hematuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, To treat people suffering from autoimmune hemolytic anemia, Evan's syndrome, factor VIII inhibitor syndrome, systemic vasculitis, dermatitis, multiple myositis and rheumatic fever It can be used.

The present invention provides a cell population comprising at least a portion of T cells, and at least one substance attached to the cell population to align the cell surface residues of at least a portion of the T cells, thereby stimulating the T cells. Contacting the cell surface, wherein the surface is present at a ratio of the surface to the cell such that at least a substantial portion of the one or more populations of antigen-specific T cells are removed after about 8 days of culture. Further provided are methods for activating and expanding the T cell population by ligation. In one embodiment of the invention, the ratio is from about 50: 1 to about 5: 1. In some embodiments, the ratio is about 100: 1 to about 2: 1. In one embodiment, the ratio is at least about 45: 1. In some embodiments, the ratio is at least about 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11: 1, 10 : 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, or 2: 1. In one particular embodiment, the ratio is about 5: 1.

The present invention exposes a cell population comprising at least a portion of T cells to one or more apoptosis-inducing compositions, thereby inducing apoptosis in at least a substantial portion of the one or more clonal T cell populations present in the mixed population of T cells from the individual. A method of removing at least a substantial portion of a clonal T cell subpopulation from a mixed population of T cells from an individual, comprising removing at least a substantial portion of the clonal T cell population from the mixed population of T cells.

1 is a dot graph showing the presence of CD3 ⁺ CD8 ⁺ HLA-A2CMVpp65 antigen specific T cells in HLA-A2-positive donors.

2 is a dot graph showing the increase in CD25 expression in CMV-activated HLA-A2CMVpp65 antigen-specific T cells.

FIG. 3 is a dot graph showing deletion of prestimulated tetramer-positive cells (ie, CMVpp65-Ag-specific) by upregulation of CD25 on restimulated cells and second strong stimulation provided by 3 × 28 beads to be. After the primary stimulation on day 14, the cultures were left unstimulated (Panels A1-A4) or restimulated using an XCELLERATE® process using 3X28 beads (Panel B1-B4). ). CD25 was upregulated on restimulated cells (Panel B2), but tetramer-positive (ie CMVpp65-Ag-specific) prestimulated cells were deleted by a second strong stimulus provided by 3 × 28 beads ( Panel B3).

4 is a histogram showing increased expression of major effector molecules, including CD95, on leukemia B-cells co-cultured with XCELLERATED T cells (registered trademark).

FIG. 5 is a dot graph showing induction of apoptosis in leukemia B-cells co-cultured with accelerated T cells (registered trademark).

FIG. 6 is a graph showing the disappearance of leukemia B-cells and accompanying expansion of T cells during the Accelrate® process.

FIG. 7 is a graph comparing fold increase in polyclonal T cells and fold increase in CMV pp65 A2-tetramer ⁺ (antigen-specific) T cells using various bead: cell ratios. Filled bars represent polyclonal T cells. Striped bars represent CMV-specific T cells.

Prior to describing the present invention, definitions of various terms to be used below may be described to help understanding the present invention.

The term “biocompatible” as used herein refers to a property that is predominantly nontoxic to viable cells.

The term “stimulus” as used herein refers to a primary response induced by ligation of cell surface residues. For example, for receptors, this stimulation involves ligation of the receptor and subsequent signal transduction responses. In the case of stimulation of T cells, such stimulation refers to the ligation of T cell surface residues which in one embodiment subsequently induce a signal transduction response, eg, binding to the TCR / CD3 complex. In addition, the stimulatory response activates cells and upregulates or downregulates the expression of cell surface molecules, such as receptors or adhesion molecules, or upregulates or downregulates the secretion of molecules, eg, tumor growth factor beta ( TGF-β) can be downregulated. Thus, ligation of cell surface residues can lead to reorganization of the cytoskeletal structure or adhesion of cell surface residues in the absence of a direct signal transduction response, each of which may augment, alter or alter subsequent cellular responses. It can work.

The term "activation" as used herein refers to a change in state of a cell that undergoes sufficient cell surface residue ligation to induce measurable morphological, phenotypic and / or functional changes. In the case of T cells, this activation may be a state of T cells stimulated sufficiently to induce cell proliferation. In addition, activation of T cells may be responsible for cytokine production and / or secretion, and upregulation or downregulation of expression of cell surface molecules such as receptors or adhesion molecules, or upregulation or downregulation of the secretion of several molecules, and It can lead to the performance of regulatory or cytolytic effector functions. For other cells, the term assumes upregulation or downregulation of certain physico-chemical processes.

The term "target cell" as used herein refers to any cell intended to be stimulated by cell surface residue ligation.

"Antibody" as used herein refers to both polyclonal and monoclonal antibodies (mAb); Primateization (eg, humanization); Rats; Mouse-human; Mouse-primates; And chimeras; Circular molecules, fragments thereof (eg, scFv, Fv, Fd, Fab, Fab 'and F (ab)' ₂ fragments), or multimers or aggregates of circular molecules and / or fragments; Can be produced in nature or can be prepared, for example, by immunization, synthesis or genetic manipulation; An “antibody fragment” is derived from or is derived from an antibody that can be derivatized to bind to an antigen as used herein and in some embodiments exhibit structural features that facilitate removal and uptake, for example by incorporation of galactose residues. Refers to the fragment associated with. Such fragments include, for example, F (ab), F (ab) ' ₂ , scFv, light chain variable region (V _L ), heavy chain variable region (V _H ), and combinations thereof.

The term "protein" as used herein includes proteins, glycoproteins, and other cell-derived modified proteins, polypeptides, and peptides, and may be multimers or aggregates of circular molecules, fragments, circular molecules, and / or fragments thereof. It can be produced in nature or produced synthetically (including chemical and / or enzymatic synthesis) or by genetic manipulation.

The term "material", "ligand" or "material that binds to cell surface residues" refers to a molecule that binds to a cell population as defined herein. This material can bind any cell surface residue, such as a receptor, antigenic determinant, or other binding site present on a target cell population. The substance may be a protein, peptide, antibody and antibody fragment thereof, fusion protein, synthetic molecule or organic molecule (eg small molecule) and the like. For T cell stimulation within the scope of the specification, antibodies are used as typical examples of such materials.

The term “cell surface residues” as used herein may refer to cell surface receptors, antigenic determinants, or any other binding site present on a target cell population.

The terms "materials that bind cell surface residues" and "cell surface residues", as used herein, should generally be considered as complementary / anti-complementary sets of molecules demonstrating relatively high affinity specific binding. .

"Co-stimulatory signal" means a signal combined with a TCR / CD3 ligation that results in a primary signal, eg, T cell proliferation and / or activation, as used herein.

"Separation" as used herein includes any means for substantially purifying one component from another component (eg, by filtration, affinity, buoyancy density or magnetic attraction).

"Surface" as used herein, means any surface to which a substance may be attached, and includes, but is not limited to, metals, glass, plastics, copolymers, colloids, lipids and cell surfaces, and the like. In essence, any surface may retain a substance bonded or attached thereto.

“Monoclonal” as used herein is a T cell with single specificity as defined by spectratype analysis (measurement of TCR Vβ, Vα, Vγ or Vδ chain hypervariable region repertoire) in the case of T cell populations. It means a group. The T cell population is monoclonal (or single specific) when the Vβ, Vα, Vγ and / or Vδ spectratype profiles for a given Vβ, Vα, Vγ and / or Vδ family have a single predominant peak. It is considered to be. Spectratype analysis distinguishes rearranged variable genes of a particular size, not sequence. Thus, a single peak has a limited number comprising four potential nucleotides (adenine (a), guanine (g), cytosine (c), or thymine (t)) or a combination of the four nucleotides in the linking region It is understood that T cell populations expressing any of the rearranged TCR variable genes (Vβ, Vα, Vγ, or Vδ) can be represented. In certain embodiments of the invention, it may be desirable to clone and sequence certain bands to determine the sequence (s) of rearranged variable gene (s) present in the bands exhibiting a particular length.

"Oligoclonal" as used herein means a T cell population with multiple but narrow antigen specificities in the case of a T cell population. This term can be defined by spectratype analysis (measurement of TCR Vβ, Vα, Vγ or Vδ) chain hypervariable region repertoire). If the Vβ spectratype profile for a given TCR Vβ, Vα, Vγ or Vδ family has about 2 to about 4 dominant peaks, the T cell population is considered to be oligoclonal. The term may also be defined by the generation and characterization of antigen-specific clones for the antigen of interest.

"Polyclonal" as used herein means a T cell population with multiple and broad antigen specificity in the case of a T cell population. This may be by spectratype analysis (measurement of TCR Vβ, Vα, Vγ or Vδ chain hypervariable region repertoire). If the Vβ spectratype profile for a given TCR Vβ, Vα, Vγ or Vδ family has multiple peaks, typically five or more dominant peaks, in most cases a Gaussian distribution, the T cell population is considered to be polyclonal. . Polyclonality can also be defined by the generation and characterization of antigen-specific clones for the antigen of interest.

"Repair or increase of polyclonality" refers to expressed TCR Vβ, Vα, Vγ in a T cell population as measured by spectratype analysis or similar analysis, eg, flow cytometry or sequencing. Migration from monoclonal profile to oligoclonal profile or polyclonal profile or from oligoclonal profile to polyclonal profile in the Vδ gene. The shift from monoclonal Vβ, Vα, Vγ and / or Vδ expression profiles in the T cell population to oligoclonal profile or polyclonal profile is generally in one or more TCR Vβ, Vα, Vγ and / or Vδ families. appear. In one embodiment of the invention, this shift is observed in two, three, four or five Vβ families. In certain embodiments of the invention, migration is observed in 6, 7, 8, 9 or 10 Vβ families. In a further embodiment of the invention, the migration is observed in 11, 12, 13 or 14 Vβ families. In other embodiments of the invention, migration is observed in 15-20 Vβ families. In a further embodiment of the invention, migration is observed in 20 to 24 Vβ families. In other embodiments, the migration is in all Vβ families. The functional significance of restoring or increasing the polyclonality of the T cell population is the recovery or increased immune potential or ability to respond to the full range of antigens of the T cell population. In some aspects of the invention, some T cells in the population (eg, T cells downregulated in TCR expression) may not have a TCR associated with the methods described herein. However, in close proximity to the T cells activated by the methods described herein and the factors secreted by them, the T cells may, in other words, further increase the polyclonality of the T cell population by upregulating TCR expression. Repair or increase in polyclonality can also be determined by determining the extent of response to a particular subject antigen, eg, by determining the number of different epitopes recognized by antigen-specific cells. This can be done using standard techniques for the generation and cloning of antigen-specific T cells in vitro.

The term "clonal T cell population" as used herein refers to a T cell population having a given range of specificity for a given target antigen. This can be measured by any number of assays known in the art, eg, by generating and determining the range of specificity (ie, number of different specificities) of antigen-specific clones in a given population. In addition, the clonal T cell population has a clonal T cell population by having monoclonal or oligoclonal specificity as defined by spectratype analysis (measurement of the TCR Vβ, Vα, Vγ or Vδ chain hypervariable region repertoire). You can also define

The term "animal" or "mammal" as used herein includes all mammals, including humans. Preferably, the animal of the present invention is a human subject.

The term "exposure" as used herein means to reach a state or condition of very close or direct contact.

The term "proliferation" as used herein means to grow or multiply by creating new cells.

"Immune response or reactivity" as used herein refers to the activation of immune system cells, including but not limited to T cells, to induce effector function (s) of a particular cell. Effector functions include, but are not limited to, the ability to induce proliferation, cytokine secretion, antibody secretion, regulation and / or expression of adhesion molecules, and cytolysis.

“Stimulation of an immune response” as used herein means any stimulation such that activation and induction of effector function of immune system cells are achieved.

"Immune response dysfunction" as used herein refers to inappropriate activation and / or proliferation or lack of immune system cells, and / or inappropriate secretion of cytokines or lack of cytokines, and / or immune system cells, as used herein. Improper or inappropriate induction of other effector functions of, eg, regulation, attachment, and / or expression of homing receptors, and induction of cytolysis.

"Particles" or "surfaces" may include colloidal particles, microspheres, nanoparticles, beads, or the like as used herein. The surface can be any surface to which ligands can bind or integrate, including cell surfaces (eg, K562 cells) and are substantially nontoxic to biocompatible, ie, target cells to be stimulated. In various embodiments, commercially available surfaces such as beads or other particles are useful (eg, Miltenyi particles (Miltenyi Biotec, Germany); Sepharose beads (Pharmacia Fine Chemicals, Sweden); Dyna DYNABEADS (R) (Dynal Inc., NewYork); PURABEADS (R) (Prometic Biosciences), magnetic beads (Immunicon, Huntingdon, Valley, PA), Bangs Laboratories, Inc., Fishers, IN)).

As used herein, “paramagnetic particle” means a particle that localizes in response to a magnetic field as defined above.

"Apoptosis-inducing composition", "apoptosis composition" or "apoptosis inducer" means any composition or stimulus that increases the apoptosis activity of a cell when administered alone or in combination with other apoptosis-inducing compositions. . Apoptosis-inducing compositions used in the methods of the invention preferably induce apoptosis in activated T cells, NKT, NK or B-cells. In certain embodiments, the apoptosis-inducing compositions of the invention will induce apoptosis without further activation / stimulation. Illustrative examples of such compositions or stimuli include growth factor depletion, oxidative conditions, heat stress, serum depletion, phorbol myristate acetate (PMA) and ionomycin, superantigens (eg, SEA and SEB, etc.), various antibodies, eg For example anti-CD2, anti-CD3, anti-CD28, anti-CD20, anti-Fas antibody, or any combination thereof, MHC-peptide tetramer or dimer, Fas ligand, IL-2, IL-4, TRAIL, rolipram, doxorubicin, chlorambucil, fludarabine, corticosteroid, glucocorticoid, cyclosporine, cyclophosphamide, FK506, azathioprine, methotrexate, mycophenolate, annexin, caspase, bcl- 2 inhibitors, topoisomerase inhibitors, interleukin-1β converting enzyme (ICE) -binding agents, Shigella IpaB protein, staurosporin, ultraviolet radiation, gamma irradiation, radiation, tumor necrosis factor, various histone deacetylase inhibitors, and Know well in the art Jeans can be other things, but not limited thereto. In certain embodiments, the apoptosis-inducing composition comprises a surface, such as magnetic beads, to which one or more substances that bind cell surface residues are attached. In this regard, the material may be any of the materials described herein. In other embodiments, at least an anti-CD3 antibody is attached to the surface. In other embodiments, the surface has anti-CD3 and anti-CD28 antibodies attached. In addition, an apoptosis stimulator may be a polypeptide capable of increasing or inducing apoptosis activity of a cell. Such polypeptides include polypeptides that directly regulate the apoptosis pathway, such as Bax, Bad, Bcl-xS, Bak, Bik, and active caspases, and polypeptides that indirectly regulate the pathway. In certain embodiments, the apoptosis-inducing composition is in particular such as an accelerated T cell (registered trademark) (e.g., a cell described in US Patent Application No. 10 / 133,236) for inducing apoptosis in a population of B-cells. Activated T cells. Other exemplary apoptosis-inducing compositions include irradiated cells (eg, donor or recipient (homologous) cells), target antigens (eg, defined autoimmune target antigens such as myelin based protein (MBP), MBP in multiple sclerosis). 84-102 or target antigen identified as MBP 143-168; pancreatic islet cell antigen; S antigen in uveitis; or type II or other types of collagen in rheumatoid arthritis; thyroid receptor in Grave disease; acetylcholine receptor in myasthenia gravis) , Cytoplasmic linker protein-170 (CLIP-170), nucleic acid molecules, proteins or peptides, and non-protein or non-polynucleotide compounds.

A "composition that sensitizes cells with further activation or stimulation" or "sensitizing composition" is any composition that sensitizes cells for later activation / stimulation, as used herein. Upon subsequent activation / stimulation, the sensitized cells undergo apoptosis. In addition, the sensitizing compositions of the present invention sensitize cells by the effect of apoptosis-inducing compositions. Exemplary compositions that sensitize cells by the effects of further activation, stimulation, or apoptosis-inducing compositions include, for example, radiation (eg, donor or recipient (allogeneic) cells), superantigens (eg, SEA and SEB, etc.). A target antigen (eg, a defined autoimmune target antigen, eg, a target antigen identified as Myelin based protein (MBP), MBP 84-102 or MBP 143-168 in multiple sclerosis; pancreatic islet cell antigen; S antigen in uveitis) Or type II or other types of collagen in rheumatoid arthritis; thyroid receptors in Grave's disease; acetylcholine receptors, nucleic acid molecules, proteins or peptides in myasthenia gravis, and non-protein or non-polynucleotide compounds), proteins, glycoproteins Derived from peptides, antibody / antigen complexes, cell lysates, non-soluble cell debris, apoptotic bodies, necrotic cells, non-sustainable cell lines Cells treated such that they cannot sustain division by whole cells, natural or synthetic complex carbohydrates, lipoproteins, transformed cells or cell lines, transfected cells or cell lines, or transduced cells or cell lines, or any combination thereof Can be mentioned.

For the purposes of the present invention, apoptosis is defined as programmed cell death. Apoptosis is programmed cell death, a variety of phenomena that play a critical role in countless physiological and pathological processes. Apoptosis is associated with embryonic development, metamorphosis, endocrine-dependent tissue atrophy, normal tissue turnover, and immune thymic cell death (either induced through its antigen-receptor complex or by glucocorticoids) (Itoh et al. , Cell 66: 233, 1991). During maturation of T cells in the thymus, T cells that recognize self-antigens are destroyed through an apoptosis process, while others are positively selected. It has been suggested that some T cells that recognize certain autotrophic epitopes (eg, ineffectively processed and presented antigenic determinants of a given autologous protein) may escape this elimination process and later work in autoimmune diseases [Gammon et al., Immunology Today 12: 193, 1991]. Necrosis is accidental cell death, a cell's response to various harmful symptoms and toxic substances. Apoptosis that is morphologically different from necrosis is a spontaneous form of cell death that occurs in many different tissues under various conditions. Apoptosis occurs in two stages. Cells undergo nuclear and cytoplasmic condensation and can eventually be broken down into a number of membrane-bound fragments containing structurally circular apoptosis bodies that are predated and rapidly degraded by neighboring cells. Alternatively, cells entering the apoptosis pathway can be phagocytized and then degraded into membrane bound sieves. Apoptosis is observed in many different healthy and neoplastic tissues of adults and embryos. Death occurs spontaneously or is induced by physiological or toxic substances. Apoptosis is a basic physiological process that plays a major role in the regulation of cell populations.

Methods of measuring apoptosis are well known in the art. Apoptosis can be measured, for example, by DNA ladders, electron or light microscopy, methods such as flow cytometry, and various commercially available kits for measuring apoptosis.

As used herein, a "growth inhibitory composition" is any that inhibits growth in a cell or renders the cell dysfunctional and incapable of division when administered alone or when administered in combination with other compositions of the invention. It's a substance. The growth inhibitory composition used in the method of the invention preferably inhibits the growth of activated T cells, NKT, NK or B-cells. Illustrative examples of such compositions or stimuli include growth factor depletion, oxidative conditions, heat stress, serum depletion, phorbol myristate acetate (PMA) and ionomycin, superantigens (eg SEA and SEB, etc.), various antibodies, eg For example anti-CD2, anti-CD3, anti-CD28, anti-CD20, anti-Fas antibody, or any combination thereof, MHC-peptide tetramer or dimer, Fas ligand, IL-2, IL-4 , TRAIL, rolipram, doxorubicin, chlorambucil, fludarabine, corticosteroid, glucocorticoid, cyclosporine, cyclophosphamide, FK506, azathioprine, methotrexate, mycophenolate, annexin, caspase, bcl -2 inhibitors, interleukin-1β converting enzyme (ICE) -binding agents, Shigella IpaB protein, staurosporin, ultraviolet radiation, gamma irradiation, radiation, tumor necrosis factor, various histone deacetylase inhibitors, and in the art Among other well-known things Can be, but is not limited to this. In certain embodiments, the growth inhibitory composition comprises a surface, such as magnetic beads, to which one or more substances that bind cell surface residues are attached. In this regard, the material may be any of the materials described herein. In one embodiment, at least an anti-CD3 antibody is attached to the surface. In other embodiments, the surface has anti-CD3 and anti-CD28 antibodies attached. In addition, the growth inhibitory composition may comprise a polypeptide capable of inhibiting the growth of cells. Such polypeptides include peptides such as Bax, Bad, Bcl-xS, Bak, Bik, and active caspases. Exemplary other growth inhibitory compositions include irradiated cells (eg, donor or recipient (homologous) cells), target antigens (eg, defined autoimmune target antigens, such as myelin based protein (MBP), MBP in multiple sclerosis). 84-102 or target antigen identified as MBP 143-168; pancreatic islet cell antigen; S antigen in uveitis; or type II or other types of collagen in rheumatoid arthritis; thyroid receptor in Grave disease; acetylcholine receptor in myasthenia gravis) , Cytoplasmic linker protein-170 (CLIP-170), nucleic acid molecules, proteins or peptides, and non-protein or non-polynucleotide compounds.

As used herein, "substantially pure" population of CD3 ⁺ / CD28 ⁺ T cells is a population of cells consisting of at least about 90% CD3 ⁺ / CD28 ⁺ T cells. In some aspects of the invention, the "substantially pure" population of CD3 ⁺ / CD28 ⁺ T cells is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%, preferably Is a cell population consisting of at least about 99%, even more preferably at least about 99.9% of CD3 ⁺ / CD28 ⁺ T cells.

Source of mixed population of cells

In one embodiment, the cells to be exposed to the apoptosis-inducing or growth-inhibiting composition and / or sensitizing composition are derived from circulating blood of an individual obtained from one or more blood units or lyochemistry or leukocytosis. The ligation product typically contains T cells, including lymphocytes, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Prior to exposure to the sensitizing composition and subsequent activation and / or stimulation, a source of T cells is obtained from the subject. The term "subject" is meant to include living organisms (eg, mammals) from which an immune response can be induced. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells are a number of sources including peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumors, lymph node tissues, intestinal associated lymphoid tissue, mucosal related lymphoid tissue, spleen tissue, or any other lymphoid tissue, and tumors Can be obtained from T cells can be obtained from T cell lines and from autologous or allogeneic sources. T cells can also be obtained from heterologous sources such as mice, rats, nonhuman primates, and swine. In certain embodiments of the invention, T cells can be obtained from units of blood collected from a subject using any number of techniques known in the art, such as ficoll separation. In one preferred embodiment, the cells from the circulating blood of the individual are obtained by constitutive or leukopenia. The ligation product typically contains T cells, including lymphocytes, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by blotting can be washed to remove plasma aliquots and the cells can be placed in appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In other embodiments, the wash liquor lacks calcium and may lack magnesium or may lack many if not all of the divalent cations. As one of ordinary skill in the art would readily expect, the washing step may be carried out by a method known in the art, for example according to the manufacturer's instructions, using a semi-automatic "flow-through" centrifuge (e.g. Cobe) 2991 cell processor, Baxter). After washing, cells can be suspended in various biocompatible buffers such as calcium (Ca) -containing, magnesium (Mg) -containing PBS. Alternatively, undesirable components of the sampling sample can be removed and the cells can be resuspended directly in the culture medium.

In other embodiments, T cells are isolated by lysing or removing erythrocyte cells, and depleting monocytes, for example, by centrifugation through a PERCOLL® gradient. Certain subpopulations of T cells, such as CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and CD45RO ⁺ T cells, can be further isolated by positive or negative selection techniques. For example, in one preferred embodiment, anti-CD3 / anti-CD28 (ie, 3x28) -conjugated beads, eg, Dynabiz® M-450, for a sufficient time for positive selection of the desired T cells. T cells are isolated by incubation with CD3 / CD28 T. In one embodiment, the time is about 30 minutes. In other embodiments, the time ranges from 30 minutes to 36 hours or longer, with all integer values in this range. In further embodiments, the time is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. In another preferred embodiment, the time is 10 hours to 24 hours. In one preferred embodiment, the incubation time is 24 hours. For isolation of T cells from patients with leukemia, longer incubation times, eg 24 hours, can be used to increase cell yield. Longer incubation times can be used to isolate T cells in any situation where there are fewer T cells compared to other cell types in isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Longer incubation times can also be used to increase the capture efficiency of CD8 ⁺ T cells. For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (eg, Dynabiz® M-450 CD3 / CD28 T cell expander). . In one aspect of the invention, enrichment of the T cell population by negative selection can be achieved using a combination of antibodies designated as surface markers specific for the cells selected negatively. Preferred methods are cell sorting and / or selection via negative autoimmune adhesion or flow cytometry using a mixture of monoclonal antibodies designated as cell surface markers present on negatively selected cells. For example, to enrich CD4 ⁺ cells by negative selection, monoclonal antibody mixtures typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

Further aspects of the invention include various markers prior to sensitization, stimulation, and expansion, such as CD62L, CD45RA or CD45RO, cytokines (eg, IL-2, IFN-γ, IL-4, IL-10), cytokines. Caine receptors (e.g. CD25), perforin, adhesion molecules (e.g. VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1), and / or homing molecules (e.g. L Provide a T cell population or composition that is depleted or enriched for a cell population expressing -selectin). In one embodiment, cells expressing any of the above markers are depleted or selected positively by an antibody or other ligand / binding agent designated for the marker. Those skilled in the art will readily identify a variety of specific methods for depleting or positively selecting a sample of cells expressing a desired marker.

Monocyte populations (i.e., CD14 ⁺ cells) may be attached by a variety of methods, including anti-CD14 coated beads or columns prior to ex vivo expansion, or by using the phagocytic activity of these cells to facilitate removal or to adhere to plastics. Can be depleted from blood products. Thus, in one embodiment, the present invention uses paramagnetic particles of sufficient size to be surrounded by phagocyte monocytes. In some embodiments, the paramagnetic particles are commercially available beads, eg, beads of the trade name Dynabiz® manufactured by Dynal AS. Exemplary Dynabiz® in this regard are M-280, M-450 and M-500. In one aspect, the other non-specific cells are removed by coating the paramagnetic particles with “unrelated” proteins (eg, serum proteins or antibodies). Unrelated proteins and antibodies include proteins and antibodies or fragments thereof that do not specifically target T cells to be expanded. In certain embodiments, unrelated beads include beads coated with both anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.

In sum, such monocyte depletion may result in the removal of PBMCs or constitutive peripheral blood isolated from whole blood using Ficoll with one or more various unrelated or non-antibody coupled paramagnetic particles, and any amount that allows removal of monocytes. Pre-incubation at 22-37 ° C. for about 30 minutes to 2 hours (bead: cell ratio of about 20: 1) followed by magnetic attachment of paramagnetic particles or magnetic removal of surrounding cells. Preincubation may be carried out at a low temperature, such as 3 to 4 ℃. Such separation can be carried out using standard methods available in the art. For example, any magnetic separation method can be used, including a variety of commercially available ones, such as DYNAL® Magnetic Particle Concentrator (DYNAL MPC®). Confirmation of required depletion can be monitored by various methods known to those of skill in the art, including flow cytometry analysis of CD14 positive cells before and after the depletion.

T cells for exposure to and subsequent stimulation of apoptosis-inducing and / or sensitizing compositions may be frozen after a wash step that does not require a monocyte-removal step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and some monocytes from the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this situation, one method uses PBS or other suitable cell freezing medium containing a final concentration of 10% DMSO and 4% human serum albumin, and then Freezing cells at −80 ° C. at a rate of 1 per minute and storing them in the vapor phase of a liquid nitrogen storage tank.

Removal of undesirable cell subpopulations from a mixed population of cells

Direct exposure to apoptosis-inducing compositions

The present invention provides a method of removing at least a portion of an undesirable clonal population of cells, typically T cells, B cells, NKT or NK cells, from a population of immune cells. The invention further provides compositions and uses thereof which comprise a cell population which no longer contains undesirable cells or which has a significantly reduced number of undesirable cells.

An undesirable cell population can be eliminated or reduced to a statistically significant amount by exposing the cells directly to the apoptosis-inducing composition. Exposure to apoptosis-inducing compositions can occur in vivo or in vitro. Without wishing to be bound by theory, it is believed that preactivated cells are more sensitive to apoptosis compositions than natural or inactivated cells. Thus, exposure to the apoptosis composition in vivo or in vitro using dosages and conditions that induce apoptosis will selectively kill highly activated cells, such as unwanted autoreactive cells, in a patient. In a preferred embodiment of the invention, the autoreactive cells to be removed comprise T cells, NKT, NK, or B cells.

Accordingly, the present invention provides a method for removing at least a substantial portion of any unwanted subpopulation of clonal cells (eg, T, B, NKT, or NK cells) from a mixed population of immune cells. For the purposes of the present invention, a substantial portion means at least 70% of the unwanted subpopulation of cells. In certain embodiments a substantial portion means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and higher percentages of unwanted subpopulations of cells. Removal of cells can be any number known in the art, including, but not limited to, flow cytometry and functional analysis using various antibodies and / or peptide-MHC tetramers such as proliferation and chromium release assays. It can be measured using the technique of.

Apoptotic inducing composition or apoptosis inducer means any composition or stimulus that increases apoptosis activity of a cell when administered alone or in combination with other apoptosis inducing compositions. Apoptosis-inducing compositions used in the methods of the invention preferably induce apoptosis in activated T cells, NKT cells, NK cells, and B cells. The amount and conditions under which the apoptosis-inducing composition induces the desired apoptosis can be different and can be determined by one skilled in the art using routine optimization. In certain embodiments, the apoptosis-inducing compositions of the invention will induce apoptosis without further activation / stimulation. Illustrative examples of such substances or stimuli include growth factor depletion, oxidative conditions, heat stress, freeze-thaw stress, serum depletion, various antibodies such as anti-CD2, anti-CD3, anti-CD28, anti-CD20, or Anti-Fas antibodies; MHC-peptide tetramers; Fas ligand, TRAIL, FR901228 (described in US Pat. No. 6,403,555), FK506, annexin, caspase, cytokines such as IL-2 or IL-4, cyclophosphamide, chemotherapeutic agents, UV, Steroids, corticosteroids, glucocorticoids, rolipram, doxorubicin, chlorambucil, fludarabine, bcl-2 inhibitors, topoisomerase inhibitors, interleukin-1β converting enzyme (ICE) -binding agents, Shigella IpaB protein, star Urosporin, ultraviolet radiation, gamma irradiation, radiation, tumor necrosis factor, histone deacetylase inhibitors, and others well known in the art, but are not limited to these. In certain embodiments, the apoptosis-inducing composition comprises a surface, such as magnetic beads, to which one or more substances that bind cell surface residues are attached. In this regard, the material may be any material as described herein. In one embodiment, at least an anti-CD3 antibody is attached to the surface. In other embodiments, the surface has anti-CD3 and anti-CD28 antibodies attached. In addition, an apoptosis stimulator may be a polypeptide capable of increasing or inducing apoptosis activity in a cell. Such polypeptides include polypeptides that directly regulate the apoptosis pathway, such as Bax, Bad, Bcl-xL, Bak, Bik, and active caspases, and polypeptides that indirectly regulate the pathway. Other exemplary apoptosis-inducing compositions include irradiated cells (eg donor or recipient (allo) cells), target antigens (eg defined autoimmune target antigens such as myelin based protein (MBP), pancreatic islet cell antigens). , Cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren lupus antigen (SL), scleroderma antigen 70 (Scl- 70)) nucleic acid molecules, proteins or peptides, and non-protein or non-polynucleotide compounds.

In one aspect of the invention, one or more apoptosis-inducing compositions are administered in vivo with the pharmaceutically acceptable excipient. Any combination of apoptosis-inducing compositions can be administered with a cytokine such as IL-2 or IL-4, for example an anti-CD3 antibody, which is described in patent application W09428926. As will be readily appreciated by those skilled in the art, testing for any apoptosis-inducing composition used in the methods of the present invention may include: 1) pharmacokinetic behavior of such materials; 2) identification of safety and any undesirable effects; And 3) usually need to be carried out in a predetermined dosage range to determine the optimal dosage for effective induction of apoptosis in the cells to be removed. This constitutes a phase 1 clinical trial. Thus, certain apoptosis-inducing compositions used in the methods described herein require individual routine optimization. The apoptosis-inducing compositions of the invention can be administered topically, parenterally or by inhalation. The term “parenteral” includes subcutaneous injection, intravenous, intramuscular, intravitreal injection or infusion techniques. Such compositions will typically contain an effective amount of apoptosis-inducing composition alone or in combination with an effective amount of any other active substance. The dosages and preferred drug concentrations contained in the compositions may vary depending on a number of factors, including the intended use, the weight and age of the mammal, and the route of administration. Preliminary dosages may be determined according to animal testing, and scaling of dosages for human administration may be performed according to practice acceptable in the art.

In one aspect of the invention, the cell population is exposed to one or more apoptosis-inducing compositions in vitro. As will be readily appreciated by those skilled in the art, testing for any apoptosis-inducing composition used in the methods of the present invention may include 1) pharmacokinetic behavior of such materials; 2) identification of safety and any undesirable effects; And 3) usually need to be carried out in a predetermined dosage range to determine the optimal dosage for effective induction of apoptosis in the cells to be removed. Thus, certain apoptosis-inducing compositions used in the methods described herein require individual routine optimization. In one particular embodiment, the remaining population of cells with the unwanted reactive subpopulation of cells removed can be administered to the patient without further stimulation / activation or expansion.

In one embodiment of the invention, the cells are exposed multiple times to the apoptosis-inducing composition alone or to a combination of this and other apoptosis-inducing compositions. In some aspects of the invention, prior to exposing the mixed population of cells to one or more apoptosis-inducing compositions, activation / discharge as described in the sections entitled “Stimulation / Activation of Cell Population” and “Expansion of Cell Population” below. It may be desirable to stimulate and in some cases expand. In one preferred embodiment, cells remaining in the population after exposure to the apoptosis-inducing composition of the invention are subjected to in vitro as described in the sections entitled "Stimulation / Activation of Cell Population" and "Expansion of Cell Population" below. Enable / stimulate on In some embodiments, the apoptosis-inducing composition and the composition used for activation / stimulation and expansion are the same composition. In one particular embodiment, the surface to which the substance is attached is used as an apoptosis-inducing composition as described herein and further used to activate / stimulate and expand the mixed population. In this regard, some clonal cells in the population are induced to perform apoptosis, while others stimulate / activate and proliferate in response to the composition. In this case, exemplary compositions that can be used to induce apoptosis in a subset of T cells and to stimulate / activate and expand a mixed population of T cells are anti-CD3 and anti-CD28 antibodies co-fixed on beads (3x28 beads) It includes.

In other embodiments of the invention, cells remaining after exposure to one or more apoptosis-inducing compositions are further stimulated / activated to expand in vivo. In vivo stimulation and expansion of the cells of the invention can be performed using any number of cytokines such as IL-2 and IL-4 or other materials described herein that stimulate cells.

In a further embodiment of the invention, cells remaining after exposure to one or more apoptosis-inducing compositions are treated as described below in sections entitled “Stimulation / Activation of Cell Population” and “Expansion of T Cell Population”. The material bound to this surface is used to further stimulate / activate and expand in vitro. Stimulation and activation of the remaining cells that do not perform apoptosis using the surface of the present invention can increase the polyclonality of the remaining populations of T cells as measured by the range of response of the population to a given antigen. Repair or increase in polyclonality can be measured by determining the extent of response to a particular subject antigen, for example by determining the number of different epitopes recognized by antigen-specific cells. This can be done using standard techniques to generate and clone antigen-specific T cells in vitro.

Stimulating and activating the remaining cells that do not perform apoptosis using the surface of the present invention restores polyclonality to the remaining populations of T cells for expressed TCR genes, as indicated by spectratype analysis. . The polyclonality of the T cell compositions of the present invention is described in US Patent Application 60 / 375,733. Spectratype analysis is a method of measuring TCR Vβ, Vα, Vγ or Vδ gene usage and nucleotide insertion levels by pools of T cells during recombination in T cell development (described in US Pat. No. 5,837,447). Spectratype analysis can be used to determine the extent or limit of T cell immune response potential. In addition, spectratype analysis can be used to determine whether certain undesirable clonal populations of T cells have been removed from the mixed population of T cells.

The ability of the V, D and J gene segments to randomly combine together introduces a large element of combinatorial diversity into the TCR repertoire. The exact point at which the V, D, and J segments connect can vary, resulting in local amino acid diversity at the junction. The exact nucleotide position of the junction can be as different as 10 residues, indicating codon changes at the junction of these fragments by deleting nucleotides from the ends of the V, D and J gene segments. When additional nucleotides that are not encoded by each gene segment are added to the linkages between the linked gene segments, the diversity increases more during the rearrangement process (the diversity produced by this process is called "N-region diversity"). (Janeway, Travers, Walport. Immunobiology. Fourth Ed., 98 and 150. Elsevier Science Ltd / Garland Publishing. 1999).

Diversity levels for the T cell repertoire assess in part that the TCR Vβ, Vα, Vγ or Vδ chain is used by individual T cells in the pool of circulating T cells, and inserted after the Vβ gene at the VDJ or VJ gene junction. The number of random nucleotides added can be measured. In general, if the circulating T cell pool contains T cells expressing the full range of the TCR Vβ, Vα, Vγ or Vδ chains and from the genetic recombination reaction using the widest array of nucleotides into which these individual V region chains are inserted When induced, the T cell arm of the immune system will have the greatest potential for recognizing a population of potential antigens. When the range of TCR V region chains expressed by circulating pools of T cells is limited or reduced, and when the chains expressed by recombinant genes with expressed TCRs with restricted nucleotide inserts are used, the range of immune response potential is Correspondingly reduced. The result is a decrease in the ability to respond to a wide variety of antigens, leading to increased risk of infection and cancer.

Methods of measuring apoptosis are known in the art and are described, for example, in Current Protocols in Immunology, John Wiley & Sons, New York, N. Y. or US Pat. No. 6,312,684. Exemplary assays for measuring apoptosis include DNA ladders, electron or light microscopy, flow cytometry, and several commercially available kits for the measurement of apoptosis. In certain embodiments, cells are observed for morphological changes such as chromatin condensation, cell contraction, increased granularity, and other signs of apoptosis known to those of skill in the art. Chromatin condensation can be detected by standard methods, for example by light microscopy on stained cell samples. Cell contractility and granularity can be easily detected by measuring the light scattering properties of the cells (Kerr, et al. Supra., And Wyllie, et al., Supra). The observation that DNA is single or double stranded into the ladder of oligonucleosomes is another indication that apoptosis was induced (Arend, et al., Am. J. Pathol, 136: 593, 1990; Wyllie, et al. , J. Pathol, 142.:67, 1984). However, sometimes apoptotic cells do not exhibit double stranded nucleosome DNA fragmentation (Collins, et al., Int. J. Rad. Biol., 62:45 1992; Cohen, et al., Biochem. J., 286: 331 1992); Instead a single DNA strand cut will be observed. Single stranded cleavage can be readily detected using methods of in situ nick end-labeling of DNA. This method is described in Wyllie, et al. (Br. J. Cancer, 67:20, 1993).

In one embodiment, the cells of the invention are exposed to a growth inhibitory composition as described herein. In some embodiments, the growth inhibitory composition of the invention is such that the growth inhibited cells do not expand and eventually do not compete with the mixed population of cells when the mixed population of cells is activated / stimulated and expanded as described herein. Inhibit growth in at least a substantial portion of one or more clonal populations of T cells. The end result is the effective removal of growth inhibited cells from the mixed population of cells.

Exposure to compositions that sensitize cells with further stimulation / activation

Alternatively, at least a substantial portion of the undesirable population of cells can be removed by first sensitizing the cells with further stimulation / activation, and then exposing the cells to the surface of the invention for further stimulation or activation. This additional stimulation / activation induces apoptosis in sensitized cells to remove cells from the population. In addition, the sensitizing compositions of the present invention sensitize cells by the effects of the apoptosis-inducing compositions described above. Thus, the present invention is one in which at least a substantial portion of an undesirable clonal population of cells, typically T cells or B cells, is sensitized by further stimulation / action or by the effect of an apoptosis-inducing composition. Provided is a method of removing said portion from a population of immune cells by exposure to said composition. In addition, the present invention provides compositions and uses thereof comprising a cell population that no longer contains undesirable cells.

In one aspect of the invention, a population of immune cells, at least a portion of a cell, such as a previously highly activated T cell or B cell, is exposed to a composition or compositions that are sensitized by further activation or stimulation. In a preferred embodiment, the sensitized cells comprise undesirable autoreactive T or B cells. In further embodiments, the sensitized cells comprise alloreactive cells present in donor hematopoietic stem cells. In another embodiment, the sensitized cells comprise alloreactive cells from potential organ transplant recipients.

In one embodiment of the invention, the sensitized composition comprises irradiated cells. In certain embodiments, the irradiated cells are cells from hematopoietic stem cell transplant recipients and the cells to be sensitized are cells from hematopoietic stem cell transplant donors. In other embodiments, the sensitizing composition comprises irradiated cells from an organ donor and the cells to be sensitized are cells from an organ recipient. In some embodiments, the cell to be sensitized is a cell derived from an organ recipient after transplantation. The cells are typically irradiated with gamma rays of about 3000 to 3600 rads, more preferably about 3300 rads. Other irradiated cells that may be useful in the present invention, such as lymphoblastoid or tumor cell lines, are typically irradiated with gamma rays of about 6000 to 10,000 rads, more preferably about 8000 rads. The cells may also be treated with other means, such as chemicals (eg, etiposide and mitomycin, etc.).

The sensitizing compositions of the present invention include any composition or combination thereof that sensitizes immune cells, such as T, NKT, NK, or B cells, with subsequent stimulation such that subsequent stimulation or activation induces apoptosis. In addition, the sensitizing compositions of the present invention include any composition or combination thereof that sensitizes immune cells, such as T or B cells, to subsequent exposure to the apoptosis-inducing composition. Sensitizing compositions of the present invention include antibodies such as anti-CD2, anti-CD3, anti-FAS; MHC-peptide dimers or tetramers, cytokines such as IL-2, TRAIL, compounds such as rolipram, doxorubicin, chlorambucil and fludarabine. Sensitizing compositions also include FAS-ligands and natural ligands for CD2 and CD3. In addition, the sensitizing compositions may include bcl-2 inhibitors such as those described in US Pat. No. 6,277,844, topoisomerase inhibitors such as etoposide, CPT-11 and topotecan, and eg US Pat. Others described in US Pat. No. 5,834,012. Other exemplary sensitizing compositions include interleukin-1β converting enzyme (ICE) -binding agents that induce apoptosis, for example Shigella IpaB protein described in US Pat. No. 5,972,899, or US Pat. No. 6,350,741, 6,294,546 and The compound of 6,329,365 is mentioned.

Exemplary sensitizing compositions of the present invention also include autoantigens. Autoantigens include defined autoimmune target antigens, eg, defined autoimmune target antigens, eg, target antigens identified as myelin based protein (MBP), MBP 84-102 or MBP 143-168 in multiple sclerosis; Pancreatic islet cell antigens; S antigen in uveitis; Or type II or other types of collagen in rheumatoid arthritis; Cytoplasmic linker protein-170 (CLIP-170) in SLE; Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren lupus antigen (SL); Scleroderma antigen 70 (Scl-70); Thyroid receptors in Grave's disease; Acetylcholine receptors, nucleic acid molecules, proteins or peptides, and non-protein or non-polynucleotide compounds in myasthenia gravis. In addition, the autoantigens of the present invention are HLA-DQ which confers sensitivity to MHC molecules known to be involved in autoimmunity, such as several common autoimmune diseases such as type 1 diabetes, rheumatoid arthritis and multiple sclerosis. And peptide mixtures eluted from -DR molecules, or HLA-B27 molecules known to confer sensitivity to reactive arthritis and ankylosing spondylitis. The autoantigens of the invention may also be synthetic peptides that are expected to bind to MHC molecules associated with autoimmune diseases.

In addition, the present invention provides a sensitizing composition that selectively removes at least a substantial portion of a T cell population that expresses specific Vβ, Vα, Vγ, or Vδ genes. For example, antibodies specific for a particular Vβ, Vα, Vγ or Vδ gene can be used to specifically sensitize T cells according to the methods of the present invention. Alternatively, at least a substantial portion of the cells can be removed from the population by negatively selecting T cells expressing a particular subject Vβ, Vα, Vγ or Vδ gene.

In a further embodiment of the invention, one or more sensitizing compositions are used simultaneously and for a time sufficient to induce the desired sensitization.

As described above for apoptosis-inducing compositions, the present invention relates to a method for administering a composition in vivo or in vitro or in combination with in vivo and in vitro, wherein the composition sensitizes cells to the effect of further stimulation / activation or apoptosis-inducing composition. to provide. As with any medical substance, biological tests for any substance to be administered in vivo that sensitize cells with further stimulation / activation, for example, a number of apoptosis-inducing compounds, antibodies, peptides and proteins used for immunization, include: 1 Pharmacokinetic behavior of these substances; 2) their immunogenicity; And 3) usually need to be carried out at a predetermined dosage range to determine safety and confirmation of any undesirable effects. This constitutes a phase 1 clinical trial. Thus, certain substances that sensitize cells to further stimulation / activation used in the methods of the invention (eg, target antigens identified as MBP 84-102 or MBP 143-168 in multiple sclerosis; S antigens in uveitis; or Type II collagen in rheumatoid arthritis) requires individual routine optimization. The sensitizing compositions of the present invention can be administered topically, parenterally or by inhalation. The term “parenteral” includes subcutaneous injection, intravenous, intramuscular, intravitreal injection or infusion techniques. Such compositions will typically contain an effective amount of a sensitizing composition alone or a combination of this composition and any other active substance in an effective amount. The dosages and preferred drug concentrations contained in the compositions may vary depending on a number of factors, including the intended use, the weight and age of the mammal, and the route of administration. Preliminary dosages can be determined according to animal testing, and scaling of dosages for human administration can be performed according to the practice accepted in the prior art.

Abundant evidence from vaccine development suggests that each synthetic peptide or recombinant DNA-derived protein is effective in inducing an immune response in humans. In addition, these studies provide guidance on dosage ranges effective for immunization (Zajoc, BA, DJ West, WJ McAleer and EM Scolnick, Overview of clinical studies with Hepatitis B vaccine made by recombinant DNA, J. Infect. 13: (Suppl A) 39-45 (1986) .Yamamoto, S., T. Kuroki, K. Kurai and S. Iino, Comparison of results for phase I studies with recombinant and Plasma-derived hepatitis B vaccines, and controlled study comparing intramuscular and subcutaneous injections of recombinant hepatitis B vaccine, J. Infect. 13: (Suppl A) 53-60 (1986). Francis, DP et al., The prevention of Hepatitis B with vaccine, Ann. Int. Med. 97: 362 -366 (1982) .Putney et al., Features of HIV envelope and development of a subunit vaccine, AIDS Vaccine Research and Clinical Trials, S. Putney and B. Bolognesi, eds. (New York: Dekker) pp. 3-62 (1990) .Steven, VC and WR Jones, Vaccines to prevent pregnancy, New Generation Vaccines, GC Woodrow and MM Levine, eds. (New Y ork: Dekker) pp. 879-900 (1990) .Herrington et al., Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium Falciparium sporozoites, Nature, 328: 257-259 (1987)).

In one embodiment of the invention, after immunization (ie, in vivo sensitization) using a substance that sensitizes the cell to further stimulation / activation or exposure to an apoptosis-inducing composition, the substance carries a reactive receptor, For example, there is a waiting period to activate a subset of B cells expressing a T cell bearing a reactive TCR or a specific antibody receptor, causing these cells to express a cytokine receptor, eg, an IL-2 receptor. . For example, this process will induce IL-2 receptor only in antigen stimulated T cells. Based on studies of both human and mouse T cells in vitro, expressing a significant number of IL-2 receptors requires about 12 hours to about 24 hours after antigen exposure and is optimal for most T cells. It takes about 72 hours to express the number of IL-2 receptors. Thus, the waiting period can be as short as about 12 hours or as long as about 72 hours, and in the case of various disease symptoms this period can be as long as 120 hours due to delayed immune responsiveness, becoming increasingly optimal towards the upper end of this range. Becomes

In one embodiment of the invention, the patient is administered IL-2, or other suitable cytokine, such as IL-4, to induce apoptosis in activated cells as described above. The administration of IL-2 to humans has been thoroughly studied in cancer patients and evaluated for various doses (Loize, MT, LW Frana, SO Sharrow, RJ Robb and SA Rosenberg, In vivo administration of purified human interleukin 2.1. -life and immunologic effects of the Jurkat cell line-derived interleukin 2.J. Immunol. 134: 157-166 (1985). Lotze, JT, YL Malory, SE Ettinghausen, AA Rayner, SO Sharrow, CAY Seipp, MC Custer and SA Rosenberg, In vivo administration of purified human interleukin 2.II.Half-life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL 2.J. Immunol. 135: 2865-2875 (1985) .Donahue, JH and SA Rosenberg, The fate of interleukin-2 after in vivo administration, J. Immunol. 130: 2203-2208 (1983). Belldegrun, A., MM Muul and S. A Rosenberg, interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization, and an titumor activity, Cancer Research 48: 206-214 (1988) .Rosenberg, SA, MT Lotze, LM Muul, S. Leitman, AE Chang, SE Ettinghausen, YL Malory, JM Skibber, E. Shiloni, JT Vetto, CA Seipp, C. Simpson and CM Reichert, Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer, New Eng. J. Med. 313: 1485-1492 (1985)). The data indicate that IL-2 should be provided to I.V. as frequent bolus administration or continuous infusion. Previously established dosages range from about 300 to about 3000 units / kg / hour continuous infusion, or 104 to 106 units / kg I.V. It is in the range of bolus.

In one aspect of the invention, the cell population is exposed to one or more sensitizing compositions in vitro. As will be readily appreciated by those skilled in the art, tests on any sensitizing composition used in the methods of the present invention include: 1) pharmacokinetic behavior of such materials; 2) identification of safety and any undesirable effects; And 3) it is usually necessary to carry out at a predetermined dosage range to determine the optimal dosage for effectively inducing apoptosis in the cells to be removed. Thus, certain sensitizing compositions used in the methods described herein require individual conventional optimization.

In one embodiment of the invention, cells are harvested from a subject previously treated in vivo with a substance that sensitizes the cells to further stimulation / activation. The cells are further stimulated / activated to induce apoptosis and then expanded in vitro as described below.

Remove cells from the mixed population by stimulating sensitized cells to induce apoptosis of the cells

In one aspect of the invention, further activating or stimulating a population of immune cells comprising sensitized cells as described above to induce apoptosis as described in the section entitled “Stimulation / Activation of a Cell Population” below. Thereby sensitizing cells, eg, autoreactive or alloreactive T- or B-cells, from the mixed population of cells. At the same time, a population of activated cells in which at least a substantial portion of the unwanted subpopulation of T (or B cells) has been removed by activating and stimulating and expanding the remaining desired cells, e. To form. As mentioned above, stimulation / activation as described herein can be performed on cells remaining after the mixed population of cells is directly exposed to the apoptosis-inducing composition. Further stimulation and activation provided by the present invention also restores polyclonality to the T cell population for the expressed TCR gene as indicated by spectratype analysis.

In one embodiment of the invention, the sensitized cells are stimulated / activated several times as described below with or without additional sensitizing composition, and the sensitized cells are removed if it is necessary to remove at least a substantial portion of the undesirable cells. Stimulate / activate multiple times. For example, for autoimmune diseases, the present invention provides a method of stimulating a cell two or more times in the presence of an antigen (ie, a sensitizing composition) after the initial round of stimulation / activation. Similarly, for hematopoietic stem cell transplantation, the present invention removes at least a substantial portion of undesirable cells from the hematopoietic stem cell donor at least twice, or in the presence of irradiated cells from hematopoietic stem cell transplant recipients. In order to provide a method for stimulating a plurality of times as necessary. In the case of organ transplantation, the present invention stimulates cells from organ recipients two or more times, or if necessary, multiple times as needed to remove at least a substantial portion of undesirable cells in the presence of irradiated cells from organ donors. Provide a way to. In one embodiment, the methods of the invention are performed on cells from a patient (eg, a host cell) after transplantation to remove undesirable cells. In certain embodiments, it may not be necessary to remove all undesirable cells, for example in the case of hematopoietic stem cell transplantation for certain types of cancer, and graft versus leukemia cell effects may be desirable.

In some aspects of the invention, sections entitled "Stimulation / Activation of Cell Population" and "Expansion of Cell Population" below, prior to exposing the cells to one or more substances that sensitize further stimulation / activation and subsequent stimulation. It may be desirable to stimulate / activate and in some cases expand the mixed population of cells as described.

In another aspect of the invention, the cells are sensitized and then exposed to the apoptosis-inducing composition to remove at least a substantial portion of the cells sensitized by the effect of the apoptosis-inducing composition. The cells remaining in the population can then be further stimulated / activated to expand as described below.

In one embodiment, the cells of the invention are exposed to a growth inhibitory composition as described herein. In some embodiments, the growth inhibitory composition of the invention is such that when the mixed population of cells is activated / stimulated and expanded as described herein, the growth inhibited cells do not expand and eventually do not compete with the mixed population of cells, Inhibit growth in at least a substantial portion of one or more clonal populations of T cells. The end result is that the growth inhibited cells are effectively removed from the mixed population of cells.

Pure CD3 ⁺ CD28 ⁺  Generation of T Cell Populations

The present invention provides a method of generating a substantially pure population of CD3 ⁺ CD28 ⁺ T cells from a population of immune cells. For the purposes of the present invention, substantially pure CD3 ⁺ CD28 ⁺ T cell populations contain less than 10% CD3 ⁺ CD28 ⁻ T cells. In certain embodiments, the substantially pure CD3 ⁺ CD28 ⁺ T cell population is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% Contains CD3 ⁺ CD28 ^- T cells.

Pure populations of CD3 ⁺ CD28 ⁺ T cells can be generated by magnetic concentration, selection, and by stimulating a mixed population of T cells with a composition capable of stimulating both CD3 and CD28 molecules on the T cell surface. Selection and stimulation of both CD3 and CD28 molecules on the cell surface results in the activation and proliferation of this subset of cells. In contrast, under the conditions described herein, exposing CD3 ⁺ CD28 ⁻ T cells to a composition capable of selecting and stimulating both CD3 and CD28 surface molecules is not sufficient to induce both activation and expansion of this T cell population. Further, if reducing the incubation time of the CD3 / CD28 beads as described herein CD3 ⁺ CD28 ^- cells instead of CD3 ⁺ CD28 ⁺ cells are selected predominantly (e. G., Followed by a 15-minute selection from 1 rpm at room temperature are magnetic concentration Leaves many or most CD3 ⁺ CD28 ^- cells). Initiation of TCR by a specific antigen, or a molecule capable of stimulating a CD3 surface molecule, such as an anti-CD3 antibody, may result in expansion and lymphokine secretion unless supplemented by a costimulatory signal, ie specific stimulation of the CD28 molecule. It is considered insufficient to induce. Indeed, in the absence of costimulation, these T cells can acquire a non-reactive or anergy state.

Thus, stimulating and selecting a mixed population of T cells using a method of the present invention, eg, a composition capable of initiating CD3 and stimulating CD28, results in a substantially pure population of CD3 ⁺ CD28 ⁺ T cells.

In one embodiment of the invention, this population of substantially pure CD3 ⁺ CD28 ⁺ T cells can be used to treat acute or chronic GVHD. In other embodiments, autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitus, Addison's disease, chronic digestive disorders, chronic fatigue syndrome using substantially pure CD3 ⁺ CD28 ⁺ T cell populations , Colitis, Crohn's disease, myofascial pain, lupus, psoriasis, Sjogren's syndrome, hyperthyroidism / grave disease, hypothyroidism / hamositopathy, insulin dependent diabetes mellitus (type 1), myasthenia gravis, endometriosis, scleroderma , Pernicious anemia, Good Pathur syndrome, Wegner's disease and rheumatic fever. In further embodiments, cells of the invention can be used to treat autoimmunity associated with giant granular lymphocytic leukemia (LGL). A mixed population of immune cells can be removed from the stimulated donor and these cells with a composition capable of stimulating CD3 and CD28 molecules. Without wishing to be bound by theory, it is believed that this stimulus results in specific activation and expansion of CD3 ⁺ CD28 ⁺ T cells and results in anergiogenesis of T cells lacking the expression of the costimulatory molecule, CD28. Once a pure population of CD3 ⁺ CD28 ⁺ T cells is generated, these cells can then be injected to treat autoimmune disease, LGL, or GVHD.

Stimulation / Activation of Cell Population

Stimulated and activated T cells of the present invention are generated by cell surface residue ligation to induce activation. In certain embodiments, stimulated and activated T cells are generated by activating only the T cell population through the involvement of TCR, eg, using anti-CD3 antibodies or natural ligands against TCR. In certain embodiments, stimulated and activated T cells are described, for example, in US Patent Application Nos. 08 / 253,694, 08 / 435,816, 08 / 592,711, 09 / 183,055, 09 / 350,202 And accessory molecules on the T cell surface with ligands that activate the T cell population and bind with accessory molecules as described in US Pat. Nos. 09 / 252,150, and US Pat. Nos. 6,352,694, 5,858,358, and 5,883,223. By stimulation. In the case of the sensitized cells described above, activating the sensitized T cell population and stimulating the accessory molecule on the sensitized T cell surface with a ligand that binds the accessory molecule leads to apoptosis and subsequent elimination of the cell.

In general, T cell activation of cells can be achieved by cell surface residue ligation, eg, by stimulating T cell receptor (TCR) / CD3 complex or CD2 surface protein. Many anti-human CD3 monoclonal antibodies are commercially available, such as hybridomas obtained from clone BC3 (XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, WA), American Type Culture Collection. OKT3 prepared from cells, and monoclonal antibody G19-4. Similarly, stimulatory forms of anti-CD2 antibodies are known and available. Stimulation via CD2 with anti-CD2 antibodies is typically accomplished using a combination of two or more different anti-CD2 antibodies. Stimulating combinations of the anti-CD2 antibodies described include the following: T11.3 antibodies in combination with T11.1 or T11.2 antibodies (Meuer et al., Cell 36: 897-906, 1984), and 9 9.6 antibody in combination with the -1 antibody (recognizes the same epitope as T11.1) (Yang et al., J. Immunol. 137: 1097-1100, 1986). Other antibodies may also be used that bind the same epitope as any of the antibodies described above. Additional antibodies or antibody combinations can be prepared and confirmed by standard techniques. In addition, the stimulus is in the superantigen (eg, Staphylococcus enterotoxin A (SEA), Staphylococcus enterotoxin B (SEB), Toxic Shock Syndrome Toxin 1 (TSST-1)), Through contact with toxins or include but are not limited to phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, lipopolysaccharides (LPS), T cell stimulators, and IL-2 It can be achieved through a variety of cleavage accelerators, which are not limited.

To further activate the T cell population, a costimulatory or accessory molecule on the T cell surface is stimulated with a ligand that binds to this accessory molecule. Thus, those skilled in the art will recognize that any substance can be stimulated using any material, including an anti-CD28 antibody or fragment thereof or a natural ligand for CD28 that can crosslink with CD28 molecules. Exemplary anti-CD28 antibodies or fragments useful in the present invention include monoclonal antibody 9.3 (IgG2 _a ) (Bristol Myers Squibb, Princeton, NJ), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1) , 248.23.2 (IgM), clones B-T3 (XR-CD28; Diaclone, Besancon, France) and EX5.3D10 (IgG2a) (ATCC HB11373). Exemplary natural ligands include B7 protein families, such as B7-1 (CD80) and B7-2 (CD86) (Freedman et al., J. Immunol. 137: 3260-3267, 1987; Freeman et al., J 143: 2714-2722, 1989; Freeman et al., J. Exp. Med. 174: 625-631, 1991; Freeman et al., Science 262: 909-911, 1993; Azuma et al., Nature 366: 76-79, 1993; Freeman et al., J. Exp. Med. 178: 2185-2192, 1993).

Other exemplary accessory molecules on the T cell surface that can be stimulated with ligands that bind the accessory molecules of the present invention include, but are not limited to, CD54, LFA-1, ICOS, and CD40.

In addition, binding homologs of natural ligands, whether natural or synthesized by chemical or recombinant techniques, can be used in accordance with the present invention. Other materials include natural and synthetic ligands. Substances include, but are not limited to, other antibodies or fragments thereof, growth factors, cytokines, chemokines, soluble receptors, steroids, hormones, promoters, such as PHAs, or other superantigens.

As described above, subsequent stimulation and activation of the remaining cells that did not undergo apoptosis or did not undergo apoptosis due to no sensitization resulted in the rest of the T cells with respect to the expressed TCR gene as indicated by spectratype analysis. Restore polyclonality to the population.

Expansion of cell population

In general, the present invention provides for the expansion of a cell population that remains after the cell population has been exposed to an apoptosis-inducing or sensitizing composition, and subsequently to the undesirable subpopulation of cells, preferably autoreactive or undesirable alloreactive T cells. Provides any induction of apoptosis in the population. In one embodiment of the invention, the remaining T cells can be stimulated with a single substance. In other embodiments, the remaining T cells can be stimulated with two or more substances, i.e., a substance that induces a primary signal and one or more substances that induce one or more costimulatory signals. Ligands useful for stimulating a single signal or for stimulating accessory molecules stimulating a primary signal and a second signal can be used in soluble form attached to the cell surface or immobilized on the surface as described herein. Ligands or substances attached to the surface act as "surrogate" antigen presenting cells (APCs). In a preferred embodiment, both the primary and secondary materials are cofixed onto the surface. In an embodiment, a molecule, eg, a CD3 ligand, and a costimulatory molecule, eg, a CD28 ligand, that provide a primary activation signal are coupled to the same surface, eg, a particle. In addition, as mentioned above, one, two or more stimulatory molecules may be used on the same or different surfaces.

The cell population is contacted with, for example, an anti-CD3 antibody or an anti-CD2 antibody immobilized on the surface, or with a protein kinase C activator (eg bryostatin) with calcium ionophores, as described herein. Can be stimulated. For costimulation of accessory molecules on the T cell surface, ligands that bind to the accessory molecules are used. For example, a population of CD4 ⁺ cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions appropriate to stimulate proliferation of T cells. Alternatively, the cell population can be contacted with PMA and ionomycin. Similarly, to stimulate proliferation of CD8 ⁺ T cells, anti-CD3 antibodies and anti-CD28 antibodies B-T3, XR-CD28 (Diaclone, Besancon, France) can be used in other methods commonly known in the art. Berg et al., Transplant Proc. 30 (8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9): 1319-1328, 1999; Garland et. al., J. Immunol Meth. 227 (1-2): 53-63, 1999).

Primary and costimulatory signals for T cells can be provided by different protocols. For example, a substance that provides each signal can be present in solution or coupled to a surface. When coupled to a surface, the material can be coupled to the same surface (ie, "cis" form) or to separate surfaces (ie, "trans" form). Alternatively, one material can be coupled to the surface and another material in solution. In one embodiment, the substance providing the costimulatory signal is bound to the cell surface and the substance providing the primary activation signal is placed in solution or coupled to the surface. In certain embodiments, two materials can be placed in solution. In other embodiments, the substance may be in soluble form and then crosslinked with a cell expressing an Fc or Sc receptor or antibody or with other binding agents that bind to the substance. In a preferred embodiment, two materials are immobilized on a spherical or hemispherical surface, typical examples being beads or cells, on the same beads, ie "cis" or in separate beads, ie "trans". For example, a substance that provides a primary activation signal is an anti-CD3 antibody, and a substance that provides a costimulatory signal is an anti-CD28 antibody and co-fixes both substances with equivalent molecular weight on the same beads. In one embodiment, each antibody bound to the beads for T cell expansion and T cell growth is used in a 1: 1 ratio. In some aspects of the invention, the ratio of anti-CD3: anti-CD28 antibody (CD3: CD28) bound to the beads is such that an increase in T cell expansion is observed compared to expansion observed using a 1: 1 ratio. use. In one particular embodiment, an increase of about 0.5 to about 3 times is observed relative to the expansion observed using a ratio of 1: 1. In one embodiment, the ratio of anti-CD3: anti-CD28 (CD3: CD28) antibody bound to the beads ranges from about 100: 1 to 1: 100, including all integer values there between. In certain embodiments, the ratio of CD3: CD28 is at least about 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50 : 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1 , 5: 1, 4: 1, 3: 1, 2: 1 or 1: 1. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, ie the ratio of CD3: CD28 is less than one. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2: 1. In one specific embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1: 200. In one specific embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1: 150. In one particular embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1: 100. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:75. In a further embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:50. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:45. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:40. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:35. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:30. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:25. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:20. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:15. In one preferred embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1:10. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1: 5. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 1: 4. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads is used at 1: 3. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads is used at 3: 1.

The ratio of particles to cells of 1: 500 to 500: 1 and any integer value within this range can be used to stimulate T cells or other target cells. As will be readily appreciated by those skilled in the art, the ratio of particle to cell may depend on the particle size relative to the target cell. For example, small size beads can bind only a few cells, while larger beads can bind multiple cells. In some embodiments, the ratio of cells to particles is in the range from 1: 100 to 100: 1 and any integer value within this range, and in other embodiments the ratio is from 1:50 to 50: 1 and any between these ranges. Contains an integer value. In other embodiments, the ratio of cells to particles in the range from 1: 9 to 9: 1 and any integer value between these ranges may be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells indicative of T cell stimulation may vary as mentioned above, but certain preferred values are at least 1: 150, 1: 125, 1: 100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2.5, 1: 2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 and 15: 1, with one preferred ratio being at least 1: 1 particles per T cell. In one particular embodiment, the preferred ratio of particles to cells is 1: 5 or 1:10. In one embodiment, less than 1: 1 particle to cell uses a ratio.

In further embodiments, the ratio of particles to cells can vary depending on the number of days of stimulation. For example, in one embodiment, the ratio of particles to cells is 1: 1 to 10: 1 on the first day, after which additional particles are added to the cells every day or every other day for up to 10 days (cells per day added). By number) in a final ratio of 1: 1 to 1:10. In other embodiments, the ratio of particles to cells is at least about 1: 2.5 on day 1 and the ratio of additional particles to cells is about 1:10, 1:25, 1:50, or 1: 100 on day 5 , At a ratio of 1:10, 1:25, 1:50, or 1: 100 on day 7, and 1:10, 1:25, 1:50, or 1: 100 on day 9. In one specific embodiment, the ratio of particles to cells is 1: 1 on the first day of stimulation and adjusted to 1: 5 on the third and fifth days of stimulation. In other embodiments, the particles are added at a final ratio of 1: 1 on the first day of stimulation on a daily or two-day basis, and at a ratio of 1: 5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, the particles are added in a final ratio of 1: 1 on the first day of stimulation on a daily or two-day basis and a final ratio of 1:10 on the third and fifth days of stimulation. Those skilled in the art will appreciate that other various ratios may be suitable for use in the present invention. In particular, the ratio will vary depending on particle size and cell size and type.

One aspect of the invention derives from the surprising findings that can result in different results for expansion of antigen-specific T cells using different bead: cell ratios. In particular, different bead: cell ratios can be used to selectively expand or delete antigen-specific (memory) T cells. In one embodiment, the specific bead: cell ratio used selectively deletes antigen-specific T cells. Specifically, high bead: cell ratios, such as about 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50 : 1 and higher ratios lead to the deletion of antigen-specific T cells. Without wishing to be bound by theory, it is believed that the antigen-specific T cells are sensitized with further stimulation. Stimulation with a high bead-to-cell ratio provides high concentrations of stimulatory antibodies to excessively stimulate antigen-specific T cells, thereby killing these cells by apoptosis or other mechanisms. Thus, in this regard, the bead compositions described herein function as apoptosis-inducing compositions. Further, in this regard, as will be appreciated by those skilled in the art, in some embodiments, the same composition used as an apoptosis-inducing composition (e.g., a surface to which a substance stimulating a cell surface residue is attached, e.g. beads described herein) Composition) to expand the remaining mixed population of cells used in any of the various immunotherapeutic situations described herein. The use of low bead: cell ratios provides a stimulus signal for antigen-specific T cells, which rather than over-stimulate rapid proliferation of the cells. In further embodiments, the specific bead: cell ratio used selectively expands antigen-specific T cells. Those skilled in the art will readily appreciate that any ratio may be used provided that the desired expansion or deletion occurs. Thus, the compositions and methods described herein can be used to expand or delete certain T cell populations for use in the various immunotherapeutic situations described herein.

Using some methods, it may be advantageous to maintain long-term stimulation of the T cell population that has undergone initial activation and stimulation by isolating T cells from the stimulus after about 7 days to about 14 days. T cell proliferation rates are monitored periodically (eg, daily), for example by examining the size of T cells or measuring the volume of these cells using a Coulter counter. In this regard, dormant T cells have an average diameter of about 6.8 microns, and in the presence of stimulatory ligands upon initial activation and stimulation, the average T cell diameter increases by more than 12 microns by day 4 and begins to decrease by approximately day 6 will be. When the average T cell diameter decreases to about 8 microns, T cells can be activated and restimulated to induce further proliferation of T cells. Alternatively, T cell proliferation rate and T cell restimulation time can be monitored by analyzing for the presence of cell surface molecules induced on activated T cells, such as CD154, CD54, CD25, CD137, CD134.

When inducing long-term stimulation of CD4 ⁺ and / or CD8 ⁺ T cell populations, stimulants such as anti-CD3 antibodies and anti-CD28 antibodies (eg B-T3, XR-CD28 (Diaclone, Besancon, France) It may be necessary to reactivate and restimulate T cells several times with)) to produce an increased number of CD4 ⁺ or CD8 ⁺ cell populations from about 10 to about 1,000 times the original T cell population. For example, in one embodiment of the invention, T cells are stimulated for 2-3 times as described. In other embodiments, T cells are stimulated for 4 or 5 times as described. Using current methods, T cell numbers with increased polyclonality of about 100 to about 100,000 times prior to stimulation can be achieved. In addition, T cells expanded by the methods of the invention secrete substantial levels of cytokines (eg, IL-2, IFN-γ, IL-4, GM-CSF and TNF-α) into the culture supernatant. For example, compared to stimulation with IL-2, CD4 ⁺ T cells expanded by the use of anti-CD3 and anti-CD28 costimulation secrete high levels of GM-CSF and TNF-α into the culture medium. Such cytokines can be purified from the culture supernatant or the supernatant can be used directly to maintain the cells in culture. Similarly, expanded T cells can be administered by the methods of the invention together with culture supernatants and cytokines to support the growth of cells in vivo.

In one embodiment, cells are stationary (hyperproliferative or non-proliferative), for example using anti-CD3 and anti-CD28 antibodies co-fixed onto beads (3x28 beads) (about 8 days to 14 days after initial stimulation) T cell stimulation is performed for a time sufficient to return to work). The stimulus signal is then removed from the cells, the cells are washed and reinjected into the patient. As demonstrated by the examples, a cell at the end of the stimulation phase is "super-induced" by the method of the present invention as evidenced by its ability to respond to antigen and the ability of the cell to demonstrate a memory-like phenotype. super-inducible) ". Thus, upon exogenous restimulation or restimulation by antigen in vivo after infusion, activated T cells demonstrate a strong response characterized by unique phenotypic properties, eg, persistent CD154 expression, increased cytokine production, and the like.

In a further embodiment of the invention, cells, such as T cells, are combined with material-coated or conjugated beads, after which the beads and cells are separated and then the cells are cultured. In other embodiments, prior to culturing, the material-coated or conjugated beads and cells are not separated, but are cultured together. In further embodiments, force is first applied to enrich the beads and cells, followed by cell surface residue ligation to induce cell stimulation and / or polarization of the activation signal.

For example, where the T cells are target cell populations, cell surface residues can be ligated by contacting paramagnetic beads (3x28 beads) with anti-CD3 and anti-CD28 antibodies attached to the prepared T cells. In one embodiment, cells (eg, 10 ⁴ to 10 ⁹ T cells) and beads (eg, Dynabiz® M-450 CD3 / CD28 T paramagnetic beads, 1: 1 ratio) are buffered, Preferably in PBS (without divalent cations such as calcium and magnesium). It will also be apparent to those skilled in the art that any cell concentration may be used. For example, target cells may be very rarely present in a sample to constitute only 0.01% of the sample or the entire sample (ie 100%) may comprise the target target cell. Therefore, any cell number is within the scope of the present invention. In certain embodiments, it may be desirable to significantly reduce (ie, increase cell concentration) the volume in which the particles and cells are mixed together to achieve maximum contact of the cells and particles. For example, in one embodiment, a concentration of about 2 billion cells per ml is used. In other embodiments, more than 100 million cells per ml are used. In further embodiments, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells per ml are used. In another embodiment, concentrations of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells per ml are used. In further embodiments, concentrations of 125 million or 150 million cells can be used per ml. High concentrations can be used to indicate increased cell yield, cell activation and cell expansion. In addition, high cell concentrations can be used to more effectively capture cells capable of weakly expressing target antigens, such as CD28-negative T cells. Such cell populations may have therapeutic value and would be desirable to obtain. For example, high cell concentrations are used to more effectively select CD8 ⁺ T cells that normally exhibit weaker CD28 expression.

In related embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and particles, the interaction between the particles and the cells is minimized. Accordingly, cells that express high amounts of the desired antigen to be bound to the particles are selected. For example, CD4 ⁺ T cells express higher levels of CD28 and capture and stimulate more effectively than diluted concentrations of CD8 ⁺ T cells. In one embodiment, the cell concentration used is about 5 × 10 ⁶ / ml. In other embodiments, the concentration used may be between about 1 × 10 ⁵ / ml and about 1 × 10 ⁶ / ml, and any integer value between these ranges.

The buffer in which the cells are suspended can be any that is appropriate for the particular cell type. When using any cell type, the buffer may contain other ingredients necessary to maintain cell integrity during the process, such as 1-5% serum. In other embodiments, cells and beads can be combined in cell culture medium. The cells and beads can be mixed for a period of time ranging from 1 minute to several hours, for example by any means for rotation, shaking, or mixing. The containers of beads and cells are then concentrated by force, for example by placing them in a magnetic field. The medium and unbound cells are removed and the cells attached to the beads or other surface are pumped, for example, washed through a peristaltic pump and then resuspended in a medium suitable for cell culture.

In one embodiment of the invention, the mixture may be incubated for 30 minutes to several hours (about 3 hours) to about 14 days, or for any time or minute that is an integer value between these ranges. In other embodiments, the mixture may be incubated for 21 days. In one embodiment of the invention, the beads and T cells are incubated together for about 8 days. In other embodiments, the beads and T cells are incubated together for 2 to 3 days. As described above, several stimulation cycles may be desirable to allow the incubation time of T cells to be greater than 60 days. Conditions suitable for T cell culture include proliferation and viability, including serum (eg, fetal bovine or human serum) or interleukin-2 (IL-2), insulin, or any other additive for the growth of cells known to those skilled in the art. Appropriate media (eg, Minimal Essential Media or RPMI media 1640 or X-vivo 15 (BioWhittaker)) which may contain the necessary factors are mentioned. The medium is supplemented with amino acids and vitamins, supplemented with no serum or an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine (s) necessary for the growth and expansion of T cells. RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20. Antibiotics such as penicillin and streptomycin are included in the experimental culture, but not in the culture of cells to be injected into the subject. Target cells are maintained under the conditions necessary to support growth, for example, at an appropriate temperature (eg 37 ° C.) and at atmospheric pressure (eg air + 5% CO ₂ ).

In one embodiment of the invention, the bead: cell ratio can be adjusted to obtain the desired T cell phenotype. In one particular embodiment, the bead: cell ratio can be varied to selectively expand or delete antigen-specific (memory) T cells. In one embodiment, the specific bead: cell ratio used selectively deletes antigen-specific T cells. In further embodiments, the specific bead: cell ratio used selectively expands antigen-specific T cells. Those skilled in the art will readily appreciate that any ratio can be used as long as the desired expansion or deletion of antigen-specific T cells occurs. Thus, the compositions and methods described herein can be used to expand or delete a specific population of T cells for use in the various immunotherapeutic situations described herein.

In other embodiments, the exposure time for stimulants, eg, anti-CD3 / anti-CD28 (ie, 3 × 28) -coated beads, can be altered or adjusted in a manner to obtain the desired T cell phenotype. Alternatively, any number of selection techniques can be used to select a desired population of T cells prior to stimulation. Since expansion of T _H cells may enhance or repair overall immune responsiveness, a larger population of helper T cells (T _H ), typically CD4 ⁺ , as opposed to CD8 ⁺ cytotoxic or regulatory T cells would be desirable. Can be. Although many specific immune responses are mediated by CD8 ⁺ antigen-specific T cells that can directly lyse or kill target cells, most immune responses are important immune-modulatory molecules, such as GM-CSF, CD40L and IL. Requires help of CD4 ⁺ T cells expressing -2. If CD4-mediated help is desired, methods such as those described herein that preserve or enhance the CD4: CD8 ratio can be quite beneficial. An increased number of CD4 ⁺ T cells can increase the amount of cell-expressed CD40L introduced into a patient, potentially improving target cell visibility (improving APC function). Similar effects can be seen by increasing the number of injected cells expressing GM-CSF or IL-2, which are mainly expressed by CD4 ⁺ T cells. Alternatively, in situations where less CD4-help is needed and an increased number of CD8 ⁺ T cells is required, for example, XCELLERATE described herein, by pre-selecting CD8 ⁺ cells prior to stimulation and / or culture. Trademark) method. This situation may be present where increased levels of IFN- [gamma] or increased cytolysis of target cells are desired. For example, by altering the time and type of exposure to stimulants, T cells with the desired TCR repertoires expressing the desired Vβ family genes can be expanded.

To achieve isolation of different T cell populations, the exposure time to the particles can be varied. For example, in one preferred embodiment, the T cells are isolated by incubating the T cells with 3 × 28 beads, eg, Dynabiz® M-450, for a time sufficient for positive selection of the desired T cells. In one embodiment, the time is about 30 minutes. In other embodiments, the time is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. In another preferred embodiment, the time is 10 hours to 24 hours, or longer. In one preferred embodiment, the incubation time is 24 hours. When isolating T cells from cancer patients, longer incubation times, such as 24 hours, can be used to increase cell yield.

In certain embodiments, the stimulation and / or extension time can be 10 weeks or less, 8 weeks or less, 4 weeks or less, 2 weeks or less, 10 days or less, or 8 days or less (the range of 4 weeks or less is 4 weeks or less). In any time range up to 1 day (24 hours) or any value between these ranges). In certain embodiments, it may be desirable to clone T cells using, for example, limited dilution or cell sorting, which may require longer stimulation times. In some embodiments, stimulation and expansion may be performed for up to 6 days, up to 4 days, up to 2 days, and in other embodiments for up to 24 hours, preferably for as little as 4 hours to 6 hours or less. (The range includes any integer value between these ranges). If T cell stimulation is performed for a shorter time, the T cell population may not increase significantly, but this population can continue to proliferate in vivo and more closely resemble a natural effector T cell pool, Will provide stronger and healthier activated T cells. Because the availability of T cell help is often a limiting factor in response to protein antigens, the ability to selectively expand or selectively inject into a CD4 ⁺ rich population of T cells is very useful. A further advantage of this rich population is that activated helper T cells that recognize antigens presented by B lymphocytes carry two types of stimulation, physical contact and cytokine production resulting in the proliferation and differentiation of B cells. Obvious.

In various embodiments, those skilled in the art will understand that the removal of stimulation signals from cells depends on the type of surface used. For example, when using paramagnetic beads, magnetic separation is a possible option. Separation techniques are described in detail in the paramagnetic bead manufacturer's instructions (e.g., DYNAL Inc., Oslo, Norway). It is also possible to use filtration which is beads large enough for the surface to separate from the cells. In addition, various injection filters are commercially available, including 20 micron and 80 micron injection filters (Baxter). Thus, if the beads are larger than the mesh size of the filter, this filtration is highly effective. In related embodiments, the beads may pass through a filter, but cells may remain, thus allowing separation. In one particular embodiment, the biocompatible surface used may degrade (ie, biocompatible) during incubation during the exposure period.

The antibodies used in the methods described herein are readily available from published sources, such as the American Type Culture Collection (ATCC), but antibodies against T cell accessory molecules and CD3 complexes can be prepared by standard techniques. Methods of producing antibodies for use in the methods of the invention are well known in the art and are discussed in further detail below.

Ligand fixation on the surface

As indicated above, the method of the present invention preferably uses a ligand bound to the surface. The surface can be any surface to which a ligand can bind or integrate, and is biocompatible, ie substantially nontoxic to the target cell to be stimulated. The biocompatible surface is biodegradable or not biodegradable. The surface may be natural or synthetic, and the synthetic surface may be a polymer. Surfaces can include collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, extracellular interstitial compositions, liposomes, or cell surfaces. Examples of the polysaccharides include cellulose, agarose, dextran, chitosan, hyaluronic acid, or alginate. Other polymers include polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylenes, polytetrafluoroethylene (PTFE) Or polyurethane. The polymer may be lactic acid or a copolymer. Copolymers may include lactic acid and glycolic acid (PLGA). Non-biodegradable surfaces can include polymers such as poly (dimethylsiloxane) and poly (ethylene-vinyl acetate). Biocompatible surfaces include, for example, glass (e.g., raw glass), collagen, chitin, metals, hydroxyapatite, aluminate, bioceramic materials, hyaluronic acid polymers, alginates, acrylic ester polymers, lactic acid polymers, glycolic acid Polymers, lactic / glycolic acid polymers, purified proteins, purified peptides, or extracellular interstitial compositions. Other polymers including the surface include glass, silica, silicon, hydroxyapatite, hydrogels, collagen, acrolein, polyacrylamide, polypropylene, polystyrene, nylon, or any number of plastic or synthetic organic polymers. have. Surfaces can include biological structures, such as liposomes or cell surfaces. The surface may be in the form of lipids, plates, bags, pellets, fibers, meshes or particles. The particles can include colloidal particles, microspheres, nanoparticles or beads, and the like. In various embodiments, commercially available surfaces, such as beads or other particles, are useful (eg, Miltenyi Biotec, Germany; Sepharose Beads (Pharmacia Fine Chemicals, Sweden); Dynabiz®) Dynal Inc., New York); Puraviz® (Prometic Biosciences).

When using beads, the beads can be of any size to perform target cell stimulation. In one embodiment, the size of the beads is preferably in the range of about 5 nm to about 500 μm. Thus, the choice of bead size depends on the particular application for which the beads will function. For example, when using beads in monocyte depletion, small sizes (eg 1.0 μm and 4.5 μm diameters or any size that can be enclosed, eg nanometer size) are selected to facilitate monocyte absorption. ; If separation of beads by filtration is desired, bead sizes of 50 μm or greater are commonly used. In addition, when using paramagnetic beads, the beads typically range in size from about 2.8 μm to about 500 μm, more preferably from about 2.8 μm to about 50 μm. Finally, one may choose to use superparamagnetic nanoparticles that can be as small as about 10 ^-5 nm. Thus, as is clear from the above discussion, virtually any particle size can be used.

The material may be attached, incorporated, coupled or integrated to the surface by a variety of available methods known in the art. The substance may be a natural ligand, a protein ligand or a synthetic ligand. Attachment can be covalent or non-covalent, electrostatic or hydrophobic attachment, and can be achieved by various methods of attachment, including, for example, chemical, mechanical, enzymatic, electrostatic or other means by which ligands can stimulate cells. . Attachment of the material may be direct or indirect (eg, tethered). For example, first attaching an antibody against a ligand to the surface (direct attachment), or avidin or streptavidin, or attaching a second antibody to the surface for binding with a biotinylated ligand (Indirect attachment). To the cell surface, attachment may be through gene expression of a substance using any number of techniques known in the art, for example, transfection or transduction of an expression vector comprising a coding region of a subject of interest. . Antibodies to the ligands can be attached to the surface via anti-idiotype antibodies. Other examples include using Protein A or Protein G, or other non-specific antibody binding molecule, attached to a surface that binds the antibody. Alternatively, the ligand can be attached to the surface by chemical or other means, such as crosslinking to the surface using commercially available crosslinking reagents (Pierce, Rockford, IL). In some embodiments, the ligand is covalently bound to the surface. In addition, in one embodiment, a commercially available tosyl-activated Dynabiz® or Dynabiz® having an epoxy-surface reactor is incubated with the subject polypeptide ligand according to the manufacturer's instructions. In sum, such conditions typically include incubation in a phosphate buffer at pH 4 to pH 9.5 at a temperature in the range of 4 to 37 ° C.

In one aspect, the substance, for example some ligands, may be of single or multiple origin and may be antibodies or fragments thereof, while in other aspects the costimulatory ligands may be B7 molecules (eg, B7-) when using T cells. 1, B7-2). This ligand is coupled to the surface by any of the different attachment means discussed above. The B7 molecule to be coupled to the surface can be isolated from cells expressing the costimulatory molecule or obtained using standard recombinant DNA techniques and expression systems that allow for the preparation and isolation of the costimulatory molecule (s) as described herein. have. Fragments, mutants or variants of the B7 molecule may also be used that retain the ability to initiate costimulatory signals in T cells when coupled to the surface of the cell. Those skilled in the art will also recognize that any ligand useful for activating and inducing proliferation of a subset of T cells may be immobilized on the bead or culture vessel surface or on any surface. In addition, covalent bonding of the ligand with the surface is one preferred method, but absorption and capture by the second monoclonal antibody can also be used. The amount of specific ligand attached to the surface is readily determined by flow cytometry analysis if the surface is a bead surface or by enzyme linked immunosorbent assay (ELISA) for the surface, for example tissue culture dishes, meshes, fibers, whites. You can decide.

In certain embodiments, the stimulatory form of the B7 molecule or anti-CD28 antibody or fragment thereof is attached to the same solid surface as the substance that stimulates the TCR / CD3 complex, eg, an anti-CD3 antibody. In addition to anti-CD3 antibodies, other antibodies that bind to receptors that mimic antigen signals can be used. For example, the beads or other surfaces can be coated with a combination of anti-CD2 antibodies and B7 molecules, in particular anti-CD3 antibodies and anti-CD28 antibodies.

When coupling to a surface, the material can be coupled to the same surface (ie, "cis" form) or to separate surfaces (ie, "trans" form). Alternatively, one material is coupled to the surface and the other material is placed in solution. In one embodiment, the substance providing the costimulatory signal is bound to the cell surface and the substance providing the primary activation signal is placed in solution or coupled to the surface. In a preferred embodiment, both materials are fixed to the same beads (ie "cis") or separate beads (ie "trans"). For example, a substance that provides a primary activation signal is an anti-CD3 antibody, and a substance that provides a co-stimulatory signal is an anti-CD28 antibody and co-fixes these two substances at equivalent molecular weight to the same beads. In one embodiment, a 1: 1 ratio of each antibody bound to the beads is used for CD4 ⁺ T cell expansion and T cell growth. In some aspects of the invention, the ratio of anti-CD3: CD28 antibody bound to the beads is the rate at which T cell expansion is observed to increase relative to expansion observed using the 1: 1 ratio. In one particular embodiment, an increase of about 0.5 to about 3 times is observed relative to the expansion observed using a ratio of 1: 1. In one embodiment, the ratio of CD3: CD28 antibody bound to the beads ranges from 100: 1 to 1: 100 and all integer values therebetween. In one aspect of the invention, more anti-CD28 antibodies bind to the particles than the anti-CD3 antibodies, ie the CD3: CD28 ratio is less than one. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2: 1. In one specific embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1: 200. In one specific embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1: 150. In one specific embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1: 100. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:75. In a further embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:50. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads uses 1:45. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:40. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:35. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:30. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads uses 1:25. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:20. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:15. In one preferred embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1:10. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads uses 1: 5. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 1: 4. In other embodiments, the CD3: CD28 ratio of the antibody bound to the beads uses 1: 3. In another embodiment, the CD3: CD28 ratio of the antibody bound to the beads uses 3: 1.

Surface-bound material

Substances contemplated in the present invention include protein ligands, natural ligands and synthetic ligands. Materials that can cause signaling by binding to cell surface residues to trigger ligation and aggregation under certain conditions include lectins (eg, phytohemagglutinin (PHA), lentil lectins, concanavalin A), antibodies , Antibody fragments, peptides, polypeptides, glycopeptides, receptors, B cell receptors and T cell receptor ligands, MHC-peptide dimers or tetramers, extracellular epilepsy components, steroids, hormones (eg, growth hormones, corticosteroids, Prostaglandins, tetra-iodo tyronine), bacterial residues (e.g. lipopolysaccharides), cleavage promoters, superantigens and derivatives thereof, growth factors, cytokines, adhesion molecules (e.g. L-selectin, LFA-3, CD54) , LFA-1), chemokines, and small molecules, but are not limited thereto. Such materials may be isolated from natural sources such as cells, blood products, and tissues by chemical synthesis or other methods known to those skilled in the art, or from cells that have been recombinantly produced and expanded in vitro.

In one aspect of the invention, where it is desirable to stimulate T cells, useful materials include ligands that can bind to CD3 / TCR complex, CD2, and / or CD28 and initiate activation or proliferation respectively. have. Thus, the term “ligand” includes proteins that are “natural” ligands for cell surface proteins, eg, B7 molecules for CD28, and antibodies that are directed against artificial ligands, eg, cell surface proteins. Such antibodies and fragments thereof can be prepared according to conventional techniques such as hybridoma methods and recombinant DNA and protein expression techniques. Useful antibodies and fragments can be derived from any species, including humans, and can be prepared as chimeric proteins using sequences from more than one species.

Methods well known in the art can be used to generate antibodies, polyclonal antisera, or monoclonal antibodies specific for a ligand. The antibody may also be prepared from genetically engineered immunoglobulins (Ig) or Ig fragments designed to have desirable properties. For example, and not by way of limitation, the antibody 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 different second mammalian species. Phosphorus recombinant IgG. Most commonly, chimeric antibodies have murine variable region sequences and human constant region sequences. The murine / human chimeric immunoglobulin implants complementarity determining regions (CDRs) that confer binding specificity for antigens derived from murine antibodies to human-derived V region framework regions and human-derived constant regions By being "humanized". In addition, antibodies containing CDRs of different specificity can be combined to generate multi-specific (such as bi- or tri-specific) antibodies. Fragments of such molecules may be generated by proteolytic cleavage, or optionally proteolytic cleavage, followed by mild reduction and alkylation of disulfide bonds or by recombinant genetic engineering techniques.

An antibody has an affinity constant (K _a ) greater than or equal to about 10 ⁴ M ⁻¹ or equivalent to an antigen, preferably an affinity constant greater than or equal to about 10 ⁵ M ⁻¹ , more preferably about “Immunospecific” when specifically binding to an affinity constant of greater than or equal to 10 ⁶ M ⁻¹ , or even more preferably greater than or equal to about 10 ⁷ M ⁻¹ or equivalent Is defined as The affinity of the binding partner or antibody is known in conventional techniques, such as those described in Scatchard et al. (Ann. NY Acad. Sci. USA 51: 660, 1949) or surface plasmon resonance (BIAcore, Biosensor, Piscataway). , NJ) (see, eg, Wolff et al., Cancer Res., 53: 2560-2565, 1993).

Antibodies can generally be prepared by any of a variety of techniques known to those of skill in the art (see, eg, Harlow et al., Antibodies: A Laboratory Manual, 1998, Cold Spring Harbor Laboratory). In one such technique, animals are immunized with a ligand that is an antigen to produce polyclonal antiserum. Suitable animals include rabbits, sheep, goats, pigs, cows, and smaller mammalian species such as mice, rats and hamsters. Antibodies of the invention are produced as described in US Pat. Nos. 6,150,584, 6,130,364, 6,114,598, 5,833,985, 6,071,517, 5,756,096, 5,736,137 and 5,837,243. You can also

An immunogen can be included in a cell expressing a ligand, a purified or partially purified ligand polypeptide or a variant or fragment thereof, or a ligand peptide. Ligand peptides can be produced by proteolytic cleavage or chemically synthesized. Peptides for immunization can be selected by analyzing the primary, secondary or tertiary structure of the ligand according to methods known to those skilled in the art to determine amino acid sequences that are more likely to produce antigenic peptides in the host animal. (See, eg, Novotny, Mol. Immunol. 28: 201-207, 1991; Berzoksky, Science 229: 932-40, 1985).

Immunogen preparation can include covalently coupling a ligand polypeptide or variant or fragment thereof, or peptide to another immunogenic protein, such as keyhole limpet hemocyanin or bovine serum albumin. In addition, peptides, polypeptides or cells can be emulsified in adjuvants (see Harlow et al., Antibodies: A Laboratory Manual, 1988 Cold Spring Harbor Laboratory). Generally, after the first injection, the animal is administered one or more booster immunizations according to the desired schedule for the animal species. The immune response can be monitored by periodically bleeding the animal, separating the serum, and analyzing the serum by immunoassay, eg, Ouchterlony assay, to assess specific antibody titers. Once antibody titers are established, animals can be periodically bled to accumulate polyclonal antisera. The polyclonal antibody that specifically binds to the ligand polypeptide or peptide is then purified from the antiserum by, for example, affinity chromatography using Protein A or using a ligand polypeptide or peptide coupled to a suitable solid support. can do.

Monoclonal antibodies that specifically bind a ligand polypeptide or fragment or variant thereof are described, for example, in Kohler and Milstein (Nature, 256: 495-497, 1975; Eur. J. Immunol. 6: 511-519, 1976) and improved techniques thereof. Hybridomas, which are immortal eukaryotic cell lines, that produce antibodies with the desired specificity for ligand polypeptides or variants or fragments thereof can be generated. Animals such as rats, hamsters, or preferably mice are immunized with ligand immunogens prepared as described above. Lymphoid cells, most commonly spleen cells, obtained from an immunized animal can be immortalized by fusing with a drug-sensitized myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. Splenocytes and myeloma cells can be combined for several minutes with a membrane fusion promoter such as polyethylene glycol or nonionic detergent, and then placed on a selection medium that supports the growth of hybridoma cells but not the growth of myeloma cells. Plate at low density. Preferred selection media is HAT (hypoxanthine, aminopterin, thymidine). After sufficient time, usually about 1 to 2 weeks, cell colonies are observed. Single colonies can be isolated and antibodies produced by the cells can be tested for binding activity with ligand polypeptides or variants or fragments thereof. Hybridomas that produce antibodies with high affinity and specificity for ligand antigens are preferred. Hybridomas that produce monoclonal antibodies that specifically bind to ligand polypeptides or variants or fragments thereof are included in the present invention.

Monoclonal antibodies can be isolated from the supernatants of hybridoma cultures. Another method of producing murine monoclonal antibodies is to inject the hybridoma cells into the abdominal cavity of syngeneic mice. Mice produce ascites fluids containing monoclonal antibodies. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation or extraction.

Human monoclonal antibodies can be produced by any number of techniques. Such methods include Epstein Barr virus (EBV) transformation of human peripheral blood cells (see US Pat. No. 4,464,456), in vitro immunization of human B cells (e.g., Boerner et al., J 147: 86-95, 1991)), fusion of spleen cells from immunized transgenic mice carrying a human immunoglobulin gene and carrying an immunoglobulin gene inserted by a yeast artificial chromosome (YAC) Fusion of spleen cells from immunized transgenic mice (see, eg, US Pat. No. 5,877,397; Bruggemann et al., Curr. Opin. Biotechnol. 8: 455-58, 1997; Jakobovits et al., Ann NY Acad. Sci. 764: 525-35, 1995), or isolation from human immunoglobulin V region phage libraries.

Chimeric antibodies and humanized antibodies used in the present invention can be produced. Chimeric antibodies have one or more constant region domains derived from a first mammalian species and one or more variable region domains derived from a different second mammalian species (eg, Morrison et al., Proc. Natl. Acad Sci. USA, 81: 6851-55, 1984). Most commonly, a polynucleotide sequence encoding one or more variable region domains derived from non-human monoclonal antibodies, eg, a variable region derived from a murine, rat or hamster monoclonal antibody, comprises one or more human constant regions. Chimeric antibodies can be prepared by cloning in a vector containing the coding sequence (see, eg, Shin et al., Methods Enzymol. 178: 459-76, 1989; Walls et al., Nucleic Acids Res. 21). : 2921-29, 1993). The human constant region selected may depend on the effector function desired for the particular antibody. Another method known in the art for the production of chimeric antibodies is homologous recombination (US Pat. No. 5,482,856). Preferably, the vector will transfect eukaryotic cells for stable expression of chimeric antibodies.

Non-human / human chimeric antibodies can be further genetically engineered to produce “humanized” antibodies. Such antibodies have a plurality of CDRs derived from immunoglobulins of a non-human mammal species, one or more human variable framework regions, and one or more human immunoglobulin constant regions. By humanization, antibodies with reduced binding affinity can be obtained as compared to non-human monoclonal antibodies or chimeric antibodies. Thus, one of ordinary skill in the art designs one or more strategies using humanized antibodies.

In certain embodiments, it may be desirable to use antigen-binding fragments of antibodies. Such fragments include Fab fragments or F (ab ') ₂ fragments, which may be prepared by proteolytic cleavage with papain or pepsin, respectively. Antigen binding fragments can be separated from Fc fragments by affinity chromatography, for example using immobilized Protein A or immobilized ligand polypeptide or variants or fragments thereof. Other methods of generating Fab fragments include mild reduction of F (ab ′) ₂ fragments followed by alkylation (see, eg, Weir, Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston).

Non-human, human, or humanized heavy and light chain variable regions of any of the Ig molecules described above can be prepared with single chain Fv (sFv) fragments (single chain antibodies) (see, eg, Bird et al., Science 242: 423). -426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). Polynucleotide sequences encoding sFv in-frame can be linked with polynucleotide sequences encoding various effector proteins to produce multifunctional fusion proteins. Such methods are known in the art and are described, for example, in EP-B1-0318554, US Pat. No. 5,132,405, 5,091,513, and 5,476,786.

An additional method of selecting antibodies that specifically bind a ligand polypeptide or variant or fragment thereof is by phage display (see, eg, Winter et al., Annul. Rev. Immunol. 12: 433-55). , 1994; Burton et al., Adv. Immunol. 57: 191-280, 1994). Human or murine immunoglobulin variable region gene combination libraries can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to ligand polypeptides or variants or fragments thereof. In phage vectors (see, eg, US Pat. No. 5,223,409; Huse et al., Science 246: 1275-81, 1989; Kang et al., Proc. Natl. Acad. Sci. USA 88: 4363-66, 1991; Hoogenboom et al., J. Molec. Biol. 227: 381-388, 1992; Schlebusch et al., Hybridoma 16: 47-52, 1997) and references cited therein. ).

How to use

In general, the compositions and methods described herein can be used to remove at least a portion of an undesirable clonal population of cells, typically T cells, B cells, NKTs, or NK cells from a population of immune cells. In addition, the present invention provides a composition and use thereof comprising a cell population which no longer contains undesirable cells or which has a significantly reduced number of undesirable cells. In addition, using the compositions and methods of the present invention, hematopoietic stem cell transplantation (including allografts and autografts from sources including blood, umbilical cord blood and bone marrow), organ transplantation (eg, acute or chronic GVHD) For use in the treatment of immune defects and autoimmune diseases, including autoimmune diseases caused by cancer or by a variety of common immunodeficiencies, such as giant granular lymphocyte (LGL) leukemia, chronic lymphocytic leukemia (CLL) The clonal population, which does not, selectively expands the cell population that is deleted. As a result, for T cells, a cell population that expresses TCR polyclonal for antigen reactivity but essentially homogeneous for CD4 ⁺ or CD8 ⁺ is not desirable for any of cells such as autoreactive cells or alloreactive cells. Can be prepared by removing unpopulated subpopulations. For B cells, one can create a cell population in which any undesirable subpopulations of B cells that produce autoreactive antibodies have been removed. In addition, by this method, the resulting population of T- or B- cells is expanded to a number sufficient to reconstruct the entire CD4 ⁺ or CD8 ⁺ T cell population or B cell population of the individual ( About 5 X 10 ¹¹ pieces). The resulting cell populations are also known to those skilled in the art and may be genetically transduced using various techniques used in immunotherapy.

In one embodiment, T or B cell compositions of the invention can be used in the context of hematopoietic stem cell transplantation. A major problem in hematopoietic stem cell transplantation is graft-versus-host disease (GVHD) caused by alloreactive T cells present in the injected hematopoietic stem cell sample. Thus, the present invention can be used to remove alloreactive T cells and to expand the remaining T cell population to infusion into the patient. The cell composition of the present invention may be used alone or in combination with other therapies.

In one embodiment, the T or B cell compositions of the invention can be used in the context of any autoimmune disease. Exemplary autoimmune diseases include systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis, progressive systemic sclerosis, Sjogren's syndrome, multiple sclerosis, multiple myositis, dermatitis, uveitis, arthritis, type I insulin-dependent Diabetes, Hashimoto thyroiditis, Grave thyroiditis, Myasthenia gravis, Autoimmune myocarditis, Vasculitis, Aplastic anemia, Autoimmune hemolytic anemia, Myelodysplastic syndrome, Evan syndrome, Stiff-person syndrome, Atopic dermatitis, Psoriasis, Behchet's syndrome, Crohn's disease, biliary cirrhosis, inflammatory bowel disease, ulcerative colitis, firmfaster syndrome, Wegner's granulomatosis, paroxysmal nocturnal hematuria, myelodysplastic syndrome, allergic diseases such as hay fever, Exogenous asthma, or insect bite and stagnation allergies, and food and drug allergies.

Other uses of the T and B cell compositions of the present invention include skin symptoms of inflammatory and hyperproliferative dermatosis and immunologically mediated diseases such as seborrheic dermatitis, angioedema, erythema, acne, and alopecia areata; Various eye diseases (autoimmune and others); Allergic reactions, eg pollen allergy, asthma (eg bronchial asthma, allergic asthma, endogenous asthma, exogenous asthma and dust asthma), especially chronic or refractory asthma (eg terminal asthma and airway hyper- Reversible obstructive airway disease, including symptoms such as reactivity), bronchitis and allergic rhinitis; Treatment and / or prevention of inflammation of mucous membranes and blood vessels.

As mentioned above, the T and B cell compositions of the invention can be used for the treatment of immune deficiencies associated with organ transplantation, such as host versus graft disease. Treatment of immune defects associated with any organ transplant is contemplated herein. For example, the methods and cells of the present invention can be used for the treatment of immune defects associated with kidney, heart, lung, and liver transplantation.

In certain embodiments of the invention, chemotherapy, radiation, immunosuppressants such as cyclosporin, azithioprine, methotrexate, mycophenolate, and FK506, an antibody, or other immunooablative agent, such as camphor Administration of cells of the invention to patients treated with radiation and substances such as anti-CD3 antibody, cyclophosphamide, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228 do. These drugs inhibit calcium dependent phosphatase calcineurin (cyclosporin and FK506) or inhibit p70S6 kinase important for signaling by growth factor induction (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993; Isoniemi (supra)). In other embodiments, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or campath is performed. The cell composition of the present invention is administered to a patient suffering from an autoimmune disease. In another embodiment, the cell composition of the invention is administered to a patient suffering from an autoimmune disease that has undergone a B-cell ablation therapy, such as Rituxan, which reacts with CD20. The dosage of the therapeutic agent to be administered to the patient will depend on the exact nature of the condition to be treated and the treatment recipient. Scaling of the dosage for human administration can be performed according to the practice to be accepted in the prior art. The dosage of campath is generally in the range of 1 mg to about 100 mg, for example in adult patients, and will usually be administered daily for a period of 1 day to 30 days. Preferred daily dosages are from 1 mg to 10 mg per day, but in some cases more dosages up to 40 mg per day can be used (as described in US Pat. No. 6,120,766).

In a further aspect of the invention, at least a substantial portion of autoreactive cells are removed from the patient in vitro using the methods of the invention, followed by further stimulation and expansion to administer to the patient. In related embodiments, the methods of the invention are used to remove at least a substantial portion of autoreactive cells from a patient in vitro and then administer to the patient to expand in vivo. It is contemplated in one aspect that the compositions of the present invention can be used with other therapies available in the art for the treatment of autoimmune diseases.

In one embodiment, T cells are stimulated and expanded as described herein to induce or enhance responsiveness in immunocompromised individuals as a result of treatment associated with hematopoietic stem cell transplantation. The present invention provides a method for reducing the risk or severity of GVHD adverse effects in a patient comprising administering a T cell of the invention to a patient performing hematopoietic stem cell transplantation. In one specific embodiment, at least a substantial portion of the alloreactive cells present in the donor hematopoietic stem cells are removed by the method of the present invention. In another embodiment, a T cell composition of the invention is administered to a patient who has undergone hematopoietic stem cell transplantation after treatment with a chemotherapeutic agent. In another embodiment, the method of the invention is used to remove at least a substantial portion of alloreactive cells from bone marrow in vitro, and then further stimulated and expanded, and then administered to the patient. In a further embodiment, the methods of the invention are used to remove at least a substantial portion of alloreactive cells from bone marrow and then administer to the patient to expand in vivo. The compositions of the present invention are administered in the art for use in hematopoietic stem cell transplantation such as administration of G-CSF, IL-2, IL-11, IL-7, IL-12 and antiviral treatment In one aspect, it can be used with.

In one embodiment, T cells are stimulated and expanded as described herein to induce reactivity in an immunocompromised individual as a result of treatment associated with organ transplantation including but not limited to kidney, heart, lung and liver transplantation, or You can increase it. In one particular embodiment, at least a substantial portion of the alloreactive cells present in the recipient are removed by the method of the present invention. Accordingly, the present invention provides a method of reducing the risk or severity of organ rejection. In a further embodiment, the T cell composition of the invention is administered to a patient who has undergone organ transplantation after treatment with a chemotherapeutic agent. In a further embodiment, the method of the invention is used to remove at least a substantial portion of the alloreactive cells from the transplant recipient in vitro, then further stimulated and expanded and then administered to the patient. It is contemplated in one aspect that the compositions of the present invention can be used in conjunction with other therapies available in the art for use in organ transplantation.

Another embodiment of the invention provides a method of selectively expanding a population of T _H1 cells from a CD4 ⁺ T cell population. In this method, CD4 ⁺ T cells are costimulated with an anti-CD28 antibody, eg, monoclonal antibody 9.3, to induce secretion of T _H1 -specific cytokines, including IFN-γ, to T compared to T _H2 cells. Enrich _H1 cells.

The invention further provides a method of selectively expanding the T _H2 cell population from the CD4 ⁺ T cell population. In this method, CD4 ⁺ T cells are co-stimulated with anti-CD28 antibodies, such as monoclonal antibodies B-T3, XR-CD28 to induce the secretion of T _H2 -specific cytokines, thereby inducing T compared to T _H1 cells. Enrich _H2 cells (see, eg, Fowler, et al. Blood 1994 Nov 15; 84 (10): 3540-9; Cohen, et al., Ciba Found Symp 1994; 187: 179-93) .

The invention further provides a method of using the cell composition of the invention with regulatory T cells. See, eg, Woo, et al., J Immunol. 2002 May 1; 168 (9): 4272-6; Shevach, EM, Annu. Rev. Immunol. 2000, 18: 423; Stephens, et al., Eur. J. Immunol. 2001, 31: 1247; Salomon, et al, Immunity 2000, 12: 431; and Sakaguchi, et al., Immunol. Rev. 2001, 182: 18). Techniques can be used to generate regulatory T cells.

The invention further provides a method of selectively expanding a T cell population expressing a particular Vβ, Vα, Vγ or Vδ gene. For example, in this method, T cells expressing certain Vβ, Vα, Vγ or Vδ genes are positively or negatively selected and then further expanded / stimulated according to the method of the present invention. Alternatively, stimuli and expanded T cells expressing certain subject Vβ, Vα, Vγ or Vδ genes may be selected for positive or negative selection to further stimulate and expand.

In another example, the sensitizing composition and / or two or more immobilizations for stimulating a receptor required for T cell activation prior to administering cells (eg, immobilized on a plastic surface or separable microparticles) to the subject Blood is drawn directly from a patient containing a loaded antibody (eg, anti-CD3 and anti-CD28) or other components into a stand-alone disposable device. In one embodiment, the disposable device can include a container (eg, a plastic bag or flask) with suitable tubing connections suitable for combining / dock with a syringe and a sterile docking device. This device will contain a solid surface for fixation of T cell activating components (eg, anti-CD3 and anti-CD28 antibodies); May be the container itself or the surface of the insert, typically a flat surface, etched flat surface, irregular surface, porous pad, filter, clinically acceptable / safe ferro-fluid, beads, etc.) . In addition, when using a stand-alone device, the subject may be connected to the device or the device may be detachable from the patient. In addition, the device can be used at room temperature or incubated at physiological temperature using a portable incubator.

Because devices and methods for the collection and processing of blood and blood products are well known, those of ordinary skill in the art can readily design various devices that meet the needs described above or modify existing devices in view of the teachings provided herein. Will be easily recognized. Thus, such devices and methods are not limited to the specific embodiments described herein and include any device or method that can maintain sterility and maintain blood in fluid form with reduced complement activation, wherein T cell activation Components necessary for (eg, anti-CD3 and anti-CD28 antibodies or ligands thereof) may be immobilized or isolated from blood or blood products prior to administration to a subject. In addition, those skilled in the art will readily appreciate that various blood products can be used with the methods and devices described herein. For example, a method and apparatus for providing rapid activation of T cells from cryopreserved whole blood, peripheral blood mononuclear cells, other cryopreserved blood-derived cells, or cryopreserved T cell lines, upon thawing and prior to subject administration. Can be used. In another example, methods and apparatus can be used to enhance the activity of a previously expanded ex vivo T cell product or T cell line prior to administration to a subject, thus providing a highly activated T cell product. Finally, as can be readily appreciated, the methods and devices can be used in autologous or allogeneic cell therapy simultaneously with subjects and donors.

In addition, the methods of the present invention may be used in conjunction with vaccines to enhance the reactivity and in vivo effects of antigens. In one embodiment, a composition of the invention is combined with a composition that enhances T cells in vivo, such as IL-2, IL-4, IL-7, IL-10, IL-12, and IL-15 To the patient. Further, given that the T cells expanded by the present invention have a relatively long half-life in the body, these cells carry a target nucleic acid sequence of interest and are potentially complete for gene therapy by homing at the site of cancer, disease or infection. Can act as a carrier. Thus, cells expanded by the present invention can be delivered to a patient in combination with a vaccine, one or more cytokines, one or more therapeutic antibodies, and the like. Indeed, any therapy that benefits from a stronger T cell population is within the scope of the methods of use described herein.

There are various in vitro animal models for testing and ascertaining the cell compositions of the invention and their application to certain immune system related diseases or conditions. Thus, those skilled in the art will be able to easily select a suitable model from those presently known in the art. This model involves the use of NOD mice, where IDDM derives from the spontaneous T cell dependent autoimmune destruction of insulin-producing pancreatic β cells that age with age (Bottazzo et al., J. Engl. J. Med , 113: 353, 1985; Miyazaki et al., Clin.Exp. Immunol., 60: 622, 1985). In NOD mice, a model of human IDDM, therapeutic strategies targeting T cells have been successful in preventing IDDM (Makino et al., Exp. Anim., 29: 1, 1980). Such strategies include neonatal thoracic extraction, cyclosporine administration and infusion of anti-pan T cells, anti-CD4, or anti-CD25 (IL-2R) monoclonal antibody (mAb) (Tarui et. al., Insulitis and Type I Diabetes Lessons from the NOD Mouse, Academic Press, Tokyo, p.143, 1986). Other models include, for example, autoimmune and inflammatory diseases such as multiple sclerosis (EAE model), rheumatoid arthritis, graft-versus-host disease (skin grafts, heart transplants, Langerhans islet transplants and colon and small intestine) Transplantation models for the study of transplant rejection, using transplantation and the like), asthma models and systemic lupus erythematosus (systemic autoimmune-lpr or NZBxNZVVF ₁ models), and the like. Takakura et al., Exp. Hematol. 27 (12): 1815-821, 1999; Hu et al., Immunology 98 (3): 379-385, 1999; Blyth et al., Am. J. Respir.Cell Mol Biol. 14 (5): 425-438, 1996; Theofilopoulos and Dixon, Adv. Immunol. 37: 269-389, 1985; Eisenberg et al., J. Immunol. 125: 1032-1036, 1980; Bonneville et al. Nature 344: 163-165, 1990; Dent et al., Nature 343: 714-719, 1990; Todd et al., Nature 351: 542-547, 1991; Watanabe et al., Biochem Genet. 29: 325 -335, 1991; Morris et al., Clin.Imm unol.Immunopathol. 57: 263-273, 1990; Takahashi et al., Cell 76: 969-976, 1994; Current Protocols in Immunology, Richard Coico (Ed.), John Wiley & Sons, Inc., Chapter 15, 1998 ) Reference).

Collagen-induced arthritis (CIA) is a T cell dependent animal model of rheumatoid arthritis (RA) (DE Trentham et al., "Autoimmunity to Type II Collagen: An Experimental Model of Arthritis," J. Exp. Med. 146) 857-868 (1977). Within two weeks after immunization with type II collagen (CII) in IFA, sensitive rats develop polyarthritis, which exhibits histological changes in pannus formation and bone / cartilage erosion. In addition, humoral and cellular responses to CII occur not only in RA but also in CIA (E. Brahn, "Animal Models of Rheumatoid Arthritis: Clues to Etiology and Treatment" in Clinical Orthopedics and Related Research (B. Hahn, ed., Philadelphia, JB Lippincott Company, 1991. In conclusion, CIA is a useful animal model of RA that functions as an in vivo system for potentially investigating new therapeutic interventions as described herein.

Pharmaceutical composition

The T cell populations of the invention may be administered alone or as a pharmaceutical composition, in combination with diluents and / or other components such as IL-2 or other cytokines or cell populations. In sum, the pharmaceutical compositions of the present invention may comprise a target cell population as described herein with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffers such as neutral buffered saline and phosphate buffered saline, and the like; Carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; Polypeptides or amino acids such as glycine; Antioxidants; Chelating agents such as EDTA or glutathione; Adjuvant (eg, aluminum hydroxide); And preservatives. The composition of the present invention is preferably formulated for intravenous administration. The present invention also provides a pharmaceutical composition comprising the sensitizing composition described herein.

Pharmaceutical compositions of the invention may be administered in a manner appropriate to the disease to be treated (or prevented). Dosage and frequency will be determined by factors such as the patient's symptoms, and the type and severity of the patient's disease, but the appropriate dosage may be determined by clinical trials. When referring to an “immunologically effective amount” or “therapeutic amount”, the precise dosage of the composition of the present invention takes into account individual differences in the age, weight, severity and symptoms of the patient, and any other factors related to the treatment of the patient. Can be determined by one skilled in the art. A pharmaceutical composition comprising a subject T or B cell may be administered at a dosage of 10 ⁴ to 10 ⁷ APC per kg body weight, preferably 10 ⁵ to 10 ⁶ APC per kg body weight (including all integer values within this range). It can generally be said that. In addition, the cell composition may be administered multiple times at such doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for disease indications and adjusting the treatment accordingly.

In some adoptive immunotherapy studies, T cells are administered to a patient at approximately 1 × 10 ⁹ to 2 × 10 ¹¹ cells (see, eg, US Pat. No. 5,057,423). In some aspects of the invention, particularly when homologous or heterologous cells are used, a small number of cells in the range of 10 ⁶ / kg (10 ⁶ -10 ¹¹ per patient) can be administered. In certain embodiments, the T or B cells are subjected to 1 X 10 ⁵ , 1 X 10 ⁶ , 1 X 10 ⁷ , 1 X 10 ⁸ , 5 X 10 ⁸ , 1 X 10 ⁹ , 5 X 10 ⁹ , 1 X 10 ¹⁰ , 5 × 10 ¹⁰ , 1 × 10 ¹¹ , 5 × 10 ¹¹ , or 1 × 10 ¹² cells. T or B cell compositions may be administered multiple times at dosages within these ranges. T or B cells can be autologous or heterologous (homologous or heterologous) to the patient performing therapy. If necessary, treatment may be performed with a scavenger (eg PHA) or lymphokine, cytokine, and / or chemokine (eg, GM-CSF, IL-4, to enhance the repair of an immune response as described herein). Administration of IL-13, Flt3-L, RANTES, MIP1α, etc.).

The invention also prevents or inhibits the severity of autoimmune disease in an animal, comprising administering to the animal an effective amount of activated polyclonal T cells in a subject from which an undesirable subset of autoreactive T cells has been removed. It provides a method to reduce or reduce. T cell compositions of the invention may be administered in combination with T cell ablation therapy and / or other therapies to treat autoimmune diseases.

In addition, the invention comprises administering an effective amount of a donor bone marrow of a subject from which an undesirable subpopulation of alloreactive T cells has been removed to an animal in need of hematopoietic stem cell transplantation, wherein the graft-versus-host disease in the animal Provided are methods for preventing, inhibiting or reducing the severity of the disease. The compositions of the present invention can be administered in conjunction with other therapies for the treatment of immune deficiencies associated with hematopoietic stem cell transplantation.

The invention also includes administering to an animal in need of organ transplantation an effective amount of a T cell composition of a subject from which an undesired subpopulation of alloreactive T cells has been removed, thereby preventing host to graft disease or transplant rejection in said animal. Provided are methods for preventing, inhibiting or reducing severity. The compositions of the present invention can be administered in conjunction with other therapies for the treatment of immune deficiencies associated with organ transplantation.

Administration of the pharmaceutical composition of the subject may be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation or implantation. The compositions of the present invention can be administered to a patient subcutaneously, intracutaneously, intramuscularly, intravenously (i.v.), intratumorally or intraperitoneally. Preferably, the T cell composition of the present invention is administered by intravenous injection. The composition of activated T cells can be injected directly into a tumor or lymph node.

In another embodiment, the pharmaceutical composition can be delivered to a controlled release system. In one embodiment, a pump can be used (Langer, 1990, Science 249: 1527-1533; Sefton 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980; Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In other embodiments, polymeric materials can be used (Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla .; Controlled Drug Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105). In another embodiment, a controlled release system can be located in the vicinity of the therapeutic target, thus requiring only a portion of the systemic dosage (eg, Medical Applications of Controlled Release, 1984, Langer and Wise ( eds.), CRC Pres., Boca Raton, Fla., vol. 2, pp. 115-138).

The T cell and / or sensitizing composition of the present invention may be administered using any number of matrices. The matrix has been used for many years in the case of tissue manipulation (see, eg, Principles of Tissue Engineering (Lanza, Langer, and Chick (eds.)), 1997). The present invention typically uses the matrix in new situations that act as artificial lymphoid organs to support, maintain or regulate the immune system through the regulation of T cells. Thus, the present invention can use matrix compositions and formulations that have proven useful in tissue manipulation. Thus, the types of matrices that can be used in the compositions, devices, and methods of the present invention are not really limited, and may include both biological matrices or synthetic matrices. In one specific example, US Pat. No. 5,980,889; 5,913,998; 5,913,998; 5,902,745; 5,902,745; 5,843,069; US Pat. 5,787,900; 5,787,900; Or the compositions and devices described in US Pat. No. 5,626,561. The matrix, when administered to a mammalian host, typically contains the features associated with biocompatibility. The matrix can be prepared from natural and synthetic materials. The matrix may be non-biodegradable or biodegradable in situations where it is desirable to leave permanent tissue or removable structures as implants in the body of the animal. The matrix may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, liposomes, cells or nanoparticles. The matrix can also be designed to allow sustained release of seeded cells or prepared cytokines or other active agents. In some embodiments, the matrix of the present invention may be described as a semisolid scaffold that is flexible and elastic and permeable to dissolved gaseous materials including inorganic salts, aqueous fluids and oxygen. .

The matrix is used herein as an example of a biocompatible material. However, the invention is not limited to matrices, so when the term matrix or matrices emerges, the term permits cell retention or cell crossing; Biocompatible; It is to be understood that the material itself is semipermeable and / or includes devices and other materials that can each allow macromolecules to traverse directly through the material for use with a particular semipermeable material.

All references mentioned in the references are hereby incorporated by reference in their entirety. In addition, all numerical ranges used herein explicitly include all integer values within this range, and the specific numerical values within this range are selected with regard to the particular application. In addition, the following examples are provided merely for the purpose of illustration, and are not intended to limit the present invention.

Example 1

Deletion of Antigen-Specific T Cells After Restimulation with CD3 / CD28 Accelerated (R) Beads

This example describes the removal of antigen-specific T cells from a mixed population of cells by restimulation with anti-CD3 / CD28 Accelerated® beads (3 × 28 beads). The production of accelerated T cells (registered trademark) using the process described herein is essentially described in US patent application Ser. No. 10 / 133,236.

Human PBMCs were screened for HLA-A2 CMVpp65 positive by flow cytometry using HLA-A2 tetramer loaded with CMVpp65 peptide (HLA-A2 CMVpp65). About 3% of CD3 ⁺ CD8 ⁺ T cells in the selected donor expressed TCR specific for HLA-A2-CMVpp65 (FIG. 1).

PBMCs from donor (donor 2) and control donor (donor 1) were activated with CMV antigen coated on paramagnetic beads, and by day 10 of culture, many cells were CD25 (IL-2R) positive by flow cytometry analysis. All HLA-A2 CMVpp65 ⁺ T cells expressed high levels of CD25, indicating activation (FIG. 2, right panel).

After 14 days from the first stimulus, cultures were left unstimulated (FIG. 3, panels A1-A4), or restimulated for 16 hours using the Accelrate® process with 3 × 28 beads ( 3, panels B1-B4). As shown in FIG. 3, CD25 is upregulated in restimulated cells (Panel B2), while tetrameric-positive (ie, CMVpp65-Ag-specific) prestimulated cells have a second strong, provided by 3 × 28 beads. Deleted by stimulation (panels B3 and B4), while other cells were not affected. The cells were attached to the beads or bound to the cells attached to the beads, magnetically selected and put back into the culture prior to restimulation with 3 × 28 beads. In a further study, cells were restimulated for an additional 4 days. Deletion of tetramer-positive cells was still observed after an additional 4 days in 3 × 28 restimulated culture.

These results demonstrate that activated CMVpp65-antigen-specific T cells restimulated with 3 × 28 beads are most likely removed from the cell population via apoptosis.

Example 2

Measurement of apoptosis

This example describes an exemplary assay for measuring apoptosis.

DNA Fragmentation Assay : Cells were lysed in 50 μl of lysis buffer (10 mM EDTA, 50 mM Tris pH 8, 0.5% sodium dodecyl sulfate, 0.5 mg / ml proteinase K). RNAse A (0.5 mg / ml) was added and the lysates were incubated at 37 ° C. for 1 hour. Two phenol extractions (equivalent volume) were performed followed by one chloroform extraction. DNA was precipitated with 2 volumes of ice cold ethanol and incubated at 80 ° C. for 1 hour. DNA was pelleted by centrifugation at 14,000 rpm for 10 minutes at 4 ° C. The pellet was dried for 30 minutes in air and resuspended in 50 μl Tris-EDTA (pH 8). DNA was 1.8% in 1 × TBE running buffer (0.05 M Tris base, 0.05 M boric acid, 1 mM disodium EDTA) according to the method of Preston, et al., Cancer Res., 1994, 54, 4214-4223. Electrophoresis on agarose gels.

Example 3

Induction of apoptosis in B-cells by co-culture with accelerated T cells (registered trademark)

This example describes the deletion of leukemia B-cells in B-CLL patient samples by co-culture with accelerated T cells (registered trademark).

Accelerated T cells (registered), essentially produced as described in US Patent Application No. 10 / 133,236, were co-cultured with unengineered autologous leukemia cells from B-CLL patients. Cell surface markers for CD54, CD80, CD95 (FAS) and CD86, and Annexin / PI (apoptosis) were measured at 24 and 48 hours by flow cytometry. Accelerated T cells (registered trademark) were shown to induce the expression of CD95 (FAS) on leukemia B cells (FIG. 4). After 48 hours of co-culture with Accelerated T cells® on Day 12, autologous leukemia B cells showed increased expression of CD95 and sensitivity to anti-FAS as measured by flow cytometry. (Figure 5). As shown in FIG. 5, the addition of anti-FAS antibodies to cocultured T: B cells resulted in increased apoptosis in leukemia B-cells. In further studies, T cells grew but leukemia B-cells were removed during the Accelrate® process (FIG. 6).

In summary, Accelerated T cells® can upregulate important effector molecules on leukemia B cells, induce functional FAS on leukemia B-cells, and induce leukemia B-cells into the apoptosis pathway. Leukemia B cells were not actually detectable at the end of the Accelrate® process. Thus, accelerated T cells (registered trademark) can be used as a sensitizing composition or apoptosis-inducing composition for the removal of leukemia B-cells from a mixed population of cells.

Example 4

Various Bead: Cell Ratios Can Selectively Expand and Delete Memory CD8 T Cells

This example shows that the bead: cell ratio can have a significant effect on the expansion of several T cell populations. In particular, high bead: cell ratios (3: 1-10: 1) tend to induce death in antigen-specific T cells, while low bead: cell ratios (1: 1-1: 10) are antigen-specific. Enemies result in expansion of T cells. In addition, the data described below show that low bead: cell ratios also result in enhanced cell expansion in the polyclonal cell population. Thus, this example shows that the low bead: cell ratio enhances overall cell expansion. In addition, this example demonstrates that the beads described herein at high bead: cell ratios can be used as apoptosis-inducing compositions.

Cells were prepared and stimulated using the Accelrate I® process essentially as described in US Patent Application No. 10 / 187,467, filed June 28, 2002. In sum, in this process, accelerated T cells (registered trademark) were prepared from peripheral blood mononuclear cell (PBMC) lysate products. After collecting from the patient at the clinical location, the PBMC component collections were washed and cryopreserved. Cells were then thawed and placed in culture for 1 hour at 37 ° C./5% CO ₂ to allow monocytes and other adherent cells to bind to the culture plate. Non-adherent cells were transferred to fresh culture plates and stimulated as follows. After this monocyte depletion step, take a volume containing a total of 5 x 10 ⁸ CD3 ⁺ T cells, and set it to 1.5 x 10 ⁹ Dynabiz® M-450 CD3 / CD28 T to accelerate. The process was initiated (approximately 3: 1 beads versus T cells). Subsequently, the mixture of cells and Dynabiz® M-450 CD3 / CD28 T was incubated at 37 ° C., 5% CO ₂ for about 8 days to accelerate the T cells (registered) for the first injection. Generated. After cryopreservation of the remaining monocyte-depleted PBMCs, upon second or additional cell product expansion (after about 21 days), the PBMCs are thawed and washed, followed by a volume containing a total of 5 × 10 ⁸ CD3 ⁺ T cells. Was taken and set to 1.5 × 10 ⁹ Dynabiz® M-450 CD3 / CD28 T to initiate the Accelrate® process for the second injection. During incubation at 37 ° C., 5% CO ₂ for about 8 days, CD3 ⁺ T cells were activated and expanded. The anti-CD3 mAb used was BC3 (XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, WA), and the anti-CD28 mAb (B-T3, XR-CD28) was from Diaaclone, Besancon, France. Obtained.

For the experiments described below, the cultures containing cells from which adherent cells had been removed had beads added in a bead: T cell ratio as shown in Table 1 below. Beads used in this example included Dynabiz® M-450 CD3 / CD28 T with a 1: 1 CD3: CD28 antibody ratio bound to the beads.

Various Bead: Cell Ratios Can Selectively Expand or Delete Memory CD8 T Cells Bead: Cell Ratio Multiple increase Polyclonal T cells CMV antigen-specific T cells 10: 1 149 0 5: 1 294 0 3: 1 346 1.4 1: 1 562 20.6 1: 5 113 53 1:10 79 45.8

The results summarized in Table 1 and graphically shown in FIG. 7 indicate that antigen-specific T cells can be selectively deleted using high bead: cell ratios and can be selectively expanded using low bead: cell ratios. Prove it. Similar results were observed using EBV-specific and influenza-specific CD8 T cells (not shown). Without wishing to be bound by theory, it is believed that antigen-specific T cells are sensitized with further stimulation. Stimulation with high bead-to-cell ratios provides high concentrations of stimulatory antibodies resulting in stimulation of antigen-specific T cells, causing these cells to be killed by apoptosis or other mechanisms. Thus, in this regard, the beads function as apoptosis-inducing compositions. The low bead: cell ratio is not used to provide stimulation signals but rather to antigen-specific T cells that induce rapid proliferation of cells. In addition, increased proliferation was also observed in the polyclonal population of T cells using a low bead: cell ratio. In particular, these results suggest that the bead: cell ratio of 1: 1 is optimal for polyclonal T cell expansion.

Thus, in this example, evidence is provided to support the use of different bead: cell ratios depending on the desired results. For expansion of antigen-specific T cells, a low bead: cell ratio is preferred. If deletion of antigen-specific T cells is a desirable result, a high bead: cell ratio is preferred.

Example 5

Deletion of Alloreactive T Cells After Restimulation Using CD3 / CD28 Accelerated (R) Beads

This example describes the deletion of alloreactive T cells after restimulation with CD3 / CD28® Accelerate® beads.

PBMCs were stimulated for 3 days with allogeneic PBMC or JY B-lymphoblastoid allogeneic cell lines. On day 3, homologous PBMC- or JY-stimulated using an Accelerate® process, essentially as described in US Patent Application 10 / 350,305, with or without positive selection for 30 minutes with CD3 / CD28 beads. PBMCs were incubated with CD3 / CD28 beads. After the Accelrate® process, cells were restimulated with homologous PBMCs or JY homologous antigens and CD25 upregulation was measured. Restimulation with allogeneic cells following the Accelrate® process did not result in upregulation of CD25 expression (measured using flow cytometry analysis), indicating that alloreactive cells were deleted. In particular, positive selection of JY stimulated CD8 ⁺ T cells during the Accelerate® process significantly reduced alloreactivity. However, T cells were excel as demonstrated by third party homologous PBMCs and JY reactions (eg, restimulation of JY-stimulated cultures with homologous PBMCs or restimulation of allo-PBMC-stimulated cultures with JY). The ability to respond to unrelated antigens remained in the lauded (R) culture.

Thus, these results indicate that activated alloreactive T cells are deleted by restimulation with CD3 / CD28 beads, while the remaining polyclonal T cells can be exponentially expanded for use in any number of immunotherapy applications. Shows that there is.

Claims (67)

At least a portion of at least a portion of the population of one or more clonal T cells present in a mixed population of T cells from an individual by exposing at least a portion of the cell population comprising T cells to one or more pro-apoptotic or growth inhibitory compositions Removing at least a substantial portion of the clonal T cell subpopulation from the mixed population of T cells by inducing apoptosis or growth inhibition in the mixed population of T cells How to. The method of claim 1, further comprising expanding the remaining mixed population of T cells. The remainder of the cell of claim 2, wherein the remaining mixed population of cells is exposed to a surface to which one or more substances ligating the cell surface residues of at least some of the remaining T cells are attached to stimulate the remaining T cells. How to expand a mixed population. The method of claim 3, wherein a first substance ligating the first T cell surface residues of the T cells is attached to the surface, and a second substance ligating the second residues of the T cells on the same surface or the second surface. To which said ligation by a first substance and a second substance induces proliferation of said T cells. A population of T cells produced according to the method of any one of claims 1 to 3. The method of claim 1, wherein the apoptosis-inducing or growth inhibitory composition comprises an autoantigen. The method of claim 6, wherein the autoantigen is myelin based protein (MBP), MBP 84-102, MBP 143-168, pancreatic islet cell antigen, collagen, thyroid antigen, Scl-70, nucleic acid, acetylcholine receptor, S antigen and type II collagen. The method of claim 1, wherein the apoptosis-inducing composition comprises allogeneic or heterologous cells. The method of claim 1, wherein said cell population comprising at least a portion of T cells is exposed to one or more apoptosis-inducing compositions in vivo. The method of claim 1, wherein said cell population comprising at least a portion of T cells is exposed ex vivo to one or more apoptosis-inducing compositions. The method of claim 3, wherein said cells are exposed to said surface for a time sufficient to increase polyclonality. 12. The method according to claim 11, wherein said increase is monoclonal to oligoclonal in the T cell population as measured by the Vβ, Vα, Vγ or Vδ spectratype profile of one or more Vβ, Vα, Vγ or Vδ family genes. Or transfer to polyclonal. A T cell population produced according to the method of claim 6 or 11. A method of treating an autoimmune disease of a patient comprising administering to the patient with the T cell population of claim 13. The method of claim 14, wherein the patient is treated with an immunooablative agent prior to administering the T cell population of claim 10. The method of claim 15, wherein the immunodepressant is selected from the group consisting of campath, anti-CD3 antibody, cyclophosphamide, fludarabine, cyclosporin, FK506, mycophenolic acid, steroids, FR901228 and irradiation. How. The method of claim 14, wherein the patient is treated with a T cell ablation therapy prior to administering the T cell population of claim 10. The method of claim 1, wherein the apoptosis-inducing or growth inhibitory composition is an anti-CD3 antibody, anti-CD2 antibody, anti-CD20 antibody, target antigen, MHC-peptide tetramers or dimers, Fas ligand, anti-Fas antibody. , IL-2, IL-4, TRAIL, rolipram, doxorubicin, chlorambucil, fludarabine, cyclophosphamide, azathioprine, methotrexate, cyclosporin, mycophenolate, FK506, bcl-2 inhibitor, Topoisomerase inhibitors, interleukin-1β converting enzyme (ICE) -binding agents, Shigella IpaB proteins, staurosporin, ultraviolet radiation, gamma irradiation, tumor necrosis factor, target antigen nucleic acid molecule, protein or peptide, and non -One or more compositions selected from the group consisting of protein or non-polynucleotide compounds. The method of claim 3, wherein the one or more agents are antibodies or antibody fragments. The method of claim 3, wherein the first substance is an antibody or fragment thereof, and the second substance is an antibody or fragment thereof. The method of claim 3, wherein the first substance and the second substance are different antibodies. The method of claim 3, wherein the first agent is an antibody fragment of an anti-CD3 antibody, an anti-CD2 antibody, or an anti-CD3 or anti-CD2 antibody. The method of claim 3, wherein the second agent is an anti-CD28 antibody or antibody fragment thereof. The method of claim 3, wherein the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody. (a) exposing a cell population comprising at least a portion of T cells to one or more compositions that sensitize at least a portion of the T cells with further activation or stimulation, and (b) inducing the apoptosis of the sensitized T cells to a surface to which the one or more substances ligating the cell surface residues of at least some of the sensitized T cells are attached to stimulate the sensitized T cells Exposing for sufficient time to remove the sensitized T cells from the population A method of removing at least a substantial portion of a clonal T cell subpopulation from an individual-derived T cell mixing population comprising a. The method of claim 25, wherein step (b) further comprises exposing the cell population to the surface for a time sufficient to stimulate at least a portion of the remaining T cells to propagate at least a portion of the remaining cells. . 27. The method of claim 25, wherein a first substance ligating the first T cell surface residues of the T cells is attached to the surface, and a second substance ligating the second residues of the T cells on the same surface or the second surface. To which said ligation by a first substance and a second substance induces proliferation of said T cells. The method of claim 26, wherein said cells are exposed to said surface for a time sufficient to increase polyclonality. 29. The method according to claim 28, wherein said increase is monoclonal to polyclonal or polyclonal in the T cell population as measured by the Vβ, Vα, Vγ or Vδ spectratype profile of one or more Vβ, Vα, Vγ or Vδ family genes. And a transfer to a castle. A T cell population produced according to the method of claim 25 or 28. The method of claim 25, wherein the subject requires hematopoietic stem cell transplantation. The method of claim 31, wherein the sensitizing composition comprises recipient PBMCs that have been treated to be unable to sustain division and wherein the cell population comprises donor T cells. A T cell population produced according to the method of claim 32. 34. A method of reducing the risk or severity of a GVHD adverse effect in a patient comprising administering a population of T cells according to claim 30 or 33 to a patient transplanting hematopoietic stem cells. The method of claim 25, wherein the subject requires organ transplantation. 36. The method of claim 35, wherein the sensitizing composition comprises donor cells that have been treated to be non-dividable and the cell population comprises recipient T cells. The method of claim 36, wherein said cells are exposed to said surface for a time sufficient to increase polyclonality. The method according to claim 37, wherein said increase is monoclonal to oligoclonal or polyclonal as measured by the Vβ, Vα, Vγ or Vδ spectratype profile of one or more Vβ, Vα, Vγ or Vδ family genes. And a transfer to a castle. A population of T cells produced according to the method of claim 36 or 37. A method of reducing the risk of organ rejection in a patient comprising administering to the patient transplanting the organ of the T cell population of claim 39. The method of claim 40, wherein the patient is treated with a T cell ablation therapy prior to administering the T cell population of claim 36. The method of claim 25, wherein the sensitizing composition comprises an autoantigen. The method of claim 42, wherein the autoantigen is selected from the group consisting of myelin based protein (MBP), MBP 84-102, MBP 143-168, Scl-70, pancreatic islet cell antigen, S antigen, and type II collagen. . The method of claim 43, wherein said cells are exposed to said surface for a time sufficient to increase polyclonality. 45. The method according to claim 44, wherein said increase is monoclonal to oligoclonal or polyclonal as measured by the Vβ, Vα, Vγ or Vδ spectratype profile of one or more Vβ, Vα, Vγ or Vδ family genes. And a transfer to a castle. 45. A T cell population produced according to the method of claim 42 or 44. The method of treating an autoimmune disease of a patient comprising administering to the patient with the T cell population of claim 46. 48. The method of claim 47, wherein the patient is treated with a T cell ablation therapy prior to administering the T cell population of claim 46. The method of claim 26, wherein the one or more agents are antibodies or antibody fragments. The method of claim 26, wherein the first substance is an antibody or fragment thereof and the second substance is an antibody or fragment thereof. 51. The method of claim 50, wherein the first substance and the second substance are different antibodies. The method of claim 26, wherein the first agent is an antibody fragment of an anti-CD3 antibody, an anti-CD2 antibody, or an anti-CD3 or anti-CD2 antibody. The method of claim 26, wherein the second agent is an anti-CD28 antibody or antibody fragment thereof. The method of claim 26, wherein the first agent is an anti-CD3 antibody and the second agent is an anti-CD28 antibody. At least a portion of the individual comprising generating a substantially pure population of CD3 ⁺ / CD28 ⁺ T cells by exposing a population of cells comprising T cells to a composition that stimulates and / or selects surface CD3 and CD28 molecules ex vivo. A method of producing a substantially pure population of CD3 ⁺ / CD28 ⁺ T cells from a derived T cell population. At least in part exposing a cell population comprising T cells to a composition that stimulates and / or selects surface CD3 and CD28 molecules ex vivo, thereby producing a substantially pure population of CD4 ⁺ / CD3 ⁺ / CD28 ⁺ T cells. Generating a substantially pure population of CD4 ⁺ / CD3 ⁺ / CD28 ⁺ T cells from an individual derived T cell population. At least in part exposing a cell population comprising T cells to a composition that stimulates and / or selects surface CD3 and CD28 molecules ex vivo, thereby producing a substantially pure population of CD8 ⁺ / CD3 ⁺ / CD28 ⁺ T cells. Generating a substantially pure population of CD8 ⁺ / CD3 ⁺ / CD28 ⁺ T cells from an individual derived T cell population. The method of any one of claims 55-57, further comprising expanding said CD3 ⁺ CD28 ⁺ T cells for a sufficient time such that the proportion of contaminating CD3 ⁺ / CD28 ⁻ T cells is less than about 5%. The method of any one of claims 55-57, further comprising expanding said CD3 ⁺ CD28 ⁺ T cells for a sufficient time such that the proportion of contaminating CD3 ⁺ / CD28 ⁻ T cells is less than about 1%. 58. The method of any one of claims 55-57, further comprising expanding said CD3 ⁺ CD28 ⁺ T cells for a sufficient time such that the proportion of contaminating CD3 ⁺ / CD28 ^- T cells is less than 0.1%. 58. The method of any one of claims 55-57, wherein the anti-CD3 antibody is used to stimulate the CD3 molecule and the anti-CD28 antibody is used to stimulate the CD28 molecule. At least in part contacting a cell population comprising T cells with a surface to which one or more substances ligating at least a portion of the cell surface residues of the T cells is attached and stimulates the T cells, the surface being antigen- At least a substantial portion of one or more populations of specific T cells are present at a ratio of said surface to said cells such that they are deleted after about 8 days of culture. 63. The method of claim 62, wherein the ratio is about 10: 1 to about 5: 1. 63. The method of claim 62, wherein the ratio is about 5: 1. 63. The method of claim 62, wherein the ratio is about 10: 1. The method of claim 1, further comprising expanding the mixed population of T cells, comprising exposing the remaining mixed population of T cells to the apoptosis-inducing composition to induce proliferation of the mixed population of T cells. 67. The method of claim 66, wherein said apoptosis-inducing composition comprises anti-CD3 and anti-CD28 antibodies co-fixed onto beads.