WO2006133398A2 - Lymphocytes t donneurs favorisant la prise de greffe - Google Patents
Lymphocytes t donneurs favorisant la prise de greffe Download PDFInfo
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- WO2006133398A2 WO2006133398A2 PCT/US2006/022426 US2006022426W WO2006133398A2 WO 2006133398 A2 WO2006133398 A2 WO 2006133398A2 US 2006022426 W US2006022426 W US 2006022426W WO 2006133398 A2 WO2006133398 A2 WO 2006133398A2
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- cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- the present invention relates generally to the use of donor allogeneic lymphocytes to promote transplant engraftment, immune reconstitution, and reduce regimen-related toxicity associated with allotransplantation.
- the present invention relates to using activated donor T lymphocytes in these settings.
- the present invention relates generally to methods for stimulating and activating cells which can then be used in such settings.
- Conditioning regimens for allogeneic transplantation are typically myeloablative and lymphoablative in order to eliminate malignant cells to eradicate disease and recipient lymphoid cells to ablate donor reactive cells in the host that cause graft failure.
- Such regimens are associated with many side effects.
- the substantial risk of morbidity and mortality associated with conditioning regimens has led to the development of reduced-intensity conditioning regimens.
- these reduced-intensity regimens are still associated with side effects and, importantly, cannot be used in mismatched transplant settings or heavily alloimmunized patients such as multiply transfused patients, thereby limiting their use.
- the present invention provides methods for promoting engraftment comprising administering allogeneic T cells to a transplant patient.
- the present invention provides methods for promoting chimerism in a transplant setting.
- the T cells are activated T cells.
- T cells are activated by a method comprising, contacting a population of allogeneic cells from a suitable donor, wherein at least a portion of the population comprises T cells, with a surface, wherein said surface has attached thereto a first agent which stimulates a TCR/CD3 complex-associated signal in the T cells and a second agent that binds the CD28 accessory molecule on the surface of the T cells, thereby activating the T cells.
- a suitable donor refers to a donor that has been suitably matched as described further herein.
- the first agent is an antibody or an antigen-binding fragment thereof and in certain embodiments, is a monoclonal antibody or antigen-binding fragment thereof.
- the antibody is an anti-CD3 antibody.
- the second agent is an antibody or an antigen- binding fragment thereof and in certain embodiments, the antibody is a monoclonal antibody or antigen-binding fragment thereof.
- the antibody is an anti-CD28 antibody.
- the first and the second agents are both antibodies or antigen-binding fragments thereof.
- the first agent may be an anti-CD3 antibody or antigen-binding fragments thereof and the second agent may be an anti-CD28 antibody or antigen-binding fragments thereof.
- the second agent is a natural ligand of CD28, such as, B7-1.
- the surface can be a solid surface, a cell surface, or a paramagnetic bead.
- the first and second agents are covalently or noncovalently attached to the surface.
- the first and second agents are indirectly attached to the surface.
- the methods described herein can be used in a setting where the transplant patient is receiving a transplant for the treatment of a malignancy.
- the malignancy can be any malignancy.
- Illustrative malignancies include non-Hodgkin's lymphoma, chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), multiple myeloma, acute myelogenous leukemia, acute lymphoblastic leukemia, and other cancers.
- the methods of the present invention can be used for a transplant patient who may be receiving a transplant for the treatment of a hematologic failure (such as aplastic anemia, beta thalassemia, sickle cell anemia,), an autoimmune disease, an immunodeficiency, or a congenital disorder.
- a hematologic failure such as aplastic anemia, beta thalassemia, sickle cell anemia,
- an autoimmune disease such as a hematologic failure
- an immunodeficiency such as a congenital disorder.
- the activated T cells of the present invention can be used to promote engraftment in any setting where a transplant may be used, such as an organ transplant setting.
- the methods described herein can be used to promote engraftment that comprises lympho-engraftment or bone marrow engraftment.
- One aspect of the present invention provides a method for promoting engraftment of a transplant, comprising administering activated allogeneic T cells to a transplant patient at the time of the transplant.
- the T cells are activated by a method comprising, contacting a population of allogeneic cells from a suitable donor, wherein at least a portion of the population comprises T cells, with a surface, wherein said surface has attached thereto a first agent which stimulates a TCR/CD3 complex-associated signal in the T cells and a second agent that binds the CD28 accessory molecule on the surface of the T cells, thereby activating the T cells.
- the transplant is selected from the group consisting of a bone marrow transplant, a hematopoetic stem cell transplant, a CD34+ cell transplant, a purified stem cell transplant, a kidney transplant, a heart transplant, a liver transplant, a lung transplant, a pancreas transplant, a pancreatic islet cell transplant, an intestine transplant, a bone transplant, a cornea transplant, a skin transplant, a heart valve transplant, and a connective tissue transplant.
- the first agent is an antibody or an antigen-binding fragment thereof and in certain embodiments, is a monoclonal antibody or antigen-binding fragment thereof.
- the antibody is an anti-CD3 antibody.
- the second agent is an antibody or an antigen-binding fragment thereof and in certain embodiments, the antibody is a monoclonal antibody or antigen-binding fragment thereof. In one embodiment, the antibody is an anti-CD28 antibody. In a further embodiment the first and the second agents are both antibodies or antigen-binding fragments thereof. In this regard, the first agent may be an anti-CD3 antibody or antigen-binding fragments thereof and the second agent may be an anti-CD28 antibody or antigen-binding fragments thereof. In certain embodiments, the second agent is a natural ligand of CD28, such as, B7-1. In another embodiment, the surface can be a solid surface, a cell surface, or a paramagnetic bead.
- the first and second agents are covalently or noncovalently attached to the surface. In a further embodiment, the first and second agents are indirectly attached to the surface.
- the transplant patient is receiving a transplant for the treatment of a malignancy.
- Illustrative malignancies include, but are not limited to NHL, CLL, multiple myeloma, myelogenous leukemia, acute lymphoblastic leukemia, and CML.
- the transplant is for the treatment of aplastic anemia, an autoimmune disease, an immunodeficiency, or a congenital disorder.
- the transplant is an organ transplant.
- engraftment comprises lympho-engraftment and/or bone marrow engraftment. In one embodiment, the engraftment comprises between about 50% and 100% donor bone marrow chimerism. In certain embodiments, the engraftment comprises greater than about 60% donor bone marrow chimerism. In another embodiment, the engraftment comprises at least about 70%, 80%, 90%, or higher donor bone marrow chimerism.
- the activated allogeneic T cells have been genetically modified to express a suicide gene.
- the suicide gene may include any one or more of the following: Herpes simplex type-1 virus (HSVl) thymidine kinase gene, a fusion between HSVl thymidine kinase and zeocin-resistance gene, E. coli Cytosine Deaminase, E. coli Cytosine Deaminase fused to Uracil Phosphoribosyltransferase, S. cerevisiae Cytosine Deaminase, S.
- HSVl Herpes simplex type-1 virus
- EHV4 herpes virus 4
- HSVl Herpes simplex virus 1
- HSVl Herpes simplex virus 1
- E. coli thymidine kinase fused to thymidylate kinase
- E. coli Uracil Phosphoribosyltransferase and dimerizable, modified human caspase 9 fused to a human FK506.
- the activated allogeneic T cells comprise CD4+ T cells.
- the T cells comprise CD8+ T cells.
- the patient is administered between about 1 X 10 9 activated T cells and 2 X 10 ⁇ activated T cells.
- the patient is administered greater than about 5 X 10 9 activated T cells, greater than about 1 X 10 10 activated T cells, greater than about 5 X 10 10 activated T cells, greater than about 1 X 10 ⁇ activated T cells, or greater than about 2 X 10 n activated T cells.
- the patient is administered greater than about 1 X 10 6 activated T cells/kilogram over the course of therapy.
- the patient is administered greater than about I X lO 7 activated T cells/kilogram over the course of therapy, greater than about 1 X 10 8 activated T cells/kilogram over the course of therapy, or greater than about I X lO 9 activated T cells/kilogram over the course of therapy.
- the activated T cells are administered in multiple infusions.
- the activated T cells are ThI -type T cells.
- the activated T cells are unpolarized T cells.
- Another aspect of the present invention provides a method for promoting transplant engraftment of a transplant derived from an allogeneic donor in a recipient of the transplant, comprising: conditioning the recipient; transplanting the transplant into the recipient; administering to the recipient a dose of activated allogeneic T cells, wherein said activated T cells are generated by contacting a population of allogeneic cells from a suitable donor, wherein at least a portion of the population comprises T cells, with a surface, wherein said surface has attached thereto a first agent which stimulates a TCR/CD3 complex-associated signal in the T cells and a second agent that binds the CD28 accessory molecule on the surface of the T cells, thereby activating the T cells; and thereby promoting engraftment of the transplant.
- the conditioning regimen comprises a regimen wherein adequate or substantial transplant engraftment would not occur in the absence of the addition of allogeneic T cells.
- the transplant can be a bone marrow transplant, a hematopoetic stem cell transplant, a CD34+ cell transplant, a purified stem cell transplant, a kidney transplant, a heart transplant, a liver transplant, a lung transplant, a pancreas transplant, a pancreatic islet cell transplant, an intestine transplant, a bone transplant, a cornea transplant, a skin transplant, a heart valve transplant, or a connective tissue transplant or a combination of one or more of these transplants.
- the transplant is a megadose purified stem cell transplant.
- the conditioning regimen comprises one of the regimens listed in Table 2 and wherein the donor and the recipient are mismatched at one or more histocompatibility antigen. In a further embodiment, the conditioning regimen comprises one of the regimens listed in Table 2 and wherein the donor and the recipient are matched and unrelated. In, yet another embodiment, the conditioning regimen comprises one of the regimens listed in Table 2 and wherein the donor and the recipient are mismatched. In an additional embodiment, the conditioning regimen comprises one of the regimens listed in Table 2 and wherein the transplant and the activated allogeneic T cells are derived from cord blood. In certain embodiments, the conditioning regimen comprises one of the regimens listed in Table 2 and wherein the transplant is derived from cord blood.
- the conditioning regimen comprises one of the regimens listed in Table 2 and wherein the recipient has been alloimmunized, such as wherein the recipient is a multiply transfused anemic patient.
- the multiply transfused anemic patient suffers from aplastic anemia, sickle cell anemia, or beta thalassemia.
- the conditioning regimen comprises a regimen that is further reduced as compared to accepted reduced- intensity regimens and wherein the donor and the recipient are matched and related.
- the accepted reduced-intensity regimen that can be reduced comprises a regimen listed in Table 2.
- the transplant patient is given no post-transplant immunosuppression.
- the transplant is a xenogeneic transplant such as a transplant derived from a non-human primate or a pig.
- the transplant is a solid organ transplant and wherein the activated allogeneic T cells are administered prior to the transplant.
- the activated allogeneic T cells target host cells that cause rejection.
- the activated allogeneic T cells are administered at the same time as the transplant.
- T cells are administered within 24 hours of the transplant, within 2 days of the transplant, within 3 days of the transplant, within 4 days of the transplant, within 5 days of the transplant, within 6 days of the transplant, or within 7 days of the transplant.
- stimulation refers to a primary response induced by ligation of a cell surface moiety.
- stimulation entails the ligation of a receptor and a subsequent signal transduction event.
- stimulation of a T cell refers to the ligation of a T cell surface moiety, such as binding the TCR/CD3 complex, that in one embodiment subsequently induces a signal transduction event.
- the stimulation event may activate a cell and upregulate or downregulate expression or secretion of a molecule, such as upregulation of the IL-2 receptor (CD25).
- a cell and upregulate or downregulate expression or secretion of a molecule such as upregulation of the IL-2 receptor (CD25).
- CD25 upregulation of the IL-2 receptor
- activation refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change.
- activation refers to, in part, the state of a T cell that has been sufficiently stimulated to induce cellular proliferation.
- Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process.
- force refers to an artificial or external force applied to the cells to be stimulated that induces cellular concentration and concentration of cells with the agent that binds a cell surface moiety.
- force includes any force greater than gravity (i.e., in addition to gravity and not solely gravitational force) that induces cell concentration and/or cell surface moiety aggregation.
- Such forces include transmembrane pressure such as filtration, a hydraulic force, an electrical force, an acoustical force, a centrifugal force, or a magnetic force.
- the force utilized drives the concentration of the target cell of interest with an agent that ligates a cell surface moiety.
- the force can be pulsed, i.e., applied and reapplied (e.g., a magnetic force could be turned off and on, pulsing the population of cells in combination with a paramagnetic particle).
- the term “simultaneous”, as used herein, refers to the fact that inherently upon concentrating cells at a surface that has cell surface moiety binding agents attached thereto, results in concentration of cells with each other and with the surface, thus ligands (i.e., agents).
- ligands i.e., agents
- the use of the term “simultaneous” does not preclude previous binding of the target cells with a surface having cell surface moiety binding agents attached thereto, as concentration and further ligand binding occurs simultaneously at the concentration surface.
- the T cells may be exposed to a surface such as a paramagnetic bead having anti-CD3 and anti-CD28 antibodies attached thereto and subsequently concentrated by a magnetic field.
- target cell refers to any cell that is intended to be stimulated by cell surface moiety ligation.
- an "antibody”, as used herein, includes both polyclonal and monoclonal antibodies; primatized (e.g., humanized); murine; mouse-human; mouse-primate; and chimeric; and may be an intact molecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab' and F(ab)' 2 fragments), or multimers or aggregates of intact molecules and/or fragments; and may occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering; an "antibody fragment,” as used herein, refers to fragments, derived from or related to an antibody, which bind antigen and which, in some embodiments, may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. This includes, e.g., F(ab), F(ab) 5 2 , scFv, light chain variable region (V L ), heavy chain variable
- protein includes proteins, polypeptides and peptides; and may be an intact molecule, a fragment thereof, or multimers or aggregates of intact molecules and/or fragments; and may occur in nature or be produced, e.g., by synthesis (including chemical and/or enzymatic) or genetic engineering.
- agent refers to a molecule that binds to a defined population of cells.
- the agent may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population.
- the agent may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), or the like.
- antibodies are used as a prototypical example of such an agent.
- a “agent that binds a cell surface moiety” and “cell surface moiety”, as used herein, are used in the context of a ligand/anti-ligand pair. Accordingly, these molecules should be viewed as a complementary/anti- complementary set of molecules that demonstrate specific binding, generally of relatively high affinity (an affinity constant, Ka 5 of about 10 6 M" 1 or greater).
- a “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation.
- receptors and other cell surface moieties are anti-ligands, while agents (e.g., antibodies and antibody fragments) reactive therewith are considered ligands.
- “Quiescent”, as used herein, refers to a cell state wherein the cell has either not been induced to actively proliferate or has been induced to proliferate at some previous time point and is no longer actively proliferating (i.e., the cell has reached a resting state post-stimulation either in vivo or in vitro due to lack of stimulation/cytokine signal).
- a "surface”, as used herein, refers to any surface capable of having an agent attached thereto and includes, without limitation, metals, glass, plastics, co- polymers, colloids, lipids, cell surfaces, and the like. Essentially any surface that is capable of retaining an agent bound or attached thereto.
- a prototypical example of a surface used herein, is a particle such as a bead.
- histocompatibility refers to the similarity of tissue between different individuals. The level of histocompatibility describes how well matched the patient and donor are.
- the major histocompatibility determinants are the human leukocyte antigens (HLA). HLA typing is performed between the potential donor and the potential recipient to determine how close a HLA match the two are. The closer the match the less the donated T cells and the patient's body will react against each other.
- HLA human leukocyte antigens
- proteins proteins (antigens) found on the surface of white blood cells and other tissues that are used to match donor and patient.
- a patient and potential donor may have their white blood cells tested for such HLA antigens as, HLA-A, B and DR.
- HLA-A, B and DR white blood cells tested for such HLA antigens as, HLA-A, B and DR.
- Each individual has two sets of these antigens, one set inherited from each parent. For this reason, it is much more likely for a brother or sister to match the patient than an unrelated individual, and much more likely for persons of the same racial and ethnic backgrounds to match each other.
- the word "match” relates to how similar the HLA typing is between the donor and the recipient.
- the best kind of match is an "identical match”. This means that all six of the HLA antigens (2 A antigens, 2 B antigens and 2 DR antigens) are the same between the donor and the recipient. This type of match is described as a “6 of 6" match. Donors and recipients who are “mismatched” at one antigen are considered a "5 of 6" match, and so forth.
- allogeneic donor cells refers to cells which are derived from an individual other than the recipient.
- allogeneic donor cells can be derived from an individual who is a family member or from an individual unrelated to the recipient.
- donor cells can refer to donor T lymphocytes and to donor bone marrow cells or other cells/organs used in a transplant setting.
- xenogenic includes cells or organs from a different species, including any mammal other than human, such as pig, nonhuman primate, etc.
- the donor can be from the same species as the subject, or from a different species.
- the donor T lymphocytes and/or the donor stem cells can be xenogenic.
- immunological reconstitution means the presence of immunological potential following a period of suppressed immune potential.
- immunological activity refers to the function of cells of the immune system, including, T cells, B cells, NK cells, macrophages, neutrophils, and the like.
- Immune reconstitution can be measured using a variety of assays known in the art, for example, a measure of T cell receptor (TCR) diversity using spectratype analysis, proliferation assays ([ 3 H]Thymidine incorporation assays), cytotoxic T cell assays ⁇ e.g., chromium release assays), intracellular cytokine release assays, response to specific antigens, and the like.
- TCR T cell receptor
- graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effect refers to an anti-leukemia or anti-tumor effect mediated by the donor lymphocytes (e.g., donor T cells ) (see e.g., A.J. Barrett, Stem Cells, Vol. 15, No. 4, 248-258, July 1997).
- GVL can be measured using techniques known in the art including a variety of in vitro T cell assays as well as known clinical evaluations of the recipient and molecular diagnostic techniques ⁇ e.g., PCR-based and flow cytometry- based methods for detecting residual disease) to determine the presence or absence and/or level of malignant cells.
- myeloablative as used herein includes any therapy that eliminates, through cell killing or cell inactivation, substantially all the cells of the myeloid lineage of host origin, including hematopoietic stem cells.
- sub-myeloablative includes any therapy that eliminates a significant fraction of, but not substantially all, cells of the myeloid lineage of host origin, including hematopoietic stem cells.
- lymphoablative includes any therapy that eliminates substantially all or part of host-origin lymphocytes, including T lymphocytes. This is accomplished through cell killing, blocking, and/or down- regulation. The elimination may be short-term or long-term.
- sub-lymphoablative includes any therapy that eliminates a significant fraction of, but not substantially all, functional lymphocytes of host origin, including T lymphocytes.
- T cells such as the XCELLERATETM T cells described herein and elsewhere (see e.g., U.S. patent application numbers 10/762210; 10/350305; 10/187467; 10/133236; 08/253,694; 08/435,816; 08/592,711; 09/183,055; 09/350,202; and 09/252,150; and patent numbers 6,352,694; 5,858,358; and 5,883,223), generally produce less GVHD than do naive T cells. At the same time, these cells can be used effectively to promote immune reconstitution and promote transplant engraftment (see e.g., Example 1).
- activated T cells such as the XCELLERATETM T cells described herein and elsewhere (see e.g., U.S. patent application numbers 10/762210; 10/350305; 10/187467; 10/133236; 08/253,694; 08/435,816; 08/592,711; 09/183,055
- XCELLERATETM T cells are generally of the ThI -type phenotype. As such, it is particularly surprising that such T cells promote engraftment and produce less GVHD. It has been shown that activated CD4 + T cells generally fall into one of two distinct subsets, the ThI or Th2 cells. ThI cells principally secrete IL-2, IFN-gamma, IL- 12 and TNF-alpha while Th2 cells principally secrete IL-4 (which stimulate production of IgE antibodies), IL-5, IL-6, and IL-10. These CD4 subsets exert a negative influence on one another; i.e., secretion of ThI lymphokines inhibits secretion of Th2 lymphokines and vice versa.
- Th2 cells In addition, it is believed that exposure of Th2 cells to CTLs also suppresses Th2 cell activity.
- confirmation of the presence and quantity of the ThI response can be determined by assaying for the presence of the cytokines associated with the ThI response, such as IL- 2, IFN-gamma, IFN-alpha., IL- 12 and TNF-beta, using methods known in the art.
- the T cells of the present invention can be used in transplant settings with minimal risk of GVHD while promoting engraftment of transplanted organs/tissues/cells and immune reconstitution.
- the present invention should not be confused with donor lymphocyte infusion, commonly referred to as DLL
- DLI is usually used in the setting of allogeneic stem cell transplant, following significant conditioning which is used to enable the donor stem cells to engraft.
- DLI is routinely used at later times following transplantation and in mini-transplants specifically to induce GVL.
- DLI is generally used no earlier than 30 days post-transplant and more typically 60-90 days post-transplant.
- allogeneic T cells and in particular, activated allogeneic T cells, have not been used such that they promote transplant engraftment.
- current methods for conditioning of patients for allogeneic BMT are particularly harsh and lead to activated inflammatory stimuli, leading to an environment that encourages the development of GVHD.
- the present invention is generally related to methods for promoting engraftment and immune reconstitution using activated donor lymphocytes with reduced conditioning and reduced GVHD.
- T regulatory cells or allo-specific suppressor T cells have been expanded for a variety of uses including to suppress GVHD and to prevent rejection (see e.g., Taylor, et ah, Blood 2002, 99(10):3493-3499; Hanash and Levy, 2005, Blood 105(4): 1828- 1836; Joffre, et ah, 2004 Blood 203(1 1):4216-4221; Hoffmann et ah, 2002 J. Exp. Med. 196:289-399; Cohen, et ah, 2002 J. Exp. Med. 196:401-406; WO 01/26470).
- the present invention represents the first use of polyclonally activated T cells to promote transplant engraftment wherein the T cells are not regulatory T cells.
- the activated T cells as described herein generally, are of the ThI phenotype, unlike other studies that have used Th2-type T cells (see e.g., Erdmann, et al, Biology of Blood and Marrow Transplantation 10:604-613 (2004); Published US Application 2004/0175827 (Application No. 10/481,913)).
- Donor Cells as described herein include donor lymphocytes, donor bone marrow cells/stem cells used for transplantation, donor organs used in organ transplantation (e.g., kidney, heart, lung, liver, etc) and any other donor tissue or cells that may be used in an allogeneic transplant setting.
- donor cells of the present invention are derived from an individual other than the recipient.
- the donor cells can be derived from related or unrelated individuals.
- the donor cells are matched donor cells.
- the donor cells may be matched at all 6 HLA alleles (HLA-A, B, and DR) or at fewer HLA alleles and in certain embodiments, may also be matched at minor loci.
- the donor cells are mismatched.
- the donor cells are derived from a sibling, an unrelated donor or a haploidentical donor.
- HLA typing can be performed using any of a variety of techniques known in the art and can also be carried out by a certified molecular diagnostic laboratory.
- Donors can also be identified through marrow registries.
- the donor cells can be xenogeneic and can be derived from any mammal, such as nonhuman primates, pigs, etc.
- the methods described herein can be used where, as between the donor and recipient, there is any degree of mismatch at MHC loci or other loci which influence graft rejection.
- mismatch may be desirable, as mismatch promotes GVL effects of donor lymphocytes.
- Methods of the invention can be used where, as between allogeneic donor and recipient, there is a mismatch at at least one MHC locus or at at least one other locus that mediates recognition and rejection, e.g., a minor antigen locus.
- the donor and recipient can be: matched at class I and mismatched at class II; mismatched at class I and matched at class II; mismatched at class I and mismatched at class II; matched at class I, matched at class II.
- Mismatched, at class I or II can mean mismatched at one or two haplotypes.
- Mismatched at MHC class I means mismatched for one or more MHC class I loci, e.g., in the case of humans, mismatched at one or more of HLA-A, HLA-B, or HLA-C.
- Mismatched at MHC class II means mismatched at one or more MHC class II loci, e.g., in the case of humans, mismatched at one or more of a DP- ⁇ , a DP- ⁇ , a DQ- ⁇ , a DQ- ⁇ , a DR- ⁇ , or a DR- ⁇ . In any of these combinations other loci which control recognition and rejection, e.g., minor antigen loci, can be matched or mismatched. In certain embodiments, it is desirable to have a mismatch at at least one class I or class II locus and, in other embodiments, a mismatch at one class I and one class II locus.
- the methods described herein for inducing tolerance to an allogeneic antigen or allogeneic graft can be used where, as between the donor and recipient, there is any degree of reactivity in a mixed lymphocyte assay, e.g., wherein there is no, low, intermediate, or high mixed lymphocyte reactivity between the donor and the recipient.
- mixed lymphocyte reactivity is used to define mismatch for class II, and the invention includes methods for performing allogeneic grafts between individuals with any degree of mismatch at class II as defined by a mixed lymphocyte assay.
- Serological tests can be used to determine mismatch at class I or II loci and the invention includes methods for performing allogeneic grafts between individuals with any degree of mismatch at class I and or II as measured with serological methods.
- the invention features methods for performing allogeneic grafts between individuals which, as determined by serological, mixed lymphocyte reactivity assay, or molecular biological techniques, are mismatched at both class I and class II.
- the donor and the subject are not related, e.g., the donor is not a sibling, the offspring of, or the parent of the recipient.
- a bank of white blood cells is established wherein donors are HLA-typed, apheresis is carried out and the resulting cells are aliquoted and stored until needed for an appropriately matched recipient.
- donor stem cells can be derived from any of a variety of sources.
- stem cells can be derived from bone marrow, peripheral blood, cord blood, and the like.
- stem cells for use in the methods described herein can be purified CD34 + stem cells (see e.g., Shizuru JA, Negrin RS, and Weissman IL, Annu Rev Med. 2005;56:509-38).
- the stem cells for use herein may be T cell- depleted.
- Donor stem cells can be either allogeneic or autologous or in certain embodiments, xenogeneic.
- the donor stem cells can be matched or mismatched as described elsewhere herein generally for donor cells/tissues/organs.
- the stem cells for use in the present invention can be derived from the same or a different donor as the donor lymphocytes.
- stem cell transplantation includes infusion into a patient of hematopoietic stem cells derived from any appropriate source of stem cells in the body.
- the stem cells may be derived, for example, from bone marrow, from the peripheral circulation following mobilization from the bone marrow, or from fetal sources such as fetal tissue, fetal circulation and umbilical cord blood.
- “Bone marrow transplantation” is considered herein to be simply one form of stem cell transplantation. Mobilization of stem cells from the bone marrow can be accomplished, for example, by treatment of the donor with granulocyte colony stimulating factor (G-CSF) or other appropriate factors (e.g., IL-8) that induce movement of stem cells from the bone marrow into the peripheral circulation.
- G-CSF granulocyte colony stimulating factor
- IL-8 granulocyte colony stimulating factor
- the stem cells can be collected from peripheral blood by any appropriate cell pheresis technique, for example through use of a commercially available blood collection device as exemplified by the CS 3000RTM Plus blood cell collection device marketed by Baxter Healthcare Corporation (Deerfield, IL). Methods for performing apheresis with the CS 3000RTM Plus machine are described in Williams et al, Bone Marrow Transplantation 5: 129-33 (1990) and Hillyer et al, Transfusion 33: 316-21 (1993).
- stem cell transplantation covers stem cell infusion into a patient resulting in either complete or partial engraftment as described above.
- Organs for use in transplantation as contemplated herein include any organ or tissue useful in the setting of allogeneic transplantation. Organs and/or tissues that can be used include but are not limited to kidney, heart, lung, liver, pancreas, pancreatic islet cells, intestines, bone, cornea, skin, heart valve, connective tissue, etc.
- donor organs/tissues are generally derived from an individual other than the recipient. In this regard, the donor organ can be derived from related or unrelated individuals. In certain embodiments, the donor organs are matched donor organs. In certain embodiments, the donor organs are from live donors ⁇ e.g., kidney donors; partial liver donation).
- the activated T cells of the present invention are generated by cell surface moiety ligation that induces activation.
- the activated T cells are generated by activating a population of T cells and stimulating an accessory molecule on the surface of the T cells with a ligand which binds the accessory molecule, referred to as the XCELLERATETM process, as described for example, in U.S. patent application numbers 10/762210; 10/350305; 10/187467; 10/133236; 08/253,694; 08/435,816; 08/592,711; 09/183,055; 09/350,202; and 09/252,150; and patent numbers 6,352,694; 5,858,358; and 5,883,223.
- the T cells are activated using other methods known in the art, for example, the rapid expansion method as described in U.S. Patent No. 5,827,642, or using methods that employ agents such as antigen, peptide, protein, peptide-MHC tetramers (see Altaian, et al.
- SEA Staphylococcus enterotoxin A
- SEB Staphylococcus enterotoxin B
- TSST-I Toxic Shock Syndrome Toxin 1
- PHA phytohemagglutinin
- PMA phorbol myristate acetate
- ionomycin Conconavalin-A, and IL-2.
- T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, cord blood, thymus, tissue biopsy, lymph node tissue, spleen tissue, or any other lymphoid tissue. T cells can also be obtained from T cell lines. T cells may also be obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig. In certain embodiments, bone marrow T cells are used. In this regard, without being bound by theory, bone marrow T cells comprise more memory T cells and home back to bone marrow. Therefore, bone marrow T cells may better promote transplant engraftment.
- cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
- the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
- the cells collected by apheresis or leukapheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
- the cells are washed with phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
- a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions.
- the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca + ⁇ Mg +"1" free PBS.
- the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
- T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells, isolating and reserving the monocytes as described previously, or for example, by centrifugation through a PERCOLLTM gradient.
- a specific subpopulation of T cells such as CD28 + , CD4 + , CD8 + , CD45RA 1" , and CD45RO T cells, can be further isolated by positive or negative selection techniques.
- CD3 + , CD28 + T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS ® M-450 CD3/CD28 T Cell Expander).
- enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
- a preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
- a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl Ib 3 CD16, HLA-DR, and CD8.
- regulatory T cells can be isolated by positive or negative selection techniques.
- T regulatory cells have a CD4 + , CD25 + , CD62L hl , GITR + , and FoxP3 + phenotype (see for example, Woo, et al, J Immunol. 2002 May 1,168(9) :4272-6; Shevach, E.M., Annu. Rev. Immunol. 2000, 18:423; Stephens, et al., Eur. J. Immunol. 2001, 31 :1247; Salomon, et al, Immunity 2000, 12:431; and Sakaguchi, et al., Immunol. Rev. 2001, 182:18).
- the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes, that are subsequently removed through magnetic separation.
- the paramagnetic particles are commercially available beads, for example, those produced by Dynal AS under the trade name DynabeadsTM. Exemplary DynabeadsTM in this regard are M-280, M-450, and M-500.
- other non-specific cells are removed by coating the paramagnetic particles with "irrelevant" proteins (e.g., serum proteins or antibodies).
- Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be expanded.
- the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.
- Another method to prepare the T cells for stimulation is to freeze the cells after the washing step, which does not require the monocyte-removal step.
- the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population.
- the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1 : 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
- the cells are then frozen to -8O 0 C at a rate of 1 ° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.
- the activated T cells of the present invention are generated by cell surface moiety ligation that induces activation.
- the activated T cells are generated by activating a population of T cells and stimulating an accessory molecule on the surface of the T cells with a ligand which binds the accessory molecule, as described for example, in U.S. patent application numbers 10/762210; 10/350305; 10/187467; 10/133236; 08/253,694; 08/435,816; 08/592,711; 09/183,055; 09/350,202; and 09/252,150; and patent numbers 6,352,694; 5,858,358; and 5,883,223.
- T cell activation may be accomplished by cell surface moiety ligation, such as stimulating the T cell receptor (TCR)/CD3 complex or the CD2 surface protein with an agent as described herein.
- agents include, but are not limited to, antibodies.
- a number of anti-human CD3 monoclonal antibodies are commercially available, exemplary are, clone BC3 (XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, WA), OKT3, prepared from hybridoma cells obtained from the American Type Culture Collection, and monoclonal antibody Gl 9-4.
- stimulatory forms of anti-CD2 antibodies are known and available. Stimulation through CD2 with anti-CD2 antibodies is typically accomplished using a combination of at least two different anti-CD2 antibodies.
- Stimulatory combinations of anti-CD2 antibodies that have been described include the following: the Tl 1.3 antibody in combination with the Tl 1.1 or Tl 1.2 antibody (Meuer et al, Cell 36:897-906, 1984), and the 9.6 antibody (which recognizes the same epitope as Tl 1.1) in combination with the 9-1 antibody (Yang et al, J. Immunol. 757:1097-1100, 1986).
- Other antibodies that bind to the same epitopes as any of the above-described antibodies can also be used.
- Additional antibodies, or combinations of antibodies can be prepared and identified by standard techniques.
- Stimulation may also be achieved through contact with agents such as antigen, peptide, protein, peptide-MHC dimers, trimers, tetramers, pentamers (see Airman, et al. Science 1996 Oct 4;274(5284):94-6 for description of tetramers), superantigens ⁇ e.g., Staphylococcus enterotoxin A (SEA), Staphylococcus enterotoxin B (SEB), Toxic Shock Syndrome Toxin 1 (TSST-I)), endotoxin, or through a variety of mitogens, including but not limited to, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, Conconavalin-A and IL-2.
- agents such as antigen, peptide, protein, peptide-MHC dimers, trimers, tetramers, pentamers (see Airman, et al. Science 1996 Oct 4;27
- a co-stimulatory or accessory molecule on the surface of the T cells such as CD28
- an agent e.g., an antibody or a ligand
- any agent including an anti-CD28 antibody or fragment thereof capable of cross-linking the CD28 molecule, or a natural ligand for CD28, such as B7-1, can be used to stimulate T cells.
- Exemplary anti-CD28 antibodies or fragments thereof useful in the context of the present invention include monoclonal antibody 9.3 (IgG2 a ) (Bristol-Myers Squibb, Princeton, NJ), monoclonal antibody KOLT-2 (IgGl), 15E8 (IgGl), 248.23.2 (IgM), clone B-T3 (XR-CD28; Diaclone, Besancon, France) and EX5.3D10 ( ⁇ gG2 a ) (ATCC HBl 1373).
- Exemplary natural ligands include the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86) (Freedman et al, J. Immunol.
- binding homologues of a natural ligand can also be used in accordance with the present invention.
- Other agents may include natural and synthetic ligands. Agents may include, but are not limited to, other antibodies or fragments thereof, a peptide, polypeptide, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens.
- the methods of the present invention use agents/ligands bound to a surface.
- the surface may be any surface capable of having an agent bound thereto or integrated into and that is biocompatible, that is, substantially non-toxic to the target cells to be stimulated.
- the biocompatible surface may be biodegradable or non-biodegradable.
- the surface may be natural or synthetic, and a synthetic surface may be a polymer.
- the surface may comprise collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions.
- a polysaccharide may include for example, cellulose, agarose, dextran, chitosan, hyaluronic acid, or alginate.
- polymers may include polyesters, polyethers, polyanhydrides, polyalkylcyanoacryllates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinylacetates, block copolymers, polypropylene, polytetrafluorethylene (PTFE), or polyurethanes.
- the polymer may be lactic acid or a copolymer.
- a copolymer may comprise lactic acid and glycolic acid (PLGA).
- Non-biodegradable surfaces may include polymers, such as poly(dimethylsiloxane) and poly(ethylene-vinyl acetate).
- Biocompatible surfaces include for example, glass (e.g., bioglass), collagen, metal, hydroxyapatite, aluminate, bioceramic materials, hyaluronic acid polymers, alginate, acrylic ester polymers, lactic acid polymer, glycolic acid polymer, lactic acid/glycolic acid polymer, purified proteins, purified peptides, or extracellular matrix compositions.
- Other polymers comprising a surface may include glass, silica, silicon, hydroxyapatite, hydrogels, collagen, acrolein, polyacrylamide, polypropylene, polystyrene, nylon, or any number of plastics or synthetic organic polymers, or the like.
- the surface may comprise a biological structure, such as a liposome, a virus-like particle, or a cell.
- the surface may be in the form of a lipid, a plate, bag, pellet, fiber, mesh, or particle.
- a particle may include, a colloidal particle, a microsphere, nanoparticle, a bead, or the like.
- commercially available surfaces such as beads or other particles, are useful (e.g., Miltenyi Particles, Miltenyi Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADSTM, Dynal Inc., New York; PURABEADSTM, Prometic Biosciences).
- the bead may be of any size that effectuates target cell stimulation.
- beads are preferably from about 5 nanometers to about 500 ⁇ m in size. Accordingly, the choice of bead size depends on the particular use the bead will serve. For example, if the bead is used for monocyte depletion, a small size is chosen to facilitate monocyte ingestion (e.g., 2.8 ⁇ m and 4.5 ⁇ m in diameter or any size that may be engulfed, such as nanometer sizes); however, when separation of beads by filtration is desired, bead sizes of no less than 50 ⁇ m are typically used.
- the beads when using paramagnetic beads, the beads typically range in size from about 2.8 ⁇ m to about 500 ⁇ m and more preferably from about 2.8 ⁇ m to about 50 ⁇ m.
- super-paramagnetic nanoparticles which can be as small as about 10 "5 nm. Accordingly, as is readily apparent from the discussion above, virtually any particle size may be utilized.
- An agent may be attached or coupled to, or integrated into a surface by a variety of methods known and available in the art.
- the agent may be an antibody, a natural ligand, a protein ligand, or a synthetic ligand.
- the attachment may be covalent or noncovalent, electrostatic, or hydrophobic and may be accomplished by a variety of attachment means, including for example, chemical, mechanical, enzymatic, electrostatic, or other means whereby an agent is capable of stimulating the cells.
- the antibody to a ligand first may be attached to a surface, or avidin or streptavidin may be attached to the surface for binding to a biotinylated ligand.
- the antibody to the ligand may be attached to the surface via an anti-idiotype antibody.
- Another example includes using protein A or protein G, or other non-specific antibody binding molecules, attached to surfaces to bind an antibody.
- the ligand may be attached to the surface by chemical means, such as cross-linking to the surface, using commercially available cross-linking reagents (Pierce, Rockford, IL) or other means.
- the ligands are covalently bound to the surface.
- commercially available tosyl-activated DYNABEADSTM or DYNABEADSTM with epoxy-surface reactive groups are incubated with the polypeptide ligand of interest according to the manufacturer's instructions. Briefly, such conditions typically involve incubation in a phosphate buffer from pH 4 to pH 9.5 at temperatures ranging from 4 to 37 degrees C.
- the agent such as certain ligands may be of singular origin or multiple origins and may be antibodies or fragments thereof while in another aspect, when utilizing T cells, the co-stimulatory ligand is a B7 molecule (e.g., B7-1, B7-2). These ligands are coupled to the surface by any of the different attachment means discussed above.
- the B7 molecule to be coupled to the surface may be isolated from a cell expressing the co-stimulatory molecule, or obtained using standard recombinant DNA technology and expression systems that allow for production and isolation of the co-stimulatory molecule(s) as described herein.
- Fragments, mutants, or variants of a B7 molecule that retain the capability to trigger a co-stimulatory signal in T cells when coupled to the surface of a cell can also be used.
- any ligand useful in the activation and induction of proliferation of a subset of T cells may also be immobilized on beads or culture vessel surfaces or any surface.
- covalent binding of the ligand to the surface is one preferred methodology, adsorption or capture by a secondary monoclonal antibody may also be used.
- the amount of a particular ligand attached to a surface may be readily determined by flow cytometric analysis if the surface is that of beads or determined by enzyme-linked immunosorbent assay (ELISA) if the surface is a tissue culture dish, mesh, fibers, bags, for example.
- ELISA enzyme-linked immunosorbent assay
- the stimulatory form of a B7 molecule or an anti-CD28 antibody or fragment thereof is attached to the same solid phase surface as the agent that stimulates the TCR/CD3 complex, such as an anti-CD3 antibody.
- the stimulatory form of a 4- IBB ligand molecule or an anti-4- IBB antibody or fragment thereof is attached to the same solid phase surface as the agent that stimulates the TCR/CD3 complex, such as an anti-CD3 antibody.
- anti-CD3 antibodies other antibodies that bind to receptors that mimic antigen signals may be used.
- the beads or other surfaces may be coated with combinations of anti-CD2 antibodies and a B7 molecule and in particular anti-CD3 antibodies and anti-CD28 antibodies.
- the surfaces may be coated with three or more agents, such as combinations of any of the agents described herein, for example, anti-CD3 antibodies, anti-CD28 antibodies, and anti-4-lBB antibodies.
- the primary stimulatory signal and the co-stimulatory signal for the T- cell may be provided by different protocols.
- the agents providing each signal may be in solution or coupled to a surface.
- the agents When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation).
- one agent may be coupled to a surface and the other agent in solution.
- surfaces may be coated or conjugated directly (including covalently) or indirectly (e.g., streptavidin/biotin and the like) with antibodies or other components to stimulate T cell activation and expansion.
- the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing FC receptors or an antibody or other binding agent which will bind to the agents.
- the two agents are immobilized or otherwise attached on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans.”
- the agent providing the primary activation signal is an anti-CD3 antibody and the agent providing the co-stimulatory signal is an anti-CD28 antibody; and both agents are co-immobilized or otherwise attached to the same bead in equivalent molecular amounts.
- a 1:1 ratio of each antibody bound to the beads for CD4 + T-cell expansion and T-cell growth is used.
- a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one particular embodiment an increase of from about .5 to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e. 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.
- a 1:200 CD3:CD28 ratio of antibody bound to beads is used. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used, hi one preferred embodiment, a 1 :10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.
- three or more agents are coupled to a surface.
- the agents may be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation).
- one or more agents may be coupled to a surface and the other agent or agents may be in solution.
- Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T-cells or other target cells.
- the ratio of particle to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many.
- the ratio of cells to particles ranges from 1:100 to 100:1 and any integer values in-between and in further embodiments the ratio comprises 1:9 to 9:1 and any integer values in between, can also be used to stimulate T-cells.
- the ratio of anti-CD3- and anti-CD28 -coupled beads particles to T-cells that result in T-cell stimulation can vary as noted above, however in certain embodiments, the ratio of anti-CD3 and anti-CD28 coupled beads to cells includes 1:50, 1:40, 1:30, 1:20, 1:15, 1:10, 1:5, 1:4, 1 :3, 1:2, 1:1, 2:1, 3:1, 4:1 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1, with one particular ratio being 3:1 beads/particles per T-cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation.
- the ratio of particles to cells is from 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell counts on the day of addition).
- 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.
- particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1 :5 on the third and fifth days of stimulation.
- the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation.
- particles are added on a daily or every other day basis to a final ratio of 1 :1 on the first day, and 1 :10 on the third and fifth days of stimulation.
- ratios will vary depending on particle size and on cell size and type.
- the rate of T-cell proliferation is monitored periodically ⁇ e.g., daily) by, for example, examining the size or measuring the volume of the T-cells, such as with a Coulter Counter.
- a resting T-cell has a mean diameter of about 6.8 microns, and upon initial activation and stimulation, in the presence of the stimulating ligand, the T-cell mean diameter will increase to over 12 microns by day 4 and begin to decrease by about day 6.
- the T-cells may be reactivated and re-stimulated to induce further proliferation of the T-cells.
- the rate of T-cell proliferation and time for T-cell re- stimulation can be monitored by assaying for the presence of cell surface molecules, such as, CD154, CD54, CD25, CD137, CD134, B7-1, B7-2, which are induced on activated T-cells.
- cell surface molecules such as, CD154, CD54, CD25, CD137, CD134, B7-1, B7-2
- T-cells it may be necessary to reactivate and re-stimulate the T-cells with a stimulatory agent such as an anti-CD3 antibody and an anti-CD28 antibody (such as B-T3, XR- CD28 (Diaclone, Besancon, France) or monoclonal antibody ES5.2D8 several times to produce a population of CD4 + or CD8 + cells increased in number from about 10 to about 1, 000-fold the original T-cell population.
- a stimulatory agent such as an anti-CD3 antibody and an anti-CD28 antibody (such as B-T3, XR- CD28 (Diaclone, Besancon, France) or monoclonal antibody ES5.2D8 several times to produce a population of CD4 + or CD8 + cells increased in number from about 10 to about 1, 000-fold the original T-cell population.
- a stimulatory agent such as an anti-CD3 antibody and an anti-CD28 antibody (such as B-T3, XR- CD28 (Diaclone, Be
- the time of exposure to stimulatory agents such as anti-CD3/anti-CD28 (i.e., CD3xCD28)-coated beads may be modified or tailored to obtain a desired T-cell phenotype.
- CD4 + T-cells express important immune-regulatory molecules, such as GM-CSF, CD40L, and IL-2, for example.
- a method, such as that described herein, which preserves or enhances the CD4:CD8 ratio could be of significant benefit.
- the present invention it may be beneficial to increase the number of infused cells expressing GM-CSF, or IL-2, all of which are expressed predominantly by CD4 + T-cells.
- the T cell activation approaches described herein can also be utilized, by for example, pre-selecting for CD8 + cells prior to stimulation and/or culture. Such situations may exist where increased levels of IFN- ⁇ is preferred. Further, in other applications, it may be desirable to utilize a population of T H I -type cells versus T ⁇ 2-type cells (or vice versa).
- times of cell surface moiety ligation that induces activation may be varied or pulsed. For example expansion times may be varied to obtain the specific phenotype of interest and/or different types of stimulatory agents may be used (e.g., antibodies or fragments thereof, a peptide, polypeptide, MHC/peptide tetramer, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens).
- stimulatory agents e.g., antibodies or fragments thereof, a peptide, polypeptide, MHC/peptide tetramer, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens.
- the expression of a variety of phenotypic markers change over time; therefore, a particular time point or stimulatory agent may be chosen to obtain a specific population of T-cells.
- the stimulation and/or expansion time may be four weeks or less, 2 weeks or less, 10 days or less, or 8 days or less (four weeks or less includes all time ranges from 4 weeks down to 1 day (24 hours)).
- stimulation and expansion may be carried out for 6 days or less, 4 days or less, 2 days or less, and in other embodiments for as little as 24 or less hours, and preferably 4-6 hours or less (these ranges include any integer values in between).
- the population of T-cells may not increase in number as dramatically, but the population will provide more robust and healthy activated T-cells that can continue to proliferate in vivo and more closely resemble the natural effector T-cell pool.
- the time of exposure to stimulatory agents such as anti-CD3/anti-CD28 (i.e., 3x28)-coated beads may be modified or tailored to obtain a desired T cell phenotype.
- a desired population of T cells can be selected using any number of selection techniques, prior to stimulation.
- CD4 + T cells which express important immune- regulatory molecules, such as GM-CSF, CD40L, and IL-2, for example.
- CD4- mediated help if preferred, a method, such as that described herein, which preserves or enhances the CD4:CD8 ratio could be of significant benefit.
- Increased numbers of CD4 + T cells can increase the amount of cell-expressed CD40L introduced into patients, potentially improving target cell visibility (improved APC function). Similar effects can be seen by increasing the number of infused cells expressing GM-CSF, or IL-2, all of which are expressed predominantly by CD4 + T cells.
- the XCELLERATE approaches described herein can also be utilized, by for example, preselecting for CD8 + cells prior to stimulation and/or culture. Such situations may exist where increased levels of IFN- ⁇ or increased cytolysis of a target cell is preferred.
- the XCELLERATETM process can be modified or tailored to promote homing of T cells to particular sites of interest, such as lymph nodes or sites of inflammation, or to bone marrow, for example.
- the XCELLERATETM approaches described herein can also be utilized for the generation of T regulatory cells and/or veto cells for use in promoting transplant engraftment and immune reconstitution. Such cells can be used to suppress the anti- alloantigen host response.
- T regulatory cells can be used for specific immunosuppression in the case of inflammatory disease, autoimmunity, and foreign graft acceptance, or any other disease setting where regulatory T cells are desired.
- T regulatory cells have a CD4 + , CD25 + , CD62L hi , GITR + , and FoxP3 + phenotype (see for example, Woo, et al, J Immunol.
- Regulatory T cells can be generated and expanded using the methods of the present invention.
- the regulatory T cells can be antigen- specific and/or polyclonal.
- the activated T cells for use in promoting transplant engraftment as described herein are activated such that the T cells are not regulatory T cells, suppressor T cells, Th2-type cells or gamma delta T cells.
- the activated T cells for use in promoting transplant engraftment are primarily ThI -type T cells.
- the activated T cells for use in promoting transplant engraftment are unpolarized T cells.
- the T cells for use in the methods described herein to promote transplant engraftment can be purified using any of a variety of methods.
- the activated T cells of the present invention are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, CD3 + T cells.
- the T cells may be purified and/or expanded CD4 T cells.
- the T cells for use in the methods described herein may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, CD4 + T cells.
- the T cells may be purified and/or expanded CDS + T cells.
- the T cells for use in the methods described herein may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, CDS + T cells.
- the T cells for use in the methods described herein may be purified and/or expanded CD4 CD25 + regulatory T cells.
- the T cells for use in the methods described herein may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, CD4 + CD25 + T cells.
- the activated T cells for use in the methods herein are activated with host antigens such that the activated T cells target host cells that cause rejection.
- the activated allogeneic T cells may be directed to host cytolytic T cells, NK cells, and the like.
- the T cells may be activated such that they target host histocompatibility antigens.
- CD25 constitutes an important part of the autocrine loop that allows rapid T- cell division.
- CD 154 has been shown to play a key role in stimulating maturation of the antigen-presenting dendritic cells; activating B-cells for antibody production; regulating T H cell proliferation; enhancing Tc cell differentiation; regulating cytokine secretion of both T H cells and antigen-presenting cells; and stimulating expression of co-stimulatory ligands, including CD80, CD86, and CD 154.
- CD62L is important for homing of T-cells to lymphoid tissues and trafficking T-cells to sites of inflammation. Because down-regulation of CD62L occurs early following activation, the T-cells could be expanded for shorter periods of time. Conversely, longer periods of time in culture would generate a T-cell population with higher levels of CD62L and thus a higher ability to target the activated T-cells to these sites under other preferred conditions.
- CD49d an adhesion molecule that is involved in trafficking lymphocytes from blood to tissues spaces at sites of inflammation. Binding of the CD49d ligand to CD49d also allows the T-cell to receive co-stimulatory signals for activation and proliferation through binding by VCAM-I or fibronectin ligands.
- the expression of the adhesion molecule CD54, involved in T-cell- APC and T-cell-T-cell interactions as well as homing to sites of inflammation, also changes over the course of expansion. Accordingly, T-cells could be stimulated for selected periods of time that coincide with the marker profile of interest and subsequently collected and infused. Activated T cells could also be applied directly to an injury site. Thus, T-cell populations could be tailored to express the markers believed to provide the most therapeutic benefit for the indication to be treated.
- one of ordinary skill in the art understands removal of the stimulation signal from the cells is dependent upon the type of surface used. For example, if paramagnetic beads are used, then magnetic separation is the feasible option. Separation techniques are described in detail by paramagnetic bead manufacturers' instructions (for example, DYNAL Inc., Oslo, Norway). Furthermore, filtration may be used if the surface is a bead large enough to be separated from the cells. In addition, a variety of transfusion filters are commercially available, including 20 micron and 80 micron transfusion filters (Baxter). Accordingly, so long as the beads are larger than the mesh size of the filter, such filtration is highly efficient.
- the beads may pass through the filter, but cells may remain, thus allowing separation.
- the antibodies used in the methods described herein can be readily obtained from public sources, such as the ATCC, antibodies to T-cell accessory molecules and the CD3 complex can be produced by standard techniques. Methodologies for generating antibodies for use in the methods of the invention are well-known in the art.
- the T cells may be genetically modified using any number of methods known in the art. For example, the T cells may be genetically modified to introduce a "suicide" gene or other molecule that allows for subsequence drug/chemical removal ("killing") of the infused donor T cells if desired.
- Suitable suicide genes include, but are not limited to, Herpes simplex type-1 virus (HSVl) thymidine kinase, a fusion between HSVl thymidine kinase and zeocin- resistance gene, E. coli Cytosine Deaminase, E. coli Cytosine Deaminase fused to Uracil Phosphoribosyltransferase, S. cerevisiae Cytosine Deaminase, S.
- HSVl Herpes simplex type-1 virus
- EHV4 Equine herpes virus 4
- HSVl thymidine kinase fused to Zeocin-resistance gene
- E. coli thymidine kinase fused to thymidylate kinase E. coli Uracil Phosphoribosyltransferase, dimerizable, modified human caspase 9 fused to a human FK506 binding protein (FKBP)
- FKBP human FK506 binding protein
- T cells may be genetically modified to introduce one or more polynucleotides encoding one or more proteins or chimeric proteins that regulate T cell function ⁇ e.g., cytokine receptors such as IL-2 receptor, specific T cell receptors (e.g., receptors that recognize host alloreactive cells such as T cells, NK cells, etc), chemokine receptors such as CCR5 and CXCR4, adhesion molecules, homing receptors, and the like).
- cytokine receptors such as IL-2 receptor
- specific T cell receptors e.g., receptors that recognize host alloreactive cells such as T cells, NK cells, etc
- chemokine receptors such as CCR5 and CXCR4, adhesion molecules, homing receptors, and the like.
- T cells may be genetically modified to target T cells to the bone marrow, such as with polynucleotides encoding P-selectin glycoprotein ligand-1 (PSGL-I) and/or other E-selectin ligands, alpha-4-integrin, CD44, etc.
- PSGL-I P-selectin glycoprotein ligand-1
- CD44 alpha-4-integrin
- the T cells may be transfected using numerous RNA or DNA expression vectors known to those of ordinary skill in the art. Genetic modification may comprise RNA or DNA transfection using any number of techniques known in the art, for example electroporation (using e.g., the Gene Pulser II, BioRad, Richmond, CA), various cationic lipids, (LIPOFECTAMINETM, Life Technologies, Carlsbad, CA), or other techniques such as calcium phosphate transfection as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
- electroporation using e.g., the Gene Pulser II, BioRad, Richmond, CA
- various cationic lipids (LIPOFECTAMINETM, Life Technologies, Carlsbad, CA)
- calcium phosphate transfection as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
- RNA or DNA in 500 ⁇ l of Opti-MEM can be mixed with a cationic lipid at a concentration of 10 to 100 ⁇ g, and incubated at room temperature for 20 to 30 minutes.
- suitable lipids include LIPOFECTINTM, LIPOFECTAMINETM.
- the resulting nucleic acid-lipid complex is then added to 1-3 X 10 6 cells, preferably 2 X 10 6 , antigen- presenting cells in a total volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37 0 C for 2 to 4 hours.
- the T cells may also be transduced using viral transduction methodologies as described below
- the retroviral vector may be an amphotropic retroviral vector, preferably a vector characterized in that it has a long terminal repeat sequence (LTR) 5 e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), human immunodeficiency virus (HIV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV). murine stem cell virus (MSCV), spleen focus forming virus(SFFV), or adeno-associated virus (AAV).
- LTR long terminal repeat sequence
- retroviral vectors are derived from murine retroviruses.
- Retroviruses adaptable for use in accordance with the present invention can, however, be derived from any avian or mammalian cell source. These retroviruses are preferably amphotropic, meaning that they are capable of infecting host cells of several species, including humans.
- the gene to be expressed replaces the retroviral gag, pol and/or env sequences.
- a number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al.
- the present invention provides T cell compositions for use in promoting transplant engraftment, reducing the need for harsh conditioning regimens, and promoting immune reconstitution in transplant patients.
- the present invention provides compositions of allogeneic donor lymphocytes for use in a variety of transplant settings to promote transplant engraftment.
- the goal of the present invention in this context is not to achieve final engraftment of the lymphocytes (e.g., T cells); the desired effect is promotion of transplant engraftment (e.g., chimerism), immune reconstitution and a decrease in the need for potent conditioning regimens.
- the present invention stems in part from the observation that activated T cells, such as the XCELLERATETM T cells described herein and elsewhere, generally generate less GVHD than do naive T cells while promoting transplant (e.g., stem cells or tissue/organ transplants) engraftment and immune reconstitution.
- transplant e.g., stem cells or tissue/organ transplants
- the percentage of alloreactive cells is lower in XCELLERATETM T cells.
- harsh conditioning regimens contribute to GVHD by producing inflammatory stimuli.
- the present invention provides for activated donor T lymphocytes in transplant settings to promote engraftment and/or immune reconstitution in the absence of or with greatly reduced conditioning.
- Conditioning regimens for allogeneic transplantation are typically myeloablative and lymphoablative in order to eliminate malignant cells (to eradicate disease) and recipient lymphoid cells (to allow durable donor hematopoietic stem-cell engraftment; to eliminate graft-reactive host cells).
- malignant cells to eradicate disease
- recipient lymphoid cells to allow durable donor hematopoietic stem-cell engraftment; to eliminate graft-reactive host cells.
- Such regimens are associated with substantial risk of morbidity and mortality. Older patients (age > 50 years) and patients with confounding medical conditions are frequently not eligible for allogeneic transplantation because of the risks associated with these regimens.
- Regimen-related toxicity is a considerable cause of morbidity and mortality in transplant patients and can include low blood counts, anemia, neutropenia, thrombocytopenia, fatigue, infection, fever, mouth sores, nausea and vomiting, hair loss, pain, depression, reproductive and sexual dysfunction. Nausea, vomiting, stomatitis, enteritis, alopecia, erythema or rash, and diarrhea occur in most graft recipients and can largely be controlled. More serious complications might include idiopathic interstitial pneumonitis, hemorrhagic cystitis, heart failure and/or pericarditis, hepatic veno-occlusive disease (VOD), and, less commonly, pulmonary hemorrhage.
- VOD hepatic veno-occlusive disease
- activated T cells can be used to promote engraftment in the presence of conditioning regimens that would, without the use of T cells, not allow for transplant engraftment.
- reduced-intensity regimens such as those described in Table 2 do not generally allow transplant engraftment in other than HLA-identical sibling grafts.
- such reduced-intensity regimens could be used in many transplant settings (e.g., in HLA mismatch settings, cord blood, matched unrelated and mismatched unrelated transplants settings) where the success of the transplant has been limited by the considerable regimen-related toxicity.
- reduced-intensity regimens in conjunction with the activated T cells of the present invention can be used for transplantation in compromised or elderly patients.
- using the activated T cells of the present invention promotes engraftment and allows for reduced intensity regimens that lead to much fewer regimen-related toxicity (RRT) and extend the patient population eligible for transplant.
- RRT regimen-related toxicity
- the present invention provides methods for using activated allogeneic T cells in a variety of transplant settings with reduced- intensity conditioning regimens, thus allowing for reduction in RRT. Reduction of RRT can be easily assessed by the skilled artisan using art-accepted systems for grading toxicity (see, e.g., S.I.
- the present invention also provides methods for using activated allogeneic T cells to further reduce the currently accepted reduced-intensity conditioning regimens, thus allowing for further reduction in RRT in transplant settings where reduced- intensity regimens are currently used, such as in matched transplants, e.g., HLA- identical sibling grafts.
- reduced- intensity regimens are currently used, such as in matched transplants, e.g., HLA- identical sibling grafts.
- any of the regimens recited in Table 2 or other reduced-intensity regimens known in the art may be further reduced when used in conjuction with activated allogeneic T cells as described herein.
- Illustrative reductions in the currently accepted reduced-intensity regimens include, but are not limited to, Fludarabine 15, 20, or 25 mg/m 2 X 4, cytosine arabinoside 1.0, 1.5 g/m 2 X 4, idarubicine 8, 9, or 10 mg/m 2 X 3, Fludarabine 30 mg/m 2 X 3 or 2 cytosine arabinoside 2 g/m 2 X 3 or 2, idarubicine 12 mg/m 2 X 2 or 1, 2-chlorodeoxyadenosine 8, 9, or 10 mg/m 2 X 5, 4, or 3, cytosine arabinaside 0.5, 0.6, 0.7, 0.8 g/m 2 X 5, 4, or 3.
- a further advantage to the present invention is that, by using T cells to facilitate engraftment, this allows for reduction and potentially the elimination of post transplant immunosuppression and the related side effects (side effects including, but not limited to, increased relapse rates, infections, hyperlipidaemia, abnormal liver enzyme, hypertension, hirsutism, neuropathy, nephrotoxicity, tremor, hyperkalemia, thrush, diabetes, bone/hip problems, gastrointestinal complications, especially diarrhea and nausea along with leukopenia).
- side effects including, but not limited to, increased relapse rates, infections, hyperlipidaemia, abnormal liver enzyme, hypertension, hirsutism, neuropathy, nephrotoxicity, tremor, hyperkalemia, thrush, diabetes, bone/hip problems, gastrointestinal complications, especially diarrhea and nausea along with leukopenia).
- post transplant immunosuppressive regimens including a reduction in the use of any one or more of the following: cyclosporine, methotrexate, sirolimus (Rapamycin), prednisone, mycophenolic acid, thymoglobulin induction, Tacrolimus, Azathioprine, steroids/corticosteroids, various antibodies such as muronomab-CD3 (anti-CD3 antibody), basiliximab and daclizumab (humanized anti-monoclonal antibodies that target the IL-2 receptor) and other immunosuppressants known in the art.
- CDl 54 is expressed on activated T cells in a temporal manner and has been shown to be a key element in T cells interactions via CD40 on APCs (see e.g., U.S. application No. 10/762,210). Blocking the interaction of these two receptors can effectively alter, and even shut-off, an immune response.
- the activated T cells of the present invention have been shown to express elevated levels of CD40L that peak at about day 3 to day 4 of activation/expansion and remains elevated out to day 6 and day 7.
- CD40L may be critical with regard to the allogeneic T lymphocytes used as described herein and thus the T cells described herein may be particularly suited to generating an effective GVL reaction and promoting transplant engraftment.
- the methods of the present invention can be used in any allogeneic transplant setting to promote transplant engraftment.
- the present invention can be used in organ, tissue, and bone marrow/stem cell transplantation, and the like.
- stem cell transplants the T cells of the present invention can be administered before, during or after stem cell transplants.
- the activated T cells are administered at the same time as the transplant.
- the activated T cells are administered within 24 hours of the transplant.
- the activated T cells are administered within less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of the transplant.
- the activated T cells are administered within 11, 12, 13, 14, 15, 16, 17.
- the activated T cells are administered within 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 days of the transplant.
- the number of T cells administered to the transplant patient will vary depending on the setting and state of the patient being treated and thus can be determined by a qualified physician.
- the number of T cells administered to the transplant patient may be between I X lO 8 activated T cells and 2 X 10 11 activated T cells.
- the number of T cells administered may be about IXlO 8 , 1 X 10 9 , 5 X 10 9 , 1 X 10 10 , 5 X 10 10 , or 1 X 10 11 cells.
- the number of T cells administered to the patient will vary depending on the conditioning regimen. In this regard, in certain embodiments, the number of T cells administered is inversely related to the level of conditioning.
- T cells of the present invention can be administered in multiple infusions.
- the T cells for use in the methods of the present invention may be third party T cells, i.e., from an individual other than the stem cell donor.
- third party T cells may facilitate engraftment by inducing graft versus marrow effect to "make space" in the marrow for donor stem cells that are unrelated to the source of the T third party T cells (see e.g., veto cells such as those described in U.S. Patent No. 6,544,506).
- the number of stem cells whatever their source e.g., bone marrow cells, purified stem cells, cord blood cells, peripheral blood stem cells, etc.
- the number of CD34+ stem cells can range from 1 million cells/kilogram to 30 million cells/kilogram.
- the number of CD34 + stem cells can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 million cells/kilogram.
- the number of total nucleated cells for use in the transplant methods described herein may vary from about 1 X 10 7 /kilogram to about 1 X 10 9 /kilogram.
- the number of total nucleated cells can be about 1 X 10 7 , 5 X 10 7 , 1 X 10 8 , 2 X 10 8 , 3 X 10 8 , 4 X 10 8 , 5 X 10 8 , 6 X 10 8 , 7 X 10 8 , 8 X 10 8 , 9 X 10 8 , or about 1 X 10 9 /kilogram.
- the number also may vary.
- the number of purified stem cells can be between about 1,000 cells /kilogram to about 300,000 cells/kilogram.
- the number of purified stem cells for use in the methods described herein can be about 1 X 10 3 , 5 X 10 3 , 1 X 10 4 , 5 X 10 4 , 1 X 10 5 , 2 X 10 5 , 3 X 10 5 , 4 X 10 5 , or about 5 X 10 5 cells/kilogram.
- high doses (megadoses) of stem cells increase engraftment (see e.g., Aversa et al, 2005 Journal of Clinical Oncology 23(15):3447-3454; Reisner, et al, 2002 Current Opinion in Organ Transplantation.
- the present invention contemplates the use of megadoses of stem cells (such as purified stem cells, T cell depleted, purified stem cells) in combination with activated allogeneic T cells to promote transplant engraftment.
- stem cells such as purified stem cells, T cell depleted, purified stem cells
- the methods of the present invention can be used in any allogeneic transplant setting to promote transplant engraftment and thus, to promote chimerism.
- transplant engraftment can be measured as % chimerism. Percent chimerism can range from 10%- 100%.
- 100% chimerism following a transplant is desired since this strongly correlates with durable remission.
- 100% chimerism is not necessarily needed; all that is needed in such a setting is enough chimerism to produce cells.
- sickle cell anemia only about 10% donor chimerism is needed (sickling cells only sickle if above a certain concentration in the blood).
- donor chimerism is sufficient (such as but not limited to, bone marrow failure, aplastic anemia and sickle cell anemia).
- Mixed chimerism is generally defined as 5% or more recipient cells using analyses known in the art (e.g., STR analysis).
- the present invention contemplates using activated T cells to promote donor chimerism wherein donor chimerism is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or higher.
- the present invention contemplates using activated T cells to promote donor chimerism wherein donor chimerism is about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or higher. In certain embodiments, the present invention contemplates using activated T cells to promote donor chimerism wherein donor chimerism is about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or higher. In certain embodiments, the present invention contemplates using activated T cells to promote donor chimerism wherein donor chimerism is about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
- Chimerism analysis from peripheral blood can be determined using techniques known in the art (see for example, Massenkeil, et ah, Bone Marrow Transplantation (2003) 31, 339-345; Thiede C, et al, Bone Marrow Transplant 1999 23:1055-1060).
- Illustrative techniques include STR analysis using commercially available kit.
- Donor allogeneic or xenogeneic T cells can be stimulated and expanded as described herein or using other methods known in the art wherein T cells are stimulated and expanded to therapeutic levels, for promoting immune reconstitution and transplant engraftment in a variety of settings, such as bone marrow transplants for the treatment of malignancies.
- T cells of the present invention are useful for promoting transplant engraftment used for treating melanoma, non-Hodgkin's lymphoma, cutaneous T cell lymphoma, Hodgkin's disease, leukemia, plasmacytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, testicular cancer, gastric cancer, esophageal cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), myelodysplastic syndrome, myeloproliferative disorders, chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), or other cancers.
- ALL acute lymphoblastic leukemia
- donor allogeneic or xenogeneic T cells can be stimulated and expanded as described herein or using other methods known in the art wherein T cells are stimulated and expanded to therapeutic levels, for promoting transplant engraftment where the transplant is used in the treatment of an autoimmune disease.
- T cells of the present invention are useful for promoting engraftment for transplants used for treating autoimmune diseases such as, but not limited to, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes, Addison's disease, celiac disease, chronic fatigue syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, fibromyalgia, systemic lupus erythematosus, psoriasis, pemphigus vulgaris, Sjogren's syndrome, hyperthyroidism/Graves disease, hypothyroidism/Hashimoto's disease, Insulin-dependent diabetes (type 1), Myasthenia Gravis, endometriosis, scleroderma, pernicious anemia, Goodpasture syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including multiply transfused aplastic anemia patients), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, a
- donor allogeneic or xenogeneic T cells can be stimulated and expanded as described herein or using other methods known in the art wherein T cells are stimulated and expanded to therapeutic levels, for promoting transplant engraftment where the transplant is used in the treatment of an immunodeficiency or a congenital disorder.
- donor allogeneic or xenogeneic T cells can be stimulated and expanded as described herein or using other methods known in the art wherein T cells are stimulated and expanded to therapeutic levels, for promoting transplant engraftment where the transplant is used in the treatment of bone marrow failure or sickle cell anemia.
- the activated allogeneic T cells are used to promote transplant engraftment where the transplant is used in the treatment of multiply transfused anemic patients, such as in aplastic anemia, sickle cell anemia, and beta thalassemia.
- multiply transfused anemic patients such as in aplastic anemia, sickle cell anemia, and beta thalassemia.
- the activated T cells described herein are used for promoting transplant engraftment where the transplant is a solid organ or tissue transplant, such as a kidney, heart, liver, lung, pancreas, pancreatic islet cells, intestines, bone, cornea, skin, heart valve, connective tissue, etc.
- a solid organ or tissue transplant such as a kidney, heart, liver, lung, pancreas, pancreatic islet cells, intestines, bone, cornea, skin, heart valve, connective tissue, etc.
- the donor T cell compositions of the present invention can be used in conjunction with a variety of anti-tumor treatment modalities, including but not limited to, GLIVEC.
- one setting where the present invention can be used is in the setting where patients have been treated to minimal residual disease, for example, after chemotherapy in CLL or NHL or after GLIVEC treatment in CML.
- patients who have less than three logs reduction of tumor cells after GLIVEC treatment or more than 0.1% tumor cells will relapse.
- these patients will benefit from treatment with the T cells of the present invention.
- certain goals of treatment with the cells of the present invention are to clear minimal residual disease, achieve durable remission and also to allow patients to stop taking drugs without which patients relapse.
- cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels are administered to a patient before, simultaneously or following any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
- agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies,
- the cell compositions of the present invention are administered to a patient before, simultaneously or following bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external -beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OK.T3, CAMPATH, or antithymocyte globulin (ATG).
- chemotherapy agents such as, fludarabine, external -beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OK.T3, CAMPATH, or antithymocyte globulin (ATG).
- the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g. Rituxan.
- expanded cells are administered before or following surgery.
- the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
- the scaling of dosages for human administration can be performed according to art-accepted practices.
- the conditioning regimens may be determined by the skilled physician according to the particular patient needs. In certain embodiments, high intensity conditioning may be used. In other embodiments reduced-intensity regimens may be used.
- the T cells of the present invention allow for a conditioning regimen that, under previously known transplant conditions, would not lead to engraftment and donor chimerism.
- use of the activated T cells of the present invention administered at the time of transplantation promotes transplant engraftment under reduced conditioning regimens that would otherwise (e.g., without co-administration of T cells) not allow transplant engraftment.
- the conditioning used in the present invention is in a dose range such that with the administration of the T cells as described herein, donor chimerism is achieved, while in the absence of administration of the T cells, chimerism would normally not be achieved.
- the dose and exact regimen can be varied depending on the disease and the condition of the patient. Summarized below are standard dose ranges for certain agents contemplated for use herein (see, e.g., N. Mounier and C. Gisselbrecht Annals of Oncology 9 (Suppl. 1):S15- S21, 1998).
- reduced intensity regimens are contemplated for use herein.
- Illustrative reduced intensity regimens are summarized in Table 2 below (taken from Urs Schanz, Swiss Med WkIy 2001; 131:59-64).
- T cells of the present invention provide transplant engraftment and immune reconstitution in conjunction with reduced-intensity conditioning regimens, further reduction of the currently accepted reduced-intensity regimens is contemplated herein.
- Agents that may be used in conditioning regimens of the present invention include, but are not limited to, busulphan, dimethyl myleran, thiotepa, cyclosporin, azathioprine, methotrexate, mycophenolate, methyl prednisolone, prednisone, and FK506, antibodies, or other immunoablative agents, CAMPATH, antithymocyte globulin (ATG), anti-CD3 antibodies, cytoxin, fludarabine, cyclosporin, FK506, cyclophosphamide, etoposide, doxorubicin, vincristine, prednisone rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
- busulphan dimethyl myleran
- thiotepa cyclosporin
- azathioprine methotrexate
- mycophenolate methyl prednisolone
- prednisone pred
- Cyclosporine and FK506 are known to inhibit the calcium dependent phosphatase calcineurin. Rapamycin is known to inhibit the p70S6 kinase that is important for growth factor induced signaling. (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)).
- conditioning comprising cyclophosphamide (CY) (60mg/kilogram/day on two occasions) in combination with a supralethal dose of total-body irradiation (1000 rads; 2.0 Gy of TBI administered on each of 6 successive days, CY-TBI) may be used.
- conditioning comprising busulfan ⁇ e.g., BU 16 mg/kg administered over 4 days) followed by 60 mg/kg CY on each of 2 successive days.
- regimens may include administration of drugs for the prevention of acute GVHD, such as methotrexate (MTX) and cyclosporine (CSP).
- MTX methotrexate
- CSP cyclosporine
- the regimen may include cyclophosphamide 60 mg/kg per day, given on days -3 and —2, followed by 550 cGy TBI administered in a single dose on day -1.
- the median dose rate may be 32.4 cGy/min (range, 26.8-36.6 cGy/min).
- Total body irradiation (TBI) may be delivered in parallel opposed lateral fields with 6-MeV photons using a Clinac 600 CD (Varian Medical Systems, Alpharetta, GA) (see e.g., Blood, 15 April 2005, Vol. 105, No. 8, pp. 3035-3041).
- the transplant recipient is conditioned under sublethal, lethal or supralethal conditions prior to transplantation.
- Such conditioning is dependent on the nature of the transplant and the condition of the recipient.
- the recipient may be conditioned under sublethal, lethal or supralethal conditions, for example, by total body irradiation (TBI) and/or by treatment with lymphoablative and/or myeloablative and immunosuppressive agents according to standard protocols.
- TBI total body irradiation
- a sublethal dose of irradiation is within the range of 1-7.5 Gy TBI
- a lethal dose is within the range of 7.5-9.5 Gy TBI
- a supralethal dose is within the range of 9.5-16.5 Gy TBI.
- myeloablative agents are busulphan, dimethyl myleran and thiotepa
- immunosuppressive agents are prednisone, methyl prednisolone, azathioprine, cyclosporine, cyclophosphamide, fludarabine, antithymocyte globulin (ATG), Campath, etc.
- the present invention also contemplates the use of post-transplant immunosuppressive regimens. Immunosuppressive regimens can be determined by the skilled physician depending on the disease and transplant setting, condition of the patient, etc.
- Immunosuppressive regimens include appropriate doses of any one or more of the following: cyclosporine, methotrexate, sirolimus (Rapamycin), prednisone, mycophenolic acid, thymoglobulin induction, Tacrolimus, Azathioprine, steroids/corticosteroids, various antibodies such as muronomab-CD3 (anti-CD3 antibody), basiliximab and daclizumab (humanized antimonoclonal antibodies that target the IL-2 receptor) and other immunosuppressants known in the art.
- compositions of the present invention further provides pharmaceutical compositions comprising the activated T cells and/or stem cells, and a pharmaceutically acceptable carrier.
- Compositions of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
- pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
- compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as ethylenediaminetetraacetic acid (EDTA) or glutathione; adjuvants ⁇ e.g., aluminum hydroxide); and preservatives.
- buffers such as neutral buffered saline, phosphate buffered saline and the like
- carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
- proteins such as glucose, mannose, sucrose or dextrans, mannitol
- proteins such as glucose, mannose, sucrose or dextrans, mannitol
- proteins such as glucose, mannose, sucrose or dextrans, mannitol
- proteins such as glucose, mannose, sucrose or dextrans,
- compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).
- the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
- the compositions of the present invention can be administered in multiple, sequential dosages as determined by a clinician.
- T cells from different donors can be used in successive cycles of treatments to reduce the risk of rejection of the infused cells.
- allogeneic cells as described herein can be administered before, at the same time, or after autologous T cell therapy.
- autologous cells are activated in the same manner as allogeneic cells as described herein.
- the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient.
- activated T cells are administered approximately at 2 X 10 9 to 2 X 10 ⁇ cells to the patient. (See, e.g., U.S. Pat. No. 5,057,423).
- the number of T cells administered to the transplant patient may be between 1 X 10 activated T cells and 2 X 10 activated T cells.
- the number of T cells administered may be about 1 X 10 s , 1 X 10 9 , 5 X 10 9 , 1 X 10 10 , 5 X 10 10 , 1 X 10 11 , or 2 X 10 11 cells.
- the number of T cells administered may be greater than about 1 X 10 9 , 5 X 10 9 , 1 X 10 10 , 5 X 10 10 , or 1 X 10 11 cells.
- the number of T cells administered may be from about 1 X 10 6 /kilogram to about 1 X 10 9 /kilogram.
- the number of T cells administered may be about 1 X 10 6 /kilogram, 5 X 10 6 /kilogram, 1 X 10 7 /kilogram, 5 X 10 7 /kilogram, 1 X 10 8 /kilogram, 5 X 10 8 /kilogram, or about 1 X 10 9 /kilogram.
- the number of T cells administered to the patient will vary depending on the conditioning regimen.
- the number of T cells administered is inversely related to the level of conditioning.
- a strong (e.g., more harsh) conditioning regimen a lower number of T cells would be infused while patients receiving a light conditioning regimen would receive a larger number of T cells.
- a particular dose for each of conditioning, T cells, and stem cells can be determined for each patient.
- T cell, or other altered post co-culture cell compositions may be administered multiple times at dosages within these ranges and those noted elsewhere herein.
- the activated T cells may be autologous or allogeneic to the patient undergoing therapy.
- the T cell compositions can be administered before, at the same time as, or after any of a variety of factors to encourage T cell growth in vivo, such as IL-2 and/or IL- 15 or other cytokines.
- the donor lymphocytes of the present invention can be administered in conjunction with donor bone marrow and/or stem cell transplantation (e.g., at the same time as). In another embodiment, the donor lymphocytes of the present invention can be administered before, at the same time, or after donor bone marrow and/or stem cell transplantation.
- compositions of the present invention may be administered to a patient subcutaneously, intradermally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
- the T cell compositions of the present invention are preferably administered by i.v. injection.
- the compositions of activated T cells may be injected directly into a site of tissue injury.
- the pharmaceutical composition can be delivered in a controlled release system.
- a pump may be used (see Langer, 1990, Science 249:1527-1533; Sefton 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980; Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321:574).
- polymeric materials can be used (see Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa.; Controlled Drug Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al, 1985, Science 228:190; During et al, 1989, Ann. Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71:105).
- a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release, 1984, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa., vol. 2, pp. 115-138).
- the compositions of the present invention may also be administered using any number of matrices. Matrices have been utilized for a number of years within the context of tissue engineering (see, e.g., Principles of Tissue Engineering (Lanza, Langer, and Chick (eds.)), 1997.
- the present invention utilizes such matrices within the novel context of acting as an artificial lymphoid organ to support, maintain, or modulate the immune system, typically through modulation of T cells. Accordingly, the present invention can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering. Accordingly, the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices. In one particular example, the compositions and devices set forth by U.S. Patent Nos: 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized. Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host.
- Matrices may be formed from both natural and synthetic materials.
- the matrices may be non-biodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant; or biodegradable.
- the matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles.
- matrices can be designed to allow for sustained release seeded cells or produced cytokine or other active agent.
- the matrix of the present invention is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.
- a matrix is used herein as an example of a biocompatible substance.
- the current invention is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi-permeable membrane or used in conjunction with a particular semi-permeable substance.
- Compositions comprising the activated T cells as described herein can be provided as pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes.
- the combinations may be administered by the topical, transdermal, oral, rectal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route.
- parenteral e.g., intravenous, subcutaneous or intramuscular
- the combinations may be incorporated into biodegradable polymers allowing for sustained release of the composition, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of tissue injury.
- biodegradable polymers and their use are described, for example, in detail in Brem et al. J. Neurosurg. 74:441-446 (1991).
- compositions will depend on the condition being treated, and other clinical factors such as weight and condition of the human or animal, the nature of the composition, and the route of administration of the composition. It is to be understood that the present invention has application for both human and veterinary use.
- the formulations include those suitable for oral, rectal, ophthalmic, (including intravitreal or intracameral) nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration.
- the formulations may conveniently be presented in a dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into associate the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
- Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier.
- a preferred topical delivery system is a transdermal patch containing the ingredient to be administered.
- Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
- Formulations suitable for nasal administration, wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is administered, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
- Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops include aqueous or oily solutions of the active ingredient.
- Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
- the formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient.
- compositions comprising cells of the present invention are targeted to the desired location through the use of paramagnetic beads and application of a magnetic force inside or outside a target tissue (as described, for example, in US patent No 6,203,487).
- the cells of the present invention are exposed to paramagnetic beads conjugated to appropriate surface markers either in vivo or in vitro or a combination of the two such that binding of the paramagnetic particle to the cells occurs.
- a composition comprising cells bound to the paramagnetic particles and a pharmaceutically acceptable excipient is administered to a mammal.
- a magnet may be placed adjacent to a target tissue, i.e., an area of the body or a selected tissue or organ into which local cell delivery is desired.
- the magnet can be positioned superficial to the body surface or can be placed internal to the body surface using surgical or percutaneous methods inside or outside the target tissue for local delivery.
- the magnetic particles bound to cells are delivered either by direct injection into the selected tissue or to a remote site and allowed to passively circulate to the target site or are actively directed to the target site with a magnet or the targeting ligand.
- This experiment was designed to test the ability of donor activated T cells to promote engraftment in a completely histoincompatible mouse model using escalating doses of conditioning and activated donor T cells.
- the results indicate that lower doses of conditioning can be used to promote mixed chimerism (engraftment) with the addition of about 5 X 10 7 donor activated T cells.
- the experiment was set up as follows: Four million T cell-depleted bone marrow cells were transferred from H-2 b , Ly5.1 + mice into H-2 d , Ly5.2 + recipient host mice that had been treated with escalating conditioning doses as shown in Table 3.
- Donor T cells were from H-2 b , Ly5.2 + mice.
- the T cells were activated in the presence of irradiated T cell depleted spleen cells, 300 ru/ml murine IL-2, 1:1 ratio of CD3/CD28 beads for four days. Overall T cell expansion was 21 fold and resulted in 24%CD4+/77% CD8+ T cells.
- the enriched na ⁇ ve T cell population was 53% CD4+/41% CD8+ T cells.
- the activated T cells were given in escalating doses for each group of conditioned mice (e.g., 100, 200, or 400 rads) according to the regimen shown in Table 3.
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Abstract
L'invention concerne normalement l'amélioration de la prise de greffe, de la reconstitution immune et de réduction de la toxicité en rapport au conditionnement associée à l'allotransplantation utilisant des lymphocytes donneurs halogéniques, plus particulièrement des lymphocytes T donneurs activés. L'invention concerne de manière générale des procédés de stimulation et d'activation des cellules susceptibles d'être utilisées dans la transplantation.
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US9434935B2 (en) | 2013-03-10 | 2016-09-06 | Bellicum Pharmaceuticals, Inc. | Modified caspase polypeptides and uses thereof |
US9913882B2 (en) | 2013-06-05 | 2018-03-13 | Bellicum Pharmaceuticals, Inc. | Methods for inducing partial apoptosis using caspase polypeptides |
US10189880B2 (en) | 2014-11-03 | 2019-01-29 | Leiden University Medical Center | T cell receptors directed against Bob1 and uses thereof |
US11344578B2 (en) | 2017-04-19 | 2022-05-31 | Board Of Regents, The University Of Texas System | Immune cells expressing engineered antigen receptors |
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