EA046588B1 - USING CART19 TO TREAT OR PREVENT GRAFT-versus-HOST DISEASE (GVHD) - Google Patents

Abstract

The invention relates to methods for treating or preventing graft-versus-host disease (GVHD) in an individual. The invention comprises administering a genetically modified T cell expressing a CAR, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a CD33 signaling domain.

Description

Cross reference to related application

The present invention claims benefit from U.S. Provisional Application No. 61/671,508, filed July 13, 2012, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Through the use of gene transfer techniques, T cells can be genetically modified to stably express antibody binding domains on their surface that provide new antigen specificity independent of the major histocompatibility complex (MHC). Chimeric antigen receptors (CARs) are an application of this approach and combine the antigen recognition domain of a specific antibody with the intracellular domain of the CD3-Z chain or FcgRI protein in a single chimeric protein (Gross et al., 1989 Proc. Natl. Acad. Sci. U.S.A. 86 : 10024-10028; Irving et al., 1991 Cell 64: 891-901). CAR trials are currently underway in a number of medical centers (Kohn et al. 2011 Mol. Ther. 19: 432-438; Jena et al. 2010 Blood 116: 1035-1044). For most malignant tumors, tumor-specific antigens have not yet been identified, but for malignant neoplasms involving B cells, CD19 is an effective tumor target. CD19 expression is restricted to normal and malignant B cells (Uckun et al., 1988 Blood 71: 1329), and CD19 is widely used as a target for safe CAR testing. Although CARs can induce T cell activation in a manner similar to the action of the endogenous T cell receptor, the main obstacle to the clinical application of this method has so far been the limited expansion of CAR + T cells in vivo, the rapid disappearance of cells after infusion, and the low clinical activity ( Jena et al., 2010 Blood 116: 1035-1044; Sadelain et al., 2009 Immunol. 21: 215-223).

CAR-mediated T cell responses can be further enhanced by the addition of co-stimulatory domains. In a preclinical model, inclusion of the CD137 signaling domain (41BB) was shown to significantly enhance the antitumor activity and survival of CARs in vivo compared with inclusion of the CD3-z chain alone (Milone et al., 2009 Mol. Ther. 17, 1453-1464; Carpenito et al. al., 2009 Natl. Acad. Sci. 106: 3360-3365). To evaluate the safety and feasibility of adoptive transfer of T cells genetically modified to express such CARs, a pilot clinical trial was conducted using autologous T cells expressing anti-OPC CARs containing both CD3-Z and costimulatory domain 4 -1BB (CART19 cells), to target CD19+ malignancies. Three patients were treated according to this protocol. Some of the results obtained from one of these patients are described in (Porter et al., 2011 N. Engl. J. Med. 365:8), which reports that the results of this treatment resulted in tumor regression, preservation of CART19 cells, and unexpected the occurrence of delayed tumor decay syndrome. It was also shown that CART19 cells had a strong clinical antitumor effect in all three patients treated. On average, each administered CAR T cell and/or its progeny killed more than 1,000 leukemia cells in vivo in patients with late-stage chemotherapy-resistant chronic lymphocytic leukemia (CLL). CART19 cells underwent active T cell expansion in vivo, persisted in blood and bone marrow (BM) at high levels for at least 6 months, continued to express functional receptors on cells with a memory cell phenotype, and maintained anti-OP^ function in vivo. However, it remains unclear how CART19 cells avoid rejection by the human host, given that CAR19 constructs contain both mouse (antibody determinant) sequences and unique junction fragments between the various components of the CAR19 construct.

Thus, there is still a need in the field for a mechanism that allows long-term persistence of CART19 cells and explains why these cells are not rejected by the human host. The present invention satisfies this need.

The essence of the invention

The invention relates to a method for depleting B cells in an individual. In one embodiment, the method includes administering to an individual an effective amount of cells genetically modified to express a CAR, wherein the CAR comprises an antigen binding domain, a co-stimulatory signaling region, and a CD3-zeta signaling domain, wherein the antigen binding domain acts on a B cell surface marker, thereby depleting B -cells of an individual.

The invention relates to a method for stimulating tolerance in an individual. In one embodiment, the method includes administering to an individual an effective amount of cells genetically modified to express a CAR, wherein the CAR comprises an antigen binding domain, a costimulatory signaling region, and a CD3-zeta signaling domain, wherein the antigen binding domain acts on a B cell surface marker, thereby promoting tolerance. at the individual.

In one embodiment, the tolerance is tolerance to the transplanted tissue.

In one embodiment, the genetically modified cell depletes B cells.

In one embodiment, the genetically modified cell is introduced simultaneously with the transplanted tissue.

- 1 046588

In one embodiment, the genetically modified cell is introduced before the transplanted tissue is introduced.

In one embodiment, the genetically modified cell is administered after the transplanted tissue has been administered.

The invention relates to a method for treating graft-versus-host disease (GVHD). In one embodiment, the method includes administering a cell genetically modified to express a CAR to an individual in need thereof, wherein the CAR comprises an antigen binding domain, a co-stimulatory signaling region, and a CD3-zeta signaling domain, wherein the antigen binding domain acts on a B cell surface marker, thereby providing treatment for GVHD in an individual.

In one embodiment, the genetically modified cell depletes B cells.

In one embodiment, the genetically modified cell is introduced simultaneously with the transplanted tissue.

In one embodiment, the genetically modified cell is introduced before the transplanted tissue is introduced.

In one embodiment, the genetically modified cell is administered after the transplanted tissue has been administered.

Brief description of drawings

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments that are currently preferred. However, it should be understood that the invention is not limited to the specific designs and methods of the embodiments shown in the drawings.

Fig. 1, including Figures 1A to 1F, is a series of images demonstrating the robust in vivo expansion and persistence in blood and bone marrow of CART19 cells. DNA was isolated from whole blood as shown in FIG. 1A to 1C, or from bone marrow, as shown in FIG. 1D to 1F, samples were obtained from UPN 01 as shown in FIG. 1A and 1D, UPN 02, as shown in FIG. 1B and 1E, and UPN 03, as shown in Figures 1C and 1F, were analyzed by quantitative PCR in bulk using an appropriate assay to detect and quantify CART19 sequences. Each point on the graph corresponds to the average of three measurements of 100-200 ng of genomic DNA, with a maximum %CV value of less than 1.56%. Pass/fail parameters for the assay included predefined ranges for slope and amplification efficiency and control sample amplification. The lower limit of quantitation, established by the standard curve range, corresponded to 2 transgene copies/μg genomic DNA; sample values below this number were considered estimates and displayed if at least 2/3 of the replicates exhibited a Ct value with %CV for values of 15%. CART19 cells were introduced on days 0, 1 and 2 for UPN 01 and UPN 03 and on days 0, 1, 2 and 11 for UPN 02.

Fig. 2, containing figures 2A to 2C, is a series of images demonstrating the prolonged surface expression of CART19 and the formation of functional CAR memory in vivo. In fig. 2A shows the detection of CAR-expressing CD3+ lymphocytes and the absence of B cells in peripheral blood and bone marrow. Freshly processed peripheral blood or bone marrow mononuclear cells obtained from UPN 03 169 days after CART19 cell infusion were assessed by flow cytometry for surface expression of CAR19 (top) or the presence of B cells (bottom); PBMCs obtained from healthy donors were stained with ND365 as a control. To assess CAR19 expression in CD3+ lymphocytes, samples were stained with antibodies to CD14PE-Cy7 and CD16-PE-Cy7 (dump channel) and CD3-FITC, which give a signal above the threshold value for CD3+, and CAR19 expression was assessed in the CD8+ and CD8- lymphocyte compartments by staining with CD8a-PE and anti-CAR19 idiotypic antibodies conjugated to Alexa-647. The data in the diagrams are selected based on the dump channel negative/CD3 positive cell population. To assess the presence of B cells, samples were stained with antibodies to CD14-APC and CD3-FITC (dump channels) and assessed for the presence of B cells in the dump channel-negative fraction by staining with antibodies to CD20-PE and CD19-PE- Cy-7. In all cases, quadrants below the threshold were set to unstained controls, as shown in Fig. 2B and 2C. Immunophenotyping of CD4+ (Fig. 2B) and CD8+ (Fig. 2C) T cell subsets is shown. Frozen peripheral blood samples from UPN 03, obtained by apheresis 56 and 169 days after T-cell infusion, were left overnight in culture medium without added factors, washed, and subjected to multiparametric immunophenotyping for expression of markers of T-cell memory, activation, and exhaustion. . The gating strategy as shown in FIG. 6, included initial gating on the dump channel (CD14, CD16, Live/Dead Aqua)-negative and CD3-positive cells and subsequent positive gating on CD4+ and CD8+ cells. Gates and quadrants were established using FMO controls (CAR, CD45RA, PD-1, CD25, CD127, CCR7) or by gating on positive cell populations (CD3, CD4, CD8) and clearly demarcated subpopulations (CD27,

- 2 046588

CD28, CD57); data were displayed after biexponential transformation to objectively visualize events. The functional competence of retained CAR cells was demonstrated in the following experiments. Frozen peripheral blood samples from UPN 03, obtained by apheresis 56 and 169 days after T cell infusion, were left overnight in culture medium without added factors, washed, and assayed directly ex vivo for the ability to recognize CD19-expressing target cells. using CD107 degranulation assays. After incubation for two hours in the presence of anti-CD28, anti-CD49d, and CD107FITC, cell mixtures were harvested, washed, and subjected to multiparametric flow cytometry analysis to assess the ability of CART19 cells to degranulate in response to CD19-expressing targets. The gating strategy included initial gating on dump channels (CD14-PE-Cy7, CD16-PE-Cy7, Live/Dead Aqua)-negative and CD3-PE-positive cells and subsequent gating on CD8-PE-Texas Red-positive cells; data are presented for the CD8+ population. In all cases, negative quadrants were established from unstained controls.

Fig. 3, containing Figures 3A to 3C, is a series of images illustrating the results of experiments assessing clinical responses following infusion of CART19 cells. In fig. Figure 3A shows that UPN 02 was treated with two cycles of rituximab and bendamustine with minimal response (R/B, arrow). CART19 T cells were infused starting at day 4 after treatment with bendamustine alone (B, arrow). Rituximab- and bendamustine-resistant leukemia cells were rapidly cleared from the blood, as indicated by a decrease in absolute lymphocyte count (ALC) from 60,600/μL to 200/μL within 18 days after infusion. Treatment with corticosteroids was started on the 18th day after infusion due to poor health and non-infectious febrile syndrome. The reference line (dashed line) indicates the upper limit of normal ALC values. In fig. 3B shows exemplary staining experiments on bone marrow samples obtained by serial biopsy or thrombus samples obtained from patients UPN 01 and 03 for CD20. Leukemic infiltration, observed before treatment in both patients, was absent in samples obtained after treatment, and was accompanied by normalization of cell saturation and trilineage hematopoiesis. UPN 01 had no CLL cells, as shown by flow cytometry, cytogenetics, and fluorescence in situ hybridization, or normal B cells, as shown by flow cytometry, in the bone marrow or blood. UPN 03 showed 5% residual normal CD5-negative B cells, confirmed by flow cytometry on day +23, which also indicates that they are polyclonal; No normal B cells were detected on day +176. In fig. Figure 3C presents experimental results using serial CT imaging to evaluate chemotherapy-resistant generalized lymphadenopathy with rapid resolution. Bilateral axillary lesions resolved 83 (UPN 01) and 31 (UPN 03) days after infusion, as indicated by the arrow and circle.

Fig. 4, containing Figures 4A to 4C, is a series of images illustrating the absolute lymphocyte count and total number of circulating CART19+ cells for UPN 01, 02, and 03. Total lymphocyte count (total normal cells and CLL cells) versus total cell count CART19+ in blood is indicated for all 3 individuals using absolute lymphocyte count based on CBC values and blood volume of 5.0 L. The total number of circulating CART19 cells was calculated by applying sequential CBC values with absolute lymphocyte counts and qPCR marker values as shown in FIG. 1, converting copies/μg DNA to average relative values as described later herein. Relative qPCR marker values were shown to correlate strongly (<2-fold variation) with flow cytometry-derived characteristics of infusion products and with data obtained from samples for which corresponding flow cytometry data were available for direct CART19 cell counting by staining .

Fig. 5, containing Figures 5A to 5D, is a series of images illustrating experiments involving direct ex vivo detection of CART19 positive cells in UPN-01 PBMC 71 days after T cell infusion. UPN-01 PBMCs obtained either immediately after apheresis at 71 days postinfusion or frozen during apheresis for T cell derivation (reference line) and thawed in a viable state before staining were analyzed by flow cytometry to detect the presence of CART19-expressing cells group CAR19 on its surface. To assess CAR19 expression in lymphocytes, samples were stained with CD3PE and idiotypic anti-CAR19 antibodies conjugated to Alexa-647 or stained with CD3-PE alone (FMO for CAR19). In fig. Figure 5A shows that initial lymphocyte gating was established based on forward and side scatter (FSC vs. SSC), followed by gating on CD3+ cells. In fig. 5B shows a CD3+ lymphocyte gate; in fig. Figure 5C shows CAR idiotype staining; in fig. Figure 5D shows the FMO of idiotype CAR. CAR19-positive gate was established on CAR19 FMO samples.

- 3 046588

Fig. 6, containing Figures 6A to 6C, is a series of images illustrating a gating strategy for identifying CART19 expression by polychromatic flow cytometry in UPN 03 blood samples. The gating strategy in FIG. 6C is shown for a sample obtained from UPN 03 on day 56 and also illustrates the strategy used for a sample obtained from UPN 03 on day 169. FIG. 6A shows the primary gate: dump gate (CD14, CD16, LIVE/dead Aqua)-negative, CD3-positive. In fig. 6B shows secondary gates: CD4-positive, CD8-positive. In fig. Figure 6C shows the tertiary gates: CAR19-positive and CAR19-negative, established according to the CAR FMO samples (right panels).

Fig. 7 is an image summarizing patient demographics and responses.

Fig. 8 is an image illustrating long-term expression of CART19.

Fig. 9, containing FIG. 9A and 9B are a series of images illustrating severe B cell deficiency.

Fig. 10 is an image showing decreased plasmacyte counts in all 3 patients.

Detailed description

The present invention is based in part on the unexpected discovery that T cells expressing anti-CD19 CAR including CD3z and the 4-1BB co-stimulatory domain (CART19 cells) persist in the mammalian host for long periods of time. For example, in the present case, cells expressing surface CAR19 were observed in the mammalian host for over 21 months after infusion of CAR19 T cells. Thus, the present invention provides a method of depleting normal B cells in a mammal by administering to the mammal in need thereof CARs that target B cells to induce tolerance in the mammal.

The invention relates to compositions and methods for depleting B cells for the purpose of inducing tolerance. The present invention relates to a method for the adoptive transfer of T cells transfected to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody-based specificity for a target antigen (eg, B cell antigen) with a T cell receptor-activating intracellular domain to produce a chimeric protein that provides specific anti-B cell immune activity.

In one embodiment, the CAR of the invention comprises an extracellular domain comprising an antigen recognition domain that interacts with a B cell antigen, a transmembrane domain, and a cytoplasmic domain.

In one embodiment, CAR T cells of the invention can be produced by introducing a lentiviral vector containing the desired CARs. The CAR T cells of the invention are capable of replicating in vivo, resulting in their long-term persistence leading to sustained B cell depletion and tolerance.

In one embodiment, the invention relates to the administration of a genetically modified T cell expressing a CAR to effectively reduce the incidence, severity or duration of graft-versus-host disease (GVHD), rejection crisis or post-transplant lymphoproliferative syndrome.

Definitions.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly accepted by those skilled in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice of testing the present invention, the preferred materials and methods are described herein. In the description and claims of the present invention, the following terminology will be used.

It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting.

Objects designated in this description may be either singular or plural. By way of example, an element means one element or more than one element.

Approximate, as used herein in relation to a measured value such as quantity, time duration, etc., includes variations within ±20% or ±10%, in some cases ±5%, in some cases ±1% and in some cases ±0.1% of the given value, since such variations are suitable for carrying out the disclosed methods.

Activation, as used herein, refers to the state of a T cell that has been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. The term activated T cells refers in particular to T cells that undergo cell division.

- 4 046588

The term antibody, as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins obtained from natural sources or from recombinant sources, and can be immunoreactive regions of intact immunoglobulins. Antibodies are often tetramers of immunoglobulin molecules. The antibodies of the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York, 1988, Proc. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term antibody fragment refers to a region of an intact antibody and refers to the antigen binding-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies and multispecific antibodies derived from antibody fragments.

The term antigen or Ag, as used herein, is defined as a molecule that provokes an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can act as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. Those skilled in the art will recognize that any DNA containing nucleotide sequences or partial nucleotide sequences encoding a protein that mediates an immune response, thus encoding an antigen, as that term is used herein. In addition, those skilled in the art will appreciate that the antigen does not need to be encoded solely by the full-length nucleotide sequence of the gene. It will be readily understood that the present invention relates to, by way of non-limiting examples, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be combined in various combinations to provide the desired immune response. In addition, it will be clear to those skilled in the art that the antigen does not need to be encoded by a gene at all. It is easy to understand that an antigen can be produced through synthesis or obtained from a biological sample. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

The term autoantigen in accordance with the present invention means any self-antigen that is recognized by the immune system as foreign. Self-antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

The term autoimmune disease, as used herein, is defined as a disorder that results in an autoimmune response. Autoimmune disease results in an inappropriate and excessive response to self antigen. Examples of autoimmune disease include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease , hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatoid arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia and ulcerative colitis, along with others.

As used herein, the term autologous is intended to refer to any material obtained from the same individual to whom the material is later administered.

Allogeneic refers to a graft obtained from another animal of the same species.

Xenogeneic refers to a graft obtained from an animal of another species.

A B cell surface marker, as used herein, is an antigen expressed on the surface of a B cell that can be targeted by an agent that binds to it. Examples of B cell surface markers include leukocyte surface markers CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD37, CD53, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 and CD86. B cell surface markers of particular interest are preferentially expressed on B cells over other non-B cell tissues of a mammal and can be expressed on both B cell precursors and mature B cells. In one embodiment, the preferred marker is CD19, which is found on B cells throughout the lineage's differentiation from the pro/pre-B cell stage to the terminally differentiated plasma cell stage.

As used herein, B cell depletion refers to a decrease in the level of B

- 5,046,588 cells in an animal or human after treatment with a drug, cell therapy or antibody compared to the level before treatment. B cell levels can be measured by well known assays such as a complete blood count, FACS assay for known B cell markers, and by the methods described herein. B cell depletion may be partial or complete. In one embodiment, the B cell depletion is 25% or more.

The terms deplete and exhaustion are used herein in relation to B cells, and for the purpose of describing the invention and claims, they are used in one or more of the following meanings: blocking B cell functions; functional inactivation of B cells; B cell cytolysis; inhibition of B cell proliferation; inhibition of B cell differentiation into plasma cells; initiation of B cell dysfunction that results in therapeutic benefit; inhibition of the production of antibodies to antigens exfoliated from the cell; decreased number of B cells; inactivation of B cells stimulated or activated by antigens shed from the cell; blocking one or more functions of B cells, stimulated or activated by antigens shed from the cell; cytolysis of B cells, stimulated or activated by antigens exfoliated from the cell; and a decrease in the number of B cells, stimulated or activated by antigens shed from the cell. B cell depletion may result from one or more mechanisms including, but not limited to, clonal inactivation, apoptosis, antibody-dependent cellular cytotoxicity, complement-mediated cytotoxicity, and signaling cascade-mediated inactivation, cell dysfunction, or cell death.

The term malignancy, as used herein, is defined as a disease that is characterized by the rapid and uncontrolled growth of abnormal cells. Malignant cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various malignancies include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and so on.

The CD19 antigen refers to an antigen of approximately 90 kDa, which can be identified, for example, by antibodies HD237 or B4 (Kiesel et al., 1987 Leukemia Research II, 12: 1119). CD19 is found on cells throughout B cell lineage differentiation from the stem cell stage to final differentiation into plasma cells, including but not limited to pre-B cells, B cells (including naïve B cells, antigen-stimulated B cells, B memory cells, plasma cells and B lymphocytes) and follicular dendritic cells. CD19 is also found on B cells of human fetal tissue. In preferred embodiments, the CD19 antigen targeted by the antibodies of the invention is a human CD19 antigen.

The term costimulatory ligand, as used herein, includes a molecule on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) that specifically binds to a recognized costimulatory molecule on a T cell, thereby providing a signal, which, in addition to the primary signal provided by, for example, the binding of the TCR/CD3 complex to a peptide-bound MHC molecule, mediates a T cell response including, but not limited to, proliferation, activation, differentiation, and the like. The costimulatory ligand may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), molecule intercellular adhesion cell (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin-beta receptor, 3/TR6, ILT3, ILT4, HVEM, agonist or antibody that binds Toll-like receptor ligand, and a ligand that specifically binds to B7-H3. A costimulatory ligand also includes, but is not limited to, an antibody that specifically binds to a costimulatory molecule found on a T cell, including but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, associated with lymphocyte function antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds to CD83.

A costimulatory molecule refers to a recognizable binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, including but not limited to proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecule, BTLA, and Toll-like receptor ligand.

A costimulatory signal as used herein refers to a signal that, in combination with a primary signal, such as TCR/CD3 binding, results in T cell proliferation and/or activation or suppression of key molecules.

Disease is a state of animal health in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if left untreated.

- 6 046588 the disease is not cured. By comparison, a disorder in an animal is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health status is less favorable than in the absence of the disorder. If left untreated, the disorder does not necessarily cause further deterioration in the animal's health.

An effective amount, as used herein, means an amount that provides a therapeutic or prophylactic effect.

As used herein, endogenous refers to any material derived from or from within an organism, cell, tissue or system.

As used herein, the term exogenous refers to any material introduced into an organism, cell, tissue or system that was obtained outside the organism, cell, tissue or system.

The term expression, as used herein, is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.

An expression vector refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed.

The expression vector contains cis-acting elements sufficient for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (eg, naked or included in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that include a recombinant polynucleotide.

Homologous refers to the sequence similarity or sequence identity of two polypeptides or two nucleic acid molecules. When a position in both of two sequences being compared is occupied by the same base or amino acid monomer, for example, if a position in each of two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions in the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, comparisons are made when two sequences are aligned to ensure maximum homology.

The term immunoglobulin or Ig, as used herein, is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD and IgE. IgA is a primary antibody found in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions from the respiratory and genitourinary tracts. IgG is the most abundant circulating antibody. IgM is the main immunoglobulin produced during the primary immune response in most individuals. It is the most effective immunoglobulin involved in agglutination, complement fixation and other antibody responses and is important for protection against bacteria and viruses. IgD is an immunoglobulin for which antibody function has not been demonstrated but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by stimulating the release of mediators from mast cells and basophils upon exposure to an allergen.

As used herein, the term immune response includes T cell and/or B cell mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly mediated by T cell activation, such as the production of antibodies (humoral responses) and activation of cytokine-secreting cells, such as macrophages. Immune cells involved in the immune response include lymphocytes such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen presenting cells (eg, professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells such as macrophages, eosinophils, mast cells, basophils and granulocytes.

As used herein, the term immunological tolerance refers to methods applied to a proportion of treated individuals, compared to untreated individuals, in which there is: a) a reduced level of specific immune response (believed to be mediated, at least in part, by antigen -specific effector T-lymphocytes, B-lymphocytes, antibodies or their equivalents); b) delay in occurrence or

- 7 046588 development of a specific immune response; or c) reduced risk of initiating or developing a specific immune response. Specific immunological tolerance occurs when immunological tolerance occurs preferentially to certain antigens over others.

As used herein, instructional material includes a publication, writing, diagram, or any other mode of expression that can be used to describe the applicability of the compositions and methods of the invention. The instructional material of a kit according to the invention may, for example, be attached to a container that contains a nucleic acid, peptide and/or composition according to the invention, or may be supplied with a container that contains a nucleic acid, peptide and/or composition. Alternatively, the instructional material may be provided separately from the container with the expectation that the instructional material and the connection will be shared by the recipient.

Isolated means altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not isolated, but the same nucleic acid or peptide, partially or completely separated from its naturally occurring materials, is isolated. The isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as, for example, a host cell.

Lentivirus, as used herein, belongs to the genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they are able to infect nondividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most effective vector gene delivery methods. Examples of lentiviruses are HIV, simian immunodeficiency virus, and feline immunodeficiency virus. Lentivirus-derived vectors provide a method for achieving significant levels of gene delivery in vivo.

The term modulation, as used herein, means providing a detectable increase or decrease in the level of response in an individual compared to the level of response in an individual in the absence of treatment or compound and/or compared to the level of response in an otherwise identical individual not receiving treatment. The term includes disruption and/or alteration of a natural signal or response that produces a beneficial therapeutic response in an individual, preferably a human.

Parenteral administration of an immunogenic composition includes, for example, subcutaneous (SC), intravenous (IV), intramuscular (IM) or intrasternal administration or infusion methods.

Terms patient, individual, subject, etc. are used interchangeably herein and apply to any animal or its cells, both in vitro and in situ, applicable to the methods described herein. In certain non-limiting embodiments, the patient, individual, or subject is a human.

The term rejection refers to a condition in which a transplanted organ or tissue is not accepted by the recipient's body. Rejection occurs when the recipient's immune system attacks the transplanted organ or tissue. Rejection may continue for several days to several weeks after transplantation (acute) or for several months to several years after transplantation (chronic).

By specifically binding, as used herein in relation to an antibody, is meant an antibody that recognizes a particular antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen of one species may also bind to the same antigen of one or more other species. However, such cross-species reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to other allelic forms of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms specific binding or specifically bind can be used in relation to the interaction of an antibody, protein or peptide with a second chemical compound, which means that the interaction depends on the presence of a specific structure (eg, an antigenic determinant or epitope) on the chemical compound; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins in general. If an antibody is specific for epitope A, then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled A and the antibody will reduce the amount of labeled A bound to the antibody.

By stimulation is meant the primary response caused by the binding of a stimulatory molecule (eg, the TCR/CD3 complex) to a recognition ligand, resulting in signal transduction, including but not limited to signaling through the TCR/CD3 complex. Stimulation may cause changes in the expression of certain molecules, such as a decrease in the expression of TGF-β, and/or reorganization of cytoskeletal structures, etc.

- 8 046588

The term stimulatory molecule, as used herein, means a molecule on a T cell that specifically binds to a recognized stimulatory ligand located on an antigen presenting cell.

A stimulatory ligand, as used herein, means a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, dendritic cell, B cell, etc.), can specifically bind to a recognized binding partner (referred to herein as stimulatory molecule) on the T cell, thereby providing a primary T cell response including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well known in the art and include, but are not limited to, MHC class I peptide-linked molecules, anti-CD3 antibody, anti-CD28 superagonist antibody, and anti-CD2 superagonist antibody.

The term individual is intended to designate living organisms in which an immune response can be elicited (eg, mammals). Examples of individuals include humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a substantially purified cell is a cell that is substantially free of other cell types. A largely purified cell also refers to a cell that has been separated from the other types of cells with which it is normally associated in its natural state. In some cases, the population of largely purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that has been separated from the cells with which it is naturally associated. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

The term therapeutic, as used herein, means treatment and/or prevention. The therapeutic effect is obtained by suppressing, weakening or eliminating the disease state.

The term therapeutically effective amount refers to the amount of a compound that produces a biological or medical response in a tissue, system, or individual desired by a researcher, veterinarian, doctor, or other clinician. The term therapeutically effective amount includes that amount of a compound that, when delivered, is sufficient to prevent the development or alleviate to some extent one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount varies depending on the compound, the disease and its severity and age, weight, etc. the individual receiving treatment.

Graft, as used herein, refers to the cells, tissue, or organ that is administered to an individual. The source of the transplanted material may be cultured cells, cells obtained from another individual, or cells obtained from the same individual (eg, after the cells are cultured in vitro). Exemplary organ transplants are kidney, liver, heart, lung, and pancreas.

The term treat a disease, as used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder observed in an individual.

The term transfected or transformed or transfected, as used herein, refers to the method by which an exogenous nucleic acid is transferred or introduced into a host cell. A transfected or transformed or transfected cell is a cell that has been transfected, transformed or transfected with an exogenous nucleic acid. A cell includes a primary individual cell and its progeny.

The term tolerant refers to an individual with a reduced or absent immune response to a particular antigen or group of antigens. In the context of the invention, an individual is considered tolerant if he or she does not reject (ie does not develop a strong immune response against) the transplanted cells. In some cases, a tolerant individual does not reject transplanted cells even in the absence of immunosuppressive therapy. In the context of the invention, an individual is considered intolerant if that individual rejects the transplanted cells. Intolerant individuals include individuals in whom rejection can be controlled by immunosuppressive therapy (eg, standard immunosuppression), as well as individuals who develop an active immune response against the transplanted cells.

As used herein, in vivo tolerance refers to the essentially absent immune response specific to a foreign tissue. The immune response may result from the recipient individual developing an immune response to the foreign tissue, or alternatively, the immune response may result from the foreign tissue developing an immune response to the recipient individual (eg, GVHD). Methods for measuring tolerance in vivo are well known in the art.

- 9 046588

Ranges: Throughout the specification, various aspects of the invention may be presented in range format. It should be understood that descriptions in range format are for convenience and brevity only and should not be construed as strictly limiting the scope of the invention. Thus, the description of a range should be viewed as including all possible portions of the range, as well as the individual numerical values that fall within that range. For example, a description of a range such as 1 to 6 should be considered to include all possible portions of the range, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numeric values within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is done regardless of the width of the range.

Description.

The present invention relates to compositions and methods for depleting normal B cells in a mammal. In one embodiment, depletion of B cells using a CAR of the invention induces tolerance in a mammal.

In one embodiment, the present invention relates to a method for inducing tolerance in vivo to transplanted foreign tissue. In some embodiments, the method can be used in part to prevent and/or treat transplant tissue rejection. In general, the method involves administering the CAR T cells of the invention to an individual who has received a foreign tissue transplant. The term foreign tissue as used herein can include a bone marrow graft, an organ transplant, a blood transfusion, or any other foreign tissue or cell that is specifically administered to an individual.

In another embodiment, the method can be used in part to prevent and/or treat graft-versus-host disease (GVHD). In general, the method involves administering the CAR T cells of the invention to an individual who has received a foreign tissue transplant. The term foreign tissue as used herein can include a bone marrow graft, an organ transplant, a blood transfusion, or any other foreign tissue or cell that is specifically administered to an individual.

In one embodiment, the CARs of the invention can be prepared to contain an extracellular domain including an antigen binding domain that interacts with a B cell antigen fused to the intracellular signaling domain of the zeta chain of a T cell antigen receptor complex (eg, CD3 zeta). An exemplary B cell antigen is CD19, since this antigen is expressed on malignant B cells. However, the invention is not limited to interaction with CD19. Rather, the invention includes any B cell antigen binding molecule that binds to a recognized antigen. The antigen-binding molecule is preferably fused to the intracellular domain of one or more co-stimulatory molecules and the zeta chain. Preferably, the antigen binding molecule is fused to one or more intracellular domains selected from the group of CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3 zeta signaling domain, and any combination thereof.

In one embodiment, the CAR of the invention comprises a CD137 (41BB) signaling domain. This is because the present invention is based in part on the discovery that CAR-mediated T cell responses can be further enhanced by the addition of co-stimulatory domains. For example, inclusion of the CD137 signaling domain (4-1BB) significantly enhances CAR-mediated activity and survival of CAR T cells in vivo compared with otherwise identical CAR T cells not engineered to express CD137 (4-1BB). However, the invention is not limited to certain CARs. In contrast, any CAR that interacts with B cells can be used in the present invention. Compositions and methods for preparing CAR are described in PCT/US11/64191, incorporated herein by reference.

Ways.

The invention relates to methods of using the CAR and CAR T cells of the invention to deplete B cells and induce tolerance. In one embodiment, the method includes inducing transplant tolerance (eg, an organ or tissue graft) in a patient. In another embodiment, the method includes preventing and/or treating GVHD. In a specific embodiment, the CAR of the invention interacts with CD19 on B cells.

In one embodiment, the ability to induce sustained donor humoral tolerance is the most important factor for achieving sustained transplant tolerance and/or preventing or treating GVHD. The present invention relates to the use of the CAR T cells of the invention for the depletion of B cells and for the induction of tolerance by administering the CAR T cells to an animal, preferably a mammal and most preferably a human, for the treatment of one or more diseases, disorders, symptoms or conditions associated with organ or tissue transplantation (eg, graft rejection, GVHD and/or associated conditions).

Organ rejection occurs as a result of the destruction of transplanted tissue by the host's immune cells during the immune response. Likewise, the immune response is also involved in GVHD, od

- 10 046588 However, in this case, foreign transplanted immune cells destroy host tissues. For example, organ rejection and/or GVHD can occur after transplantation of the heart, heart valve, lung, kidney, liver, pancreas, intestine, skin, blood vessels, bone marrow, stem cells, bone, or islet cells. However, the invention is not limited to specific types of transplantation. By way of non-limiting example, islet cell transplantation may be performed to prevent the onset of diabetes or to treat diabetes. Administration of the CAR T cells of the invention, which inhibit the immune response, in particular the proliferation, differentiation or survival of B cells, is an effective therapy for preventing organ and/or tissue rejection or GVHD. Administration of CAR T cells according to the invention can also be used to induce transplantation tolerance after organ and/or tissue transplantation.

The CAR T cells of the invention can also be used to induce transplantation tolerance; to treat, reduce, inhibit and/or prevent organ and/or tissue transplant rejection; and/or to reduce the antibody titer in a patient who has received an organ or tissue transplant. In one embodiment, the CAR T cells of the invention can be used to induce transplant tolerance in a patient by administering to the patient an effective amount of the CAR T cells of the invention, thereby preventing or delaying transplant rejection. In another embodiment, the CAR T cells of the invention can be used to treat organ or graft rejection in a patient by administering to the patient an effective amount of the CAR T cells of the invention, thereby inhibiting organ or tissue rejection. In yet another embodiment, the CAR T cells of the invention can be used to lower the antibody titer in a patient who has received, or will undergo, an organ or tissue transplant by administering to the patient an effective amount of the CAR T cells of the invention, thereby lowering the antibody titer.

In one embodiment, the invention relates to a method of inducing transplant tolerance in a patient, comprising administering to the patient an effective amount of CAR T cells of the invention that delays graft rejection in the patient.

In another embodiment, the invention provides a method of treating organ or tissue transplant rejection in a patient, comprising administering to the patient an effective amount of CAR T cells of the invention that inhibits organ or tissue transplant rejection in the patient.

In another embodiment, the invention relates to a method of reducing the antibody titer in a patient who has undergone, or will undergo, an organ or tissue transplantation, comprising administering to the patient an effective amount of CAR T cells according to the invention, which reduces the antibody titer in the patient.

In one embodiment, the invention provides a method of inhibiting or reducing immunoglobulin production in a patient, comprising administering to the patient an effective amount of CAR T cells of the invention.

In one embodiment, the CAR T cells of the invention reduce or inhibit the functioning of B cells. In another embodiment, the CAR T cells of the invention deplete or destroy the B cells of an individual. For example, CAR T cells of the invention can be engineered to interact with a B cell surface antigen, allowing T cell effector functions against B cells.

Therapy to inhibit adverse immune responses after transplantation.

The present invention relates to a method of using the CAR T cells of the invention as a therapy for inhibiting GVHD or graft rejection after transplantation. Thus, the present invention relates to a method of exposing a donor graft, eg a biocompatible array or donor tissue, organ or cell, to CAR T cells of the invention before, simultaneously with or after transplantation of the graft into a recipient. The CAR T cells of the invention serve to mitigate, inhibit or reduce the adverse graft response of the donor against the recipient, providing the prevention or treatment of GVHD.

As discussed elsewhere in this document, T cells can be obtained from any source, such as a tissue donor, transplant recipient, or other unrelated source (another individual or another species), to produce the CAR T cells of the invention for use in exclusion or reducing an unwanted graft immune response against the transplant recipient. Thus, the CAR T cells of the invention may be autologous, allogeneic, or xenogeneic to a tissue donor, transplant recipient, or other unrelated source.

In one embodiment of the present invention, the graft is exposed to the CAR T cells of the invention before, simultaneously with, or after transplantation of the graft into the recipient. In this situation, the immune response against the graft caused by any alloreactive

- 11 046588 mi cells of the recipient will be suppressed by the CAR T cells of the invention present in the graft, since CAR T cells can deplete B cells and promote tolerance.

In another embodiment of the present invention, the donor graft may be pretreated or preconditioned by treating the graft prior to transplantation into the recipient to reduce the immunogenicity of the graft against the recipient, thereby reducing and/or preventing GVHD or graft rejection. The graft may be brought into contact with cells or tissue of the recipient prior to transplantation to activate T cells that may be associated with the graft. After the graft has been treated with the recipient's cells or tissue, the cells or tissue can be removed from the graft. The treated graft is then contacted with the CAR T cells of the invention to reduce, inhibit or eliminate the activity of T and/or B cells that have been activated by treating the recipient cells or tissue. After such treatment of the graft with the CAR T cells of the invention, the CAR T cells can be removed from the graft prior to transplantation into the recipient. However, some CAR T cells may adhere to the graft and thus may be injected into the recipient along with the graft. In such a situation, CAR T cells administered to the recipient can suppress the immune response against the recipient caused by any graft-associated cells. Without wishing to be bound by any particular theory, treatment of the graft with CAR T cells prior to transplantation of the graft into the recipient serves to reduce, inhibit or eliminate the activity of activated T and/or B cells, thereby preventing re-stimulation or inducing reduced T cell responsiveness. or B cells in relation to antigenic stimulation from recipient tissue and/or cells. It will be clear to those skilled in the art based on the present invention that pre-treatment or pre-conditioning of the graft prior to transplantation can reduce or eliminate graft-versus-host disease.

Therapeutic use.

In one embodiment, the present invention provides a method of cell therapy in which T cells are genetically modified to express a CAR and the CAR T cells are administered to a recipient in need thereof. The introduced cell is capable of killing the cell of interest. In one embodiment, the cell of interest is a B cell. Unlike antibody therapies, CAR T cells are able to replicate in vivo, allowing for long-term persistence that can lead to sustained B cell depletion and tolerance.

In one embodiment, the CAR T cells of the invention can undergo sustained T cell expansion in vivo and can persist for an extended period of time. In another embodiment, the CAR T cells of the invention are converted into specific memory T cells that can be reactivated to inhibit B cell proliferation. For example, CART19 cells induce an immune response specific to cells expressing CD19.

The CAR-modified T cells of the invention may also serve as a vaccine for ex vivo immunization and/or in vivo therapy in a mammal. Preferably the mammal is a human.

For ex vivo immunization, at least one of the following is performed in vitro before introducing the cells into the mammal: i) cell expansion, ii) introduction of the CAR-encoding nucleic acid into the cells, and/or iii) cryopreservation of the cells.

Ex vivo methods are well known in the art and are described in more detail below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (ie, transfected or transfected in vitro) using a vector expressing the CARs described herein. The CAR-modified cell can be administered to a mammalian recipient to obtain a therapeutic effect. The mammalian recipient may be a human, and the CAR-modified cell may be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic to the recipient.

In addition to the use of a cell-based vaccine for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against a B cell antigen in a patient.

In general, cells activated and expanded as described herein can be used to deplete B cells and induce tolerance. In particular, the CAR modified T cells of the invention are used to treat one or more diseases, disorders, symptoms or conditions associated with a transplanted organ or tissue (eg, GVHD and/or associated conditions). Thus, the present invention provides methods for treating or preventing organ rejection and GVHD, comprising administering to an individual in need thereof a therapeutically effective amount of CAR-modified T cells of the invention.

- 12 046588

In one embodiment, the CAR T cells of the invention are administered in combination with an immunosuppressive agent. Any immunosuppressive agent known in the art may be used. For example, the immunosuppressive agent may be Cyclosporine, Azathioprine, Rapamycin, Mycophenolate mofetil, Mycophenolic acid, Prednisone, Sirolimus, Basiliximab or Daclizumab, or any combination thereof. Additional specific immunosuppressive agents that may be used include, but are not limited to, ORTHOCLONE OKT™ 3 (muromonab^3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF™ (FK506, tacrolimus), CELLCEPT™ (mycophenolate mofetil, active metabolite of which is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocorticosteroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrexate), OXSORALEN-ULTRA™ (methoxalene), RITUXAN ™ (rituximab) and RAPAMUNE™ (sirolimus).

The CAR T cells of the invention can be administered to a patient before, after, or simultaneously with an immunosuppressive agent. For example, the CAR T cells of the invention may be administered after an immunosuppressive agent is administered to a patient, or the CAR T cells of the invention may be administered before an immunosuppressive agent is administered to a patient. Alternatively, or in addition, the CAR T cells of the invention are administered to a patient concurrently with an immunosuppressive agent.

The CAR T cells of the invention and/or an immunosuppressive agent can be administered to a patient after transplantation. Alternatively, or in addition, the CAR T cells of the invention and/or an immunosuppressive agent can be administered to a patient prior to transplantation. The CAR T cells of the invention and/or an immunosuppressive agent can also be administered to a patient during a transplant operation.

In some embodiments, a method of administering CAR T cells of the invention to a patient is administered after initiation of immunosuppressive therapy. In some embodiments, the method is used more than once, for example, to monitor a transplant recipient over time, and, if necessary, using different immunosuppressive therapy regimens. In some embodiments, immunosuppressive therapy is reduced if the transplant recipient is expected to be tolerant to the transplant. In some embodiments, no immunosuppressive therapy is prescribed, for example, no immunosuppressive therapy is administered if the transplant recipient is expected to be tolerant to the transplant. If the transplant recipient demonstrates biomarkers of intolerance, immunosuppressive therapy can be reinstituted or continued at standard levels.

The transplanted organ or tissue may be a heart, heart valve, lung, kidney, liver, pancreas, intestine, skin, blood vessels, bone marrow, stem cells, bone, or islet cells.

The CAR T cells of the invention can be administered after a diagnosis of transplant organ or tissue rejection has been made, followed by dosing of both the CAR T cells of the invention and an immunosuppressive agent until symptoms of organ or tissue rejection resolve.

In some embodiments, the CAR T cells of the invention are administered following a diagnosis of elevated antibody titers, followed by dosing of both the CAR T cells of the invention and an immunosuppressive agent until the antibody titers decrease.

Preferably, treatment using the CAR T cells of the invention is carried out by administering an effective amount of the CAR T cells of the invention to a patient.

The CAR T cells of the present invention can be administered alone or as part of a pharmaceutical composition in combination with diluents and/or other components, such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may include target cell populations as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain 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 EDTA or glutathione; auxiliary agents (for example, aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease being treated (or prevented). The amount and frequency of administration should be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages may be determined through clinical trials.

When an effective amount is indicated, the exact amount of the compositions of the present invention to be administered may be determined by the physician, taking into account individual differences in age, weight, antibody titer, and condition of the patient (individual). In general, it can be stated that a pharmaceutical composition containing T cells described herein can be

- 13 046588 administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight, including all intermediate values within these ranges. T cell compositions at these doses can also be administered multiple times. The cells can be administered by infusion methods well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Optimal dosages and treatment regimens for a particular patient can be easily determined by those skilled in the art by monitoring the patient for signs of disease and modifying treatment based on this.

In certain embodiments, it may be necessary to deliver activated T cells to an individual and then collect blood (or perform apheresis), activate the T cells contained therein according to the present invention, and reinfuse these activated and expanded T cells into the patient. This method can be repeated many times every few weeks. In certain embodiments, T cells can be activated in a blood sample of 10 to 400 ^cc . In certain embodiments, T cells are activated in 20, 30, 40, 50, 60, 70, 80, 90, or 100 ^cc blood samples. Without wishing to be bound by any particular theory, this repeated blood draw/reinfusion protocol can be used to select specific T cell populations.

Administration of the composition to an individual may be accomplished by any suitable route, including aerosol inhalation, injection, oral administration, blood transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intracerebrally, intramuscularly, by intravenous (IV) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient via intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. T cell compositions can be injected directly into a tumor, lymph node or site of infection.

In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art in which T cells are expanded to therapeutic levels are administered to a patient in combination with (e.g., concomitantly or subsequent to) any number of suitable treatments, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab for patients with multiple sclerosis or efalizumab for patients with psoriasis or other treatments for patients with progressive multifocal leukoencephalopathy. In additional embodiments, T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressive agents such as CAM PATH, anti-CD3 antibodies or other therapies using antibodies, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase, which plays an important role in growth factor-induced molecular cascades (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al. ., Immun. 73:316-321, 1991; Curr. In a further embodiment, the cellular compositions of the present invention are administered to a patient in combination with (e.g., before, concomitantly, or after) a bone marrow transplant, T-cell depleting therapy using either chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or KEMPAS. In another embodiment, the cellular compositions of the present invention are administered following B-cell depleting therapy, such as therapy with CD20-interacting agents, such as Rituxan. For example, in one embodiment, individuals may undergo standard treatment with high-dose chemotherapy following a peripheral blood stem cell transplant. In certain embodiments, following transplantation, individuals receive an infusion of expanded immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

The dosages of the above agents to be delivered to the patient vary depending on the particular type of condition being treated and the recipient of the treatment. Determination of dosages for administration to humans can be carried out according to methods accepted in the art. For example, the dose for CEMPAS typically ranges from 1 to about 100 mg for an adult patient and is typically administered daily for 1 to 30 days. The preferred daily dose is 1 to 10 mg per day, although higher doses of up to 40 mg per day may be used in some cases (described in US Pat. No. 6,120,766).

Experimental examples

The invention is further described in detail by reference to the following experimental examples. These examples are for illustrative purposes only and are not intended to be limiting unless otherwise noted. Accordingly, the invention should not be construed as being limited to the examples below, but should be construed to include any and all variations resulting from the inventive concept set forth herein.

Without further description, it is believed that those skilled in the art can, using the above description and the following illustrative examples, prepare and use the compounds of the present invention and use the given methods. The following exemplary examples, therefore, specifically indicate preferred embodiments of the present invention and should not be construed as limiting the remaining portions of the specification.

Example 1: T cells expressing chimeric receptors deplete normal B cells and promote tolerance.

The results described herein indicate that CART19 cells persist and provide therapeutic benefit to the patient for at least 18 months. The engineered T cells, which had undergone over 1,000-fold expansion in vivo, were transported to the bone marrow and continued to express functional CARs at high levels for at least 6 months. On average, each CAR+ T cell administered killed at least 1,000 CLL cells. A CD19-specific immune response was observed in the blood and bone marrow and was accompanied by complete remission in two of three patients. A portion of the cells persisted as memory CAR+ T cells, indicating the potential of this MHC-non-restricted approach for the effective treatment of B-cell malignancies.

The materials and methods used in these experiments are described below.

Materials and methods.

Protocol design.

The clinical trial (NCT01029366) was conducted as described in PCT/US11/64191, which is incorporated herein by reference in its entirety.

Getting a vector.

The CD19-BB-z transgene (GeMCRIS 0607-793) was designed and constructed as described (Milone et al., 2009, Mol Ther. 17: 1453-1464). The lentiviral vector was prepared according to current drug manufacturing and quality control regulations using the three plasmid method at Lentigen Corporation as described (Zufferey et al., 1997, Nature biotechnol 15:871-875).

Preparation of CART19 cell product.

Methods for generating T cells using paramagnetic polystyrene beads coated with monoclonal antibodies to CD3 and CD28 have been described (Laport et al., 2003, Blood 102: 2004-2013). Lentiviral transduction was performed as described (Levine et al., 2006, Proc Natl Acad Sci USA 103: 1737217377).

The results of the experiments are described below.

In vivo expansion and persistence of CART19 and trafficking to the bone marrow.

CAR+ T cells, which have undergone expansion using CD3/CD28 beads and express the 4-1BB signaling domain, are considered an improvement on CARs lacking 4-1BB. qPCR analysis was performed to monitor the number of CART19 cells in the blood and bone marrow. All patients experienced expansion and persistence of CART19 cells in the blood for at least 6 months, as shown in FIG. 1A and 1C. It should be noted that in patients UPN 01 and UPN 03, a 1000-10000-fold expansion of CAR+ T cells in the blood was observed during the first months after infusion. The highest levels of expansion coincided with the appearance of post-infusion clinical symptoms in patient UPN 01 (day 15) and patient UPN 03 (day 23). Additionally, after initial decay, which can be modeled using first-order kinetics, CART19 T cell levels stabilized in all 3 patients 90 to 180 days after infusion. Importantly, CART19 T cells were also transported into the bone marrow in all patients, although at levels 5-10 times lower than those observed in the blood, as shown in FIG. from 1D to 1F. Patients UPN 01 and 03 showed linear-logarithmic decay in the bone marrow, with disappearance of ^T1 / ₂ within about 35 days.

Prolonged expression and formation of a population of CART19 memory cells in the blood.

A major question in CAR-mediated immunotherapy of malignancies is whether optimized cell processing and the use of co-stimulatory domains can improve the survival of genetically engineered T cells and enable the generation of memory CAR+ T cells in patients. Previous studies have not demonstrated sustained expansion, prolonged survival, and/or CAR expression on T cells following infusion (Kershaw et al., 2006, Clin Cancer Res 12:6106-6115; Lamers et al., 2006, J Clin Oncol 24:e20 -e22; Till et al., 2008, Blood, 112, 22612271; Savoldo et al., 2011, J Clin Invest doi: 10.1172/JCI461 10). Analysis of blood and bone marrow samples 169 days after infusion by flow cytometry revealed the presence of CAR19-expressing cells in UPN 03 (Figs. 2A and 2B) and the absence of B cells, as shown in Figs. 2A. Need

- 15 046588 but note that, through qPCR analysis, persisting CAR+ cells were detected in all three patients after 4 months or more, as shown in FIG. 1 and fig. 4. The in vivo occurrence of CAR+ cells calculated by flow cytometry closely matched the values obtained by PCR for the CART19 transgene. It is important to note that in patient UPN 03, only CD3+ cells expressed CAR19, since CAR19+ cells were not detected in the CD16- or CD14-positive subsets, as shown in Fig. 2A. CAR expression was also detected on the surface of 4.2% of T cells in the blood of patient UPN 01 71 days after infusion, as shown in FIG. 5.

Polychromatic flow cytometry was then used to perform detailed analysis to further examine the expression, phenotype and function of CART19 cells in UPN 03 using an anti-CAR idiopathic antibody (MDA-647) and the gating strategy shown in FIG. 6. Marked differences were observed in the expression of memory markers and activation by CD8+ and CD4+ cells based on CAR19 expression. After 56 days, CART19 CD8+ cells exhibited a predominantly effector memory phenotype (CCR7-CD27-CD28-), consistent with prolonged and sustained exposure to the antigen, as shown in Fig. 2C. In contrast, CAR-negative CD8+ cells consisted of a mixture of effector and central memory cells, with CCR7 expression in a subpopulation of cells and significant amounts in the CD27+/CD28− and CD27+/CD28+ fractions. While CART19 and CAR-negative cell populations significantly expressed CD57, this molecule was coexpressed equally with PD-1 in CART19 cells, presumably reflecting the long replicative history of these cells. In contrast to the CAR-negative cell population, the entire CART19 CD8+ population was devoid of CD25 and CD127 expression. At day 168, while the phenotype of the CAR-negative cell population remained the same as in the sample taken on day 56, the CART19 population began to contain a small population with features of central memory cells, prominent expression of CCR7, higher levels of CD27 and CD28, as well as CAR+ cells that were PD-1 negative, CD57 negative and CD127 positive.

In the CD4+ compartment at day 56 CART19 cells were characterized by a uniform absence of CCR7 and a predominance of CD27+/CD28+/PD-1+ cells distributed among the CD57+ and - compartments, and a significant lack of expression of CD25 and CD127, as shown in FIG. 2B. In contrast, CAR-negative cells at this time point were heterogeneous with respect to the expression of CCR7, CD27, and PD-1, expressed CD127, and also contained a large population of CD25+/CD127− (potential regulatory T cells). On day 169, while CD28 expression remained stably positive in all CAR+CD4+ cells, the CART19 fraction of CD4+ cells acquired a central memory phenotype with CCR7 expression, a higher percentage of CD27 cells, the appearance of a PD-1 negative subpopulation and the appearance of CD127. CAR-negative cells remained largely consistent with their day 56 counterparts, with the exception of decreased CD27 expression and decreased percentages of CD25+/CD127- cells.

CART19 cells can maintain effector function after 6 months in the blood.

In addition to short persistence and poor proliferation in vivo, a limitation of previous studies using CAR+ T cells has been the rapid loss of functional activity of administered T cells in vivo. The high levels of CART19 cell survival and surface expression of the CAR19 molecule in UPN 01 and 03 patients provided an opportunity to directly analyze anti-CD19-specific effector functions in cells recovered from cryopreserved peripheral blood samples. PBMCs from patient UPN 03 were cultured with target cells that were either positive or negative for CD19 expression (Fig. 2D). Robust CD19-specific effector function of CART19 T cells was demonstrated through specific degranulation against CD19-positive, but not CD19-negative, target cells, as assessed by surface expression of CD107a. It should be noted that exposure of the CART19 population to CD19-positive targets induced rapid internalization of surface CAR-19, as shown in FIG. 6 for surface expression of CAR19 in the same effector cells using standard flow cytometry staining. The presence of co-stimulatory molecules on target cells was not necessary to trigger degranulation of CART19 cells, since the NALM-6 line does not express CD80 or CD86 (Brentjens et al., 2007, Clin Cancer Res 13:5426-5435). The effector function was expressed on the 56th day after infusion and persisted on the 169th day. Robust effector function of CAR+ and CAR-T cells has also been demonstrated through pharmacological stimulation.

Clinical activity of CART19 cells.

No significant toxic effects were observed in any of the patients within four days after infusion, other than transient febrile reactions. However, all patients then developed significant clinical and laboratory toxic effects between 7 and 21 days after the first infusion. These toxic effects were short-lived and reversible. Of the 3 patients treated to date, 2 had a complete response and 1 had a partial response >6 months after CART19 infusion, according to standard criteria

- 16 046588 (Hallek et al., 2008, Blood 111:5446). Details of the medical history and response to treatment for each patient are shown in FIG. 7.

Briefly, patient UPN 01 developed a febrile syndrome with shivering and transient hypertension beginning 10 days after infusion. The fever lasted for about 2 weeks and then ended; the patient did not experience any systemic symptoms thereafter. The patient developed a rapid and complete response as shown in FIG. 3. 1-6 months after infusion, flow cytometry demonstrated no circulating CLL cells in the blood. Bone marrow 1, 3, and 6 months after CART19 cell infusion demonstrated a consistent absence of lymphocytic infiltrate as demonstrated by morphological analysis and flow cytometry as shown in FIG. 3B. CT slices 1 and 3 months after infusion demonstrated resolution of adenopathy, as shown in Fig. 3C. Complete remission at the time of writing of this invention was observed for 10+ months.

Patient UPN 02 received 2 cycles of bendamustine with rituximab, resulting in stable disease as shown in FIG. 3A. The patient received a third dose of bendamustine as lymphocyte-depleting chemotherapy before CART19 T-cell infusion. The patient developed a fever reaching 40°C, shivering, and shortness of breath, requiring 24-hour hospitalization on the 11th day after the first infusion and on the day of receiving the second dose of CART19 cells. Fever and systemic symptoms persisted on day 15, and the patient experienced temporary cardiac dysfunction; all symptoms disappeared after corticosteroid treatment was started on day 18. Following CART19 infusion, which coincided with an increase in body temperature, the patient experienced rapid clearance of p53-deficient CLL cells from the peripheral blood, as shown in FIG. 3A, and partial reduction of adenopathy, bone marrow showed persistently strong CLL infiltration one month after treatment, despite significant cellular reduction in peripheral blood. The patient remains clinically healthy.

Patient UPN 03 received pentostatin and cyclophosphamide as lymphocyte-depleting chemotherapy before infusion of CART19 cells. Three days after chemotherapy, but before cell infusion, the bone marrow was hypercellular (60%) with approximately 50% CLL involvement. The patient received a low dose of CART19 cells (1.5 x 10 ⁵ CAR+ T cells/kg, distributed over 3 days). Likewise, no acute toxic effects of the infusion were observed. However, 14 days after the first infusion, the patient developed tremors, fever, nausea, and diarrhea. 22 days after the infusion, tumor lysis syndrome was diagnosed, requiring hospitalization. The patient experienced resolution of systemic symptoms, and over the course of 1 month of CART19 infusion, the patient experienced clearance of circulating CLL from the blood and bone marrow as demonstrated by morphologic analysis, flow cytometry, cytogenetic analysis, and fluorescence in situ hybridization. CT slices demonstrated resolution of the abnormal adenopathy as shown in Fig. 3B and 3C. Complete remission was maintained for more than 8 months after the first infusion of CART19 cells.

Discussion of the relationship of the CART19 effector to CLL target cells in vivo.

Preclinical studies have shown that large tumors can be destroyed and that infusion of 2.2 x 10 ⁷ CAR can kill tumors consisting of 1 x 10 cells with an in vivo E:T ratio of 1:42 in humanized mice (Carpenito et al., 2009 , Proc Natl Acad Sci USA 106:3360–3365), although these calculations did not take into account T cell expansion after injection. Assessment of CLL tumor burden over time allowed calculation of tumor size reduction and CART19 E:T ratios achieved in vivo in three individuals based on the number of CAR+ T cells administered. Tumor burdens were calculated by measuring CLL mass in bone marrow, blood, and secondary lymphoid tissues. Baseline tumor mass values as shown in FIG. 7 indicate that each patient had approximately 10 ¹² CLL cells (ie 1 kg of tumor mass) before CART19 infusion. Patient UPN 03 had a baseline tumor mass of 8.8x1011 CLL cells in the bone marrow on day -1 (i.e., post-chemotherapy and before CART19 infusion), and a tumor mass in secondary lymphoid tissue of 3.3-5 ,5x1011 CLL cells, depending on the method of obtaining volumetric KT sections. Given that UPN 03 was administered to only 1.4 x ^{10 7} CART19 cells, using an estimate of total tumor burden (1.9 x 10 12 CLL cells), and given that no CLL cells were observed after treatment, a significant E: ratio was achieved: T equal to 1:93000. Using similar calculations, effective in vivo E:T ratios of 1:2200 and 1:1000 were obtained for UPN 01 and UPN 02. Finally, killing of CART19 by T cells coupled with >1000-fold expansion of CART19 in vivo presumably contributed to the potent anti-leukemia effect mediated by CART19 cells.

T cells expressing chimeric receptors provide memory and potent antitumor effects in patients with advanced leukemia.

Limited in vivo expression and effector function of CARs was a major limitation in first-generation CAR trials (Kershaw et al., 2006, Clin Cancer Res 12:6106–6115; Lamers et al., 2006, J Clin Oncol 24:e20–e22; Till et al., 2008, Blood, 112, 2261-2271; Park et al., 2007, Mol Ther 15:825-833;

- 17 046588 et al., 2008, Nat Med 14: 1264-1270). Based on preclinical modeling demonstrating increased survival of CARs containing the 4-1BB signaling module (Milone et al., 2009, Mol Ther. 17: 1453-1464; Carpenito et al., 2009, Proc Natl Acad Sci USA 106:3360-3365 ), experiments were designed to produce second-generation CARs constructed using lentiviral vector technology. These second-generation CARs have been shown to be safe in chronic HIV infection studies (Levine et al., 2006, Proc Natl Acad Sci USA 103: 17372-17377). These results indicate that when these second generation CARs were expressed in T cells and cultured under conditions designed to promote engraftment of central memory T cells (Rapoport et al., 2005, Nat Med 11: 1230-1237; Bondanza et al. al., 2006, Blood 107: 1828-1836), there was increased expansion of CAR T cells after infusion compared to previous studies. CART19 cells acquired CD19-specific cellular memory and killed tumor cells with previously unreachable E:T ratios in vivo.

CART19 represents the first CAR trial using the 4-1BB signaling domain and the first trial using lentiviral vector technology. The results presented demonstrate efficient trafficking of CARs to tumor sites, as well as the de facto emergence of tumor-infiltrating lymphocytes that exhibit CD19 specificity. The dramatic expansion in vivo allowed us to demonstrate for the first time that CARs directly derived from patients can maintain effector function in vivo for several months. Previous research has suggested that introduction of first-generation CARs into virus-specific T cells is preferable for primary T cells (Pule et al., 2008, Nat Med 14: 1264-1270), however, the results obtained for second-generation CARs introduced into optimally co-stimulated primary T cells call this assumption into question. Without wishing to be bound by any particular theory, it was cautioned that the clinical effects were dramatic and unprecedented with lysis of 1 kg tumor masses in all three patients, accompanied by delayed production of cytokines at potentially dangerously high levels in two patients. The classic cytokine storm effect was not observed. However, the present study was designed to reduce this possibility by slowly infusing CART19 over three days.

It has been shown that very low doses of CAR can induce strong clinical responses. A pilot study demonstrated the safety of the CART19 vector. The fact that doses of CART19 cells several orders of magnitude lower than those tested in previous trials may have a clinical benefit has important implications for the future implementation of CAR therapies on a larger scale, as well as for the design of trials investigating CARs targeting against targets other than CD19.

The present studies also indicate that CART19 is expressed in both central memory cells and effector T cells, and this presumably contributes to their long-term survival compared with previous studies. Without wishing to be bound by any particular theory, CAR T cells can differentiate in vivo into central memory cell-like cells upon encountering and subsequently killing target cells (eg, CLL tumor cells or normal B cells) expressing this surrogate antigen. In fact, activation of 4-1BB has been shown to stimulate memory development in relation to the TCR signaling pathway (Sabbagh et al., 2007, Trends Immunol 28:333-339).

The enhanced proliferation and survival of CART19 has revealed aspects of CAR T cell pharmacokinetics that were previously unknown. The kinetics of cytokine production in plasma and bone marrow have been shown to correlate with peak levels of CART19, so it is possible that degradation is initiated when CD19-expressing target cells become limiting. The mechanism of increased survival of CART19 may relate to the above inclusion of the 4-1BB domain or to the natural TCR and/or CAR signaling pathway. A promising possibility is that increased survival is associated with the CART19 population identified in bone marrow samples, leading to the hypothesis that CD19 CAR may be maintained through interaction with B cell precursors in the bone marrow. Related to this question is the question of what exactly stimulates the initial expansion of CART19 cells in vivo. With some rare exceptions (Savoldo et al., 2011, J Clin Invest doi: 10.1172/JCI46110; Pule et al., 2008, Nat Med 14: 1264-1270), the present study is the only study that did not use IL infusions -2, whereby CART19 cells presumably underwent expansion under the influence of homeostatic cytokines or, more likely, under the influence of CD19 expressed on target leukemia cells and/or normal B cells. In the latter case, this may represent a beneficial property for CARs directed against targets on normal APCs such as CD19 and CD20, since it is possible that self-renewal of CART19 occurs on normal cells, providing a mechanism for CAR memory formation through self-vaccination/re-immunization and, accordingly, long-term immunological surveillance of the tumor. The mechanisms of CART19 homeostasis may require further studies to clarify the internal

-

Claims (1)

and extrinsic cellular survival mechanisms. Prior to these findings, many investigators considered CAR therapy as a temporary form of immunotherapy, however, CARs with optimized signaling domains may contribute to both induction and consolidation of remission, as well as long-term immunosurveillance. Strong anti-leukemic effects were observed in all three patients, including two patients with p53-deficient leukemia. Previous studies using CARs have had difficulty isolating the antitumor effects of lymphocyte-depleting chemotherapy. However, the delayed cytokine production coupled with tumor lysis kinetics in fludarabine-resistant patients, coincident with and possibly dependent on in vivo CAR expansion shown in the present study, indicates that CART19 mediate potent antitumor effects. The present results do not exclude a role for chemotherapy in enhancing the effects of CAR. To fully understand the key factors required to obtain robust CAR T cell function in vivo, careful comparison of vector, transgene, and cell production methods may be required with results from studies occurring at other centers. Unlike antibody therapies, CAR-modified T cells are capable of replicating in vivo, and their long-term persistence can provide sustained tumor control. The availability of a ready-to-use treatment that includes killer T cells without cross-resistance is potentially useful in improving the clinical outcome of patients with B-cell malignancies. A limitation of an antibody treatment method, such as that of rituximab and bevacizumab, is that it requires multiple antibody infusions, which is inconvenient and expensive. The use of a prolonged antibody treatment regimen (in this case for at least 6 months in 3 of 3 patients currently treated) with anti-CD19 scFv expressed on T cells after a single infusion of CART19 cells has a number of practical implications. benefits including convenience and cost savings. Sustained detection of CART19 eighteen months after infusion. The results reported here demonstrate long-lasting CART19 expression and profound B-cell aplasia (Figs. 8 and 9), as well as a decrease in plasma cell numbers in all 3 patients (Fig. 10). The most surprising result of the CART19 trial is that CART19 cells containing a murine scFv exhibiting highly immunogenic phenotypes were actually not rejected by the host patient's immune system. This suggests that CART19 cells deplete normal B cells in the host patient and, as a result, induce tolerance. Without wishing to be bound by any particular theory, CART19 cells can be used in the following applications: 1) cross-match positive solid organ transplant patients; destruction of pre-existing memory B cells may allow organ transplantation not currently possible in such immunized patients; 2) induction of tolerance to immunogenic proteins that are administered to patients (for example, hemophilia); 3) rituximab shows therapeutic efficacy in arthritis and other autoimmune disorders; CART19 may exhibit equal or greater efficacy. In some cases, CART19 cells can be used to kill all subpopulations of B cells (ie, naïve cells, memory cells, plasma cell precursors, and suppressor B regulatory cells). B-regulatory cells may contribute to the immunosuppression of some malignancies, and thus CART19 may improve immune responses by removing B-regulatory cells. The descriptions of any and all patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been described with reference to specific embodiments, it should be understood that other embodiments and variations of the present invention may be practiced by others skilled in the art without departing from the spirit and scope of the invention. The appended claims are intended to include all such embodiments and equivalent variations. CLAIM 1. A method of treating or preventing graft-versus-host disease (GVHD) in an individual in need thereof, comprising administering to the individual an effective amount of an agent selected from the group consisting of at least one of bendamustine, rituximab, OKT3, CEMPAS, fludarabine and cyclophosphamide; and administering to the individual T cells genetically modified to express a CAR after administration of an effective amount of the agent; wherein the CAR contains an antigen binding domain, an intracellular co-stimulatory signaling region 4-1BB and a CD3 zeta signaling domain; -