Use of cart19 to deplete normal b cells to induce tolerance

Abstract

To provide compositions and methods for inducing tolerance in a human.SOLUTION: The invention includes administering a genetically modified T cell expressing a CAR, where the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3ζ signaling domain.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to 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 technology]

[0002] Background of the Invention Using gene transfer technology, T cells can be genetically engineered to stably express antibody binding domains on their surface, conferring novel antigen specificities independent of major histocompatibility complex (MHC). Chimeric antigen receptors (CARs) are one application of this approach, combining the antigen recognition domain of a specific antibody with the intracellular domain of the CD3-z chain or FcgRI protein into a single chimeric protein (Gross et al., 1989 Proc. Natl. Acad. Sci. USA 86:10024-10028 (Non-Patent Document 1); Irving et al., 1991 Cell 64:891-901 (Non-Patent Document 2)). Clinical trials testing CARs are currently underway at several academic medical centers (Kohn et al. 2011 Mol. Ther. 19:432-438 (Non-Patent Document 3); Jena et al., 2010 Blood 116:1035-1044 (Non-Patent Document 4)). While tumor-specific antigens remain poorly defined in most cancers, CD19 is a prominent tumor target in B-cell malignancies. CD19 expression is restricted to normal and malignant B cells (Uckun et al., 1988 Blood 71: 13-29 (Non-Patent Document 5)), and therefore CD19 is widely accepted as a target for safely testing CARs. Although CARs can induce T cell activation in a manner similar to endogenous T cell receptors, limited in vivo expansion of CAR+ T cells, rapid loss of cells after infusion, and disappointing clinical activity represent major obstacles to the clinical application of this technology (Jena et al., 2010 Blood 116: 1035-1044 (Non-Patent Document 4); Sadelain et al., 2009 Curr. Opin. Immunol. 21: 215-223 (Non-Patent Document 6)).

[0003] CAR-mediated T cell responses may be further enhanced by the addition of a costimulatory domain. In one preclinical model, the inclusion of a CD137 (4-1BB) signaling domain significantly increased the antitumor activity and in vivo persistence of CARs compared with those containing only the CD3-z chain (Milone et al., 2009 Mol. Ther. 17, 1453-1464 (Non-Patent Document 7); Carpenito et al., 2009 Proc. Natl. Acad. Sci. USA 106: 3360-3365 (Non-Patent Document 8)). 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 (CART19 cells) expressing an anti-CD19 CAR containing both the CD3-z and 4-1BB costimulatory domains to target CD19+ malignant tumors. Three patients have been treated under this protocol. Some findings from one of these patients have been described (Porter et al., 2011 N. Engl. J. Med. 365: 8), reporting that the treatment resulted in tumor regression, persistence of CART19 cells, and the unexpected development of delayed tumor lysis syndrome. It was also observed that CART19 cells mediated potent clinical antitumor effects in all three treated patients. Each infused CAR T cell and / or their progeny eliminated an average of more than 1,000 leukemia cells in vivo in patients with advanced, chemotherapy-resistant chronic lymphocytic leukemia (CLL). CART19 cells underwent robust in vivo T-cell expansion, persisted at high levels in the blood and bone marrow (BM) for at least 6 months, continued to express functional receptors on cells with a memory phenotype, and maintained anti-CD19 effector function in vivo. However, given that the CAR19 construct contains both mouse sequences (antibody determinants) and unique junction fragments between different components of the CAR19 construct, it remains unclear how CART19 cells avoid rejection by the human host.

[0004] Thus, there remains a need in the art for the mechanism of long-term persistence of CART19 cells and why these cells are not rejected by the human host. The present invention addresses this need. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Gross et al., 1989 Proc. Natl. Acad. Sci. USA 86:10024-10028 [Non-patent document 2] Irving et al., 1991 Cell 64:891-901 [Non-patent document 3] Kohn et al. 2011 Mol. Ther. 19:432-438 [Non-patent document 4] Jena et al., 2010 Blood 116:1035-1044 [Non-Patent Document 5] Uckun et al., 1988 Blood 71: 13-29 [Non-patent document 6] Sadelain et al., 2009 Curr. Opin. Immunol. 21: 215-223 [Non-Patent Document 7] Milone et al., 2009 Mol. Ther. 17, 1453-1464 [Non-patent document 8] Carpenito et al., 2009 Proc. Natl. Acad. Sci. USA 106: 3360-3365 [Non-Patent Document 9] Porter et al., 2011 N. Engl. J. Med. 365: 8 Summary of the Invention

[0006] The present invention provides a method for depleting B cells in a subject. In one embodiment, the method comprises administering to the subject 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 CD3ζ signaling domain, and the antigen binding domain targets a B cell surface marker, thereby depleting B cells in the subject.

[0007] The present invention provides a method for promoting tolerance in a subject. In one embodiment, the method comprises administering to the subject 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 CD3ζ signaling domain, and the antigen binding domain targets a B cell surface marker, thereby promoting tolerance in the subject.

[0008] In one embodiment, the tolerance is transplant tolerance to the transplanted tissue.

[0009] In one embodiment, the genetically modified cells deplete B cells.

[0010] In one embodiment, the genetically modified cells are administered simultaneously with the transplant tissue.

[0011] In one embodiment, the genetically modified cells are administered prior to administration of the transplant tissue.

[0012] In one embodiment, the genetically modified cells are administered after administration of the transplant tissue.

[0013] The present invention provides a method for treating graft-versus-host disease (GVHD). In one embodiment, the method comprises administering to a subject in need thereof cells genetically modified to express a CAR, wherein the CAR comprises an antigen-binding domain, a costimulatory signaling region, and a CD3ζ signaling domain, and the antigen-binding domain targets a B-cell surface marker, thereby treating GVHD in the subject.

[0014] In one embodiment, the genetically modified cells deplete B cells.

[0015] In one embodiment, the genetically modified cells are administered simultaneously with the transplant tissue.

[0016] In one embodiment, the genetically modified cells are administered prior to administration of the transplant tissue.

[0017] In one embodiment, the genetically modified cells are administered after administration of the transplant tissue. [The present invention 1001] 1. A method of depleting B cells in a subject, comprising: administering to a subject 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 CD3ζ signaling domain, wherein the antigen binding domain targets a B cell surface marker. thereby depleting B cells in said subject. [The present invention 1002] 1. A method of promoting tolerance in a subject, comprising: administering to a subject 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 CD3ζ signaling domain, wherein the antigen binding domain targets a B cell surface marker. thereby promoting tolerance in said subject. [The present invention 1003] 1002. The method of claim 1002, wherein said tolerance is transplant tolerance to a transplanted tissue. [The present invention 1004] 1003. The method of claim 1002, wherein said genetically modified cells deplete B cells. [The present invention 1005] 1003. The method of claim 1002, wherein said genetically modified cells are administered simultaneously with the transplanted tissue. [The present invention 1006] 1003. The method of claim 1002, wherein said genetically modified cells are administered prior to administration of the transplant tissue. [The present invention 1007] 1003. The method of claim 1002, wherein said genetically modified cells are administered after administration of the transplant tissue. [The present invention 1008] 1. A method for treating graft-versus-host disease (GVHD), comprising: administering to a subject in need thereof cells genetically modified to express a CAR, wherein the CAR comprises an antigen binding domain, a costimulatory signaling region, and a CD3ζ signaling domain, wherein the antigen binding domain targets a B cell surface marker. thereby treating GVHD in said subject. [The present invention 1009] The method of claim 1008, wherein said genetically modified cells deplete B cells. [The present invention 1010] 1009. The method of claim 1008, wherein said genetically modified cells are administered simultaneously with the transplanted tissue. [The present invention 1011] 1009. The method of claim 8, wherein said genetically modified cells are administered prior to administration of the transplant tissue. [The present invention 1012] 1009. The method of claim 8, wherein said genetically modified cells are administered after administration of the transplant tissue. [Brief explanation of the drawings]

[0018] 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, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1A] This series of images demonstrates the sustained in vivo expansion and persistence of CART19 cells in blood. DNA isolated from whole blood samples obtained from UPN 01 was subjected to Q-PCR analysis using a certified assay to detect and quantify CART19 sequences. Each data point represents the average of triplicate measurements on 100-200 ng of genomic DNA, with a maximum % CV of less than 1.56%. Assay pass / fail parameters included the slope and efficiency of amplification, as well as a predefined range for amplification of a reference standard sample. The lower limit of quantification for the assay, as determined by the range of the standard curve, was 2 transgene copies / μg genomic DNA; values ​​for samples below that number were considered estimates and presented if at least two of three replicates generated Ct values ​​using a % CV of 15%. CART19 cells were infused on days 0, 1, and 2. [Figure 1B]This series of images demonstrates the sustained in vivo expansion and persistence of CART19 cells in blood. DNA isolated from whole blood samples obtained from UPN 02 was subjected to Q-PCR analysis using a certified assay to detect and quantify CART19 sequences. Each data point represents the average of triplicate measurements on 100-200 ng of genomic DNA, with a maximum % CV of less than 1.56%. Assay pass / fail parameters included the slope and efficiency of amplification, as well as a predefined range for amplification of a reference standard sample. The lower limit of quantification for the assay, as determined by the range of the standard curve, was 2 transgene copies / μg genomic DNA; values ​​for samples below that number were considered estimates and presented if at least two of three replicates generated Ct values ​​using a % CV of 15%. CART19 cells were infused on days 0, 1, 2, and 11. [Figure 1C] This series of images demonstrates the sustained in vivo expansion and persistence of CART19 cells in blood. DNA isolated from whole blood samples obtained from UPN 03 was subjected to Q-PCR analysis using a certified assay to detect and quantify CART19 sequences. Each data point represents the average of triplicate measurements on 100-200 ng of genomic DNA, with a maximum % CV of less than 1.56%. Assay pass / fail parameters included the slope and efficiency of amplification, as well as a predefined range for amplification of a reference standard sample. The lower limit of quantification for the assay, as determined by the range of the standard curve, was 2 transgene copies / μg genomic DNA; values ​​for samples below that number were considered estimates and presented if at least two of three replicates generated Ct values ​​using a % CV of 15%. CART19 cells were infused on days 0, 1, and 2. [Figure 1D]This series of images demonstrates the sustained in vivo expansion and persistence of CART19 cells in bone marrow. DNA isolated from bone marrow, a sample obtained from UPN 01, was subjected to Q-PCR analysis using a certified assay to detect and quantify CART19 sequences. Each data point represents the mean of triplicate measurements on 100–200 ng of genomic DNA, with a maximum % CV of less than 1.56%. Assay pass / fail parameters included the slope and efficiency of amplification, as well as a predefined range for amplification of the reference standard sample. The lower limit of quantification for the assay, as determined by the range of the standard curve, was 2 transgene copies / μg genomic DNA; values ​​for samples below that number were considered estimates and presented if at least two of three replicates generated Ct values ​​using a % CV of 15%. CART19 cells were infused on days 0, 1, and 2. [Figure 1E] This series of images demonstrates the sustained in vivo expansion and persistence of CART19 cells in bone marrow. DNA isolated from bone marrow samples obtained from UPN 02 was subjected to Q-PCR analysis using a certified assay to detect and quantify CART19 sequences. Each data point represents the mean of triplicate measurements on 100–200 ng of genomic DNA, with a maximum % CV of less than 1.56%. Assay pass / fail parameters included the slope and efficiency of amplification, as well as a predefined range for amplification of the reference standard sample. The lower limit of quantification for the assay, as determined by the range of the standard curve, was 2 transgene copies / μg genomic DNA; values ​​for samples below that number were considered estimates and presented if at least two of three replicates generated Ct values ​​using a % CV of 15%. CART19 cells were infused on days 0, 1, 2, and 11. [Figure 1F]This series of images demonstrates the sustained in vivo expansion and persistence of CART19 cells in bone marrow. DNA isolated from bone marrow samples obtained from UPN 03 was subjected to Q-PCR analysis using a certified assay to detect and quantify CART19 sequences. Each data point represents the mean of triplicate measurements on 100–200 ng of genomic DNA, with a maximum % CV of less than 1.56%. Assay pass / fail parameters included the slope and efficiency of amplification, as well as a predefined range for amplification of the reference standard sample. The lower limit of quantification for the assay, as determined by the range of the standard curve, was 2 transgene copies / μg genomic DNA; values ​​for samples below that number were considered estimates and presented if at least two of three replicates generated Ct values ​​using a % CV of 15%. CART19 cells were infused on days 0, 1, and 2. [Figure 2A]Figure 2, comprised of Figures 2A-2C, is a series of images depicting long-term surface CART19 expression and the establishment of functional memory CARs in vivo. Figure 2A depicts the detection of CAR-expressing CD3+ lymphocytes and the absence of B cells in the periphery and bone marrow. Freshly processed peripheral blood or bone marrow mononuclear cells obtained from UPN 03 on day 169 post-CART19 cell infusion were assessed by flow cytometry for surface expression of CAR19 (top) or the presence of B cells (bottom); as a control, PBMCs obtained from healthy donor ND365 were stained. To assess CAR19 expression in CD3+ lymphocytes, samples were co-stained with antibodies against CD14-PE-Cy7 and CD16-PE-Cy7 (dump channel) and CD3-FITC, with positive gating on CD3+. CAR19 expression in the CD8+ and CD8- lymphocyte compartments was assessed by co-staining with CD8a-PE and an Alexa-647-conjugated anti-CAR19 idiotype antibody. Data in the plots are gated on the dump channel-negative / CD3-positive cell population. To assess the presence of B cells, samples were co-stained with antibodies against CD14-APC and CD3-FITC (dump channel) and assessed for the presence of B cells in the dump channel-negative fraction by co-staining with antibodies against CD20-PE and CD19-PE-Cy-7. In both cases, the negative gate quadrant was established relative to the unstained control, as depicted in Figures 2B and 2C. T cell immunophenotyping of CD4+ (Figure 2B) and CD8+ (Figure 2C) T cell subsets is shown. Frozen peripheral blood samples from UPN 03 obtained by apheresis on days 56 and 169 after T cell infusion were left overnight in medium without added factors, washed, and subjected to multiparameter immunophenotyping for the expression of T cell memory, activation, and exhaustion markers. The gating strategy, as depicted in Figure 6, involved initial gating on dump channel (CD14, CD16, Live / Dead Aqua)-negative and CD3-positive cells, followed by a positive gate 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 delineated subsets (CD27, CD28, CD57); data were presented after bi-exponential transformation for objective visualization of events. The functional competence of surviving CAR cells was demonstrated in the following experiment. Frozen peripheral blood samples from UPN 03 obtained by apheresis on days 56 and 169 after T cell infusion were incubated overnight in medium without added factors, washed, and then directly assessed ex vivo for their ability to recognize CD19-expressing target cells using a CD107 degranulation assay. After a 2-hour incubation in the presence of anti-CD28, anti-CD49d, and CD107-FITC, the cell mixture was harvested, washed, and subjected to multiparameter flow cytometry analysis to assess the ability of CART19 cells to degranulate in response to CD19-expressing targets. The gating strategy included an initial gate on dump channel (CD14-PE-Cy7, CD16-PE-Cy7, Live / Dead Aqua)-negative and CD3-PE-positive cells, followed by gating on CD8-PE-Texas Red-positive cells; data presented are for the CD8+ gated population. In each case, the negative gate quadrant was established relative to an unstained control. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A]Images depict the results of an experiment evaluating clinical response after CART19 cell infusion. UPN 02 was treated with two cycles of rituximab and bendamustine, resulting in a modest response (R / B, arrow). CART19 T cells were infused 4 days after bendamustine administration alone (B, arrow). Leukemia resistant to rituximab and bendamustine was 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 of infusion. Corticosteroid treatment was initiated 18 days after infusion due to fatigue and a noninfectious fever syndrome. The baseline (dotted line) indicates the upper limit of normal for ALC. [Figure 3B] This image depicts the results of a study evaluating clinical response after CART19 cell infusion. This image depicts the results of an example study in which serial bone marrow biopsy or clot specimens from patients UPN 01 and 03 were stained for CD20. The pretreatment leukemic infiltrates present in both patients were absent in the posttreatment specimens, accompanied by normalization of cellularity and trilineage hematopoiesis. UPN 01 had no CLL cells in the bone marrow or blood, as assessed by flow cytometry, cytogenetics, and fluorescent in situ hybridization, or normal B cells, as detected by flow cytometry. UPN 03 was confirmed to have 5% residual normal CD5-negative B cells by flow cytometry on day +23, which were also shown to be polyclonal; no normal B cells were detected on day +176. [Figure 3C] 1A-1C are images depicting the results of an experiment evaluating clinical response after infusion of CART19 cells.

[0023] Figure 1B depicts the results of an experiment using serial CT imaging to assess rapid resolution of chemotherapy-resistant generalized lymphadenopathy. Bilateral axillary masses resolved by day 83 (UPN 01) and day 31 (UPN 03) after infusion, as indicated by the arrows and circles. [Figure 4]Figure 4, consisting of Figures 4A-4C, is a series of images depicting absolute lymphocyte counts and total CART19+ cells in circulating blood for UPNs 01, 02, and 03. For all three subjects, the total number of lymphocytes in circulating blood (normal cells plus CLL cells) is plotted against the total CART19+ cells, using absolute lymphocyte counts from CBC values ​​and assuming a blood volume of 5.0 L. The total number of CART19 cells in circulating blood was calculated using tandem CBC values ​​along with absolute lymphocyte counts and Q-PCR readouts, as depicted in Figure 1, and converted to a mean % readout of copies / µg DNA as described elsewhere herein. The Q-PCR % readouts were found to correlate well (with less than a 2-fold deviation) with the flow cytometry characteristics of the infused product and with data from samples for which simultaneous flow cytometry data for direct enumeration of CART19 cells by staining was available. [Figure 5] Figure 5, consisting of Figures 5A-5D, is a series of images depicting an experiment involving direct ex vivo detection of CART19-positive cells in UPN-01 PBMCs 71 days after T cell infusion. UPN-01 PBMCs, either collected immediately after apheresis at day 71 after infusion or frozen for T cell product manufacturing at the time of apheresis (baseline) and thawed viably before staining, were subjected to flow cytometry analysis to detect the presence of CART19 cells expressing the CAR19 moiety on their surface. To assess CAR19 expression in lymphocytes, samples were co-stained with CD3-PE and an Alexa-647-conjugated anti-CAR19 idiotypic antibody or with CD3-PE alone (FMO for CAR19). Figure 5A depicts an initial lymphocyte gate established based on forward and side scatter (FSC vs. SSC), followed by gating on CD3+ cells. Figure 5B depicts the CD3+ lymphocyte gate; Figure 5C depicts CAR idiotype staining; Figure 5D depicts CAR idiotype FMO. A CAR19 positive gate was established for the CAR19 FMO sample. [Figure 6]Figure 6, comprised of Figures 6A-6C, is a series of images depicting the gating strategy for identifying CART19 expression by polychromatic flow cytometry in UPN 03 blood specimens. The gating strategy for Figure 6C is shown for the UPN 03 day 56 sample, which is representative of the strategy used for the UPN 03 day 169 sample. Figure 6A depicts the primary gate: Dump (CD14, CD16, LIVE / dead Aqua) negative, CD3 positive. Figure 6B depicts the secondary gate: CD4 positive, CD8 positive. Figure 6C depicts the tertiary gate: CAR19 positive and CAR19 negative, which was established for the CAR FMO sample (right-most panel). [Figure 7] This is an image summarizing patient demographics and response. [Figure 8] 1 is an image depicting long-term expression of CART19. [Figure 9A] A series of images depicting severe B-cell hypoplasia. [Figure 9B] A series of images depicting severe B-cell hypoplasia. [Figure 10] Images demonstrating plasma cell depletion in all three patients. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description The present invention is based, in part, on the surprising discovery that T cells expressing an anti-CD19 CAR (CART19 cells) containing both CD3z and 4-1BB costimulatory domains persisted for extended periods within a mammalian host. For example, it has now been observed that cells expressing surface CAR19 persisted within a mammalian host for 21 months after infusion of CAR19 T cells. Thus, the present invention provides a method for depleting normal B cells in a mammal by administering to a mammal in need thereof a CAR that targets B cells to induce tolerance in the mammal.

[0020] The present invention relates to compositions and methods for depleting B cells and thus inducing tolerance. The present invention relates to methods for adoptive cell transfer of T cells transduced to express chimeric antigen receptors (CARs), which are molecules that combine antibody-based specificity for a target antigen (e.g., a B cell antigen) with an intracellular domain that activates the T cell receptor to create a chimeric protein that exhibits specific anti-B cell immune activity.

[0021] In one embodiment, the CAR of the present invention comprises an extracellular domain having an antigen recognition domain that targets a B cell antigen, a transmembrane domain, and a cytoplasmic domain.

[0022] In one embodiment, the CAR T cells of the present invention can be generated by introducing a lentiviral vector containing the desired CAR. The CAR T cells of the present invention can replicate in vivo, resulting in long-term persistence that can lead to sustained B cell depletion and tolerance.

[0023] In one embodiment, the invention relates to the administration of genetically modified T cells expressing a CAR to effectively reduce the incidence, severity, or duration of graft-versus-host disease (GVHD), rejection episodes, or post-transplant lymphoproliferative disorder.

[0024] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0025] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0027] As used herein, "about" when referring to a measurable value, such as an amount, duration, or the like, is intended to encompass a range of variation from the specified value of ±20% or ±10%, sometimes ±5%, sometimes ±1%, and sometimes ±0.1%, as such variation is reasonable in practicing the disclosed methods.

[0028] As used herein, "activation" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be accompanied by the induction of cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division.

[0029] The term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or may be the immune-responsive portion of an intact immunoglobulin. Antibodies are often tetramers of immunoglobulin molecules. Antibodies in the present invention may exist in various 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; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0030] The term "antibody fragment" refers to a portion of an intact antibody, including the antigen-determining variable region of the 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 formed from antibody fragments.

[0031] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production and activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Moreover, antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be synthetically produced or obtained from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0032] The term "autoantigen," according to the present invention, refers to any self-antigen that is recognized by the immune system as if it were foreign. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins, including cell surface receptors.

[0033] As used herein, "autoimmune disease" is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0034] As used herein, the term "autologous" is intended to refer to any material originating from the same individual that is later reintroduced into that individual.

[0035] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0036] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0037] As used herein, a "B cell surface marker" refers to an antigen expressed on the surface of a B cell that can be targeted by an agent that binds to it. Exemplary B cell surface markers include 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 leukocyte surface markers. B cell surface markers of particular interest are those that are preferentially expressed on B cells compared to other non-B cell tissues in mammals and may be expressed on both precursor B cells and mature B cells. In one embodiment, a preferred marker is CD19, which is found on B cells throughout the differentiation of this lineage from the pro / pre-B cell stage to the terminally differentiated plasma cell stage.

[0038] As used herein, "B cell depletion" refers to a reduction in B cell levels in an animal or human after drug, cell, or antibody therapy compared to pre-treatment levels. B cell levels can be measured using well-known assays, for example, by performing a complete blood count, by FACS analysis staining for known B cell markers, and by methods described elsewhere herein. B cell depletion can be partial or complete. In one embodiment, B cell depletion is 25% or greater.

[0039] The terms "deplete" and "depletion," as used herein in reference to B cells, mean one or more of the following in this specification and the appended claims: blocking B cell function; functional inactivation of B cells; cytolysis of B cells; inhibiting B cell proliferation; inhibiting B cell differentiation into plasma cells; causing B cell dysfunction to provide a therapeutic benefit; inhibiting the production of anti-shed antigen antibodies; reducing the number of B cells; inactivating shed antigen-primed or activated B cells; blocking one or more functions of shed antigen-primed or activated B cells; cytolysis of shed antigen-primed or activated B cells; and reducing the number of shed antigen-primed or activated B cells. B cell depletion may be the result of one or more mechanisms, including, but not limited to, clonal inactivation, apoptosis, antibody-dependent cellular cytotoxicity, complement-mediated cytotoxicity, and signal pathway-mediated inactivation, dysfunction, or cell death.

[0040] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrollable growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers 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 malignancies, lymphoma, leukemia, lung cancer, etc.

[0041] The "CD19" antigen refers to an antigen of approximately 90 kDa, which can be identified, for example, by the HD237 or B4 antibody (Kiesel et al., 1987 Leukemia Research II, 12:1119). CD19 is found on cells throughout B-lineage cell differentiation, from the stem cell stage to terminal differentiation into plasma cells, including, but not limited to, pre-B cells, B cells (including naive B cells, antigen-stimulated B cells, memory B cells, plasma cells, and B lymphocytes), and intrafollicular dendritic cells. CD19 is also found on B cells in human fetal tissues. In a preferred embodiment, the CD19 antigen targeted by the antibody of the present invention is human CD19 antigen.

[0042] "Costimulatory ligand," as that term is used herein, includes a molecule on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.) that can specifically bind to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, among others, and ligands that specifically bind to CD83.

[0043] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0044] "Costimulatory signal," as used herein, refers to a molecule that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0045] "Disease" refers to an animal's health condition in which the animal is unable to maintain homeostasis and the animal's health will continue to deteriorate unless the disease is improved. In contrast, an animal's "disorder" is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is more unfavorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause the animal's health condition to further deteriorate.

[0046] As used herein, an "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0047] As used herein, "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0048] As used herein, the term "exogenous" refers to any material produced outside of an organism, cell, tissue, or system that is introduced into that organism, cell, tissue, or system.

[0049] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0050] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide.

[0051] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, comparisons are performed by aligning the two sequences to maximize homology.

[0052] The terms "immunoglobulin" or "Ig" as used herein are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this protein class are IgA, IgG, IgM, IgD, and IgE. IgA is the predominant antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most mammals. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function but may function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0053] As used herein, the term "immune response" includes T cell-mediated 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 also includes immune responses indirectly caused by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in immune responses include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells, such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, etc., and non-professional antigen-presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, etc.); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0054] As used herein, the term "immune tolerance" refers to a procedure performed on a proportion of treated subjects compared to untreated subjects, in which a) the level of a specific immune response (believed to be mediated at least in part by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or their equivalents) is reduced; b) the initiation or progression of a specific immune response is delayed; or c) the risk of initiation or progression of a specific immune response is reduced. "Specific" immune tolerance occurs when immune tolerance is preferentially elicited to one antigen over others.

[0055] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the utility of the compositions and methods of the invention. The instructional materials of the kits of the invention may, for example, be affixed to a container that contains the nucleic acids, peptides, and / or compositions of the invention, or may be shipped together with a container that contains the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compounds be used together by the recipient.

[0056] "Isolated" means altered or removed from the 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 the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0057] As used herein, "lentivirus" refers to a genus in the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.Vector derived from lentiviruses provide a means to achieve a fairly high level of gene transfer in vivo.

[0058] The term "modulate," as used herein, means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in that subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical subject not receiving the treatment. The term encompasses perturbing and / or affecting a native signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0059] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0060] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof, whether in vitro or in situ, to which the methods described herein may be applied. In one non-limiting embodiment, the patient, subject, or individual is a human.

[0061] The term "rejection" refers to the failure of a transplanted organ or tissue to be accepted by the recipient's body. Rejection is caused by the recipient's immune system attacking the transplanted organ or tissue. Rejection can occur within days to weeks after transplantation (acute) or months to years after transplantation (chronic).

[0062] The term "specifically binds," as used herein with respect to antibodies, refers to an antibody that recognizes a specific antigen in a sample but does not substantially recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in itself, alter the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in itself, alter the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction involving labeled "A" and the antibody.

[0063] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation may mediate altered expression of certain molecules, such as downregulation of TGF-β and / or remodeling of cytoskeletal structure.

[0064] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0065] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), is capable of specifically binding to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0066] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0067] As used herein, "substantially purified" cells are cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that naturally accompany them in their natural state. In some cases, a population of substantially purified cells refers to a homogeneous cell population. In other cases, the term simply refers to cells that have been separated from the cells that naturally accompany them in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0068] As used herein, the term "therapeutic" refers to treatment and / or prophylactic treatment. A therapeutic benefit is achieved by suppressing, ameliorating, or eradicating the disease state.

[0069] The term "therapeutically effective amount" refers to that amount of a compound of interest that will elicit the biological or medical response in a tissue, system, or subject that a researcher, veterinarian, physician, or other clinical professional is seeking to investigate. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the occurrence of, or ameliorate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0070] The term "graft," as used herein, refers to cells, tissues, or organs that are introduced into an individual. The source of the transplant material can be cultured cells, cells from another individual, or cells from the same individual (e.g., after culturing the cells in vitro). Exemplary organ transplants include kidney, liver, heart, lung, and pancreas.

[0071] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder from which a subject suffers.

[0072] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0073] The term "tolerant" refers to an individual who has a reduced or absent immune response to a particular antigen or group of antigens. In the context of the present invention, an individual is considered to be tolerant if he or she does not reject the transplanted cells (i.e., does not mount an overt immune response thereto). In some cases, a tolerant individual does not reject the transplanted cells even in the absence of immunosuppressive therapy. In the context of the present invention, an individual is considered "non-tolerant" if they reject the transplanted cells. Non-tolerant individuals include those in which an active immune response to the transplanted cells is mounted, as well as those in which rejection is controlled using immunosuppressive therapy (e.g., standard immunosuppression).

[0074] As used herein, "in vivo tolerance" refers to the substantial lack of an immune response specific to foreign tissue. The immune response may result from the recipient subject initiating an immune response against the foreign tissue, or conversely, the immune response may result from the foreign tissue initiating an immune response against the recipient subject (e.g., GVHD). Methods for measuring in vivo tolerance are generally known in the art.

[0075] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges within that range, as well as each individual numerical value. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed each individual number within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the broadness of the range.

[0076] explanation The present invention provides compositions and methods for depleting normal B cells in a mammal. In one embodiment, depletion of B cells using a CAR of the present invention induces tolerance in a mammal.

[0077] In one embodiment, the present invention provides a method for inducing in vivo tolerance to transplanted foreign tissue. In some embodiments, the method can be used, in part, to prevent and / or treat rejection of the transplanted tissue. Generally speaking, the method includes administering CAR T cells of the present invention to a subject exposed to transplanted foreign tissue. The term "foreign tissue," as used herein, can include bone marrow transplants, organ transplants, transfused blood, or any other foreign tissue or cells that are intentionally introduced into a subject.

[0078] In another embodiment, the method can be used, in part, to prevent and / or treat graft-versus-host disease (GVHD). Generally speaking, the method includes administering the CAR T cells of the present invention to a subject exposed to transplanted foreign tissue. The term "foreign tissue," as used herein, can include bone marrow grafts, organ transplants, transfused blood, or any other foreign tissue or cells that are intentionally introduced into a subject.

[0079] In one embodiment, the CAR of the present invention can be engineered to include an extracellular domain having an antigen-binding domain targeting a B cell antigen fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3ζ). An exemplary B cell antigen is CD19, as this antigen is expressed on malignant B cells. However, the present invention is not limited to targeting CD19. More precisely, the present invention encompasses any B cell antigen-binding moiety when bound to its cognate antigen. Preferably, the antigen-binding moiety is fused to an intracellular domain from one or more of a costimulatory molecule and the zeta chain. Preferably, the antigen-binding moiety is fused to one or more intracellular domains selected from the group consisting of the CD137 (4-1BB) signaling domain, the CD28 signaling domain, the CD3ζ signaling domain, and any combination thereof.

[0080] In one embodiment, the CAR of the present invention comprises a CD137 (4-1BB) 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 a costimulatory domain. For example, the inclusion of a CD137 (4-1BB) signaling domain significantly increased the CAR-mediated activity and in vivo persistence of CAR T cells compared to identical CAR T cells that were not engineered to express CD137 (4-1BB). However, the present invention is not limited to a specific CAR. More precisely, any CAR that targets B cells can be used in the present invention. Compositions and methods for producing CARs are described in PCT / US11 / 64191, which is incorporated herein by reference.

[0081] method The present invention relates to a method of using the CAR and CAR T cells of the present invention to deplete B cells and promote tolerance. In one embodiment, the method comprises promoting transplant tolerance (e.g., of an organ or tissue transplant) in a patient. In another embodiment, the method comprises preventing and / or treating GVHD. In one specific embodiment, the CAR of the present invention targets CD19 on B cells.

[0082] In one embodiment, the ability to induce durable humoral tolerance in the donor is key to achieving robust transplant tolerance and / or preventing or treating GVHD. The present invention encompasses the use of the CAR T cells of the invention to deplete B cells and induce tolerance by administering the CAR T cells to an animal, preferably a mammal, and most preferably a human patient, for the purpose of treating one or more diseases, disorders, symptoms, or conditions associated with organ or tissue transplants (e.g., transplant rejection, GVHD, and / or conditions associated therewith).

[0083] Organ rejection occurs through the destruction of transplanted tissue by host immune cells through an immune response. Similarly, an immune response is also involved in GVHD, in which foreign transplanted immune cells destroy host tissue. 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 pancreatic islet cells. However, the present invention is not limited to a particular type of transplant. As a non-limiting example, pancreatic islet cell transplantation can be performed to prevent the onset of diabetes or as a treatment for diabetes. Administration of CAR T cells of the present invention that inhibit immune responses, particularly B cell proliferation, differentiation, or survival, is an effective therapy for preventing organ and / or tissue rejection or GVHD. Administration of CAR T cells of the present invention can also be used to promote transplant tolerance after organ and / or tissue transplantation.

[0084] The CAR T cells of the present invention can also be used to promote transplant tolerance; treat, reduce, inhibit, and / or prevent organ and / or tissue transplant rejection; and / or reduce antibody titers in patients who have received an organ or tissue transplant. In one embodiment, the CAR T cells of the present invention can be used to promote transplant tolerance in a patient by administering an effective amount of the CAR T cells of the present invention to the patient, thereby preventing or delaying transplant rejection. In another embodiment, the CAR T cells of the present invention can be used to treat organ or transplant rejection in a patient by administering an effective amount of the CAR T cells of the present invention to the patient, thereby inhibiting rejection of the transplanted organ or tissue. In yet another embodiment, the CAR T cells of the present invention can be used to reduce antibody titers in a patient who has received or will receive an organ or tissue transplant by administering an effective amount of the CAR T cells of the present invention to the patient, thereby reducing antibody titers.

[0085] In one embodiment, the invention provides a method of promoting transplant tolerance in a patient, the method comprising administering to the patient an effective amount of a CAR T cell of the invention, thereby delaying transplant rejection in the patient.

[0086] In another embodiment, the invention provides a method of treating rejection of a transplanted organ or tissue in a patient, the method comprising administering to the patient an effective amount of a CAR T cell of the invention, thereby inhibiting rejection of the transplanted organ or tissue in the patient.

[0087] In another embodiment, the invention provides a method of reducing antibody titers in a patient who has received or will receive an organ or tissue transplant, the method comprising administering to the patient an effective amount of a CAR T cell of the invention, thereby reducing antibody titers in the patient.

[0088] In one embodiment, the invention provides a method of inhibiting or reducing immunoglobulin production in a patient, the method comprising administering to the patient an effective amount of a CAR T cell of the invention.

[0089] In one embodiment, the CAR T cells of the present invention reduce or inhibit B cell function. In another embodiment, the CAR T cells of the present invention deplete or eliminate B cells from a subject. For example, the CAR T cells of the present invention can be engineered to target B cell surface antigens to enable the T cells to exert effector functions on B cells.

[0090] Therapies to inhibit harmful immune responses after transplantation The present invention includes methods of using the CAR T cells of the present invention as a therapy for inhibiting GVHD or graft rejection after transplantation. Thus, the present invention encompasses methods of contacting a donor graft, such as a biocompatible lattice or donor tissue, organ, or cells, with the CAR T cells of the present invention before, simultaneously with, or after transplantation of the graft into a recipient. The CAR T cells of the present invention are useful for preventing or treating GVHD by alleviating, inhibiting, or reducing adverse reactions of the donor graft to the recipient.

[0091] As discussed elsewhere herein, T cells can be obtained from any source, e.g., a tissue donor, a graft recipient, or other unrelated source (all from a different individual or species) for making the CAR T cells of the invention, for use in eliminating or reducing an unwanted immune response by the graft to the recipient of the graft. Thus, the CAR T cells of the invention can be autologous, allogeneic, or xenogeneic to the tissue donor, graft recipient, or other unrelated source.

[0092] In one embodiment of the present invention, a graft is exposed to the CAR T cells of the present 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 recipient cells is believed to be suppressed by the CAR T cells of the present invention present in the graft because the CAR T cells can deplete B cells and induce tolerance.

[0093] In another embodiment of the present invention, a donor graft can be "preconditioned" or "pretreated" by treating the graft prior to transplantation into a recipient with the aim of reducing the immunogenicity of the graft to the recipient and thereby reducing and / or preventing GVHD or graft rejection. The graft can be contacted with cells or tissues from the recipient prior to transplantation with the aim of activating T cells that may be associated with the graft. After treating the graft with cells or tissues from the recipient, the cells or tissues may be removed from the graft. The treated graft is then further contacted with CAR T cells of the present invention with the aim of reducing, inhibiting, or eliminating the activity of T cells and / or B cells activated by treatment with cells or tissues from the recipient. Following this treatment of the graft with CAR T cells of the present invention, the CAR T cells may be removed from the graft prior to transplantation into the recipient. However, it is believed that some CAR T cells will adhere to the graft and therefore be introduced into the recipient along with the graft. In this situation, the CAR T cells introduced into the recipient can suppress the immune response against the recipient caused by any cells associated with the graft. Without wishing to be bound by any particular theory, treating 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 cells and / or B cells, thereby preventing restimulation or inducing hyporesponsiveness of T and / or cells to subsequent antigenic stimulation by tissues and / or cells derived from the recipient. Based on the present disclosure, one skilled in the art will understand that preconditioning or pretreatment of the graft prior to transplantation can reduce or eliminate the graft-versus-host response.

[0094] therapeutic application In one embodiment, the present invention comprises a type of cell therapy, in which T cells are genetically modified to express CAR, and the CAR T cells are transfused into recipients in need thereof. The transfused cells can kill target cells. In one embodiment, the target cells are B cells. Unlike antibody therapy, CAR T cells can replicate in vivo, resulting in long-term persistence, which can lead to sustained B cell depletion and tolerance.

[0095] In one embodiment, the CAR T cells of the present invention can undergo robust in vivo T cell expansion and persist for extended periods of time. In another embodiment, the CAR T cells of the present invention can become specific memory T cells that can be reactivated to inhibit B cell proliferation. For example, CART19 cells elicit a specific immune response against cells expressing CD19.

[0096] The CAR-modified T cells of the present invention can also serve as a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. Preferably, the mammal is a human.

[0097] For ex vivo immunization, at least one of the following is performed in vitro before the cells are administered to a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0098] Ex vivo procedures are well known in the art and will be discussed in more detail below. Briefly, cells are isolated from a mammal (preferably human) and then genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to achieve therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0099] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against B cell antigens in a patient.

[0100] Generally, cells activated and expanded as described herein can be used for B cell depletion and tolerance induction. In particular, the CAR-modified T cells of the present invention are used to treat one or more diseases, disorders, symptoms, or conditions associated with organ or tissue transplants (e.g., GVHD and / or associated conditions). Accordingly, the present invention provides a method for treating or preventing organ rejection and GVHD, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0101] In one embodiment, the CAR T cells of the present invention are administered together with immunosuppressants.Any immunosuppressant known in the art can be used.For example, the immunosuppressant can be cyclosporine, azathioprine, rapamycin, mycophenolate mofetil, mycophenolic acid, prednisone, sirolimus, basiliximab or daclizumab, or any combination thereof. Other specific immunosuppressive agents that may be used include ORTHOCLONE OKT™ 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF™ (FK506, tacrolimus), CELLCEPT™ (mycophenolate mofetil, the active metabolite of which is mycophenolic acid), IMURAN™ (azathioprine), glucocorticoids, and steroids. Steroids, including but not limited to corticosteroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisone), FOLEX™ and MEXATE™ (methotrexate), OXSORALEN-ULTRA™ (methoxsalen), RITUXAN™ (rituximab), and RAPAMUNE™ (sirolimus).

[0102] The CAR T cells of the present invention can be administered to a patient before, after, or simultaneously with an immunosuppressant. For example, the CAR T cells of the present invention can be administered after an immunosuppressant is administered to a patient, or the CAR T cells of the present invention can be administered before an immunosuppressant is administered to a patient. Alternatively, or additionally, the CAR T cells of the present invention are administered simultaneously with the administration of an immunosuppressant to a patient.

[0103] The CAR T cells and / or immunosuppressants of the present invention can be administered to the patient after transplantation. Alternatively or additionally, the CAR T cells and / or immunosuppressants of the present invention can be administered to the patient before transplantation. The CAR T cells and / or immunosuppressants of the present invention can also be administered to the patient during the transplant procedure.

[0104] In some embodiments, the method of the present invention for administering CAR T cells to a patient can be performed once after immunosuppressive therapy has begun. In some embodiments, the method is performed multiple times, for example, to monitor the transplant recipient over time, and, if applicable, is performed under different immunosuppressive therapy regimens. In some embodiments, if the transplant recipient is expected to tolerate the transplant, immunosuppressive therapy is weakened. In some embodiments, if the transplant recipient is expected to tolerate the transplant, immunosuppressive therapy is not prescribed at all, for example, immunosuppressive therapy is stopped. If the transplant recipient shows signs of intolerance biomarkers, immunosuppressive therapy can be resumed or continued at a standard level.

[0105] The organ or tissue transplant may be a heart, heart valve, lung, kidney, liver, pancreas, intestine, skin, blood vessels, bone marrow, stem cells, bone, or pancreatic islet cells.

[0106] The CAR T cells of the invention can be administered after a diagnosis of organ or tissue rejection of the transplant, and the CAR T cells of the invention and an immunosuppressant can be administered multiple times until the symptoms of organ or tissue rejection subside.

[0107] In some embodiments, the CAR T cells of the invention can be administered after a diagnosis of elevated antibody titers, and the CAR T cells of the invention and the immunosuppressant can be administered multiple times until the antibody titer decreases.

[0108] Treatment with the CAR T cells of the invention is preferably achieved by administering to the patient an effective amount of the CAR T cells of the invention.

[0109] The CAR T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with other components, such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention can comprise the target cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or additives. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0110] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, but the appropriate amount can be determined by clinical trials.

[0111] When an "effective amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, antibody titer, and condition of the patient (subject). Generally, pharmaceutical compositions comprising T cells as described herein are administered in an amount of 100 mg / kg of cells. 4 ~10 9 cells / kg body weight, preferably 10 cells 5 ~10 6The T cell compositions can be administered at doses of 1000 cells / kg body weight, including all integer values ​​within these ranges. The T cell compositions can also be administered multiple times at these doses. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting treatment accordingly.

[0112] In some embodiments, it may be desirable to administer activated T cells to a subject, followed by subsequent blood collection (or apheresis), activation of T cells therefrom according to the present invention, and reinfusion of these activated and expanded T cells back into the patient. This process can be performed multiple times, every 2-3 weeks. In some embodiments, T cells can be activated from a blood collection of 10 cc to 400 cc. In some embodiments, T cells are reactivated from a blood collection of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Without being bound by theory, the use of this multiple blood collection / multiple reinfusion protocol may be useful for selecting specific populations of T cells.

[0113] The compositions can be administered in any convenient manner, including aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously (i.v.), or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to patients by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions may also be injected into tumors, lymph nodes, or directly into the site of infection.

[0114] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any of a variety of reasonable therapeutic modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients, efalizumab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative drugs such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signal transduction (Liu et al., Cell, 66:807-815, 1991; Henderson et al., Immun., 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously with, or after) T cell depletion therapy using bone marrow transplantation, chemotherapy drugs such as fludarabine, external beam radiation (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered after B cell depletion therapy with an agent reactive with CD20, such as Rituxan. For example, in one embodiment, a subject can receive standard treatment of high dose chemotherapy followed by a peripheral blood stem cell transplant.In certain embodiments, after transplantation, the subject receives a transfusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0115] The dosages of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dosages for human administration can be increased or decreased according to accepted practices in the art. For example, doses of CAMPATH generally range from 1 to about 100 mg for adult patients, usually administered daily for a period of 1 to 30 days. A preferred daily dose is 1 to 10 mg / day, although higher doses up to 40 mg / day can be used in some cases (see U.S. Patent No. 6,120,766). [Example]

[0116] Experimental Example The invention will now be described with reference to the following experimental examples, which are provided for illustrative purposes only, and the present invention should in no way be considered limited to these examples, but should also be considered to encompass any and all variations that become evident as a result of the teachings provided herein.

[0117] Without further description, one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following examples, therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0118] Example 1: T cells expressing chimeric receptors deplete normal B cells , induce tolerance The results presented herein demonstrate that the CART19 cells persist in patients for at least 18 months, providing therapeutic benefit. The engineered T cells expanded more than 1,000-fold in vivo, trafficked to the bone marrow, and continued to express high levels of functional CARs for at least six months. On average, each infused CAR+ T cell eradicated at least 1,000 CLL cells. CD19-specific immune responses were demonstrated in the blood and bone marrow, accompanied by complete remissions in two of three patients. A portion of the cells persisted as memory CAR+ T cells, indicating the potential of this MHC-unrestricted approach for the effective treatment of B-cell malignancies.

[0119] The materials and methods used in these experiments are described below.

[0120] material and method Protocol Design A clinical trial (NCT01029366) was conducted as described in PCT / US11 / 64191, which is incorporated herein by reference in its entirety.

[0121] Vector construction The CD19-BB-z transgene (GeMCRIS 0607-793) was designed and constructed as described (Milone et al., 2009, Mol Ther. 17:1453-1464). Lentiviral vectors were produced according to good manufacturing practice using a three-plasmid construction approach at Lentigen Corporation as described (Zufferey et al., 1997, Nature biotechnol 15:871-875).

[0122] Preparation of CART19 cell products The method for T cell preparation using paramagnetic polystyrene beads coated with anti-CD3 and anti-CD28 monoclonal antibodies was described (Laport et al., 2003, Blood 102:2004-2013). Lentiviral transduction was performed as described (Levine et al., 2006, Proc Natl Acad Sci USA 103:17372-17377).

[0123] The results of the experiment are given below.

[0124] In vivo expansion and persistence of CART19 and trafficking to bone marrow CAR+ T cells expressing the 4-1BB signaling domain and expanded using CD3 / CD28 beads appear to be an improvement over CARs lacking 4-1BB. We developed a Q-PCR assay that allows for quantitative tracking of CART19 cells in the blood and bone marrow. As depicted in Figures 1A and 1C, all patients experienced expansion and persistence of CART19 cells in the blood for at least 6 months. Notably, patients UPN 01 and UPN 03 experienced a 1,000- to 10,000-fold expansion of CAR+ T cells in the blood during the first month after infusion. The peak expansion levels coincided with the onset of post-infusion clinical symptoms in patients UPN 01 (day 15) and UPN 03 (day 23). Furthermore, in all three patients, CART19 T cell levels stabilized between days 90 and 180 after infusion, following an initial decline that could be modeled using first-order kinetics. Importantly, in all patients, CART19 T cells also trafficked to the bone marrow, although at levels 5- to 10-fold lower than those observed in the blood, as depicted in Figures 1D-1F. Patients UPN 01 and 03 showed log-linear decay in the bone marrow, with a T1 / 2 of approximately 35 days.

[0125] Long-term expression and establishment of a population of memory CART19 cells in the blood A key issue in CAR-mediated cancer immunotherapy is whether optimized cell manufacturing and costimulatory domains can enhance the persistence of genetically modified T cells and enable the establishment of CAR+ memory T cells in patients. Previous studies have not demonstrated robust expansion, long-term persistence, and / or expression of CARs on T cells after 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, 2261-2271; Savoldo et al., 2011, J Clin Invest doi:10.1172 / JC146110). Flow cytometry analysis of samples from both blood and bone marrow 169 days after infusion revealed the presence of CAR19-expressing cells in UPN 03 (Figures 2A and 2B) and the absence of B cells, as depicted in Figure 2A. Of note, as depicted in Figures 1 and 4, all three patients had persistent CAR+ cells by Q-PCR assay at 4 months and beyond. The in vivo frequency of CAR+ cells by flow cytometry closely matched the values ​​obtained from PCR assays for the CART19 transgene. Importantly, as depicted in Figure 2A, in patient UPN 03, only CD3+ cells expressed CAR19, as no CAR19+ cells were detectable in the CD16+ or CD14+ subsets. As depicted in Figure 5, CAR expression was detected on the surface of 4.2% of T cells in the blood of patient UPN 01 even at 71 days after infusion.

[0126] Next, we performed detailed studies to further characterize the expression, phenotype, and function of CART19 cells in UPN 03 using polychromatic flow cytometry with an anti-CAR idiotypic antibody (MDA-647) and the gating strategy shown in Figure 6. Based on CAR19 expression, we observed striking differences in the expression of memory and activation markers in both CD8+ and CD4+ cells. As depicted in Figure 2C, at day 56, CART19 CD8+ cells displayed a predominantly effector memory phenotype (CCR7-CD27-CD28-), consistent with prolonged and robust exposure to antigen. In contrast, CAR-negative CD8+ cells consisted of a mixture of effector and central memory cells, with a subset of cells expressing CCR7 and presenting significant numbers in the CD27+ / CD28- and CD27+ / CD28+ fractions. Although both the CART19 and CAR-negative cell populations expressed significant amounts of CD57, this molecule was uniformly coexpressed with PD-1 in CART19 cells, which may reflect the advanced replicative history of these cells. In contrast to the CAR-negative cell population, all CART19 CD8+ populations lacked expression of both CD25 and CD127. By day 169, the phenotype of the CAR-negative cell population remained similar to the day 56 samples, but the CART19 population contained a minority population with central memory cell characteristics, particularly CCR7 expression and higher levels of CD27 and CD28, as well as CAR+ cells that were PD-1-negative, CD57-negative, and CD127-positive.

[0127] In the CD4+ compartment, as depicted in Figure 2B, at day 56, CART19 cells were characterized by a uniform lack of CCR7 and a predominance of CD27+ / CD28+ / PD-1+ cells distributed in both the CD57+ and CD57- compartments, as well as the essentially absence of CD25 and CD127 expression. In contrast, CAR-negative cells at this time point were heterogeneous with respect to CCR7, CD27, and PD-1 expression, expressed CD127, and also contained a significant population of CD25+ / CD127- (potential regulatory T cells). By day 169, while all CAR+ CD4+ cells remained uniformly positive for CD28 expression, a fraction of CART19 CD4+ cells trended toward a central memory phenotype with CCR7 expression, a higher percentage of CD27- cells, the emergence of a PD-1-negative subset, and the acquisition of CD127 expression. CAR-negative cells remained fairly consistent with their day 56 counterparts, with the exception of decreased CD27 expression and a reduced percentage of CD25+ / CD127- cells.

[0128] CART19 cells can retain effector function after six months in the blood Limitations of previous studies using CAR+ T cells include short persistence and poor in vivo expansion, as well as the rapid loss of infused T cell functional activity in vivo. The high levels of CART19 cell persistence and surface expression of the CAR19 molecule in patients UPN 01 and 03 offered the opportunity to directly examine anti-CD19-specific effector function 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. Robust CD19-specific effector function of CART19 T cells was demonstrated by specific degranulation of CD19-positive target cells, as distinct from CD19-negative target cells, as assessed by surface CD107a expression. Notably, exposure of the CART19 population to CD19-positive targets induced rapid internalization of surface CAR-19, as depicted in Figure 6 for surface expression of CAR19 on the same effector cells by standard flow cytometry staining. Given that the NALM-6 line does not express CD80 or CD86, the presence of costimulatory molecules on target cells was not required to induce degranulation of CART19 cells (Brentjens et al., 2007, Clin Cancer Res 13:5426-5435). Effector function was evident at 56 days post-infusion and was maintained at 169 days. Robust effector function of CAR+ and CAR-T cells may also be demonstrated by pharmacological stimulation.

[0129] Clinical activity of CART19 cells No significant toxicity was observed in any patient during the first 4 days after infusion, other than transient febrile reactions. However, all patients subsequently experienced significant clinical and laboratory toxicities between days 7 and 21 after the initial infusion. These toxicities were short-lived and reversible. According to standard criteria (Hallek et al., 2008, Blood 111:5446), of the three patients treated to date, two achieved a complete response and one achieved a partial response more than 6 months after CART19 infusion. Details of each patient's medical history and response to therapy are depicted in Figure 7.

[0130] Briefly, patient UPN 01 developed a fever syndrome with rigors and transient hypotension beginning 10 days after infusion. The fever persisted for approximately 2 weeks and then resolved; the patient experienced no further constitutional symptoms. This patient achieved a rapid and complete response, as depicted in Figure 3. Between 1 and 6 months after infusion, no circulating CLL cells were detected in the blood by flow cytometry. As depicted in Figure 3B, morphological and flow cytometric examinations demonstrated a persistent absence of lymphoid infiltrates in the bone marrow 1, 3, and 6 months after CART-19 cell infusion. As depicted in Figure 3C, CT scans 1 and 3 months after infusion showed resolution of lymphadenopathy. The complete response has persisted for more than 10 months since the time of this report.

[0131] Patient UPN 02 was treated with two cycles of bendamustine and rituximab, resulting in stable disease, as depicted in Figure 3A. The patient received a third dose of bendamustine as lymphodepleting chemotherapy before CART19 T cell infusion. The patient developed fever reaching 40°C, rigors, and dyspnea, requiring 24 hours of hospitalization on day 11 after the first infusion, the day of the second booster dose of CART19 cells. The fever and constitutional symptoms persisted, and the patient experienced transient cardiac failure on day 15; all symptoms resolved after corticosteroid therapy was initiated on day 18. Following the CART19 infusion, concomitant with the onset of high fever, the patient experienced rapid clearance of p53-deficient CLL cells from the peripheral blood and partial reduction of lymph node enlargement, as depicted in Figure 3A. The bone marrow showed persistent widespread CLL infiltration 1 month after treatment, despite extensive peripheral blood cell depletion. The patient remained asymptomatic.

[0132] Patient UPN 03 received lymphodepleting chemotherapy with pentostatin and cyclophosphamide prior to CART19 cell infusion. Three days after chemotherapy but prior to cell infusion, the bone marrow was hypercellular (60%) with approximately 50% CLL involvement. This patient received a low dose of CART19 cells (1.5 × 10 5 The patient received 10 CAR+ T cells / kg divided over 3 days. Again, no acute infusion toxicity was observed. However, 14 days after the first infusion, the patient began to experience rigors, fever, nausea, and diarrhea. By day 22 after infusion, tumor lysis syndrome was diagnosed, requiring hospitalization. The patient's constitutional symptoms resolved, and within 1 month of CART19 infusion, circulating CLL had disappeared from the blood and bone marrow by morphology, flow cytometry, cytogenetics, and FISH analysis. As depicted in Figures 3B and 3C, a CT scan showed resolution of the abnormal lymph node enlargement. Complete remission persisted for more than 8 months after the first infusion of CART19 cells.

[0133] Examining the ratio of CART19 effector to CLL target cells in vivo Preclinical studies have demonstrated the ability to eliminate large tumors in humanized mice, and 7 Infusion of 1 x 10 CARs resulted in an in vivo E:T ratio of 1:42. 9 While it has been shown that tumors composed of CAR+ T cells can be eradicated (Carpenito et al., 2009, Proc Natl Acad Sci USA 106:3360-3365), these calculations do not take into account T cell expansion after injection. Estimation of CLL tumor burden over time allowed calculation of the tumor shrinkage achieved in vivo in three subjects and the estimated CART19 E:T ratio based on the number of CAR+ T cells infused. Tumor burden was calculated by measuring CLL load in bone marrow, blood, and secondary lymphoid tissues. Baseline tumor burden, shown in Figure 7, was calculated based on the number of CLL T cells in each patient at 10 CAR+ T cells prior to CART19 infusion. 12 Patient UPN 03 had an estimated baseline total tumor burden in the bone marrow of 8.8 x 10 CLL cells on day -1 (i.e., after chemotherapy and before CART19 infusion). 11 and measurements of tumor burden in secondary lymphoid tissue ranged from 3.3 to 5.5 × 10 CLL cells depending on the method of volumetric CT scan analysis. 11 UPN 03 contained 1.4 × 10 CART19 cells. 7 The estimated initial tumor burden (1.3 × 10 CLL cells) was 12 Considering the complete lack of detectable CLL cells after treatment, a remarkable E:T ratio of 1:93,000 was achieved. Similar calculations yielded effective in vivo E:T ratios of 1:2200 and 1:1000 for UPN 01 and UPN 02. Ultimately, the contribution of sequential killing by CART19 T cells, combined with the >1,000-fold expansion of CART19 in vivo, likely accounts for the potent anti-leukemic effect mediated by CART19 cells.

[0134] T cells expressing chimeric receptors mediate memory and potent antitumor effects in patients with advanced leukemia Limited in vivo expression and effector function of CARs has been a central limitation in clinical trials testing first-generation CARs (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 15:825833; Pule et al., 2008, Nat Med 14:1264-1270). Based on preclinical modeling demonstrating the enhanced persistence 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 develop second-generation CARs engineered with lentiviral vector technology, which were found to be safe in the setting of chronic HIV infection (Levine et al., 2006, Proc Natl Acad Sci USA 103:17372-17377). Our results demonstrate that when this second-generation CAR is expressed in T cells and cultured under conditions designed to promote central memory T cell engraftment (Rapoport et al., 2005, Nat Med 11:1230-1237; Bondanza et al., 2006, Blood 107:1828-1836), improved CAR T cell expansion after infusion is observed compared to previous reports. CART19 cells establish CD19-specific memory cells and kill tumor cells at previously unachieved E:T ratios in vivo.

[0135] CART19 is the first CAR clinical trial incorporating the 4-1BB signaling domain and the first to use lentiviral vector technology. Our results demonstrate efficient delivery of the CAR to tumor sites, accompanied by the virtual establishment of "tumor-infiltrating lymphocytes" expressing CD19 specificity. Significant in vivo expansion allows for the first demonstration that CARs harvested directly from patients can maintain in vivo effector function for several months. Previous studies have suggested that delivery of first-generation CARs to virus-specific T cells is preferable to primary T cells (Pule et al., 2008, Nat Med 14:1264-1270), but our results using second-generation CARs delivered to optimally costimulated primary T cells challenge this notion. While not wishing to be bound by any particular theory, the clinical efficacy was striking and unprecedented, resulting in kilogram-scale tumor mass lysis in all three patients, with the caveat that two of the patients experienced delayed release of potentially dangerously high levels of cytokines. The classic cytokine storm effect was not observed, but the study was designed to mitigate this possibility by careful infusion of CART19 over a 3-day period.

[0136] It was found that extremely low doses of CARs can induce strong clinical responses. This was a pilot study that demonstrated the safety of the CART19 vector design. The observation that doses of CART19 cells several orders of magnitude lower than those examined in previous studies can have clinical benefit will have important implications for the future implementation of CAR therapy on a larger scale and for the design of trials testing CARs directed against targets other than CD19.

[0137] This study further indicates that CART19 is expressed in both central memory T cells and effector T cells, which likely contributes to their prolonged survival compared to previous reports. Without wishing to be bound by any particular theory, CAR T cells can differentiate into a central memory-like state upon encountering target cells (e.g., CLL tumor cells or normal B cells) that express a surrogate antigen in vivo and subsequently eliminate them. In fact, 4-1BB signaling has been reported to promote memory development in conjunction with TCR signaling (Sabbagh et al., 2007, Trends Immunol 28:333-339).

[0138] The expansion of CART19 proliferation and viability revealed previously unreported aspects of CAR T cell pharmacokinetics. The kinetics of cytokine release in serum and bone marrow correlated with peak CART19 levels, allowing for a delay that could be initiated when CD19-expressing cellular targets become limited. The mechanism of CART19 viability expansion could be related to the incorporation of the 4-1BB domain or signaling through the native TCR and / or CAR. An intriguing possibility is that viability expansion is related to the population of CART19 identified in bone marrow specimens, raising the hypothesis that CD19 CARs may be maintained by encountering B cell progenitors within the bone marrow. Related to this question is what drives the initial expansion of CART19 cells in vivo. With rare exceptions (Savoldo et al., 2011, J Clin Invest doi:10.1172 / JCI46110; Pule et al., 2008, Nat Med 14:1264-1270), our study is the only one that omitted IL-2 infusion, so CART19 cells likely expanded in response to homeostatic cytokines or, more likely, to CD19 expressed on leukemia targets and / or normal B cells. In the latter case, CART19 self-renewal could occur on normal cells, providing a mechanism of CAR memory via "self-vaccination / boost" and, therefore, long-term tumor immune surveillance, which could be considered a notable feature of CARs directed against targets on normal APCs, such as CD19 and CD20. The mechanism of CART19 homeostasis will require further study to elucidate the mechanisms of intrinsic and extrinsic cell persistence. Prior to these advances, most researchers viewed CAR therapy as a transient form of immunotherapy, but CARs equipped with optimized signaling domains may play a role in both inducing and enhancing remission and in long-term immune surveillance.

[0139] Potent anti-leukemic effects were observed in all three patients, including two with p53-deficient leukemia. Previous studies using CARs have made it difficult to distinguish the antitumor effects from lymphodepleting chemotherapy. However, the delayed cytokine release combined with the kinetics of tumor lysis in fludarabine-resistant patients, which occurred simultaneously with, and possibly dependent on, in vivo CAR expansion in this study, indicates that CART19 mediates potent antitumor effects. These results do not negate the role of chemotherapy in enhancing CAR efficacy.

[0140] A thorough comparison of vector, transgene, and cell manufacturing procedures with results from ongoing studies at other institutions will be necessary to fully understand the key characteristics required for sustained CAR T cell function in vivo. Unlike antibody therapies, CAR-modified T cells have the potential to replicate in vivo, and long-term persistence could lead to sustained tumor control. The availability of off-the-shelf therapies composed of non-cross-resistant killer T cells could potentially improve outcomes for patients with B-cell malignancies. A limitation of antibody therapies using drugs such as rituximab and bevacizumab is that the treatment requires repeated antibody infusions, which are both inconvenient and costly. Long-term delivery of antibody therapy (in this case, for at least 6 months in 3 of 3 patients treated to date) via anti-CD19 scFv expressed on T cells after a single infusion of CART19 cells offers numerous practical advantages, including convenience and cost savings.

[0141] Persistent detection of CART19 18 months after infusion The results presented herein demonstrate long-term expression of CART19 and severe B-cell aplasia (Figures 8 and 9), as well as plasma cell depletion (Figure 10) in all three patients. One of the major surprises in the CART19 trial was that CART19 cells bearing murine scFvs, which exhibited a highly immunogenic phenotype, were not effectively rejected by the host patient's immune system. This suggests that the CART19 cells depleted normal B cells in the host patient, thereby inducing tolerance.

[0142] Without wishing to be bound by any particular theory, CART19 cells can be used in the following applications: 1) solid organ transplant patients who are "crossmatch" positive; elimination of pre-existing memory B cells may allow organ transplants that are not currently possible in these immunized patients; 2) induction of tolerance to immunogenic proteins administered to patients (hemophilia being one example); 3) rituximab has therapeutic efficacy in arthritis and other autoimmune disorders; CART19 may work as well or better.

[0143] In some cases, CART19 cells can be used to eliminate all B cell subsets (e.g., naive, memory, plasma cell precursors, and "suppressive Bregs"). Bregs contribute to immune suppression in certain cancers, and thus CART19 may improve immune responses by eliminating Bregs.

[0144] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to cover all such embodiments and equivalent variations.

Claims

1. 1. A method of depleting B cells in a subject, comprising: administering to a subject 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 CD3ζ signaling domain, wherein the antigen binding domain targets a B cell surface marker. thereby depleting B cells in said subject.

2. 1. A method of promoting tolerance in a subject, comprising: administering to a subject 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 CD3ζ signaling domain, wherein the antigen binding domain targets a B cell surface marker. thereby promoting tolerance in said subject.

3. The method of claim 2, wherein said tolerance is transplant tolerance to the transplanted tissue.

4. The method of claim 2, wherein the genetically modified cells deplete B cells.

5. The method of claim 2, wherein the genetically modified cells are administered simultaneously with the transplanted tissue.

6. The method of claim 2, wherein the genetically modified cells are administered prior to administration of the transplant tissue.

7. The method of claim 2, wherein the genetically modified cells are administered after administration of the transplant tissue.

8. 1. A method for treating graft-versus-host disease (GVHD), comprising: administering to a subject in need thereof cells genetically modified to express a CAR, wherein the CAR comprises an antigen binding domain, a costimulatory signaling region, and a CD3ζ signaling domain, wherein the antigen binding domain targets a B cell surface marker. thereby treating GVHD in said subject.

9. The method of claim 8, wherein the genetically modified cells deplete B cells.

10. The method of claim 8, wherein the genetically modified cells are administered simultaneously with the transplanted tissue.

11. The method of claim 8, wherein the genetically modified cells are administered prior to administration of the transplant tissue.

12. The method of claim 8, wherein the genetically modified cells are administered after administration of the transplant tissue.

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