Toxicity management for the antitumor activity of CAR

Abstract

The present invention provides a method for treating a patient having a disease, disorder, or condition associated with increased expression of a tumor antigen. [Solution] A method for treating a patient having a disease, disorder, or condition associated with increased expression of a tumor antigen, comprising administering a first-line therapy and a second-line therapy to a patient in need thereof, wherein the first-line therapy comprises administering to the patient an effective amount of cells genetically modified to express a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 671,482, filed Jul. 13, 2012, and U.S. Provisional Application No. 61 / 782,982, filed Mar. 14, 2013, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background of the Invention Patients with relapsed and chemotherapy - refractory acute lymphoblastic leukemia (ALL) have a poor prognosis despite the use of aggressive therapies such as allogeneic hematopoietic stem cell transplantation (Barrett et al., 1994, N Engl J Med 331:1253 - 8 (Non - Patent Document 1); Gokbuget et al., 2012, Blood 120:2032 - 41 (Non - Patent Document 2)) and bispecific CD19 antibody fragments (Bargou et al., 2008, Science 321:974 - 7 (Non - Patent Document 3)). Chimeric antigen receptor - modified T cells targeting lineage - specific antigens CD19 and CD20 have been reported to be effective in adults with CLL and B - cell lymphoma (Till et al., 2008, Blood 112:2261 - 71 (Non - Patent Document 4); Kochenderfer et al., 2010, Blood 116:4099 - 102 (Non - Patent Document 5); Brentjens et al., 2011, Blood 118:4817 - 28 (Non - Patent Document 6); Porter et al., 2011, N Engl J Med 365:725 - 33 (Non - Patent Document 7); Kalos et al., 2011, Science Translational Medicine 3:95ra73 (Non - Patent Document 8); Savoldo et al., 2011, J Clin Invest 121:1822 - 5 (Non - Patent Document 9)). However, the effect of CAR T cells on ALL blasts, a more immature leukemia with more rapid progression, has not been fully investigated.

[0003] The development of tumor lysis syndrome and delayed cytokine secretion, in combination with increased chimeric antigen receptor T cells in vivo, has been reported (Porter et al., 2011, N Engl J Med 365:725-33 (Non-Patent Document 7); Kalos et al., 2011, Science Translational Medicine 3:95ra73 (Non-Patent Document 8)). However, the effects of cytokine secretion and disorders associated with increased chimeric antigen receptor T cells in vivo have not been fully investigated.

[0004] Therefore, there is an urgent need in the art for compositions and methods for the treatment of cancer that use CARs and address the toxicity of CARs. The present invention meets this need.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

[0006] The present invention provides a method for treating a patient having a disease, disorder, or medical condition associated with enhanced expression of a tumor antigen. In one aspect, the method includes administering to a patient in need thereof a first-line therapy and a second-line therapy, wherein the first-line therapy includes administering to the patient an effective amount of cells genetically modified to express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0007] In one aspect, cytokine levels in the patient are monitored to determine the appropriate type of second-line therapy to be administered to the patient after administration of the first-line therapy, and the appropriate second-line therapy is administered to the patient in need thereof.

[0008] In one aspect, an increase in cytokine levels identifies the type of cytokine inhibitory therapy to be administered to the patient in need thereof.

[0009] In one aspect, the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factor, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and any combination thereof.

[0010] In one embodiment, the cytokine inhibition therapy is selected from the group consisting of small interfering RNA (siRNA), microRNA, antisense nucleic acids, ribozymes, expression vectors encoding dominant negative mutants, antibodies, peptides, small molecules, cytokine inhibitors, and any combination thereof.

[0011] In one embodiment, the cytokine level is monitored by detecting the protein level of the cytokine in a patient-derived biological sample.

[0012] In one embodiment, the cytokine level is monitored by detecting the nucleic acid level of the cytokine in a patient-derived biological sample.

[0013] The present invention provides a method for reducing or avoiding adverse effects associated with the administration of cells genetically modified to express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the method comprising monitoring the level of cytokines in a patient to determine the appropriate type of cytokine therapy to be administered to the patient, and administering the appropriate cytokine therapy to the patient.

[0014] In one embodiment, an increase in the level of a cytokine identifies the type of cytokine inhibition therapy to be administered to the patient.

[0015] In one embodiment, the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and any combination thereof.

[0016] In one embodiment, the cytokine inhibition therapy is selected from the group consisting of small interfering RNA (siRNA), microRNA, antisense nucleic acids, ribozymes, expression vectors encoding dominant negative mutants, intracellular antibodies, peptides, small molecules, cytokine inhibitors, and any combination thereof.

[0017] In one embodiment, the cytokine level is monitored by detecting the protein level of the cytokine in a patient-derived biological sample.

[0018] In one embodiment, the cytokine level is monitored by detecting the nucleic acid level of the cytokine in a patient-derived biological sample. [Inventive Concept 1001] A method of treating a patient having a disease, disorder, or medical condition associated with enhanced expression of a tumor antigen, the method comprising administering to the patient in need thereof an effective amount of cells genetically modified to express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, in a first selection therapy and a second selection therapy. [Inventive Concept 1002] The method of Inventive Concept 1001, further comprising monitoring a cytokine level in the patient to determine an appropriate type of second selection therapy to be administered to the patient after administration of the first selection therapy, and administering the appropriate second selection therapy to the patient in need thereof. [Inventive Concept 1003] The method of Inventive Concept 1002, wherein an increase in the level of the cytokine identifies the type of cytokine inhibition therapy to be administered to the patient in need thereof. [Inventive Concept 1004] The method of the present invention 1003, wherein the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factor, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and any combination thereof. [The present invention 1005] The method of the present invention 1003, wherein the cytokine inhibition therapy is selected from the group consisting of small interfering RNA (siRNA), microRNA, antisense nucleic acid, ribozyme, expression vector encoding a dominant negative mutant, antibody, peptide, small molecule, cytokine inhibitor, and any combination thereof. [The present invention 1006] The method of the present invention 1002, wherein the cytokine level is monitored by detecting the protein level of the cytokine in a biological sample derived from the patient. [The present invention 1007] The method of the present invention 1002, wherein the cytokine level is monitored by detecting the nucleic acid level of the cytokine in a biological sample derived from the patient. [The present invention 1008] A method for reducing or avoiding adverse effects associated with the administration of cells genetically modified to express a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the method comprising monitoring the level of cytokines in a patient to determine the appropriate type of cytokine therapy to be administered to the patient, and administering the appropriate cytokine therapy to the patient. [The present invention 1009] The method of the present invention 1008, wherein an increase in the level of the cytokine identifies the type of cytokine inhibition therapy to be administered to the patient. [The present invention 1010] The method of the present invention 1009, wherein the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factor, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and any combination thereof. [The present invention 1011] The method of the present invention 1009, wherein the cytokine inhibition therapy is selected from the group consisting of small interfering RNA (siRNA), microRNA, antisense nucleic acid, ribozyme, expression vector encoding a dominant negative mutant, intracellular antibody, peptide, small molecule, cytokine inhibitor, and any combination thereof. [The present invention 1012] The method of the present invention 1008, wherein the cytokine level is monitored by detecting the protein level of the cytokine in a biological sample derived from a patient. [The present invention 1013] The method of the present invention 1008, wherein the cytokine level is monitored by detecting the nucleic acid level of the cytokine in a biological sample derived from a patient.

Brief Description of the Drawings

[0019] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the present invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and instrumentalities of the embodiments shown in the drawings.

[0020]

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Modes for Carrying Out the Invention

[0021] Detailed Description The present invention relates to compositions and methods for treating cancers including, but not limited to, hematological malignancies and solid tumors. The present invention also encompasses methods for treating and preventing certain types of cancers, including primary and metastatic cancers, as well as cancers refractory or resistant to conventional chemotherapy. The method includes administering a therapeutically or prophylactically effective amount of T cells transduced to express a chimeric antigen receptor (CAR) to a patient in need of such treatment or prevention. A CAR is a molecule that combines antibody-based specificity for a desired antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to create a chimeric protein that exhibits specific anti-tumor cell immune activity.

[0022] As part of an overall treatment regimen, the present invention encompasses a method of managing (e.g., preventing or delaying recurrence of, or prolonging remission time for) a particular cancer by evaluating the soluble factor profile in a patient after T cell infusion. Preferably, the soluble factor profile includes an evaluation of the cytokine profile. If the cytokine profile shows an increase in a particular cytokine after T cell infusion as compared to before T cell infusion, one of ordinary skill in the art can choose to administer to a patient in need of such management an effective amount of a cytokine inhibitory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, inclusion compound, or prodrug thereof to manage the elevated levels of the cytokine after T cell infusion.

[0023] The present invention is based in part on the discovery of the identification of a unique combination of factors whose modulation from baseline or existing levels at baseline can help track T cell activation, target activity, and potentially harmful side effects after CAR T cell infusion to help manage cancer treatment. Exemplary factors include, without limitation, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and the like.

[0024] The present invention relates to an adoptive cell transfer strategy of T cells transduced to express a chimeric antigen receptor (CAR), in combination with toxicity management, for treating cancer by generating a soluble factor profile from a patient after T cell infusion and treating elevated soluble factors. For example, generating a real-time soluble factor profile allows for intervention with an appropriate inhibitor of the elevated soluble factor to bring the levels down to normal levels.

[0025] In one aspect, the CAR of the present invention includes an extracellular domain having an antigen recognition domain that targets a desired antigen, a transmembrane domain, and a cytoplasmic domain. The present invention is not limited to a particular CAR. Rather, any CAR that targets a desired antigen can be used in the present invention. The compositions and methods for making CARs are described in PCT / US11 / 64191, which is hereby incorporated by reference in its entirety.

[0026] In any of several aspects of the above methods, the methods result in a measurable reduction in tumor size or evidence of disease or disease progression, complete response, partial response, stable disease, increased or prolonged progression-free survival, increased or prolonged overall survival, or reduced toxicity.

[0027] Definition Unless defined otherwise, 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. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but the preferred materials and methods are described herein. The following specialized terms are used in the description and claims of the present invention.

[0028] It is also to be understood that the specialized terms used herein are for the purpose of describing particular aspects only and are not intended to be limiting.

[0029] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0030] As used herein, when referring to measurable values such as amounts and lengths of time, "about" includes variations of ±20%, ±10%, in some cases ±5%, in some cases ±1%, and in some cases ±0.1% from the specified value, as such variations are reasonable in carrying out the disclosed methods.

[0031] As used herein, "activation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be accompanied by induction of cytokine production and detectable effector function. The term "activated T cell" refers, inter alia, to a T cell that is undergoing cell division.

[0032] As used herein, an "activator" or "agonist" of a soluble factor refers to a molecule of an agent that can activate the soluble factor or increase its level. An activator is a compound, such as an agonist, that increases, promotes, induces activation of, activates, or upregulates the activity or expression of a soluble factor. Assays for detecting an activator include, as described elsewhere herein, for example, the steps of expressing the soluble factor in vitro, in a cell, or in a cell membrane, applying a putative agonist compound, and then determining the functional effect on the activity of the soluble factor.

[0033] The term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from a natural or recombinant source, or it may be an immunoreactive portion of an intact immunoglobulin. Antibodies are often tetramers of immunoglobulin molecules. Antibodies in the present invention include, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab) 2and can exist in various forms, including 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).

[0034] The term "antibody fragment" refers to a portion of an intact antibody and also to the antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0035] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can include either or both antibody production or activation of specific immune competent cells. One of ordinary skill in the art will understand that virtually any macromolecule, including substantially all proteins or peptides, can serve as an antigen. Moreover, an antigen may be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will therefore encode an "antigen" as the term is used herein. Moreover, one of ordinary skill 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 immediately apparent that the present invention includes, without limitation, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is immediately apparent that an antigen can be produced synthetically or obtained from a biological sample. Such biological samples can include, without limitation, tissue samples, tumor samples, cells or biological fluids.

[0036] As used herein, the term "self-antigen" means, according to the present invention, any self-antigen that is recognized by the immune system as if it were foreign. Self-antigens include, without limitation, cellular proteins, including cell surface receptors, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins.

[0037] As used herein, "autoimmune disease" is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive reaction against 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 (type I), dystrophic epidermolysis bullosa, orchitis, glomerulonephritis, Graves' disease, Guillain-Barré 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, ulcerative colitis.

[0038] As used herein, the term "self" shall refer to any material derived from the same individual that is later re-introduced into that individual.

[0039] "Allogeneic" refers to a graft derived from different animals of the same species.

[0040] "Xenogeneic" refers to a graft derived from animals of different species.

[0041] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or, through the bloodstream and lymphatic system, to other parts of the body. 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 malignancy, lymphoma, leukemia, lung cancer, etc.

[0042] As used herein, "combination therapy" means administering a first agent together with another agent. "Together with" refers to administering one treatment modality in addition to another treatment modality. Thus, "together with" refers to administering one treatment modality to an individual before, during, or after the delivery of another treatment modality. Such combinations are considered part of a single treatment regimen or regime.

[0043] As used herein, the term "co - administration" means that the administration of the first therapy and the administration of the second therapy in combination therapy overlap with each other.

[0044] As used herein, "co - stimulatory ligand" refers to a molecule on an antigen - presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) that can specifically bind to a cognate co - stimulatory molecule on a T cell and thereby provide a signal that mediates a T - cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided, for example, by the binding of a peptide - loaded MHC molecule to the TCR / CD3 complex. Co - stimulatory ligands include, without limitation, CD7, B7 - 1 (CD80), B7 - 2 (CD86), PD - L1, PD - L2, 4 - 1BBL, OX40L, inducible co - stimulatory ligand (ICOS - L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA - G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to the Toll - ligand receptor, and a ligand that specifically binds to B7 - H3. Co - stimulatory ligands also include, among others, but not limited to, antibodies that specifically bind to co - stimulatory molecules present on T cells such as CD27, CD28, 4 - 1BB, OX40, CD30, CD40, PD - 1, ICOS, lymphocyte function - associated antigen - 1 (LFA - 1), CD2, CD7, LIGHT, NKG2C, B7 - H3, etc., and a ligand that specifically binds to CD83.

[0045] "Co - stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co - stimulatory ligand and thereby mediates a co - stimulatory response by the T cell, including, but not limited to, proliferation. Co - stimulatory molecules include, without limitation, MHC class I molecules, BTLA, and the Toll - ligand receptor.

[0046] As used herein, the term "costimulatory signal" refers to a molecule that, in combination with a primary signal such as TCR / CD3 ligation, leads to the proliferation of T cells and / or the upregulation or downregulation of key molecules.

[0047] As used herein, the term "disease" refers to the health state of an animal in which the animal is unable to maintain homeostasis and its health continues to deteriorate unless the disease is improved. In contrast, a "disorder" in an animal refers to a health state in which the animal can maintain homeostasis, but its health state is more disadvantageous than in the absence of the disorder. A disorder does not necessarily cause a further decline in the health state of the animal even if left untreated.

[0048] As used herein, the term "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0049] As used herein, the term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue or system.

[0050] As used herein, the term "exogenous" refers to any material that is produced outside of an organism, cell, tissue or system and introduced into the organism, cell, tissue or system.

[0051] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence that is operably linked to its promoter.

[0052] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains cis-acting elements sufficient 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) that incorporate the recombinant polynucleotide.

[0053] "Homologous" refers to sequence similarity or identity between two polypeptides or between two nucleic acid molecules. In both of the two sequences being compared, if a position is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function obtained by dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and then multiplying by 100. For example, if 6 out of 10 positions in two sequences match or are homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, the comparison is made after aligning the two sequences so as to obtain the maximum homology.

[0054] The term "immunoglobulin" or "Ig", as used herein, is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes also referred to as BCR (B cell receptor) or antigen receptor. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common antibody in the bloodstream. IgM is the major immunoglobulin produced in the primary immune response in most mammals. It is the most efficient immunoglobulin in agglutination reactions, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is not known, but it may act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to allergens.

[0055] As used herein, the term "immune response" includes T cell-mediated immune responses and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses such as cytokine production and cytotoxicity. In addition, the term "immune response" includes immune responses indirectly brought about by the activation of T cells, such as antibody production (humoral response), and the activation of cytokine-responsive cells such as macrophages. Immune cells involved in the immune response include lymphocytes such as B cells and T cells (CD4+, CD8+, Th1, and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; and myeloid cells such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0056] As used herein, the "inhibitor" or "antagonist" of a soluble factor refers to a molecule of an agent that can inhibit, inactivate, or reduce the level of the soluble factor. An inhibitor, for example, is a compound such as an antagonist that binds to a soluble factor, partially or completely blocks its activity, reduces it, prevents it, delays its activation, inactivates it, desensitizes it, or downregulates its activity or expression. Inhibitors include polypeptide inhibitors such as antibodies, soluble receptors, etc., as well as nucleic acid inhibitors such as siRNA or antisense RNA, genetically modified forms of soluble factors, e.g., forms having altered activity, and naturally occurring and synthetic soluble factor antagonists, small chemicals, etc. An assay for detecting an inhibitor includes, as described elsewhere herein, for example, the steps of expressing the soluble factor in vitro, in cells, or in the cell membrane, applying a putative antagonist compound, and then determining the functional effect on the activity of the soluble factor.

[0057] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, intending that the instructional material and the compound be used together as a unit by the recipient.

[0058] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the substances that coexist in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in an unnatural environment such as a host cell, for example.

[0059] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; because they can deliver a significant amount of genetic information into the DNA of host cells, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve fairly high levels of gene transfer in vivo.

[0060] As used herein, the phrase "level of soluble factor" in a biological sample typically refers to the level of the amount of protein, protein fragment, or peptide of the soluble factor present in the biological sample. The "level of soluble factor" need not be quantified, but can be simply detected, for example, by subjective visual detection by a human, with or without comparison to the level from a control sample or the expected level of a control sample.

[0061] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in that subject in the absence of treatment or compound and / or as compared to the level of response in a subject that is otherwise identical but untreated. This term encompasses mediating a beneficial therapeutic response by disturbing and / or affecting a natural signal or response in a subject, preferably a human.

[0062] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion methods.

[0063] The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, to which the methods described herein can be applied. In one non-limiting aspect, the patient, subject or individual is human.

[0064] As used herein, the term "co-administration" means administering a first therapy and a second therapy in a combination therapy at time intervals of about 15 minutes or less, for example, any of about 10, 5, or 1 minute or less. When the first and second therapies are co-administered, the first and second therapies may be contained in the same composition (e.g., a composition containing both the first and second therapies), or in separate compositions (e.g., the first therapy is contained in one composition and the second therapy is contained in another composition).

[0065] As used herein, the term "co-administration" means administering a first therapy and a second therapy in a combination therapy at time intervals of about 15 minutes or less, for example, any of about 10, 5, or 1 minute or less. When the first and second therapies are co-administered, the first and second therapies may be contained in the same composition (e.g., a composition containing both the first and second therapies), or in separate compositions (e.g., the first therapy is contained in one composition and the second therapy is contained in another composition).

[0066] As used herein with respect to an antibody, the term "specifically binds" means an antibody that recognizes a specific antigen in a sample but substantially does not recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species may bind to that antigen from one or more species. However, such interspecies reactivity by itself does not alter the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may bind to different allelic forms of that antigen. However, such cross-reactivity by itself does not alter the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) in 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", the presence of a molecule containing epitope A (or free unlabeled A) is thought to decrease the amount of labeled A bound to the antibody in a reaction containing the labeled "A" and that antibody.

[0067] As used herein, the term "stimulates" means the primary response induced by a stimulatory molecule (e.g., the TCR / CD3 complex) binding to its cognate ligand, thereby mediating signal transduction events such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation may mediate a change in the expression of certain molecules such as, for example, down-regulation of TGF-β and / or reorganization of the cytoskeletal structure.

[0068] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0069] As used herein, the term "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), specifically binds 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, inter alia, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0070] The term "subject" is intended to include a living organism (e.g., a mammal) capable of mounting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0071] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types that are normally associated with it in its native state. In some cases, a substantially purified population of cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that has been separated from the cells that are normally associated with it in its native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0072] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0073] The term "therapeutically effective amount" refers to the amount of a subject compound that is believed to induce a biological or medical response in a tissue, system, or subject that a researcher, veterinarian, physician, or other clinical specialist is investigating. The term "therapeutically effective amount" includes an amount of the compound that, when administered, is sufficient to prevent the occurrence of one or more of the signs or symptoms of the disorder or disease being treated, or to ameliorate it to some extent. The therapeutically effective amount is believed to vary depending on the compound, the disease and its severity, as well as the age, weight, etc. of the subject being treated.

[0074] As used herein, "graft" refers to cells, tissues, or organs introduced into an individual. The source of the material to be grafted can be cultured cells, cells from another individual, or cells from the same individual (e.g., after culturing the cells in vitro). Exemplary organ grafts are the kidney, liver, heart, lung, and pancreas.

[0075] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder that a subject is afflicted with.

[0076] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected with exogenous nucleic acid, transformed by exogenous nucleic acid, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0077] Scope: Throughout the present disclosure, various aspects of the invention can 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 considered as an inflexible limitation on the scope of the invention. Thus, a description of a range should be considered to specifically disclose all possible sub-ranges within that range, as well as individual numerical values. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0078] Explanation The present invention provides compositions and methods for treating cancer in a patient. In one aspect, the treatment method includes a first-choice therapy that involves administering the CAR of the present invention to the patient to introduce an anti-tumor immune response, and a step of monitoring the levels of soluble factors in the patient after T cell infusion to determine the type of a second-choice therapy that is appropriate for the treatment of the patient as a result of the first-choice therapy.

[0079] In one aspect, the second-choice therapy includes a step of evaluating the soluble factor profile in a patient after receiving an appropriate CAR T infusion (referred to elsewhere in this specification as "after T cell infusion"), and in this step, when the soluble factor profile shows an increase in a specific soluble factor after T cell infusion compared to before T cell infusion, one skilled in the art can choose to administer an effective amount of a soluble factor inhibitory compound to the patient in need to manage the elevated levels of soluble factors after T cell infusion. Thus, the second-choice therapy in one aspect includes administering a certain type of soluble factor inhibitory therapy to manage the elevated levels of a specific soluble factor resulting from the first-choice therapy using CAR T cells.

[0080] In yet another aspect, the second alternative therapy related to the step of administering a soluble factor inhibitory compound to a patient can be combined with other conventional therapies used to treat, prevent, or manage a disease or disorder associated with or characterized by unwanted angiogenesis. Examples of such conventional therapies include, but are not limited to, surgery, chemotherapy, radiation therapy, hormone therapy, biotherapy, and immunotherapy.

[0081] In one aspect, the CAR of the present invention can be engineered to include an extracellular domain having an antigen-binding domain targeting a tumor antigen fused to an intracellular signaling domain of a T cell antigen receptor complex ζ chain (e.g., CD3ζ). An exemplary tumor antigen, a B cell antigen, is CD19, because this antigen is expressed on malignant B cells. However, the present invention is not limited to targeting CD19. Rather, the present invention includes any tumor antigen-binding moiety. The antigen-binding moiety is preferably fused to an intracellular domain derived from one or more of a costimulatory molecule and the ζ chain. Preferably, the antigen-binding moiety is fused to one or more intracellular domains selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3ζ signaling domain, and any combination thereof.

[0082] In one embodiment, the CAR of the present invention includes a CD137 (4-1BB) signaling domain. This is because the present invention is based in part on the discovery that the T cell response mediated by the CAR can be further enhanced by the addition of a co-stimulatory domain. For example, by including the CD137 (4-1BB) signaling domain, the CAR-mediated activity and in vivo persistence of CAR T cells were significantly increased compared to the same CAR T cells that had not been engineered to express CD137 (4-1BB). However, the present invention is not limited to a particular CAR. Rather, any CAR that targets a tumor antigen can be used in the present invention. The compositions and methods for making and using CARs are described in PCT / US11 / 64191, which is incorporated herein by reference in its entirety.

[0083] Method The treatment regimen of the present invention results in a measurable reduction in tumor size or evidence of disease or disease progression, complete response, partial response, stable disease, increased or prolonged progression-free survival, increased or prolonged overall survival, or reduced toxicity.

[0084] As part of the overall treatment regimen, the present invention includes a first-line therapy and a second-line therapy, the first-line therapy including administering the CAR T cells of the present invention to a patient in need thereof. The treatment regimen of the present invention enables the management of cancer and its treatment by evaluating the soluble factor profile in patients after T cell infusion. Appropriate second-line therapies include administering to the patient an appropriate soluble factor inhibitor to reduce elevated levels of soluble factors resulting from the first-line therapy. In some cases, appropriate second-line therapies include administering to the patient an appropriate soluble factor activator to increase suppressed levels of soluble factors resulting from the first-line therapy.

[0085] In one aspect, an appropriate second - line therapy includes administering to the patient an appropriate cytokine inhibitor to reduce the elevated levels of cytokines resulting from the first - line therapy. In some cases, an appropriate second - line therapy includes administering to the patient an appropriate cytokine activator to increase the suppressed levels of cytokines resulting from the first - line therapy.

[0086] In one aspect, the different levels are overexpression (high expression) or underexpression (low expression) compared to the expression levels of normal or control cells, a predetermined patient population, or an internal control. In some aspects, the levels are compared between a patient and a normal individual, between after and before T - cell infusion in a patient, or between a first time point and a second time point after T - cell infusion in a patient.

[0087] In one aspect, the present invention includes the step of assessing different levels of one or more cytokines in a patient after T - cell infusion to generate a cytokine profile in order to determine the type of cytokine therapy to be applied to the patient for the purpose of controlling cytokine levels back to normal levels. Thus, the present invention may be applied to identify elevated cytokine levels as a result of the presence of the CAR T - cells of the present invention in a patient, thereby enabling a patient - specific treatment with a cytokine inhibitor to reduce the elevated levels of cytokines. In another aspect, the present invention may be applied to identify decreased cytokine levels as a result of the presence of the CAR T - cells of the present invention in a patient, thereby enabling a patient - specific treatment with a cytokine activator to increase the decreasing levels of cytokines.

[0088] In one aspect, cytokine levels that are elevated as a result of receiving CAR T cell infusion include, without limitation, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and the like. However, the present invention should not be limited to these listed cytokines. Rather, the present invention includes any cytokine identified as being elevated in a patient as a result of receiving CAR T cell infusion.

[0089] In one aspect, cytokine levels that are decreased as a result of receiving CAR T cell infusion include, without limitation, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and the like. However, the present invention should not be limited to these listed cytokines. Rather, the present invention includes any cytokine identified as being decreased in a patient as a result of receiving CAR T cell infusion.

[0090] Detection and treatment of cytokines This section describes the detection and treatment of cytokines as part of a second-line therapy, but the present invention encompasses the detection and treatment of any soluble factor as part of a second-line therapy. Thus, the description in the context of "cytokines" can apply equally to "soluble factors".

[0091] In one aspect, as part of a second - line therapy, the invention includes a method of detecting cytokine levels in a patient who has received an infusion of the CAR T cells of the invention. In some aspects, the presence or level of a cytokine can be used to select a candidate treatment. In some other aspects, the presence or level of a cytokine can be used to determine success during or after a first - line, second - line, or both first - line and second - line therapies.

[0092] Biological samples in which cytokines can be detected include, for example, serum. In some aspects, biological samples include tissue biopsy materials which may or may not have a liquid component.

[0093] Immunoassays can be used to qualitatively or quantitatively analyze cytokine levels in biological samples. A general overview of applicable techniques can be found in numerous readily available manuals, such as Harlow & Lane, Cold Spring Harbor Laboratory Press, Using Antibodies: A Laboratory Manual (1999).

[0094] In addition to using immunoassays to detect cytokine levels in patient - derived biological samples, the evaluation of cytokine expression and levels can be performed based on the level of gene expression of a particular cytokine. RNA hybridization techniques for measuring the presence and / or level of mRNA expression are well - known to those skilled in the art and can be used to evaluate the presence or level of gene expression of a cytokine of interest.

[0095] In some embodiments, the methods of the invention utilize a selective binding partner for a cytokine to identify the presence or measure the level of a cytokine in a biological sample. The selective binding partners used in the methods and kits of the invention can be, for example, antibodies. In some aspects, monoclonal antibodies specific for a particular cytokine can be used. In some other aspects, polyclonal antibodies specific for a particular cytokine can be used to practice the methods of the invention and in kits.

[0096] Commercially available antibodies to cytokines are available and can be used in the methods and kits of the invention. It is well known to those skilled in the art that the type, source, and other aspects of the antibody to be used should be considered in light of the assay in which the antibody is to be used. In some cases, an antibody that recognizes an antigen target on a Western blot may not be applicable to an ELISA or ELISpot assay, and vice versa.

[0097] In some embodiments, the antibodies used in the assays of the invention can be produced using techniques well known in the art for producing monoclonal or polyclonal antibodies (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975)). Such techniques include the preparation of antibodies by selection from libraries of recombinant antibodies in phage vectors or similar vectors, as well as the preparation of polyclonal or monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). Such antibodies can be used for therapeutic and diagnostic applications, for example, in the treatment and / or detection of any of the specific cytokine-related diseases or conditions described herein.

[0098] Detection methods using immunoassays are particularly suitable for point-of-care implementation in patients. Such methods allow for immediate diagnosis and / or prognostic evaluation of patients. Point-of-care diagnostic systems are described, for example, in U.S. Patent No. 6,267,722, which is incorporated herein by reference. Other immunoassay formats are also available that allow for the evaluation of biological samples without the need to send the sample to a laboratory for evaluation. Typically, these assays are formulated as solid assays in which a reagent, such as an antibody, is used to detect the cytokine. Exemplary test devices suitable for use in immunoassays, such as the assays of the invention, are described, for example, in U.S. Patent Nos. 7,189,522; 6,818,455 and 6,656,745.

[0099] In some aspects, the present invention provides a method for the detection of polynucleotide sequences encoding cytokines in a biological sample. As described above, a "biological sample" refers to cells or a population of cells derived from a patient or an amount of tissue or body fluid. In most cases, the sample is taken from a patient, but the term "biological sample" can also refer to cells or tissues that are analyzed in vivo, i.e., without being removed from the patient. Typically, a "biological sample" is considered to contain cells derived from a patient, but the term can also refer to acellular biological material.

[0100] In one aspect, an amplification-based assay is used to measure the level of the desired cytokine. In such an assay, the nucleic acid sequence of the desired cytokine functions as a template in an amplification reaction (e.g., polymerase chain reaction, i.e., PCR). In quantitative amplification, the amount of the amplification product is considered to be proportional to the amount of the template in the original sample. By comparison with appropriate controls, measurement of the copy number of the cytokine-related gene is provided. Methods of quantitative amplification are well known to those skilled in the art. A detailed protocol for quantitative PCR is provided, for example, in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. The RT-PCR method is well known to those skilled in the art (see, for example, Ausubel et al. supra). In some aspects, quantitative RT-PCR, e.g., TaqMan™ assay, is used, thereby enabling comparison of the level of mRNA in the sample with that of a control sample or value. A known nucleic acid sequence for the desired cytokine is sufficient to enable one of ordinary skill in the art to routinely select primers that amplify any part of the gene. Primers suitable for the amplification of specific sequences can be designed using principles well known in the art (see, for example, Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)).

[0101] In some embodiments, hybridization-based assays can be used to detect the amount of a desired cytokine in the cells of a biological sample. Such assays include dot blot assays of RNA and other assays, such as fluorescence in situ hybridization performed on a sample containing cells. Other hybridization assays are readily available in the art.

[0102] In many embodiments of the invention, the level and / or presence of a cytokine polynucleotide or polypeptide is detected in a biological sample, thereby detecting differential expression of the cytokine and generating a cytokine profile from a biological sample derived from a patient infused with the CAR T cells of the invention, compared to a control biological sample.

[0103] The amount of cytokine polynucleotide or polypeptide detected in the biological sample indicates the presence of the cytokine for the purpose of generating a cytokine profile to classify the patient for appropriate cytokine therapy. For example, if the cytokine profile shows an increase in a particular cytokine after T cell infusion compared to a control (e.g., before T cell infusion), one of ordinary skill in the art can select to administer an effective amount of a cytokine inhibitory compound to a patient in need of such treatment. Alternatively, if the cytokine profile shows a decrease in a particular cytokine after T cell infusion compared to a control (e.g., before T cell infusion), one of ordinary skill in the art can select to administer an effective amount of a cytokine activator compound to a patient in need of such treatment.

[0104] In some embodiments, the difference in cytokine levels between a sample after T cell infusion and a control sample is at least about 0.5, 1.0, 1.5, 2, 5, 10, 100, 200, 500, 1000-fold.

[0105] This method can also be used to evaluate the effectiveness of the treatment process. For example, in a patient after T cell infusion containing an elevated amount of cytokine IL-6, the effectiveness of anti-IL-6 treatment can be evaluated by monitoring IL-6 over time. For example, a reduction in the level of IL-6 polynucleotide or polypeptide in a biological sample taken from a patient after treatment, compared to the level in a sample taken from a mammalian subject before or at the beginning of treatment, indicates effective treatment.

[0106] In one aspect, the treatment regimen can be based on neutralization of the elevated cytokine. For example, an antagonist of the cytokine can be selected for treatment. Antibodies are examples of suitable antagonists and include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies, or fragments thereof. A chimeric antibody is an antibody in which the genes for the light and heavy chains are typically constructed from immunoglobulin gene segments belonging to different species by genetic engineering (see, for example, Boyce et al., Annals of Oncology 14:520-535 (2003)). For example, the variable (V) segments of the genes derived from a murine monoclonal antibody may be linked to human constant (C) segments. Thus, a typical chimeric antibody is a hybrid protein consisting of the V or antigen-binding domain derived from a murine antibody and the C or effector region derived from a human antibody.

[0107] A humanized antibody has substantially the variable region framework residues from a human antibody (referred to as the acceptor antibody) and the complementarity determining regions substantially from a mouse antibody (referred to as the donor immunoglobulin). See Queen et al., Proc. NatL. Acad. Sci. USA 86:10029-10033 (1989) and International Publication No. 90 / 07861, U.S. Patent No. 5,693,762, U.S. Patent No. 5,693,761, U.S. Patent No. 5,585,089, U.S. Patent No. 5,530,101, and Winter's U.S. Patent No. 5,225,539. The constant region, when present, is also substantially or entirely from human immunoglobulins. Antibodies can be obtained, inter alia, from the source by conventional hybridoma approaches, phage display (see, e.g., Dower et al., International Publication No. 91 / 17271 and McCafferty et al., International Publication No. 92 / 01047), the use of transgenic mice having a human immune system (Lonberg et al., International Publication No. 93 / 12227 (1993)). Nucleic acids encoding the immunoglobulin chains can be obtained from a hybridoma or cell line producing the antibody or based on the nucleic acid or amino acid sequences of immunoglobulins in published literature.

[0108] Other antagonists of the desired cytokine can also be used for therapeutic purposes. For example, a class of antagonists that can be used for the purposes of the present invention are soluble forms of receptors for cytokines. By way of mere illustration, an IL-6 antagonist is an anti-IL-6 antibody that specifically binds to IL-6. Specific antibodies have the ability to inhibit or antagonize the action of IL-6 systemically. In some embodiments, the antibody binds to IL-6 and prevents or inhibits its interaction with or activation of its receptor (e.g., IL-6Rα or IL-6Rβ). In some embodiments, the activity of IL-6 can be antagonized by using an antagonist against the interleukin-6 receptor (IL-6R). U.S. Patent Application No. 2006251653 describes methods for treating interleukin-6 related diseases and discloses numerous interleukin-6 antagonists, including, for example, humanized anti-IL-6R antibodies and chimeric anti-IL-6R antibodies. In some embodiments, derivatives of IL-6 or IL-6R can be used to block and antagonize the interaction between IL-6 / IL-6R.

[0109] The present invention is not limited to the cytokines and their corresponding activators and inhibitors described herein. Rather, the present invention includes the use of any cytokine activator and / or inhibitor used in the art to modulate cytokines. This is because the present invention is based on managing cancer treatment in patients who have received an infusion of the CAR T cells of the present invention, and the infused CAR T cells result in increases and decreases in the levels of various cytokines. One of ordinary skill in the art can, based on the disclosure presented herein, target differential expression levels of cytokines in post-T cell infusion samples compared to control samples to normalize increased or decreased cytokine levels.

[0110] Therapeutic application The present invention encompasses cells (e.g., T cells) transduced with a lentiviral vector (LV). For example, the LV encodes a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domains of CD3-ζ, CD28, 4-1BB, or any combination thereof. Thus, in some cases, the transduced T cells can induce a T cell response mediated by the CAR.

[0111] The present invention provides the use of a CAR for re-directing the specificity of primary T cells towards tumor antigens. Thus, the present invention also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, the method comprising administering to the mammal T cells that express the CAR, wherein the CAR comprises a binding moiety that specifically interacts with a predetermined target, such as a ζ-chain portion comprising the intracellular domain of human CD3ζ, and a co-stimulatory signaling region.

[0112] In one aspect, the present invention includes a type of cell therapy in which T cells are genetically modified to express a CAR and the CAR T cells are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR T cells can replicate in vivo and provide long-term persistence that can lead to sustained tumor control.

[0113] In one aspect, the CAR T cells of the present invention can cause robust in vivo T cell expansion and can survive for a longer period of time. In another aspect, the CAR T cells of the present invention can become specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth. For example, it was unexpected that the CAR T19 cells of the present invention can cause robust in vivo T cell expansion, can survive at high levels in blood and bone marrow for a long time, and can form specific memory T cells. Without wishing to be bound by any particular theory, CAR T cells can differentiate into a central memory-like state when they encounter target cells expressing a surrogate antigen in vivo and can then eliminate it.

[0114] Without wishing to be bound by any particular theory, the anti-tumor immune response induced by CAR-modified T cells can be either an active immune response or a passive immune response. In addition, the immune response mediated by the CAR can be part of an adoptive immunotherapy approach that induces a specific immune response against the antigen-binding moiety in the CAR by the CAR-modified T cells. For example, CAR T19 cells induce a specific immune response against cells expressing CD19.

[0115] The data disclosed herein specifically disclose a lentiviral vector comprising an anti-CD19 scFv derived from the FMC63 mouse monoclonal antibody, the hinge and transmembrane domains of human CD8α, and the signaling domains of human 4-1BB and CD3ζ, but the present invention should be considered to include all various modifications of each of the components of the construct as described elsewhere herein. That is, the present invention includes the use of any antigen-binding moiety in the CAR to generate a CAR-mediated T cell response specific for the antigen-binding moiety. For example, the antigen-binding moiety in the CAR of the present invention can target tumor antigens for the treatment of cancer.

[0116] Treatable cancers include tumors that are not vascularized or not yet substantially vascularized, as well as tumors that are vascularized. The cancer may include non-solid tumors (hematological tumors, such as leukemia and lymphoma), or may include solid tumors. The types of cancers treated with the CARs of the present invention include, without limitation, carcinomas, blastomas and sarcomas, certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0117] Hematological malignancies are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) malignancies include leukemia, acute leukemia (acute lymphoblastic leukemia, acute myeloblastic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia and erythroleukemia, etc.), chronic leukemia (chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia, etc.), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (asymptomatic and high-grade types), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0118] A solid tumor is an abnormal tumor of tissue that usually does not include cysts or liquid regions. Solid tumors can be either benign or malignant. Various types of solid tumors are named according to the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (such as brainstem gliomas and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases, etc.) are included.

[0119] In one aspect, the antigen that binds to the moiety of the CAR of the present invention is designed to treat a specific cancer. For example, a CAR designed to target CD19 can be used to treat cancers and disorders including, but not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage after allogeneic bone marrow transplantation, etc.

[0120] In another aspect, the CAR can be designed to target CD22 for treating diffuse large B-cell lymphoma.

[0121] In one aspect, cancers and disorders that can be treated using a combination of CARs targeting CD19, CD20, CD22, and ROR1 include, but are not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage after allogeneic bone marrow transplantation, etc.

[0122] In one embodiment, the CAR can be designed to target mesothelin for treating mesothelioma, pancreatic cancer, ovarian cancer, and the like.

[0123] In one embodiment, the CAR can be designed to target the target CD33 / IL3Ra for treating acute myeloid leukemia and the like.

[0124] In one embodiment, the CAR can be designed to target c-Met for treating triple-negative breast cancer, non-small cell lung cancer, and the like.

[0125] In one embodiment, the CAR can be designed to target PSMA for treating prostate cancer and the like.

[0126] In one embodiment, the CAR can be designed to target glycolipid F77 for treating prostate cancer and the like.

[0127] In one embodiment, the CAR can be designed to target EGFRvIII for treating glioblastoma and the like.

[0128] In one embodiment, the CAR can be designed to target GD-2 for treating neuroblastoma, melanoma, and the like.

[0129] In one embodiment, the CAR can be designed to target NY-ESO-1 TCR for treating multiple myeloma, sarcoma, melanoma, and the like.

[0130] In one embodiment, the CAR can be designed to target MAGE A3 TCR for treating multiple myeloma, sarcoma, melanoma, and the like.

[0131] However, the present invention should not be considered limited to only the antigen targets and diseases disclosed herein. Rather, the present invention should be considered to include any antigen target associated with a disease in which CARs can be used to treat the disease.

[0132] 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 mammals. Preferably, the mammal is a human.

[0133] Regarding ex vivo immunization, prior to administering the cells to a mammal, at least one of the following is performed in vitro: (i) expansion of the cells, (ii) introduction of a nucleic acid encoding the CAR into the cells, and / or (iii) cryopreservation of the cells.

[0134] 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 a human) and then genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. A therapeutic benefit can be obtained by administering the CAR-modified cells to a mammalian recipient. The mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0135] Procedures for the ex vivo expansion of hematopoietic stem cells and progenitor cells are described in U.S. Patent No. 5,199,942, which is incorporated herein by reference and can be applied to the cells of the present invention. Other suitable methods are known in the art, and thus the present invention is not limited to any particular method of ex vivo cell expansion. Briefly, ex vivo culture and expansion of T cells includes: (1) collecting CD34+ hematopoietic stem cells and progenitor cells from a mammal, from a peripheral blood harvest or a bone marrow explant; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used for cell culture and expansion.

[0136] In addition to using cell-based vaccines for ex vivo immunotherapy, the present invention also provides compositions and methods for in vivo immunotherapy for inducing an immune response against an antigen in a patient.

[0137] Generally, the cells activated and expanded as described herein can be used for the treatment and prevention of diseases that occur in individuals with reduced immune function. In particular, the CAR-modified T cells of the present invention are used for the treatment of CCL. In one embodiment, the cells of the present invention are used for the treatment of patients at risk of developing CCL. Thus, the present invention provides a method for the treatment or prevention of CCL, the method comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.

[0138] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention can include the target cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or additives. Such compositions can include 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.

[0139] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, but an appropriate amount can be determined by clinical trials.

[0140] When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount" or "therapeutic 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, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). Generally, the pharmaceutical composition containing the T cells described herein contains from 10 4 to 10 9 cells / kg body weight, preferably from 10 5 to 10 6It can be administered at a dose of [[ID=]], including all integer values within these ranges, per kg body weight. Also, the T cell composition can be administered multiple times at these dosages. The cells can be administered by using infusion techniques generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0141] In one embodiment, it is desirable to administer activated T cells to a subject, subsequently collect blood again (or perform apheresis), activate the T cells derived therefrom according to the present invention, and then re-infuse these activated and expanded T cells into the patient. This process can be performed multiple times every 2 or 3 weeks. In one embodiment, the T cells can be activated from 10 cc to 400 cc of collected blood. In one embodiment, the T cells are re-activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc or 100 cc of collected blood. Without being bound by theory, using this multiple blood collection / multiple re-infusion protocol may help to select a particular population of T cells.

[0142] Administration of the present composition can be carried out in any convenient manner, including aerosol inhalation, injection, oral ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal injection or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of T cells may be directly injected into the tumor, lymph node or site of infection.

[0143] In one aspect of the invention, cells activated and expanded using the methods described herein, or other methods known in the art for expanding T cells to therapeutic levels, are administered to a patient in any of a variety of suitable treatment modalities (e.g., before, simultaneously, or after) including, but not limited to, antiviral therapy for MS patients, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment, efalizumab treatment for psoriasis patients, or treatment with other agents such as for PML patients. In a further aspect, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodepleting agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytokines, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for signal transduction induced by growth factors (rapamycin) (Liu et al., Cell, 66:807-815, 1991; Henderson et al., Immun., 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In one further aspect, the cell compositions of the invention are administered to a patient in combination with (e.g., before, simultaneously, or after) T cell depletion therapy using any of bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam irradiation (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another aspect, the cell compositions of the invention are administered after B cell depletion therapy with an agent that reacts with CD20, such as rituxan. For example, in one aspect, a subject can receive standard treatment with high-dose chemotherapeutic agents followed by peripheral blood stem cell transplantation.In one aspect, after transplantation, the subject receives an infusion of the increased immune cells of the present invention. In one additional aspect, the increased cells are administered before or after surgery.

[0144] The dosage of the above treatment administered to a patient is thought to vary depending on the medical condition being treated and the exact nature of the recipient of the treatment. Adjustments in dosage for administration to humans can be made according to accepted practice in the art. For example, the dosage of CAMPATH is generally in the range of 1 to about 100 mg for adult patients and is usually administered daily over a period of 1 to 30 days. A preferred daily dose is 1 to 10 mg / day, although in some cases higher dosages up to 40 mg / day can also be used (described in U.S. Patent No. 6,120,766).

[0145] Treatment of cytokine release syndrome (CRS) The present invention is based in part on the discovery that the in vivo expansion of CART19 cells and the associated potent anti-tumor activity are also associated with cytokine release syndrome (CRS) that results in hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Without wishing to be bound by any particular theory, MAS / HLH is a unique biomarker associated with the potent anti-tumor activity of CART19 and may be required for the potent anti-tumor activity of CART19.

[0146] Accordingly, the present invention provides a first-line therapy comprising administering the CAR of the present invention to a patient, and a second-line therapy comprising administering a certain type of therapy to manage elevated levels of certain soluble factors resulting from the first-line therapy using CAR T cells.

[0147] In one aspect, the second alternative therapy includes compositions and methods for the treatment of CRS. Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and the like. The present invention is based on the observation that CART19 cells induce elevated levels of soluble factors including, but not limited to, IFN-γ, TNFα, IL-2, and IL-6 in patients. Thus, the second alternative therapy includes compounds and methods for neutralizing the effects on elevated cytokines resulting from the administration of CART19 cells. In one aspect, the neutralizing agent can counteract the unwanted concerted burst of cytokine expression / activity and is therefore useful for the prevention, amelioration, and treatment of CRS associated with CART19 therapy.

[0148] In one aspect, the treatment of CRS is performed around the 10th to 12th day after the infusion of CART19 cells.

[0149] In one aspect, the second alternative therapy includes administering a steroid to the patient. In another aspect, the second alternative therapy includes administering one or more of a steroid, an inhibitor of TNFα, and an inhibitor of IL-6. An example of a TNFα inhibitor is etanercept. An example of an IL-6 inhibitor is tocilizumab (toc).

Examples

[0150] Experimental Examples The present invention will now be described with reference to the following experimental examples. These examples are provided for illustrative purposes only and the present invention should in no way be considered limited to these examples, but should be considered to include any and all modifications that become apparent as a result of the teachings provided herein.

[0151] Even without further explanation, one of ordinary skill in the art can make and use the compounds of the present invention and practice the claimed methods using the foregoing description and the following illustrative examples. The following examples are, therefore, illustrative of preferred embodiments of the invention and should in no way be construed as limiting the remainder of the disclosure.

[0152] Example 1: Cytokine therapy in combination with CAR T cell infusion The results presented herein demonstrate that patients following CAR T cell infusion exhibit differential expression levels of various cytokines. In some cases, elevated levels of some cytokines are the result of the toxicity of the infused CAR T cells (Figure 1). It was observed that in 2 out of 2 patients, tocilizumab (anti-IL-6) was able to improve CAR toxicity and seemingly preserve the anti-tumor effect (Figure 2). Without wishing to be bound by any particular theory, anakinra, which blocks IL-1, and other reagents may also be useful in this regard. The data presented herein also demonstrate that IL-1 is elevated in patients and may lead to subsequent elevation of IL-6. Anakinra is an IL-1Ra recombinant protein that binds to the IL1 receptor and blocks signaling of both IL-1α and β. Anakinra has a short half-life. Since both IL-1α and β are blocked, there is an advantage in initiating treatment of patients with anakinra and reducing the cytokine storm while preserving the anti-tumor effect.

[0153] It was also observed that antibody intervention did not affect the functionality of CART19 cells as measured by perforin and IFN-γ (Figure 3).

[0154] Example 2: Chimeric antigen receptor T (CART19) cells redirected to CD19 induce cytokine release syndrome (CRS) and treatable macrophage activation syndrome (MAS), which can be managed by tocilizumab (toc), an IL-6 antagonist Infusion of CART19 cells results in in vivo expansion by 100 - 100,000 fold, tumor lysis syndrome with persistent anti - tumor activity, and long - term persistence in patients with B - cell tumors. The results presented herein demonstrate an association between in vivo expansion of CART19 cells and the resulting potent anti - tumor activity with cytokine release syndrome (CRS) that leads to macrophage activation syndrome (MAS), also known as hemophagocytic lymphohistiocytosis (HLH). Without wishing to be bound by any particular theory, MAS / HLH is considered to be an inherent biomarker associated with potent anti - tumor activity and may be required for potent anti - tumor activity.

[0155] Autologous T cells were transduced with a CAR consisting of anti - CD19 scFv / 4 - 1BB / CD3 - ζ by lentivirus, activated / enlarged ex vivo with anti - CD3 / anti - CD28 beads, and then infused into ALL or CLL patients with a disease that persists after 2 - 8 prior treatments. The anti - ALL activity of CART19 was also simulated in a xenograft mouse model with a high level of human ALL / human T - cell grafts, and two - color bioluminescence imaging was used to simultaneously detect CAR T cells and ALL.

[0156] The results presented herein provide the latest results of 10 patients, including 9 patients with CLL and 1 pediatric patient with relapsed refractory ALL who received CART19 cells. 6 / 9 evaluable patients achieved complete remission (CR) or partial remission (PR), including 4 patients with durable CR. There was no acute infusion toxicity, but all responding patients also developed CRS. All had fever and grade 3 or 4 hypotension / hypoxemia. CRS preceded the peak blood expression of CART19 cells and then increased in intensity until the peak of CART19 cells (days 10 - 31 post-infusion). The ALL patient experienced the most significant toxicity with grade 4 hypotension and respiratory failure. Steroid therapy on day 6 did not result in improvement. On day 9, in view of high levels of TNFα and IL-6 (peak increases above baseline: IFNγ 6040-fold; IL-6 988-fold; IL-2R 56-fold, IL-2 163-fold, and TNFα 17-fold), antagonists of TNFα and IL-6 (etanercept and toc) were administered. This resulted in dissipation of fever and hypotension within 12 hours, and the patient was able to rapidly be weaned from mechanical ventilation to room air. These interventions had no apparent effect on the expansion or efficacy of CART19 cells: the peak of CAR T cells (2539 CAR+ cells / uL; 77% of CD3 cells by flow) occurred on day 11, and the bone marrow on day 23 showed CR with negative minimal residual disease (MRD) compared to the bone marrow at the first study with 65% blasts. She had no history of CNS ALL, but detectable CART19 cells (21 lymphocytes / mcL; 78% CAR+) were shown in the spinal fluid. Four months post-infusion, this patient remained in CR with 17 CART19 cells / uL in the blood and 31% CAR+ CD3 cells in the bone marrow.

[0157] Subsequent clinical evaluation of the responding patients has shown that all had evidence of MAS / HLH, including dramatic elevation of ferritin, and histological evidence of HLH. Peak ferritin levels ranged from 44,000 to 605,000 and preceded and persisted through peak T cell proliferation. Other consistent findings included rapid onset of hepatosplenomegaly unrelated to the disease and moderate DIC.

[0158] Subsequently, three CLL patients were also treated with toc, and again immediate and significant resolution of fever, hypotension, and hypoxia was seen. One patient received toc on day 10 and achieved CR with an increase in CART19. Another patient showed rapid resolution of CRS after toc administration on day 9, and follow-up for response was too short. The third CLL patient received toc on day 3 due to initial fever and did not show CART-19 proliferation or response.

[0159] To simulate the timing of cytokine blockade in experimental studies, xenografts using bioluminescent primary pediatric ALL were established and then treated with excess cells from clinical-grade production. CART19 cells proliferated and resulted in long-term survival. Cytokine blockade with toc and / or etanercept prior to T cell infusion decreased in vivo proliferation of the infused CART19 cells and abrogated disease control, corroborating the results seen in one patient given toc early (day 3).

[0160] CART19 T cells can give rise to large-scale in vivo expansion, long-term persistence, and anti-tumor efficacy, but can also induce significant CRS with features suggesting a rapid response to cytokine blockade with MAS / HLH. When given prior to the onset of significant CART19 proliferation, blockade of TNFα and / or IL-6 can interfere with proliferation and effector function, but when given at a time when cell proliferation is ongoing, toc has been observed to improve symptoms correlating with a robust clinical response.

[0161] Example 3: Remission of ALL by chimeric antigen receptor-expressing T cells The results presented herein demonstrate that CAR T cells have clinical activity in acute lymphoblastic leukemia (ALL). Briefly, two pediatric patients with relapsed and refractory pre-B cell ALL were treated with 10 6 to 10 7 / kg of T cells (CTL019 CAR T cells; also called CART19) transduced with an anti-CD19 antibody and T cell signaling molecules. The CTL019 T cells expanded more than 1000-fold in both patients and were transported to the bone marrow. In addition, the CAR T cells were able to cross the blood-brain barrier and persisted at high levels for at least 6 months as measured in the cerebrospinal fluid. Eight serious adverse events were noted. Cytokine release syndrome (CRS) and B cell aplasia occurred in both patients. In one child, CRS was severe and cytokine blockade with etanercept and tocilizumab was effective in reversing the symptoms, yet still did not impede the expansion of CAR T cells and anti-leukemia efficacy. Complete remission was observed in both patients, and in one patient it persisted 9 months after treatment. The other patient relapsed at approximately 2 months after treatment with blasts that no longer expressed CD19.

[0162] The results presented herein demonstrate that T cells modified with a CAR can kill even actively treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates the need to target other molecules in addition to CD19 in some patients with ALL.

[0163] Increased in vivo expansion and potent anti-leukemia effects of CTL019 (CART19) cells have been reported in three patients with CLL (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73). CTL019 is a CAR containing the CD137 (4-1BB) signaling domain and is expressed using lentiviral vector technology (Milone et al., 1009, Mol Ther 17:1453-64). The results presented herein demonstrate the use of CTL019 in two pediatric patients with refractory and relapsed ALL. Both patients showed remission of leukemia with robust in vivo expansion of CTL019 along with transport to the bone marrow and CNS. One patient had a chemotherapy-refractory disease precluding allogeneic donor stem cell transplantation, and the other patient relapsed after allogeneic umbilical cord blood transplantation and was resistant to blinatumomab (blinatumomab) (chimeric bispecific anti-CD3 and anti-CD19) therapy, so the anti-leukemia effect was powerful.

[0164] The materials and methods used in these experiments are described below.

[0165] Materials and methods CART19 The production of CTL019 (CART19) has been previously reported (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73). CTL019 was detected and quantified in patient specimens as previously reported (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73).

[0166] Sample collection and processing Samples (peripheral blood, bone marrow) were collected in lavender top (K2EDTA) or red top (no additive) vacutainer tubes (Bacton Dickinson). Lavender top tubes were delivered to the laboratory within 2 hours of collection or, prior to processing, shipped overnight at room temperature in insulated containers essentially as described (Olson et al., 2011, J Transl Med 9:26). Samples were processed within 30 minutes of receipt according to established laboratory standard operating procedures (SOP). As described (Kalos et al., 2011, Science Translational Medicine 3:95ra73), peripheral blood and bone marrow mononuclear cells were purified, processed, and stored in liquid nitrogen. Red top tubes were processed within 2 hours of collection including clotting time; serum was isolated by centrifugation, aliquoted into single-use 100 μL aliquots, and stored at -80°C. CSF was delivered to the laboratory within 30 minutes of aspiration, and cells in the CSF were collected by centrifugation of the CSF fluid and processed for DNA and flow cytometry.

[0167] Q-PCR analysis Whole blood or bone marrow samples were collected in lavender top (K2EDTA) BD vacutainer tubes (Becton Dickinson). Genomic DNA was isolated directly from whole blood and, as described (Kalos et al., 2011, Science Translational Medicine 3:95ra73), the integrated CD19 CAR transgene sequence was detected for peripheral blood and bone marrow samples using 200 ng of genomic DNA per time point and for CSF samples using 18 - 21.7 ng of genomic DNA per time point by Q-PCR analysis of genomic DNA samples in bulk using ABI Taqman technology and a validated assay. To measure the copy number per unit DNA, 5 - 10 spiked into 100 ng of non-transduced control genomic DNA 6An eight-point standard curve consisting of the copy of CTL019 lentiviral plasmid was generated. Each data point (sample, standard curve) was evaluated in triplicate, and the positive Ct values in the 3 / 3 replicates had a %CV of less than 0.95% for all quantifiable values. Parallel growth reactions were performed to control for the quality of the DNA being examined using 20 ng of input genomic DNA from peripheral blood and bone marrow (2 - 4.3 ng for CSF samples), as well as a primer / probe combination specific for the non-transcribed genomic sequence upstream of the CDKN1A gene as described (Kalos et al., 2011, Science Translational Medicine 3:95ra73). These amplification reactions were used to generate a correction factor (CF) to correct the calculated amount against the actual DNA input. The copy of the transgene per microgram of DNA was calculated according to the formula: copy calculated from the CTL019 standard curve per ng of input DNA × CF × 1000 ng. The accuracy of this assay was determined by the ability to quantify the marking of the infused cell product by Q-PCR. These blinded determinations resulted in Q-PCR and flow marking values of 11.1% and 11.6% respectively for CHOP-100, and markings of 20.0% and 14.4% respectively for the CHOP-101 infusion product.

[0168] Soluble factor analysis Whole blood was collected into red-top (additive-free) BD Vacutainer tubes (Becton Dickinson) and processed using established laboratory standard test procedures to obtain serum, which was aliquoted for single use and stored at -80°C. Quantification of soluble cytokine factors was performed using Luminex bead array technology and kits purchased from Life technologies (Invitrogen). The assay was performed with nine-point standard curves prepared using a three-fold dilution series according to the manufacturer's protocol. Two external standard points were evaluated in duplicate, and five internal standards were evaluated in a series; all samples were evaluated in duplicate at a 1:2 dilution; the %CV calculated for duplicate measurements was less than 15%. Data were acquired as a percentage on FlexMAP-3D and analyzed using XPonent 4.0 software and five-parameter logistic regression analysis. The quantification range of the standard curve was determined by the range of 80-120% (observed / expected value). Reported values included those within the range of the standard curve and those calculated by logistic regression analysis.

[0169] Antibody reagents The following antibodies were used for these studies: MDA-CAR, a mouse antibody against CD19 CAR conjugated to Alexa647 (Jena and Copper, 2013, L. Anti-idiotype antibody for CD19. PlosONE 2013; in press). Antibodies for multi-parameter immunophenotyping: T cell detection panel: anti-CD3-FITC, anti-CD8-PE, anti-CD14-PE-Cy7, anti-CD16-PE-Cy7, anti-CD19-PE-Cy7, anti-CD16-PE-Cy7, B cell detection panel: anti-CD20-FITC, anti-CD45-PE, anti-CD45-APC, anti-CD19-PE-Cy7, anti-CD19-PE, anti-CD34-PCP-e710, and anti-CD34-APC were procured from e-Biosciences.

[0170] Multi-parameter flow cytometry Cells were evaluated by flow cytometry directly after Ficoll-Paque treatment, except for baseline samples of CHOP-101 that were evaluated immediately after thawing of cryopreserved samples. Multiparameter immunophenotyping was performed using approximately 0.2 - 0.5×10 6 total cells per condition (depending on the cell yield in the sample) for peripheral blood and bone marrow samples, and using a small number of cells collected after centrifugation of the CSF fluid for CSF samples, as described in the textbook, using fluorescence minus one (FMO) staining. Cells were stained in 100 μL PBS on ice for 30 minutes using the antibody and reagent concentrations recommended by the manufacturer, washed, and resuspended in 0.5% paraformaldehyde and captured using an Accuri C6 cytometer equipped with blue (488) and red (633 nm) lasers. Accuri files were exported in FCS file format and analyzed using FlowJo software (version 9.5.3, Treestar). Compensation values were established using single antibody staining and BD compensation beads (Becton Dickinson) and calculated by the software. The gating strategy for T cells was as follows: live cells (FSC / SSC) > dump channel (CD14+CD16+CD19-PECy7) vs CD3+ > CD3+. The general gating strategy for B cells was as follows: live cells (FSC / SSC) > SSC low events > CD19+. More detailed gating for CHOP-100 and CHOP-101 samples is described in the individual figures.

[0171] Molecular MRD analysis MRD analysis of the molecule was performed by high-throughput next-generation sequencing of the BCR IGH CDR3 region using the Adaptive Biotechnologies (Seattle, WA) and immunoSEQ assay based on the Illumina HiSeq / MiSeq platform (Larimore et al., 2012, J. Immunol 189:3221-30). For these analyses, 701-6,000 ng (approximately 111,000-950,000 genome equivalents) of genomic DNA isolated from whole blood or bone marrow samples obtained from patients was subjected to combined multiplex PCR and sequencing, and then algorithmic analysis was performed to quantify the individual IGH CDR3 sequences in the samples. To evaluate the quality of the DNA samples, parallel amplification and sequencing of the TCRB CDR3 region (Robins et al., 2009, Blood 114:4099-107) were performed in each sample. For each patient, the IGH CDR3 nucleotide sequences assayed from samples at different time points were aligned using the EMBL-EBI multiple sequence alignment tool (Goujon et al., 2010, Nucleic Acids Res 38:W695-9; Sievers et al., 2011, Mol Syst Biol 7:539). The dominant clone from the sample at the earliest time point was then tracked bioinformatically across the IGH CDR3 sequences assayed in samples at subsequent time points to identify the presence of sequences with 95% or greater pair sequence identity. The total sequence determination reads for those sequences similar to the dominant clone are reported for each time point.

[0172] The results of the experiment are described below.

[0173] Case report CHOP-100 was a 7-year-old girl with ALL who had relapsed for the second time. She was diagnosed 2 years ago and achieved remission with minimal residual disease (MRD) negativity, but relapsed 17 months after diagnosis. She entered remission again after re-induction chemotherapy but relapsed 4 months later and then did not respond to clofarabine / etoposide / cyclophosphamide. Her baseline karyotype was 48,XX,del(9)(p21.3),+11,del(14)(q2?q24),+16 / 46,XX[4]. Anticipating that there might be insufficient circulating T cells available for hematopoiesis after intensive chemotherapy, peripheral blood mononuclear cells (PBMCs) were collected by apheresis before such intensive treatment. This patient was transduced with lentivirus to expand with anti-CD3 / CD28 and express anti-CD19 CAR CTL019 cells, as previously described (Porter et al., 2011, N Engl J Med 365:725-33;Kalos et al., 2011, Science Translational Medicine 3:95ra73), at a total dose of 3.8×10 8 cells / kg (1.2×10 7 CTL019 cells / kg) over 3 consecutive days and infused. She did not receive lymphodepleting chemotherapy before CTL019 infusion, and the most recent cytotoxic therapy was given 6 weeks before CTL019 infusion. Immediate infusion toxicity was not confirmed, but she was hospitalized for mild fever, which progressed to high fever by day 4 and the patient was transferred to the pediatric intensive care unit on day 5 (CHOP-100, Figure 4A). Subsequently, she rapidly progressed to severe respiratory and cardiovascular impairment requiring mechanical ventilation and blood pressure support.

[0174] The second ALL patient was a 10-year-old girl (CHOP-101) who experienced a second relapse 28 months after diagnosis and 10 months before CTL019 infusion, following a 4 / 6 matched unrelated cord blood transplant. She had experienced graft-versus-host disease (GVHD) after transplantation, which resolved with treatment; she was not immunosuppressed at the time of relapse. She did not subsequently achieve remission despite multiple cytotoxic and biologic therapies. Her baseline karyotype was 46 XX, del(1)(p13), t(2;9)(q?21;q?21), t(3;17)(p24;q23), del(6)(q16q21), del(9)(q13q22), der(16)t(1;?;16)(p13;?p13.3)[9], / / 46, Xy[1]. Prior to PBMC collection, she was treated with 2 cycles of blinatumomab (Bargou et al., 2008, Science 321:974-7) without response. 68% of her peripheral blood cells were of donor origin at the time of PBMC collection. CTL019 T cells were generated and infused at a total dose of 10 7 cells / kg (1.4×10 6 CTL019 cells / kg) following etoposide / cyclophosphamide chemotherapy given the previous week for lymphodepletion. Her bone marrow on the day prior to CTL019 infusion was replaced by a population of CD19+ / CD34+ ALL cells with variable CD19 expression as determined by standard clinical flow cytometry (Figure 7). She had no immediate infusion toxicities but developed fever and was hospitalized on day +6. She did not experience cardiopulmonary toxicity and did not receive glucocorticoid or anti-cytokine therapy. CHOP-101 experienced fever of unknown origin (Figure 4B), myalgia, and a 2-day confusional state (grade 3), presumably due to cytokine release, which resolved spontaneously. She did not show evidence of GVHD following CTL019 cell infusion. These cells were collected from the patient but were mostly of donor (cord blood) origin.

[0175] Induction of remission in both subjects For both subjects, circulating lymphocytes and neutrophils increased during the two weeks following CTL019 infusion, as shown by plots depicting total WBC, ALC, and ANC against the timing of CTL019 infusion (Figure 4C). The majority of lymphocytes were composed of T cells expressing chimeric antigen receptors, as shown in more detail in Figure 5 (Figure 8). In both subjects, non-infectious fever was recorded following elevation of LDH (Figure 4A). The elevation of LDH and fever were similar to those previously described in CLL patients following CTL019 infusion (Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science Translational Medicine 3:95ra73). Approximately one month after infusion, morphological remission with MRD negativity (<0.01%) of leukemia was achieved in both subjects (Table 1).

[0176] Clinical remission in CHOP-100 was associated with deep molecular remission lasting at least nine months as in January 2013 (Table 1). High-throughput DNA sequencing of the IGH locus revealed a marked decrease in total IGH reads on day +23 in blood and bone marrow of CHOP-100. No malignant clone was detected in blood or bone marrow in more than 1 million cell equivalents sequenced on day +180. In contrast, T cell receptor sequences were readily detected in blood and bone marrow, indicating the integrity of DNA tested at all time points.

[0177] (Table 1) Induction of molecular remission in the blood and bone marrow of CHOP-100 and 101 Molecular analysis of minimal residual disease was performed on DNA isolated from whole blood or bone marrow.

[0178] Toxicity of CTL019 Summarize the grade 3 and 4 adverse events in Table 2. Acute toxicity consisting of fever and cytokine release syndrome (CRS) that progressed to macrophage activation syndrome (MAS) was observed in both patients. Both patients were monitored for the prevention of tumor lysis syndrome. Both experienced a substantial increase in LDH, the cause of which was likely multifactorial and was thought to include tumor lysis syndrome. Each uric acid value in CHOP-100 was either below normal or within the normal range, and she received allopurinol only on days 5-6. CHOP-101 received prophylactic allopurinol from days 0-14 and had abnormal uric acid values of 4.8-5.7, consistent with mild tumor lysis syndrome, on days 8-10.

[0179] (Table 2) Adverse events (grades 3 and 4) in CHOP-100 and CHOP-101 TIFF0007682820000002.tif78150The adverse events were graded according to the Common Terminology Criteria for Adverse Events 3.0.

[0180] In CHOP-100, glucocorticoids were administered on day +5, with a short-term response in the fever curve but without remission of hypotension. Administration of a single course of anti-cytokine therapy consisting of etanercept and tocilizumab on day +8 led to a continued rapid clinical effect: within hours, she became afebrile, and as her clinical and radiological ARDS resolved, vasoactive agents and ventilatory support were removed. She had no laboratory evidence of tumor lysis syndrome; however, biochemical evidence of MAS was represented by an increase in ferritin to 45,529 ng / dl on day +11, coagulopathy with elevated d-dimer and hypofibrinogenemia, hepatosplenomegaly, elevated transaminases, elevated LDH (Figure 4C), and elevated triglycerides, as well as a cytokine profile consistent with MAS. Her ferritin decreased to 2,368 by day +26, and the clinical and laboratory abnormalities of MAS resolved.

[0181] In CHOP-101, there was no direct evidence of tumor lysis syndrome other than changes in heat and LDH (Figure 4C), but she also developed features of MAS with a ferritin increase to 74,899 at peak on day 11, up from 33,360 on day +7, transaminases reaching grade 4 for 1 day, and elevated d-dimer in the serum. These biochemical changes were reversible, as the transaminases improved to grade 1 and the ferritin decreased to 3,894 by day +21. She was discharged from the hospital on day +16.

[0182] Both subjects developed marked elevations of numerous cytokines and cytokine receptors in the serum (Figure 1B). In both patients, the increases in interferon-γ and IL-6 were most prominent. These observations are similar to patterns previously observed in patients with CLL who also experienced remission of leukemia after CTL019 infusion (Kalos et al., 2011, Science Translational Medicine 3:95ra73). The peak cytokine elevations were temporally correlated with systemic inflammation as judged by changes in core body temperature (Figure 4C).

[0183] In vivo expansion of CTL019 in patients with ALL The fraction of circulating CTL019 T cells gradually increased to 72% (CHOP-100) and 34% (CHOP-101) of T cells in vivo (Figure 5A). The initial transduction efficiencies were 11.6% and 14.4% in the infused T cells in CHOP-100 and -101, respectively. Considering that the total ALC increased substantially in both patients (Figure 4C) and the frequency of CTL019 cells gradually increased in vivo from the baseline frequency (Figure 8), there was a robust and selective expansion of CTL019 cells in both patients. The selective increase in T cells expressing CTL019 in both patients is consistent with an anti-leukemia mechanism involving CD19-driven expansion and subsequent elimination of cells expressing CD19 in both patients (Figures 6 and 9).

[0184] On day +23, when more than 65% of CD3+ cells in peripheral blood and bone marrow were shown to be CTL019+ by flow cytometry, molecular deep sequencing analysis of TCRs in peripheral blood and bone marrow samples in CHOP-100 revealed that there were no dominant T cell TCR clone types in any compartment, and that the 10 most abundant T cells were present in the bone marrow at frequencies between 0.18 and 0.7% and in peripheral blood at frequencies between 0.19 and 0.8%. Six of the 10 dominant clones were shared between the two compartments. In addition, CAR T cells of both CD4 and CD8 were present. Thus, CAR T cells appear to proliferate after expansion stimulated by CD19 and not by clone-specific events such as TCR signaling or activation by lentiviral integration.

[0185] Transport and morphology of CTL019 CAR T cells in the bone marrow and CNS CTL019 cells expanded more than 1000-fold in peripheral blood and bone marrow (Figure 5). The frequency of CTL019 cells increased to more than 10% of circulating T cells in both subjects by day +20 (Figure 8), and was of an absolute magnitude of CTL019 expansion similar to that observed in patients with CLL (Kalos et al., 2011, Science Translational Medicine 3:95ra73). Unexpectedly, cells in the CSF also showed high levels of CTL019 gene marking and persisted at high frequency over 6 months (Figure 5B). Considering that none of the patients had detectable CNS leukemia at the time of infusion or by cytospin processing at the 1-month post-treatment assessment, the transport of CTL019 cells to the CSF was surprising. Furthermore, previous reports of CAR therapy for B cell malignancies have not observed transport of CAR T cells to the CNS (Till et al., 2008, Blood 112:2261-71; Brentjens et al., 2011, Blood 118:4817-28; Savoldo et al., 2011, J Clin Invest 121:1822-5; Jensen et al., 2010, Biol Blood Marrow Transplant 16:1245-56; Till et al., 2012, Blood 119:3940-50; Kochenderfer et al., 2012, Blood 119:2709-20). The morphology of lymphocytes in blood and CSF is shown in Figure 5D for CHOP-100 and 101. Since more than 70% of the circulating lymphocytes on day +10 were CTL019 cells (Figure 5A and Figure 5B), most of the large granular lymphocytes shown in the left panel of Figure 5D are likely to be CTL019 cells. Similarly, since many of the lymphocytes in the CSF obtained from CHOP-101 on day +23 were CTL019 cells (Figure 5B and Figure 5C), the cytospin preparation of CSF lymphocytes in Figure 5D most likely represents the morphology of in vivo CTL019 cells transported to the CNS.

[0186] Induction of B cell aplasia Both subjects showed elimination of CD19⁺ cells in the bone marrow and blood within 1 month after CTL019 infusion (Figs. 6 and 9). In CHOP-100, a large proportion of the cells remaining in the bone marrow on day +6 after infusion were CD19⁺CD20⁺ leukemic blasts. This cell population became undetectable by day +23, which is an effect maintained for over 9 months in this patient (Fig. 9A). Considering that CHOP-100 did not receive chemotherapy during the 6 weeks prior to CTL019 infusion, this indicates that CTL019 cells were sufficient to eliminate normal and leukemic B cells in this case.

[0187] Appearance of CD19 escape variants in CHOP-101 CHOP-101 experienced a clear clinical relapse in the peripheral blood 2 months after CTL019 infusion, as demonstrated by the reappearance of blasts in the circulation. These cells were weakly positive for CD45, positive for CD34, and did not express CD19 (Fig. 6). The absence of the original dominant CD34dim⁺CD34⁺CD19dim⁺ cells is consistent with the strong anti-leukemic selection pressure of CD19-directed CTL019 CAR T cells (Fig. 9B). Despite the initial flow cytometry-based MRD-negative clinical assessment on day +23 (Fig. 7), deep IGH sequencing revealed the presence of malignant clones in the peripheral blood and bone marrow at this time point (Table 1). Additionally, deep sequencing of the material obtained at clinical relapse showed that CD45dimCD34⁺CD19⁻ cells were clonally related to the original dominant CD45dim⁺CD34⁺CD19dim⁺ cells as they shared the same IGH sequence.

[0188] Remission of ALL by chimeric antigen receptor-expressing T cells The results presented in this specification demonstrate the induction of remission of relapsed and refractory leukemia in the first two patients treated with this protocol. Remission was sustained in one subject and was accompanied by relapse due to the emergence of CD19-negative blasts in the other subject. Genetically modified CTL019 cells were transported to the CNS at high levels in both patients. Cytokine elevation was as expected, reversible, and was observed to be temporally associated with the elimination of blast cells expressing CD19 in both subjects. In particular, given the low frequency of remission after infusion of allogeneic donor lymphocyte infusions that do not express the CAR, the induction of complete remission in refractory CD19-positive ALL after CAR T cell infusion is promising (Kolb et al., 1995, Blood 86:2041-50; Collins et al., 1997, J Clin Oncol 15:433-44; Collins et al., 2000, Bone Marrow Transplant 26(5):511-6). Deep sequencing technology has shown that the infusion of CTL019 CAR is associated with a 5-log reduction in the frequency of malignant B cells in CHOP-100 and further demonstrates a potent antitumor effect in chemotherapy-refractory leukemia.

[0189] The unfortunate emergence of CD19-negative blasts in one subject is consistent with previous reports documenting the presence of CD19-negative progenitor cells in some cases of ALL (Hotfilder et al., 2005, Cancer Research 65:1442-9; le Viseur et al., 2008, Cancer Cell 14:47-58). The infusion of CAR T cells redirected to novel specificities, in addition to CD19, is expected to reduce the likelihood of this event. To date, relapse due to CD19-negative escape cells after treatment with CTL019 cells in adults with CLL has not been observed (Kalos et al., 2011, Science Translational Medicine 3:95ra73), suggesting that this problem may be specific to a subset of acute leukemias. Induction of remission in CHOP-100 does not require concomitant chemotherapy and is consistent with previous reports showing that remission in CLL can be delayed by several weeks after chemotherapy (Porter et al., 2011, N Engl J Med 365:725-33). Thus, administration of concomitant cytotoxic chemotherapy may not be necessary for the CAR-mediated antitumor effect.

[0190] Both pediatric ALL patients experienced substantial toxicity following CTL019 infusion. Induction of B cell aplasia was observed, indicating that CAR T cells can function in the setting of relapsed acute leukemia. Both patients also developed clinical and laboratory evidence of cytokine release syndrome and macrophage activation syndrome within one week of infusion. The cytokine profiles observed in these patients were similar to those previously reported in children with hemophagocytosis and macrophage activation syndrome, i.e., hemophagocytic lymphohistiocytosis (Tang et al., 2008, Br J Haematol 143:84-91; Behrens et al., 2011, J Clin Invest 121(6):2264-77). Macrophage activation syndrome is characterized by prolonged fever with excessive inflammation, hepatosplenomegaly, and cytopenia. Laboratory findings characteristic of this syndrome include elevated ferritin, triglycerides, transaminases, bilirubin (mostly conjugated), and soluble interleukin-2 receptor alpha chain, and decreased fibrinogen (Janka et al., 2012, Annu Rev Med 63:233-46). Recent studies have shown that tocilizumab (anti-IL6) is promising for glucocorticoid-resistant GVHD (Drobyski et al., 2011, Biol Blood Marrow Transplant 17(12):1862-8; Le Huu et al., 2012, J Invest Dermatol 132(12):2752-61; Tawara et al., 2011, Clinical Cancer Research 17:77-88), and the results presented herein are consistent with these data.

[0191] The robust in vivo expansion, persistent B cell aplasia, and prominent anti-leukemia activity of CTL019 imply substantial and persistent effector functions of CTL019 cells in pediatric patients with advanced ALL. The high efficiency of CAR T cell transport to the CNS is promising as a surveillance mechanism to prevent relapse in sanctuary sites such as the CNS (Pullen et al., 1993, J Clin Oncol 11(5):839-49) and supports testing of CAR T cell-mediated therapies for CNS lymphoma and primary CNS malignancies. Except for B cell aplasia, whose duration is currently uncertain, the use of immune-based therapies such as CTL019 may have a favorable profile of long-term adverse effects compared to high-dose chemotherapy and radiation, which are currently used as standard treatments for the majority of cases of pediatric leukemia (Garcia-Manero and Thomas, 2001, Hematol Oncol Clin North Am 15(1):163-205).

[0192] Induction of complete remission of ALL by chimeric antigen receptor-expressing T cells Tocilizumab (anti-IL6) is promising for glucocorticoid-resistant GVHD, and the results presented herein are consistent with these data. Furthermore, in CHOP 100, the manifestation of CRS such as high fever, hypotension, and multiple organ failure was resistant to high-dose glucocorticoids administered 2 days prior to cytokine-targeted therapy. Finally, in CHOP-100, the biphasic changes in IL-1β, IL-1RA, and IL-2 shown in Figure 4B may be associated with cytokine-targeted therapy with etanercept and tocilizumab.

[0193] Induction of remission in patients refractory to blinatumomab therapy further underscores the potential of CTL019 cells. The high efficiency of CAR T cell transport to the CNS is promising as a surveillance mechanism to prevent recurrence in sanctuary sites such as the CNS, and supports testing of CAR T cell-mediated therapies for CNS lymphoma and primary CNS malignancies. Neither patient experienced cognitive effects attributable to transport of T cells to the CNS.

[0194] Example 4: Summary information Various markers were measured in patients who received CAR T cells. As non-limiting examples, ferritin, myoglobin, and plasminogen activator inhibitor 1 (PAI-1) were measured; see Figures 10, 11, and 12, respectively. Elevated levels of these markers correlated with outcome. Patients designated -01, -03, -09, -100, and -101 were classified as complete responders. Patients designated -02, -05, -10 (second infusion and response at approximately day 70), and -12 were classified as partial responders. Patients designated -06, -07, and -14 were classified as non-responders.

[0195] The disclosure of each and every patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety. Although the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention may be devised without departing from the true spirit and scope of the invention. The appended claims are intended to cover all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition for treating a harmful effect in a patient, comprising a cytokine inhibitor, wherein the harmful effect is associated with administration of cells genetically modified to express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain to the patient, the cytokine inhibitor is selected from the group consisting of an IL-6 inhibitor and an IL-1β inhibitor, further wherein the IL-6 inhibitor is an anti-IL-6 antibody or an anti-IL-6R antibody, and the IL-1β inhibitor is an anti-IL-1β antibody, an anti-IL-1R antibody, or anakinra.

2. The pharmaceutical composition according to claim 1, wherein the harmful effect comprises cytokine release syndrome (CRS).

3. The pharmaceutical composition according to claim 1 or 2, wherein the patient has cancer and the cells treat the cancer.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antigen-binding domain targets a tumor antigen.

5. The pharmaceutical composition according to claim 4, wherein the tumor antigen is selected from the group consisting of one or more of CD19, CD20, CD22, EGFRvIII, and IL3Ra.

6. The pharmaceutical composition according to claim 5, wherein the tumor antigen is CD19.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of a 4-1BB co-stimulatory domain, a CD28 co-stimulatory domain, a CD3ζ signaling domain, and any combination thereof.

8. The pharmaceutical composition according to any one of claims 2 to 7, wherein the CRS results in hemophagocytic lymphohistiocytosis / macrophage activation syndrome.

9. The cancer is (i) a hematological malignancy selected from the group consisting of acute leukemia, acute lymphoblastic leukemia, acute myeloid acute leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia, (ii) further, the CAR is an anti-CD-19 CAR, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, (iii) solid tumors, (iv) primary or metastatic cancer, and (v) cancer that is refractory or resistant to conventional chemotherapy The pharmaceutical composition according to any one of claims 3 to 8, which is selected from the group consisting of.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the cell is a human T cell (CAR T cell) transfected with a vector expressing CAR in vitro, and the CAR T cell is autologous to the patient.

11. The pharmaceutical composition according to any one of claims 1 to 10, further comprising one or more pharmaceutically acceptable carriers, diluents or additives.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the IL-1β inhibitor is anakinra.

13. The pharmaceutical composition according to any one of claims 1 to 11, wherein the IL-6 inhibitor is an anti-IL-6R antibody.

14. The pharmaceutical composition according to claim 13, wherein the IL-6 inhibitor is tocilizumab.

15. The pharmaceutical composition according to claim 14, wherein the cancer is selected from the group consisting of pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, and diffuse large B-cell lymphoma.

16. The pharmaceutical composition according to claim 14 or 15, wherein the tocilizumab is administered at a dose of 8 mg / kg.

17. The pharmaceutical composition according to any one of claims 1 to 16, which is used in combination with a corticosteroid for treating the adverse effect.

18. The pharmaceutical composition according to claim 17, wherein the corticosteroid is methylprednisolone.

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