Administration of Recombinant T Cells for the Treatment of Cancer of the Central Nervous System

Abstract

Improved methods for treating cancer using recombinant T cells are described. The present invention relates to a method for treating malignant tumors of the central nervous system by administering a composition comprising T cells (e.g., CAR T cells, tumor infiltrating lymphocytes ("TILs"), TCR-recombined T cells, or T cell clones) into the cerebrospinal fluid ("CSF") of a patient.

Description

[Technical field]

[0001] The present specification relates to methods of treating malignancies of the central nervous system by administering a composition comprising T cells (e.g., CAR T cells, tumor infiltrating lymphocytes ("TIL"), TCR-recombinant T cells, or T cell clones) into the cerebrospinal fluid ("CSF") of a patient. [Background technology]

[0002] Tumor-specific T cell-based immunotherapy, including recombinant T cells and treatment methods using ex vivo expanded or selected T cells, is being investigated for anti-tumor therapy. The T cells used in such therapy may not maintain activity in vivo for a long enough period of time. The tumor specificity of T cells may be relatively low. Recombinant T cells may have poor access to tumors. Therefore, there is a need in the art for tumor-specific cancer therapy with more effective anti-tumor function. Treating cancers of the central nervous system can be particularly challenging. For example, the treatment of high-grade malignant gliomas (MG), including anaplastic astrocytoma (AA-grade III) and glioblastoma multiforme (GBM grade IV), remains a significant therapeutic challenge. Currently available treatment options have limited curative potential, with only less than 5% of patients surviving more than 5 years after initial diagnosis. Summary of the Invention [Problem to be solved by the invention]

[0003] Described herein are methods of treating malignancies of the central nervous system by administering a composition comprising T cells (e.g., CAR T cells, tumor infiltrating lymphocytes ("TIL"), TCR modified T cells, or T cell clones) into the cerebrospinal fluid ("CSF") of a patient. T cells include modified T cells, e.g., by introduction of a nucleic acid molecule expressing a desired receptor, by ex vivo expansion of isolated or genetically modified T cells, or by ex vivo selection of a subset of T cells obtained from a patient or donor, or a combination of two or more of these techniques. Administration to the CNS can be accomplished, for example, by administration to the ventricular system or the central cavity of the spinal column. As that term is used herein, administration to the CNS is distinct from intratumoral administration (injection or infusion into the tumor itself) and administration to a cavity created by resection of a tumor. However, the CNS administration methods described herein can be used in conjunction with intratumoral administration and / or intracavitary administration following resection.

[0004] CNS administration as described herein may involve the injection of relatively large volumes of compositions containing T cells, e.g., 1 ml to 2 ml or more in a single injection, thus allowing millions of T cells to be administered in a single injection. [Means for solving the problem]

[0005] Thus, disclosed is a method of treating a patient diagnosed with a central nervous system malignancy, comprising injecting an effective amount of a composition comprising T cells into an anatomical compartment of the patient diagnosed with a central nervous system malignancy, the anatomical compartment containing cerebrospinal fluid (CSF). The method includes, for example, injecting the composition into a portion of the ventricular system or the central canal of the spinal cord. In one embodiment of the disclosed method, a central nervous system malignancy includes a primary or metastatic tumor found anywhere in the central nervous system, including portions of the brain, spinal column, etc. Preferably, the anatomical compartment contains a contiguous volume of cerebrospinal fluid of at least about 50, 100, or 150 mL.

[0006] The recombinant T cells injected by the methods described herein target tumor antigens, including surface and intracellular proteins. The malignant tumors treated may be primary tumors or secondary tumors resulting from cancers originating elsewhere in the body. Because administration into the cerebrospinal fluid may allow T cells access to areas beyond the local injection site, the methods described herein may be used to attack and reduce the size of tumors that are distant from the injection site but within the CNS. TCR-recombinant T cells are prepared by introduction of the TCRαβ gene into T cells (e.g., autologous T cells) followed by ex vivo expansion of the T cells, and the T cells are injected into patients. Infusion of the TCR-recombinant T cells confers tumor reactivity in patients whose tumors express the appropriate antigens and HLA restriction elements. The TCR can target any of a variety of tumor antigens, including, for example, melanoma-associated antigen recognized by T cells 1 (MART-1), glycoprotein (gp)100, carcinoembryonic antigen (CEA), p53, melanoma-associated antigen (MAGE-A3), and New York esophageal squamous cell carcinoma antigen (NYESO).

[0007] Described herein is a method of treating a patient diagnosed with a malignancy of the central nervous system, comprising introducing into the patient's cerebrospinal fluid (CSF) an effective amount of a composition comprising T cells.

[0008] In various embodiments, the following: the T cells are autologous or allogeneic T cells; the T cells are modified ex vivo by one or more of expansion, fractionation, or transfection with a recombinant nucleic acid molecule; the T cells comprise cells transfected with a recombinant nucleic acid molecule encoding a polypeptide that binds to a tumor cell antigen; the polypeptide is a chimeric antigen receptor; The composition is administered intraventricularly; The composition is administered into the central canal of the spinal cord; the administration is into the left or right ventricular ventricle; The composition has a molecular weight of at least 1×10 6 Contains cells; The composition comprising T cells is administered at least twice; The administrations vary in the total number of T cells administered; said administration escalates in dose; said administration de-escalates the dose; the T cells comprise CAR T cells; the T cells comprise autologous tumor-infiltrating lymphocytes; the T cells comprise TCR-recombinant T cells; the malignant tumor is a diffuse infiltrative tumor; the malignant tumor is a primary brain tumor; a reduction in size of one or more tumor lesions by at least 25%; the malignancy arises from a primary cancer selected from breast cancer, lung cancer, head and neck cancer, and melanoma; The method is performed after tumor resection; further comprising intratumoral administration of a composition comprising the T cells; the malignant tumor is a secondary brain tumor; further comprising intratumoral administration of a composition comprising therapeutic T cells that express a chimeric antigen receptor that binds to a protein expressed on the surface of glioblastoma cells; The patient has previously undergone resection of a tumor lesion; the tumor antigen is selected from the group consisting of IL13Rα2, HER2, PSCA, EGFR, EGFRvIII, EphA2, NY-ESO-1, and CD19; the T cells comprise both CD4+ and CD8+ cells; the T cells were subjected to ex vivo expansion; The T cells are at least 10% T CM Contains cells; at least 40%, 50%, 60%, 70% or more of the infused cells are CD4+; At least 40%, 50%, 60%, 70% or more of the injected cells express a cell surface receptor that targets the tumor antigen (e.g., IL13Rα2); and The dose of cells is based on the number of injected cells expressing a cell surface receptor that targets the tumor antigen (e.g., IL13Rα2). It is.

[0009] In one embodiment, the CAR T cells target IL13Rα2; and a nucleic acid molecule encoding a chimeric antigen receptor comprising human IL-13 or a variant thereof having 1 to 10 amino acid modifications; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having from 1 to 10 amino acid modifications, the transmembrane domain of CD8 or a variant thereof having from 1 to 10 amino acid modifications, the transmembrane domain of CD28 or a variant thereof having from 1 to 10 amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having from 1 to 10 amino acid modifications; at least one costimulatory domain; and the CD3ζ signaling domain in its variants with 1–10 amino acid modifications; In one embodiment, the costimulatory domain is selected from the group consisting of a CD28 costimulatory domain or a variant thereof having 1 to 10 amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1 to 10 amino acid modifications, an OX40 costimulatory domain or a variant thereof having 1 to 10 amino acid modifications; said variant of human IL-13 has 1 to 10 amino acid modifications that enhance the binding specificity of IL13Rα2 versus IL13Rα1; the human IL-13 or variant thereof is an IL-13 variant comprising the amino acid sequence of SEQ ID NO: 3 with 1 to 5 amino acid modifications, with the proviso that position 11 of SEQ ID NO: 3 is other than E; the chimeric antigen receptor comprises two different costimulatory domains selected from the group consisting of a CD28 costimulatory domain or a variant thereof having 1 to 10 amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1 to 10 amino acid modifications, and an OX40 costimulatory domain or a variant thereof having 1 to 10 amino acid modifications; the chimeric antigen receptor comprises two different costimulatory domains selected from the group consisting of a CD28 costimulatory domain or a variant thereof with one to two amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with one to two amino acid modifications, and an OX40 costimulatory domain or a variant thereof with one to two amino acid modifications; The chimeric antigen receptor Human IL-13 or a variant thereof having one or two amino acid modifications; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having one to two amino acid modifications, the transmembrane domain of CD8 or a variant thereof having one to two amino acid modifications, the transmembrane domain of CD28 or a variant thereof having one to two amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having one to two amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain in its variants with one or two amino acid modifications; Contains; the CAR comprises a spacer region located between the IL-13 or variant thereof and the transmembrane domain; the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14-20, 50, and 52; The chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 31-48.

[0010] In one embodiment, the T cell expresses a chimeric antigen receptor that binds to HER2, a nucleic acid molecule encoding a chimeric antigen receptor comprising a HER2 targeting domain; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having one to five amino acid modifications, the transmembrane domain of CD8 or a variant thereof having one to five amino acid modifications, the transmembrane domain of CD28 or a variant thereof having one to five amino acid modifications, and the transmembrane domain of CD3s or a variant thereof having one to five amino acid modifications; a costimulatory domain selected from a CD28 costimulatory domain or a variant thereof having one to five amino acid modifications and a 4-1BB costimulatory domain or a variant thereof having one to five amino acid modifications; and the CD3s signaling domain and its variants with one to five amino acid modifications; In certain embodiments, the HER2 targeting domain is a HER2 scFv; The HER2 scFv has the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 49) or a variant thereof having 1-5 amino acid modifications; The chimeric antigen receptor HER2 targeting sequence; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof with one to two amino acid modifications, the transmembrane domain of CD8 or a variant thereof with one to two amino acid modifications, the transmembrane domain of CD28 or a variant thereof with one to two amino acid modifications, and the transmembrane domain of CD3s or a variant thereof with one to two amino acid modifications; a costimulatory domain selected from a CD28 costimulatory domain or a variant thereof having one to two amino acid modifications and a 4-1BB costimulatory domain or a variant thereof having one to two amino acid modifications; and the CD3s signaling domain in its variants with one or two amino acid modifications; The nucleic acid molecule expresses a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 26 and 27, or a variant thereof having from 1 to 5 amino acid modifications.

[0011] Also described herein is a method of treating a patient diagnosed with a central nervous system malignancy, comprising injecting an effective amount of a composition comprising T cells into an anatomical compartment of the patient diagnosed with a central nervous system malignancy, the anatomical compartment containing cerebrospinal fluid (CSF). In various embodiments, said anatomical compartment comprising a portion of the ventricular system; said anatomical compartment comprising a portion of the central canal of the spinal cord; said malignant tumors of the central nervous system include brain tumors; said malignant tumor of the central nervous system including metastatic tumors; said anatomical compartment contains a continuous volume of at least about 50 mL of cerebrospinal fluid; said anatomical compartment contains a continuous volume of at least about 100 mL of cerebrospinal fluid; The anatomical compartment contains a continuous volume of at least about 150 mL of cerebrospinal fluid.

[0012] Cancers that can be treated by the methods described herein include: Tumors include the following: primary CNS malignancies and secondary malignancies arising from cancer elsewhere, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratocarcinoma / rhabdoid tumor, central nervous system, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, bronchial tumor, Burkitt's lymphoma, calcified leukemia, leukemia, and leukemia. tinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, fibrous histiocytoma of bone, malignant and osteosarcoma, gallbladder cancer, gastric,stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, hepatocellular carcinoma (primary), pancreatic cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, bone cancer fibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with primary midline carcinoma associated with the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumor / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity) cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma (pineoblastoma), cerebellar tentorial epithelial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell neck cancer, gastric (stamach, Examples of cancers that may be treated include cancers selected from the group consisting of gastric cancer, supratentorial ventricular primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, ureteral cancer, renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0013] In some embodiments, the malignancy treated by the disclosed methods comprises a tumor. In some embodiments, the treatment results in at least a 50% reduction in tumor volume, at least a 60% reduction in tumor volume, at least a 70% reduction in tumor volume, at least an 80% reduction in tumor volume, at least a 90% reduction in tumor volume. And in some embodiments, the treatment results in elimination of the malignancy.

[0014] In one embodiment, the patient does not experience grade 3 or higher toxicity.

[0015] In some embodiments, the patient is administered a steroid regimen prior to treatment with the composition comprising an effective amount of T cells, and in some embodiments, the steroid regimen is reduced to a lower dose following the treatment.

[0016] In some embodiments, the patient has a longer life expectancy compared to patients receiving standard of care ("SOC") treatment, including radiation therapy, small molecule drug therapy, antibody therapy, or a combination thereof. In some embodiments, the patient receiving standard of care ("SOC") treatment can expect to survive approximately 15 months from initial diagnosis (overall survival or OS), whereas patients receiving the disclosed treatment can expect an OS of 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more. In some embodiments, patients receiving the claimed treatment can expect an OS of 42, 48, 54, 60, 66, 72, 78, 84, 90 months or more.

[0017] In one embodiment, the composition has at least 2×10 6 2 x 10 T cells or 2 x 10 expressing cell surface receptors targeting tumor antigens 6 While in one embodiment the composition comprises at least 1×10 T cells, 6 1 x 10 T cells or 1 x 10 expressing cell surface receptors targeting tumor antigens 6 In one embodiment, the composition comprises at least 5×10 T cells. 65 x 10 T cells expressing cell surface receptors targeting tumor antigens 6 While in one embodiment the composition comprises at least 10×10 T cells, 6 10 x 10 T cells expressing cell surface receptors targeting tumor antigens 6 In one embodiment, the disclosed method includes administering the composition repeatedly, e.g., administering at least 5 times the composition repeatedly, administering at least 10 times the composition repeatedly, or administering at least 90×10 T cells to the patient. 6 and repeating the administration until a full dose of T cells or T cells expressing a cell surface receptor targeting a tumor antigen has been received. In one embodiment, the administration is repeated once per week or once every two weeks. In one embodiment, the repeated administration continues for 15 weeks.

[0018] Also disclosed herein is a method of increasing the level of at least one cytokine or chemokine in cerebrospinal fluid (CSF) of a patient, comprising: administering an effective amount of a composition comprising T cells to the CSF of a patient with a central nervous system malignancy; The method, wherein the level of at least one cytokine or chemokine in the CSF is increased after administration of the composition comprising an effective amount of T cells compared to a baseline level of the at least one cytokine or chemokine before administration.

[0019] In certain embodiments of the disclosed methods, the level of the at least one cytokine or chemokine in the CSF following said administration is increased 10-fold or 5-fold compared to the baseline level.

[0020] In some embodiments, the levels of at least five cytokines or chemokines are increased after administration of the composition comprising an effective amount of T cells, as compared to baseline levels of the at least five cytokines or chemokines prior to administration. In some embodiments, the at least one cytokine or chemokine comprises EGF, eotaxin, FGF, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Rα, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, TNF-α, or VEGF. In some embodiments, the increase in cytokine or chemokine expression is localized (i.e., specific to the CSF).

[0021] Also disclosed herein is a method for maintaining elevated numbers of T cells for at least about 5 days relative to baseline numbers observed in cerebrospinal fluid (CSF) of a patient diagnosed with a central nervous system malignancy, comprising: injecting an effective amount of T cells into the CSF of a patient diagnosed with a malignancy of the central nervous system; The observed number of T cells compared to the baseline number observed prior to said infusion step. The increase is sustained for at least about five days.

[0022] In one embodiment, an effective amount of T cells (or T cells expressing a cell surface receptor that targets the tumor antigen) is about 1×10 6 From 100 x 10 cells 6 and in one embodiment, an effective amount of T cells ranges from about 2×10 6 Approximately 50 × 10 cells 6 The range is 10 cells.

[0023] In one embodiment, the increase in observed T cell numbers is sustained for at least about 6 days, or the observed T cell numbers do not return to the baseline numbers for about 7 days.

[0024] In some embodiments, the observed T cells include infused T cells (e.g., CAR-expressing T cells), and in some embodiments, the observed T cells include endogenous T cells.

[0025] Also disclosed herein is a method of increasing the number of T cells in cerebrospinal fluid (CSF) of a patient, comprising: administering an effective amount of a composition comprising T cells to the CSF of a patient with a central nervous system malignancy; The method wherein the number of T cells detectable in the CSF is increased compared to pre-administration levels.

[0026] In some embodiments, the number of T cells detectable in the CSF is elevated compared to pre-administration levels for up to 7 days post-administration, hi some embodiments, the T cells detectable in the CSF comprise endogenous T cells and CAR-expressing T cells, and / or type 1 T cells, and / or type 2 T cells.

[0027] In one embodiment, the T cells detectable in the CSF comprise CD3+ T cells, and in one embodiment, the T cells detectable in the CSF comprise a CD14+ CD11b+ HLA-DR+ mature myeloid population, and in one embodiment, CD19+ B cells and CD11b+ CD15+ granulocytes can be detected in the CSF following administration of the composition.

[0028] In certain embodiments, reactive lymphocytes, monocytes, and macrophages can be detected in the CSF following administration of the composition.

[0029] Also disclosed herein is a method of determining the suitability of a patient diagnosed with a malignant tumor for treatment with IL-13Rα2-specific CAR T cells, comprising determining whether a score resulting from a sample from said patient indicates IL-13Rα2 expression above a predetermined threshold.

[0030] In one embodiment, the score attributed to a resected tumor sample from a patient diagnosed with a malignant tumor is calculated by determining the immunoreactivity of the sample by immunohistochemically staining with a marker for IL-13Rα2, analyzing the intensity of the staining, and calculating a score based on the intensity of the staining; A score corresponding to moderate to strong staining intensity in the sample indicates that the patient is suitable for treatment with IL-13Rα2-specific CAR T cells. In one embodiment, the score involves counting the number of cells with weak, moderate or strong staining intensity and assigning a weight to each intensity (H score, a method for quantifying immunohistochemical results using the following formula: (3× percentage of cells with strong staining) + (2× percentage of cells with moderate staining) + (1× percentage of cells with weak staining), (Based on the above, the range is 0-300). The patient may have an H-score for the relevant tumor-associated antigen of greater than 50, 50-100, greater than 100, 100-200, greater than 200, 100-300, or greater than 250. In one embodiment, expression of Ki67 in the sample is also determined by immunohistochemical staining.

[0031] Also disclosed herein is a method of treating a patient with a malignancy comprising administering to a patient diagnosed with a malignancy a composition comprising an effective amount of IL-13Rα2-specific CAR T cells, wherein the patient expresses IL-13Rα2 above a predetermined threshold.

[0032] In one embodiment, said predetermined threshold of IL-13Rα2 expression has been previously identified as suitable for treatment, including IL-13Rα2-specific CAR T cell therapy.

[0033] TILs are tumor-infiltrating lymphocytes that can be isolated from a patient or donor, expanded ex vivo, and reinfused into a patient in need thereof.

[0034] CAR T cells express a chimeric T cell receptor comprising an extracellular domain, a transmembrane region and an intracellular signaling domain. The extracellular domain comprises a portion that binds to a target cell and optionally a spacer, e.g., a portion of a human Fc domain. The transmembrane portion comprises a suitable transmembrane domain, e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 transmembrane domain or a 4IBB transmembrane domain. The intracellular signaling domain comprises a signaling domain derived from the zeta chain of the human CD3 complex (CD3zeta) and one or more costimulatory domains, e.g., a 4-1BB costimulatory domain. The target cell binding portion of the extracellular domain (e.g., scFv or ligand) can direct T cell activity when the CAR is expressed on the surface of a T cell to cells expressing a target cell surface molecule, e.g., HER2 or IL13Rα2, receptors expressed on the surface of tumor cells, including malignant glioma cells.

[0035] A variety of different T cells, e.g., patient-specific autologous T cells, can be engineered to express a TCR or CAR. A variety of T cell subsets can be used. Additionally, CARs can be expressed on other immune cells, such as NK cells. When a patient is treated with immune cells expressing a CAR or TCR, the cells can be autologous or allogeneic T cells. In some cases, the cells used are CD4+ and CD8+ central memory T cells (T CM ), and the use of such cells can improve the long-term persistence of the cells following adoptive transfer compared to the use of other types of patient-specific T cells. CM The cells can include CD4+ cells and CD8+ cells.

[0036] Among the CARs useful in the methods described herein are those that target IL13Rα2. Such CARs may include IL13 with amino acid modifications (e.g., E13Y mutations) that enhance binding specificity.

[0037] The T cells used in the methods described herein can contain a nucleic acid molecule encoding a chimeric antigen receptor (CAR), in which the chimeric antigen receptor is human IL-13 or a variant thereof having 1 to 10 (e.g., 1 or 2) amino acid modifications; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications, the transmembrane domain of CD8 or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications, the transmembrane domain of CD28 or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain of a variant thereof having 1 to 10 (e.g., 1 or 2) amino acid modifications; Includes.

[0038] The inclusion of a costimulatory domain, for example a 4-1BB (CD137) or CD28 costimulatory domain in tandem with CD3ζ in the intracellular region, allows the T cell to receive a costimulatory signal. Thus, in various embodiments, the costimulatory domain is selected from the group consisting of a CD28 costimulatory domain or a variant thereof with 1-10 (e.g. 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with 1-10 (e.g. 1 or 2) amino acid modifications, an OX40 costimulatory domain or a variant thereof with 1-10 (e.g. 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof with 1-10 (e.g. 1 or 2) amino acid modifications is present.

[0039] In a further embodiment of the method, the CAR expressed by the T cell comprises a variant of human IL-13 with 1-10 amino acid modifications that enhance binding specificity for IL13Rα2 versus IL13Rα1; the human IL-13 or variant thereof is an IL-13 variant comprising the amino acid sequence of SEQ ID NO: 3 with 1 to 5 amino acid modifications, with the proviso that position 11 of SEQ ID NO: 3 is other than E; two different costimulatory domains selected from the group consisting of a CD28 costimulatory domain or a variant thereof having 1-10 (e.g. 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-10 (e.g. 1 or 2) amino acid modifications, an OX40 costimulatory domain or a variant thereof having 1-10 (e.g. 1 or 2) amino acid modifications; the chimeric antigen receptor comprises two different costimulatory domains selected from the group consisting of a CD28 costimulatory domain or a variant thereof with one to two amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with one to two amino acid modifications, and an OX40 costimulatory domain or a variant thereof with one to two amino acid modifications; Human IL-13 or a variant thereof having one or two amino acid modifications; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having one to two amino acid modifications, the transmembrane domain of CD8 or a variant thereof having one to two amino acid modifications, the transmembrane domain of CD28 or a variant thereof having one to two amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having one to two amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain in its variants with one or two amino acid modifications; a spacer region located between the IL-13 or variant thereof and the transmembrane domain, the spacer region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14 to 20, 50, and 52; the spacer comprises an IgG hinge region; the spacer region comprises 10 to 150 amino acids; the 4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO:6; the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:7; and A linker of 3 to 15 amino acids located between the costimulatory domain and the D3ζ signaling domain or a variant thereof. In certain embodiments where there are two costimulatory domains, one is a 4-1BB costimulatory domain and the other is a costimulatory domain selected from CD28 and CD28gg.

[0040] In certain embodiments of the methods described herein, the T cell harbors a nucleic acid molecule that expresses a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 10 and 31-48; The chimeric antigen receptor an IL-13 / IgG4 / CD4t / 41-BB region comprising the amino acid sequence of SEQ ID NO:11, and a CD3ζ signaling domain comprising the amino acid sequence of SEQ ID NO:7; The chimeric antigen receptor comprises the amino acid sequences of SEQ ID NOs: 10 and 31-48.

[0041] Also disclosed is a method comprising intraventricular administration of a population of human T cells transduced with a vector comprising an expression cassette encoding a chimeric antigen receptor. Human IL-13 or a variant thereof having 1 to 10 amino acid modifications; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having from 1 to 10 amino acid modifications, the transmembrane domain of CD8 or a variant thereof having from 1 to 10 amino acid modifications, the transmembrane domain of CD28 or a variant thereof having from 1 to 10 amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having from 1 to 10 amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain in its variants with 1–10 amino acid modifications; In various embodiments, the population of human T cells comprises: A vector expressing a chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NOs: 10 and 31 to 48; The population of human T cells is classified as central memory T cells (T CMcells) (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T CM a T cell; CM At least 10%, 15%, 20%, 25%, 30% or 35% of the cells are CD4+, and CM At least 10%, 15%, 20%, 25%, 30% or 35% of the cells are CD8+ cells).

[0042] Also described are populations of autologous or allogeneic human T cells transduced with a vector containing an expression cassette encoding a chimeric antigen receptor (e.g., T CM autologous or allogeneic T cells; at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of said cells being T CM The T cells CM At least 15%, 20%, 25%, 30%, 35% of the cells are CD4+, CM In one embodiment, the method comprises CNS administration of a chimeric antigen receptor (at least 15%, 20%, 25%, 30%, 35% of the cells are CD8+ cells), wherein the chimeric antigen receptor comprises a selected amino acid sequence selected from SEQ ID NOs: 10 and 31-48. said population of human T cells comprises central memory T cells; the cancer is glioblastoma; and the transduced human T cells when prepared by a method comprising obtaining T cells from the patient, treating the T cells to isolate central memory T cells, and transducing at least a portion of the central memory cells with a viral vector comprising an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NOs: 10 and 31-48. It is.

[0043] Also described is a method, wherein the T cells administered to the patient comprise: SEQ ID NO: 10 and SEQ ID NO: 10 and 31 to 48 a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID NOs: 10 and 31-48, except for the presence of five or fewer amino acid substitutions, deletions, or insertions; A nucleic acid molecule encoding a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID NO: 10 and SEQ ID NO: 10 and 31-48, except for the presence of five or fewer amino acid substitutions; and a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID NO: 10 and SEQ ID NO: 10 and 31-48, except for the presence of no more than two amino acid substitutions; is inside.

[0044] Described herein is a method of treating a patient by CSF administration of T cells harboring a nucleic acid molecule encoding a chimeric antigen receptor (CAR). HER2-targeting scFv (e.g., amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS; comprising SEQ ID NO: 49) or a variant thereof having 1 to 10 (e.g. 1 or 2) amino acid modifications; spacer region; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications, the transmembrane domain of CD8 or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications, the transmembrane domain of CD28 or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having from 1 to 10 (e.g. 1 or 2) amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain of a variant thereof having 1 to 10 (e.g., 1 or 2) amino acid modifications; Includes.

[0045] In various embodiments, the costimulatory domain is selected from the group consisting of a CD28 costimulatory domain or a variant thereof with 1-10 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with 1-10 (e.g., 1 or 2) amino acid modifications, an OX40 costimulatory domain or a variant thereof with 1-10 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof with 1-10 (e.g., 1 or 2) amino acid modifications is present.

[0046] In an additional embodiment, the T cells administered to the patient express a CAR, said CAR comprising: scFv targeting HER2 (e.g., humanized scFv); two different costimulatory domains selected from the group consisting of a CD28 costimulatory domain or a variant thereof having 1-10 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-10 (e.g., 1 or 2) amino acid modifications, and an OX40 costimulatory domain or a variant thereof having 1-10 (e.g., 1 or 2) amino acid modifications; two different costimulatory domains selected from the group consisting of a CD28 costimulatory domain or a variant thereof with one to two amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with one to two amino acid modifications, and an OX40 costimulatory domain or a variant thereof with one to two amino acid modifications; Human IL-13 or a variant thereof having one or two amino acid modifications; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having one to two amino acid modifications, the transmembrane domain of CD8 or a variant thereof having one to two amino acid modifications, the transmembrane domain of CD28 or a variant thereof having one to two amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having one to two amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain in its variants with one or two amino acid modifications; a spacer region located between the IL-13 or variant thereof and the transmembrane domain (e.g., the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 14-20, 50, and 52); the spacer comprises an IgG hinge region; the spacer region comprises 10 to 150 amino acids; the 4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO:6; the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:7; a linker of 3 to 15 amino acids located between the costimulatory domain and the CD3ζ signaling domain or variant thereof; In certain embodiments where there are two costimulatory domains, one is a 4-1BB costimulatory domain and the other is a costimulatory domain selected from CD28 and CD28gg.

[0047] In one embodiment, the T cells administered to the patient harbor a nucleic acid molecule that expresses a polypeptide comprising an amino acid sequence selected from SEQ ID NOs:53-56.

[0048] Also disclosed is a method comprising intraventricular administration of a population of human T cells transduced with a vector comprising an expression cassette encoding a chimeric antigen receptor, HER2-targeting scFv; a transmembrane domain selected from the transmembrane domain of CD4 or a variant thereof having from 1 to 10 amino acid modifications, the transmembrane domain of CD8 or a variant thereof having from 1 to 10 amino acid modifications, the transmembrane domain of CD28 or a variant thereof having from 1 to 10 amino acid modifications, and the transmembrane domain of CD3ζ or a variant thereof having from 1 to 10 amino acid modifications; A costimulatory domain; and the CD3ζ signaling domain in its variants with 1–10 amino acid modifications; In various embodiments, the population of human T cells comprises: A vector expressing a chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NOs: 53 to 56; The population of human T cells is classified as central memory T cells (T CM cells) (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T CM a T cell; CM At least 10%, 15%, 20%, 25%, 30%, 35% of the cells are CD4+, and / or the T cells or T CM At least 10%, 15%, 20%, 25%, 30%, 35% of the cells are CD8+ cells).

[0049] Also described are populations of autologous or allogeneic human T cells transduced with a vector containing an expression cassette encoding a chimeric antigen receptor (e.g., T CM Cells autologous or allogeneic T cells; e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T CM The T cells CM At least 15%, 20%, 25%, 30%, 35% of the cells are CD4+, and / or the T CM In one embodiment, the method comprises administering to a patient a chimeric antigen receptor (CAR) comprising administering to the patient a chimeric antigen receptor (CAR) comprising at least 15%, 20%, 25%, 30%, 35% of the cells being CD8+ cells), wherein the CAR comprises an amino acid sequence selected from SEQ ID NOs: 53-56. said population of human T cells comprises central memory T cells; the cancer is glioblastoma; and the transduced human T cells when prepared by a method comprising obtaining T cells from the patient, treating the T cells to isolate central memory T cells, and transducing at least a portion of the central memory cells with a viral vector comprising an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NOs: 53-56.

[0050] Also described is a method of treating cancer in a patient comprising intracranial administration of a population of autologous or allogeneic human T cells. CM Autologous or allogeneic T cell containing cells A nucleic acid molecule encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 53 to 56; a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID NOs: 53-56, except for the presence of five or fewer amino acid substitutions, deletions, or insertions; A nucleic acid molecule encoding a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID NOs: 53-56, except for the presence of five or fewer amino acid substitutions; and a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence identical to an amino acid sequence selected from SEQ ID NOs: 53-56, except for the presence of no more than two amino acid substitutions; is inside.

[0051] Certain CARs described herein, such as the IL13(EQ)BBζ CAR and the IL13(EQ)CD28-BBζ CAR, have certain advantageous characteristics compared to certain other IL13-targeted CARs, for example, they have improved selectivity for IL13Rα and elicit low Th2 cytokine production, particularly low IL13 production.

[0052] T cells expressing CARs that target IL13Rα2 may be useful in treating cancers such as glioblastoma, as well as other cancers that express IL13Rα2. Thus, the present disclosure encompasses methods for treating cancer using T cells expressing the CARs described herein.

[0053] T cells expressing CARs that target HER2 may be useful in treating cancers such as glioblastoma, as well as other cancers that express HER2, such as breast cancer that has spread to the central nervous system. Thus, the present disclosure encompasses methods for treating cancer using T cells expressing the CARs described herein.

[0054] The CARs described herein can include a spacer region located between the targeting domain and the transmembrane domain. A variety of different spacers can be used. Some of them include at least a portion of a human Fc region, such as a hinge portion or a CH3 domain of a human Fc region or a variant thereof. Table 1 below provides various spacers that can be used in the CARs described herein.

[0055] Table 1: Examples of spacers [Table 1]

[0056] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that corresponds between the CH1 and CH2 domains of an immunoglobulin, such as an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain or both the CH3 and CH2 domains. The immunoglobulin-derived sequence can include one or more amino acid modifications, such as 1, 2, 3, 4, or 5 substitutions, such as substitutions that reduce off-target binding.

[0057] "Amino acid modification" refers to amino acid substitution, insertion, and / or deletion. "Amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position, and "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position in a parent peptide or protein sequence with another amino acid. Substitutions can be made to change the amino acids of the resulting protein non-conservatively (i.e., by changing the codon from an amino acid belonging to a group of amino acids of a particular size or characteristic to an amino acid belonging to another group) or conservatively (i.e., by changing the codon from an amino acid belonging to a group of amino acids of a particular size or characteristic to an amino acid belonging to the same group). Such conservative changes generally result in less change in the structure and function of the resulting protein. Below are examples of various groups of amino acids: 1) Amino acids with nonpolar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; 2) Amino acids with uncharged polar R groups: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): aspartic acid, glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (at pH 6.0). Another group may be amino acids with a phenyl group: phenylalanine, tryptophan, and tyrosine.

[0058] The CAR expressed by the cells used in the methods described herein may be used with a variety of transmembrane domains. Table 2 includes examples of suitable transmembrane domains. When a spacer region is present, the transmembrane region is located at the carboxy terminus of the spacer region.

[0059] Table 2: Examples of transmembrane domains [Table 2]

[0060] Many of the CARs expressed by the cells used in the methods described herein contain one or more (e.g., two) costimulatory domains. The costimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable costimulatory domains along with the sequence of the CD3ζ signaling domain.

[0061] Table 3: Examples of costimulatory domains [Table 3] [Brief description of the drawings]

[0062] [Figure 1] Schematic diagram of the IL13(E13Y)-zetakine CAR (left), which is composed of an IL13Rα2-specific human IL-13 variant (huIL-13(E13Y)), a human IgG4 Fc spacer (huγ4Fc), a human CD4 transmembrane (huCD4 tm) and a human CD3 cytoplasmic (huCD3 ζcyt) protein. Also shown is the IL13(EQ)BBζCAR, which is identical to the IL13(E13Y)-zetakine except for two point mutations located in the CH2 domain of the IgG4 spacer, L235E and N297Q shown in red, and the addition of a costimulatory 4-1BB cytoplasmic domain (4-1BBcyt). [Diagram 2]Specific vectors and open reading frames are shown. Figure 2A is a diagram of the cDNA open reading frame of the 2670 nucleotide IL13(EQ)BBZ-T2ACD19t construct, in which the IL13Rα2 specific ligand IL13(E13Y), IgG4(EQ)Fc hinge, CD4 transmembrane, 4-1BB cytoplasmic signaling, 3-glycine linker, and CD3ζ cytoplasmic signaling domain of the IL13(EQ)BBζCAR, as well as the T2A ribosomal skip and truncated CD19 sequences are shown. Also shown is the human GM-CSF receptor α and CD19 signal sequence that drives surface expression of L13(EQ)BBζCAR and CD19t. Figure 2B is a diagram of the long terminal repeat (indicated with "R") flanked sequences that will be integrated into the host genome. Figure 2C is a map of the IL13(EQ)BBZ-T2A-CD19t_epHIV7 plasmid. [Diagram 3] 1 shows the construction of pHIV7. [Figure 4] The elements of pHIV7 are shown. [Diagram 5] The production plan for IL13(EQ)BBζ / CD19t+ TCM is shown. [Figure 6] Flow cytometry analysis of surface transgene and T cell marker expression is shown. IL-13(EQ)BBζ / CD19t+ TCM HD006.5 and HD187.1 were co-stained with anti-IL-13-PE and anti-CD8-FITC to detect CD8+ CAR+ and CD4+ (i.e., CD8 negative) CAR+ cells (A) or with anti-CD19-PE and anti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4 negative) CAR+ cells (B). IL13(EQ)BBζ / CD19t+ TCM HD006.5 and HD187.1 (C) stained with fluorochrome-conjugated anti-CD3, TCR, CD4, CD8, CD62L, and CD28 (grey histograms) or with isotype controls (black histograms). In all cases, percentages are based on viable lymphocytes (DAPI negative) stained with the isotypes indicated. [Figure 7]Results of an experiment comparing the route of CAR+ T cell delivery to large established tumors (ic vs iv) are shown. EGFP-ffLuc+ PBT030-2 TS (1x105) were implanted into the right forebrain of NSG mice. On days 19 and 26, mice were injected iv via the tail vein with either 5x106 CAR+ IL13(EQ)BBζ+ TCM (11.8x106 total cells; n=4) or mock TCM (11.8x106 cells; n=4). Alternatively, on days 19, 22, 26, and 29, mice were injected ic with either 1x106 CAR+ IL13(EQ)BBζ+ TCM (2.4x106 total cells; n=4) or mock TCM (2.4x106 cells; n=5). Mean ffLuc flux (photons / sec) over time shows that ic delivered IL13(EQ)BBζ TCM mediates tumor regression of tumors at day 19. In comparison, iv delivered T cells showed no reduction in tumor burden compared to untreated or mock TCM controls (A). Kaplan Meier survival curves show improved survival for mice treated ic with IL13(EQ)BBZ TCM compared to mice treated with iv administered CAR+ TCM (p=0.0003 log-rank test) (B). Representative H&E and CD3 IHC of mice treated ic with IL13(EQ)BBZ+ TCM (C) vs. mice treated ic with IL13(EQ)BBZ+ TCM (D). CD3+ T cells were only detected in the ic treated group with no CD3+ cells detected in the tumor or surrounding brain parenchyma of iv treated mice. [Figure 8]Figure 1 shows results of a study demonstrating that CAR+ T cells injected intracranially, either intratumorally (ict) or intracerebroventricularly (icv), can traffic to tumors on the contralateral hemisphere. EGFP-ffLuc+ PBT030-2 TS (1x105) were stereotactically implanted into the left and right forebrain of NSG mice. On day 6, mice were injected ic with 1.0x106 IL13(EQ)BBζ+ TCM (1.6x106 total cells; 63% CAR; n=4) into the right tumor site. Schematic of multifocal glioma experimental model (A). CD3 IHC showing T cells infiltrating both the left and right tumor sites (B). [Figure 9] The amino acid sequence of IL13(EQ)BBζ / CD19t+ is shown (SEQ ID NO: 10). [Figure 10] 1 shows a sequence comparison of IL13(EQ)41BBζ [IL13(EQ)41BBζ T2A-CD19t_epHIV7; pF02630] (SEQ ID NO: 12) and CD19Rop_epHIV7 (pJ01683) (SEQ ID NO: 13). [Figure 11] 1 shows the amino acid sequence of IL13(EmY)-CD8h3-CD8tm2-41BBzeta (SEQ ID NO: 31 with GMCSFRa signal peptide; SEQ ID NO: 39 without GMCSFRa signal peptide). [Figure 12] 1 shows the amino acid sequence of IL13(EmY)-CD8h3-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO: 32 with GMCSFRa signal peptide; SEQ ID NO: 40 without GMSCFRa signal peptide). [Figure 13] 1 shows the amino acid sequence of IL13(EmY)-IgG4(HL-CH3)-CD4tm-41BB-Zeta (SEQ ID NO: 33 with GMCSFRa signal peptide; SEQ ID NO: 41 without GMCSFRa signal peptide). [Figure 14] 1 shows the amino acid sequence of IL13(EmY)-IgG4(L235E,N297Q)-CD8tm-41BB-Zeta (SEQ ID NO: 34 with GMCSFRa signal peptide; SEQ ID NO: 42 without GMCSFRa signal peptide). [Figure 15] 1 shows the amino acid sequence of IL13(EmY)-linker-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO: 35 with GMCSFRa signal peptide; SEQ ID NO: 43 without GMCSFRa signal peptide). [Figure 16] 1 shows the amino acid sequence of IL13(EmY)-HL-CD28m-CD28gg-41BB-Zeta (SEQ ID NO: 36 with GMCSFRa signal peptide; SEQ ID NO: 44 without GMSCFRa signal peptide). [Figure 17] 1 shows the amino acid sequence of IL13(EmY)-IgG4(HL-CH3)-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO: 37 with GMSCFRa signal peptide; SEQ ID NO: 45 without GMCSFRa signal peptide). [Figure 18] 1 shows the amino acid sequence of IL13(EmY)IgG4(L235E,N297Q)-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO: 38 with GMCSFRa signal peptide; SEQ ID NO: 46 without GMCSFRa signal peptide). [Figure 19] 1 shows the amino acid sequence of IL13(EmY)-CD8h3-CD8tm-41BBzeta (SEQ ID NO: 47 with GMCSFRa signal peptide; SEQ ID NO: 48 without GMCSFRa signal peptide). [Figure 20] 2 shows the amino acid sequence of Her2scFv-IgG4(L235E,N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t. The various domains are listed in order below the sequence and are indicated by alternating underlining and non-underlining. The mature CAR sequence (SEQ ID NO:26) does not include the GMCSFRa signal peptide, the T2A skip sequence or the truncated CD19. [Figure 21]2 shows the amino acid sequence of Her2scFv-IgG4(L235E,N297Q)-CD8tm-41BB-Zeta-T2A-CD19t. The various domains are listed in order below the sequence and are indicated by alternating underlining and non-underlining. The mature CAR sequence (SEQ ID NO:27) does not include the GMCSFRa signal peptide, the T2A skip sequence or the truncated CD19. [Figure 22] HER2-specific CAR constructs and CAR T cell expansion data are shown. [Diagram 23] Shows in vitro characterization of HER2-CAR T cells against breast cancer cell lines. [Figure 24] 1 shows the results of a study on the in vitro antitumor activity of HER2-CAR T cells. [Diagram 25] 1 shows the results of a study on the in vivo antitumor efficacy of locally intratumorally delivered HER2-CAR T cells. [Figure 26] 1 shows the results of a study on local delivery of HER2-CAR T cells in a human orthotopic BBM xenograft model. [Figure 27] 1 shows the results of a study on intraventricular delivery of HER2-CAR T cells. [Figure 28] Schematic showing the locations of tumor cell injection and CAR T cell injection for a study of intratumoral and intraventricular injection of CAR T cells targeting IL13α2R in a murine model of glioblastoma. [Figure 29]Results of a study demonstrating regression of established glioma tumor xenografts following administration of IL13(EQ)BBζ / CD19t+ TCM are shown. ffLuc+ PBT030-2 tumor cells (1×105) were stereotactically implanted into the left and right forebrain of NSG mice. On day 6, mice were ict or icv injected with 1×106 IL13(EQ)BBζ+ TCM (1.6×106 total cells; 63% CAR+) as described in Figure 4.1 above. A, Representative mice from each group showing relative tumor burden using Xenogen Living Image. B, Average Xenogen flux in left and right brain hemispheres (regions of interest, ROI) from mice in each group (n=4-5). Each continuous bar represents days 5, 9, 12, 15, and 19, respectively. *, p<0.05 when compared to the respective ROI and day / bar of the untreated PBT030-2 group using unpaired Student's t-test. C, Mean xenogen flux in the whole brain 13 days after T cell injection. *, p=0.0407 when comparing the icv group to the untreated PBT030-2 group using unpaired Student's t-test. These data are representative of three separate multifocal GBM experiments. [Diagram 30] Results of a study demonstrating that huCD3+ cells are detected in left and right brain tumors / hemispheres after ict and icv administration of IL13(EQ)BBζ / CD19t+ TCMs are shown. ffLuc+ PBT030-2 tumor cells (1×105) were stereotactically implanted into the left and right forebrain of NSG mice. On day 6, mice were either ict) injected (left image) or icv) injected (right image) with 1×106 IL13(EQ)BBζ+ TCMs (1.6×106 total cells; 63% CAR+). One week (top image) and two weeks (bottom image) after T cell administration, 2-3 mice from each group were euthanized, brains were harvested, embedded in paraffin, and IHC was performed using an anti-human CD3 antibody to detect T cells. Representative IHC images of left and right tumor sites from mice in each group (ict: m406 and m410; icv: m414 and m415) are shown. Inlays show xenogen flux images of mice on the day of euthanasia and brain harvest. [Diagram 31] Schematic diagram of CAR T cell preparation and treatment time course for clinical trials of CAR T cells for the treatment of glioblastoma (A) and provides several dosing regimens (B). [Diagram 32] Analysis of CAR T cell persistence monitored by CD19 (A) and presence of GBM cells monitored by IL13Rα2 expression (B). [Diagram 33] 1 shows imaging results for patient UPN097 in the region of the catheter used for intratumoral administration. [Diagram 34] 1 is a series of graphs showing the levels of various cytokines over the course of treatment in one patient. [Diagram 35] Images showing regression of recurrent multifocal glioblastoma including spinal metastases after intraventricular delivery of IL13Rα2-targeted CAR T cells. A patient presenting with recurrent multifocal GBM and extensive leptomeningeal disease, including one metastatic site in the spinal cord, was treated with six local injections of IL13BBζ Tcm into the resection cavity of the largest recurrent tumor focus (1 cycle of 2M and 5 cycles of 10M CAR+ T cells). The CAR T cell injection site remained stable without evidence of disease recurrence for over 7 weeks, while other lesions distant from the CAR T cell injection site continued to progress (data not shown). The patient then received five weekly intraventricular (icv) injections of IL13BBζ Tcm (1 cycle of 2M and 4 cycles of 10M CAR+ T cells). MRI and / or PET images of (A) transverse brain sections, (B) saggital brain sections, and (C) lateral (top) and anterior (bottom) sections of the spine before (left) and one week after (right) icv therapy are shown, with the tumor lesion site indicated by a red arrow in each image. [Diagram 36]Enrollment in NCT02208362 and treatment regimen using compassionate use protocol are shown. Enrollment in NCT02208362 was set on day 0 (A). Compassionate use protocol was initiated on day 107 (B). NovoTTF-100A, portable medical device delivering low intensity, intermediate frequency, alternating electric field via non-invasive, disposable scalp electrodes; FGFR, fibroblast growth factor receptor; MRI, magnetic resonance imaging, all of which were performed in the brain unless otherwise indicated; ICT, intracavitary; PET, positron emission tomography performed at designated sites; ICV, intracerebroventricular. [Figure 37] Immunohistochemistry of primary and recurrent tumors is shown. Tumor resected at initial diagnosis (A) and recurrent tumor (T1) resected at Rickham placement under NCT02208362 (B, C). Immunochemical staining with either IL13Rα2-specific or Ki67-specific DAB with hematoxylin counterstain is depicted. Successive magnified images from left to right are outlined with red boxes. [Figure 38] Identification of tumor lesions. (A) Identifying GBM lesions T1-T8 features. (B) Brain MRI scan depicting T1-T7 regions. [Figure 39]Resected tumor areas are stable without evidence of disease progression / recurrence following intracavitary delivery of IL13BBζ TCM. (A) Flow cytometry analysis of IL13BBζ TCM cell product. Top row, transduction with the indicated constructs drove coordinate surface expression of IL13BBζCAR (detected with anti-IL13) and CD19t marker (detected with anti-CD19) via the T2A ribosomal skip sequence. Staining profiles of T cell markers TCR-α / β, CD3, CD4, and CD8 and exhaustion markers LAG-3, TIM-3, KLRG1, and PD-1 are depicted in the middle row. Staining profiles of memory markers CD62L, CD45RO, CCR7, CD27, and CD28 and naive T cell marker CD45RA are shown in the bottom row. (B) Treatment scheme of NCT02208362. The patient experienced a recurrence and underwent tumor resection via placement of an intraluminal (ICT) Rickham catheter, after which he received six cycles of ICT cell doses (one cycle of 2x106 and five cycles of 10x106 CAR+ cells). There is a one-week break between cycles 3 and 4. Red arrows, IL13BBζ TCM delivery site. (C) Yellow circles on brain MRI scan indicate the site of the resected tumor where the catheter was placed (T1), as well as the resected tumor sites in the frontal lobe (T2, T3), and the newly arising tumor sites (T6, T7) over the 51-day ICT treatment period. [Diagram 40]Regression of recurrent multifocal glioblastoma encompassing spinal metastases after intraventricular delivery of IL13Rα2-targeted CAR T cells. (A) Treatment scheme under the exception use protocol. The patient underwent placement of an intraventricular (ICV) Rickham catheter followed by five cycles of ICV cell doses (1 cycle of 2×106 and 4 cycles of 10×106 CAR+ cells, indicated as cycles 7-11). There is a one-week break between cycles 9 and 10. Red arrow, IL13BBζ TCM delivery site. MRI and / or PET images of (B) transverse brain sections, (C) sagittal brain sections, and (D) lateral (top) and anterior (bottom) sections of the spine before (left) and 1 week after (right) ICV therapy. Tumor lesion sites are indicated by yellow circles in each image. (E) Maximum lesion area of ​​unresected tumors T4-T8 shows their respective decrease over time with ICV therapy. [Diagram 41] Figure 1 shows regression of recurrent multifocal GBM following ICV delivery of IL13Rα2-targeted CAR T cells. Maximum disease area of ​​unresected tumors T4-T7 is shown. [Diagram 42] Analysis of cell infiltration and cytokines from cerebrospinal fluid (CSF) samples. (A) CSF cell infiltration counts spiked after ICV cycles 9, 10, and 11 with flow cytometry evidence of both CD19+ B cells, CAR+ (i.e., CD19t+) and non-modified CD3+ T cells, CD11b+ CD15+ granulocytes, and CD11b+ CD14+ HLA-DR+ monocytes. (B) Assessment of CD3+ T cell populations in CSF for the presence of genetically modified (i.e., CD19t+) T cells. CD3 gated cells from CSF collected on selected days at cycles 9, 10, and 11 were co-stained for CD19 and CD8 (top histograms). The percentage of immunoreactive cells was then used to calculate the total CD3+ T cell counts and CD19+ CD3+ (IL13BBζ TCM) cell counts per mL of CSF fluid at each time point. (C) Fold change in cytokine levels during ICV treatment cycles 7 to 11. Only those cytokines from the 30-plex analysis showing a 10-fold or greater change compared to pre-treatment levels are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The following describes the structure, construction and characterization of various CAR T cells and their use in treating cancers of the central nervous system. Chimeric antigen receptors (CARs) are recombinant biological molecules that contain, at a minimum, an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain. Thus, the term "antigen" is not limited to molecules that bind to antibodies, but to any molecule that can specifically bind to a target. For example, CARs can encompass ligands that specifically bind to cell surface receptors. The extracellular recognition domain (also referred to as the extracellular domain or simply the recognition element it contains) contains a recognition element that specifically binds to a molecule present on the cell surface of a target cell. The transmembrane region anchors the CAR in the membrane. The intracellular signaling domain contains a signaling domain derived from the zeta chain of the human CD3 complex, and optionally contains one or more co-stimulatory signaling domains. CARs can bind antigens and transduce T cell activation, both independent of MHC restriction. Thus, CARs are "universal" immune receptors that can treat patient populations with antigen-positive tumors, regardless of their HLA genotype. Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for cancer treatment.

[0064] The CARs described herein may be made using vectors in which the CAR open reading frame is followed by a truncated CD19 (CD19t) lacking the T2A ribosomal skip sequence and cytoplasmic signaling tail (truncated at amino acid 323). With this arrangement, co-expression of CD19t provides an inert, non-immunogenic surface marker that allows accurate measurement and positive selection of genetically modified cells, as well as efficient cell tracking and / or imaging of therapeutic T cells in vivo after adoptive transfer. Co-expression of CD19t provides a marker for immunological targeting of transduced cells in vivo using clinically available antibodies and / or immunotoxin reagents to selectively eliminate therapeutic cells, thereby acting as a suicide switch.

[0065] The disclosed therapeutic methods using CAR T cells can be carried out at various doses and over various time frames. For example, a patient receiving an infusion, administration, or injection of CAR T cells (e.g., IL-13Rα2-specific CAR T cells) may receive 1×10 6 ~15×10 6 In other words, a single dose for use in the disclosed methods may comprise 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 10×10 6 , 11×10 6 , 12×10 6 , 13×10 6 , 14×10 6 , or 15 x 10 6 Over the entire treatment period, patients will receive 20×10 6 ~150×10 6 For example, the patient may receive a cumulative or total dose of about 20×10 T cells over the course of treatment. 6 , about 25×106 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6 , about 90×10 6 , about 95×10 6 , about 100×10 6 , about 105×10 6 , about 110×10 6 , about 115×10 6 , about 120×10 6 , about 125×10 6 , about 130×10 6 , about 140×10 6 , about 145×10 6 , or about 150 x 10 6 In one embodiment, a patient can receive at least 90×10 T cells. 6 In one embodiment, a patient can receive a total dose of 94×10 T cells. 6 can receive a total dose of T cells.

[0066] Furthermore, doses can be administered according to different regimens and timetables. For example, the disclosed methods can include infusions, administrations, or injections once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every other month, once every 3 months, or once every 6 months. In some embodiments, the disclosed methods can include continuous infusions, for example, from a wearable pump. Similarly, the total time course of treatment can be about 5 weeks, about 10 weeks, about 15 weeks, about 20 weeks, about 25 weeks, about 30 weeks, about 35 weeks, about 40 weeks, about 45 weeks, about 50 weeks, about 55 weeks, about 60 weeks, about 65 weeks, about 70 weeks, about 75 weeks, or more. The patient may receive 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more infusions, administrations, or injections of T cells over the course of treatment with the disclosed methods. For example, in one embodiment, a patient may receive 11 infusions of T cells over a 15 week period.

[0067] Treating cancer, and more specifically gliomas such as glioblastoma, according to the disclosed methods can provide a number of therapeutic benefits, for example, treatment with the disclosed CAR T cells can increase the levels of cytokines and chemokines in the CSF of patients treated according to the disclosed methods. Cytokine and / or chemokine expression can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%, or cytokine and / or chemokine expression can be increased by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold as compared to baseline levels measured prior to treatment with a composition comprising a CAR. This increased expression can be observed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more cytokines or chemokines.

[0068] In particular, expression of at least one of EGF, eotaxin, FGF, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Rα, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, TNF-α, and VEGF may be increased as a result of treatment with the CAR T cells disclosed herein. Furthermore, the increase in cytokine and / or chemokine expression may be localized (i.e., an increase is only observed in the CNS and CFS, while serum levels of cytokines and chemokines remain unchanged).

[0069] Treatment with the disclosed methods may also increase T cells detectable in the CSF. While it is likely that at least some of the T cells detectable in the CSF after treatment are CAR-expressing T cells, there may also be an increase in endogenous T cells recruited to the CSF. Without wishing to be bound by theory, the increase in endogenous T cells may be the result of recruitment of type 1 and type 2 T helper cells due to increased local cytokine levels. Additionally, detectable T cells may include CD3+ T cells, as well as CD14+ CD11b+ HLA-DR+ mature myeloid populations, CD19+ B cells and CD11b+ CD15+ granulocytes, and / or reactive lymphocytes, monocytes and macrophages.

[0070] An increase in the number of T cells in the CSF may be detectable for a certain period of time following treatment with the disclosed methods. A detectable increase in T cells in the CSF may persist or persist for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days or more following administration of a composition comprising the T cells. For example, the increase in the number of T cells observed in the CSF may not return to baseline levels (i.e., the number of T cells detectable prior to treatment) for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. The number of T cells detectable in the CSF may be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%, or by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, or at least 15-fold compared to baseline levels measured prior to treatment with a composition comprising CAR T cells.

[0071] The time course of CAR T cell preparation and treatment is shown in Figure 31. Concurrent with the manufacturing process, study participants underwent tumor resection followed by placement of a Rickham catheter and baseline imaging.

[0072] Patient UPN097 underwent tumor resection and received 2 × 10 6 cells, 10 × 10 in the second cycle 6In both cycles, cells were administered into the cavity remaining after resection. After cycle 2, patient UPN097 was withdrawn from the study due to rapid tumor progression.

[0073] Patient UPN109 received 2 × 10 6 cells, 10 × 10 in cycles 2 and 3 6 After a rest period, patient UPN109 was treated with 10 × 10 cells in cycles 4, 5, and 6. 6 In cycles 1-6, the cells were administered intratumorally. In cycle 7, the patient was treated with 2×10 6 In cycles 8 and 9, the patient was treated with 10×10 6 In cycles 7-10, administration was intracerebroventricularly.

[0074] The term "intraventricular" as used herein refers to the space inside the ventricular system, specifically the ventricles. Thus, the terms "intraventricular" and "intraventricular" are used interchangeably throughout this disclosure. Thus, "intraventricular administration" or "intraventricular injection" refers to the delivery of a composition to the ventricles of the brain (i.e., the cerebral ventricles). The ventricles are a series of interconnected fluid-filled spaces located in the core of the forebrain and brainstem. This system includes four ventricles, namely the left and right lateral ventricles (one of which is found in each hemisphere of the brain), the third ventricle, and the fourth ventricle.

[0075] The disclosed methods include various routes of administering the composition comprising T cells. For example, in certain embodiments, the disclosed compositions can be delivered or administered intraventricularly. In certain embodiments, the disclosed compositions can be delivered or administered into the spinal canal (i.e., intrathecal delivery). In certain embodiments, the disclosed compositions can be delivered or administered into the spinal epidural space (i.e., epidural delivery). In certain embodiments, the disclosed compositions can be delivered or administered directly to the tumor (i.e., intratumoral delivery). In certain embodiments, the disclosed compositions can be delivered or administered into a cavity formed by the resection of tumor tissue (i.e., intracavitary delivery). Furthermore, in certain embodiments, the disclosed methods can include a combination of the aforementioned routes of administration. For example, a patient can receive at least one dose of the composition comprising T cells via intracavitary delivery, followed by at least one dose of the composition via intraventricular delivery.

[0076] Figure 32A shows an analysis of CAR T cell persistence monitored by CD19. This analysis shows good T cell persistence 8 days after the second cycle. Figure 32B shows the reduced presence of GBM cells monitored by IL13Rα2 expression.

[0077] Figures 33A and 33B show imaging results for patient UPN097 in the area of ​​the catheter used for intratumoral administration. In Figure 33A, it can be seen that few CD3+ or CD8+ T cells are present before treatment. Figure 33B, a sample taken from the left frontal tumor cavity wall 16 days after treatment, shows a large area of ​​necrotic tumor and significant presence of CD3+ and CD8+ cells.

[0078] Gliomas express IL13 receptors, and in particular the high affinity IL13 receptor. However, unlike the IL13 receptor, glioma cells overexpress a unique IL13Rα2 chain that can bind IL13 independent of the requirement for IL4Rβ or γc44. Like its homolog IL4, IL13 has pleiotropic immunomodulatory activity outside the CNS. Both IL13 and IL4 stimulate IgE production by B lymphocytes and suppress proinflammatory cytokine production by macrophages. Detailed studies using autoradiography with radiolabeled IL13 showed abundant IL13 binding in almost all malignant glioma tissues studied. This binding is highly uniform within tumor sections and in single cell analysis. However, molecular probe analysis specific for IL13Rα2 mRNA did not detect expression of the glioma-specific receptor by normal brain elements, and autoradiography with radiolabeled IL13 also failed to detect specific IL13 binding in normal CNS. These studies suggest that the shared IL13Rα1 / IL4β / γc receptor is not detectably expressed in normal CNS. Thus, IL13Rα2 is a highly specific cell surface target for gliomas and a suitable target for CARs designed for the treatment of gliomas.

[0079] Certain patients may be more suitable than others to receive the disclosed therapeutic methods. For example, patients with malignant tumors that highly express IL-13Rα2 may particularly benefit from treatment with the disclosed CAR T cells. Patient suitability can be determined by staining tumor samples excised from the patient to determine the expression level of IL-13Rα2. The samples can be scored based on the number of cells that show weak, moderate, or strong staining intensity. Determining the expression level of Ki67 can also be useful for determining the aggressiveness of the disease. Once a patient is determined to be suitable to receive the disclosed CAR T cells, the patient can be treated according to the disclosed methods.

[0080] However, binding of IL13-based therapeutic molecules to the widely expressed IL13Rα1 / IL4β / γc receptor complex has the potential to mediate undesired toxicity to normal tissues outside the CNS and therefore limits the systemic administration of these agents. An amino acid substitution in IL13 alpha helix A at amino acid 13 of tyrosine to native glutamic acid selectively reduces the affinity of IL13 for the IL13Rα1 / IL4β / γc receptor. However, binding of this mutant (termed IL13(E13Y)) to IL13Rα2 was increased compared to wild-type IL13. Thus, IL13 analogs with minimal modification simultaneously increase the specificity and affinity of IL13 for glioma cells. Thus, the CARs described herein encompass IL13 that contain a mutation (E to Y, or E to other amino acids, e.g., K or R or L or V) at amino acid 13 (according to the numbering of Debinski et al. 1999 Clin Cancer Res 5:3143s). However, IL13 with the native sequence can also be used and may be particularly useful when the modified T cells are administered locally, such as by direct injection into a tumor mass.

[0081] Furthermore, gliomas are known to generally have poor patient prognosis. For example, glioblastoma multiforme (GBM) is a common malignant cancer of the CNS. The 1-year and 2-year relative survival rates for GBM are 29.6% and 9.0%, respectively. Only 3.4% of patients diagnosed with GBM survive for more than 5 years. Furthermore, recurrence after treatment with surgical resection and / or other conventional therapeutic methods is common. Current conventional treatments include, but are not limited to, radiotherapy, small molecules (e.g., temozolomide, irinotecan, imatinib mesylate, erlotinib, and hydroxyurea), and biologics such as antibodies (e.g., bevacizumab).

[0082] The disclosed treatment methods improve clinical outcomes in patients compared to current standards. For example, the disclosed methods can increase 1-year, 2-year, and 5-year survival rates. In certain embodiments, the 1-year survival rate of patients treated according to the disclosed methods can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%. In certain embodiments, the 2-year survival rate of patients treated according to the disclosed methods may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%. In certain embodiments, the 5-year survival rate of patients treated according to the disclosed methods may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%.

[0083] In some embodiments, the disclosed methods also increase the life expectancy of a patient compared to another patient undergoing conventional or SOC therapy, including radiation therapy, small molecule drug therapy, therapeutic biologics such as therapeutic antibodies, or combinations thereof. In some embodiments, where a patient undergoing SOC therapy can expect to survive approximately 15 months from initial diagnosis (overall survival or OS), patients undergoing the disclosed therapy can expect an OS of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more. In some embodiments, patients undergoing the claimed therapy can expect an OS of 42, 48, 54, 60, 66, 72, 78, 84, 90 months or more.

[0084] The disclosed methods can improve patient prognosis through various clinical outcomes. For example, the disclosed methods can reduce tumor volume in patients treated with compositions comprising T cells. In some embodiments, the disclosed treatment methods can reduce tumor volume by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%. In some embodiments, the patient's tumor can be completely eliminated and the patient can be cured of malignant tumors.

[0085] Moreover, the disclosed methods are safe and well tolerated. Patients treated with the disclosed methods may not experience significant side effects, and may be able to discontinue administration of adjunctive medications. For example, in certain embodiments, the disclosed methods do not result in toxicity of grade 3 or higher according to the NCI Common Toxicity Criteria (CTC). The CTC provides a quantifiable scale of 0-5, with 0 meaning no adverse events, 1 meaning mild, 2 meaning moderate, 3 meaning severe and undesirable, 4 meaning life threatening or incapacitating, and 5 meaning death. Thus, side effects and / or toxicity may include mild nervous system disorders such as mild or moderate headache, fatigue, myalgia, and olfactory aura, while high-grade toxicity is avoided.

[0086] Steroids such as dexamethasone are commonly used in the clinical management of gliomas to prevent neurological side effects such as cerebral edema. The disclosed treatment methods can reduce the need for such adjunctive treatment. For example, if a patient is receiving a regimen of steroids (e.g., dexamethasone) prior to treatment with the disclosed methods, the patient can reduce the dose of the steroid regimen or discontinue the steroid regimen entirely without experiencing clinically adverse effects.

[0087] Breast cancer brain metastases can express HER2. Certain of the CARs described herein that are useful for treating malignant gliomas target HER2.

[0088] The CAR described herein can be produced by any means known in the art, but is preferably produced using recombinant DNA technology. The nucleic acid encoding several regions of the chimeric antigen receptor can be prepared and assembled into a complete coding sequence by standard molecular cloning techniques known in the art as is convenient (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T-lymphocyte cell line, most preferably an autologous T-lymphocyte cell line.

[0089] Various T cell subsets isolated from patients, including unselected PBMCs or enriched CD3 T cells or enriched CD3 or memory T cell subsets, can be transduced with vectors for CAR expression. Central memory T cells are one useful T cell subset. Central memory T cells can be isolated from peripheral blood mononuclear cells (PBMCs) by selecting CD45RO+ / CD62L+ cells, for example using a CliniMACS® instrument, to immunologically select cells expressing the desired receptor. Cells enriched for central memory T cells can be activated with, for example, a SIN lentiviral vector directing expression of the CAR, and anti-CD3 / CD28 transduced with truncated human CD19 (CD19t), a non-immunogenic surface marker for both in vivo detection and potential ex vivo selection. Activated / genetically modified central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. EXAMPLES

[0090] Example 1: Construction and structure of an IL13Rα2-specific CAR The structure of a useful IL13Rα2-specific CAR is described below. The codon-optimized CAR sequence contains a membrane-tethered IL-13 ligand mutated at a single site (E13Y) to reduce potential binding to IL13Rα1, an IgG4 Fc spacer containing two mutations (L235E; N297Q) that greatly reduce the Fc receptor-mediated recognition model, a CD4 transmembrane domain, a costimulatory 4-1BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain. A T2A ribosomal skip sequence separates this IL13(EQ)BBζ CAR sequence from CD19t, an inactive and non-immunogenic cell surface detection / selection marker. This T2A binding results in synchronous expression of both IL13(EQ)BBζ and CD19t from a single transcript. Figure 1A is a schematic diagram of the 2670 nucleotide open reading frame encoding the IL13(EQ)BBZ-T2ACD19t construct. In this figure, the IL13Rα2-specific ligand IL13(E13Y), IgG4(EQ)Fc, CD4 transmembrane, 4-1BB cytoplasmic signaling, 3-glycine linker, and CD3ζ cytoplasmic signaling domains of the IL13(EQ)BBZ CAR are all shown, as well as the T2A ribosomal skip and truncated CD19 sequences. Also shown is the human GM-CSF receptor alpha, and the CD19 signal sequence that drives surface expression of the IL13(EQ)BBZ CAR and CD19t. Thus, the IL13(EQ)BBZ-T2ACD19t construct encompasses an IL13Rα2-specific hinge-optimized co-stimulatory chimeric immune receptor sequence (referred to as IL13(EQ)BBZ), a ribosomal skip T2A sequence, and a CD19t sequence.

[0091] The IL13(EQ)BBZ sequence was generated by fusion of the human GM-CSF receptor alpha leader peptide with the IL13(E13Y) ligand 5L235E / N297Q modified IgG4 Fc hinge (double mutation prevents FcR recognition), CD4 transmembrane, 4-1BB cytoplasmic signaling domain, and CD3 zeta cytoplasmic signaling domain sequences. This sequence was synthesized de novo after codon optimization. The T2A sequence was obtained from digestion of a T2A-containing plasmid. The CD19t sequence was obtained from spanning the leader peptide sequence to the transmembrane component (i.e., base pairs 1-972) of a CD19-containing plasmid. All three fragments, 1) IL13(EQ)BBZ, 2) T2A, and 3) CD19t, were cloned into the multiple cloning site of the epHIV7 lentiviral vector. When transfected into an appropriate cell, the vector integrates into the host cell genome the sequences shown diagrammatically in Figure 1B. Figure 1C provides a schematic representation of the 9515 base pair IL13(EQ)BBZ-T2A-CD19t_epHIV7 plasmid itself.

[0092] As shown diagrammatically in FIG. 2, the IL13(EQ)BBZ CAR differs in several important ways from a previously described IL13Rα2-specific CAR called IL13(E13Y)-zetakine (Brown et al. 2012 Clinical Cancer Research 18:2199). IL13(E13Y)-zetakine is composed of an IL13Rα2-specific human IL-13 mutein (huIL-13(E13Y)), a human IgG4 Fc spacer (huγ4Fc), a human CD4 transmembrane (huCD4 tm) and a human CD3ζ chain cytoplasmic (huCD3ζ cyt) portion, as shown. In contrast, IL13(EQ)BBζ) has two point mutations, L235E and N297Q, located in the CH2 domain of the IgG4 spacer, and a co-stimulatory 4-1BB cytoplasmic domain (4-1BB cyt).

[0093] Example 2: Construction and structure of epHIV7 used to express IL13Rα2-specific CAR The pHIV7 plasmid is the parental plasmid from which the clinical vector IL13(EQ)BBZ-T2A-CD19t_epHIV7 was derived at the T Cell Therapy Laboratory (TCTRL) at City of Hope (COH). The epHIV7 vector used for expression of the CAR was generated from the pHIV7 vector. Importantly, this vector uses the human EF1 promoter to drive expression of the CAR. The 5' and 3' sequences of the vector were derived from pv653RSN, previously derived from the HXBc2 provirus. The polypurine tract DNA flap sequence (cPPT) was derived from the HIV-1 pNL4-3 strain from the NIH AIDS Reagent Repository. The woodchuck posttranscriptional regulatory element (WPRE) sequence has been described previously.

[0094] The construction of pHIV7 is shown diagrammatically in Figure 3. Briefly, pv653RSN, containing 653 bp from gag-pol plus 5' and 3' long terminal repeats (LTRs) with an intervening SL3-neomycin phosphotransferase gene (Neo), was subcloned into pBluescript as follows: In step 1, the sequence from the 5'LTR to the rev response element (RRE) was modified to create p5'HIV-1 51, then the 5'LTR was modified by removing the sequence upstream of the TATA box and ligated first to the CMV enhancer and then to the SV40 origin of replication (p5'HIV-2). In step 2, the 3'LTR was cloned into pBluescript to create p3'HIV-1, followed by the removal of the cis-regulatory elements in HIV U3 and a 400 bp deletion in the 3'LTR enhancer / promoter to form p3'HIV-2. In step 3, the fragments isolated from p5'HIV-3 and p3'HIV-2 were ligated to create pHIV-3. In step 4, p3'HIV-2 was further modified by removing the extra upstream HIV sequence to generate p3'HIV-3, and a 600 bp BamHI-SalI fragment containing the WPRE was added to p3'-3 to create p3'HIV-4. In step 5, pHIV-3RRE was reduced in size by PCR and ligated to the 5' fragment from pHIV-3 (not shown) and to p3'HIV-4 to generate pHIV-6. In step 6, a 190 bp BglII-BamHI fragment containing the cPPT DNA flap sequence from HIV-1 pNL4-3 (55) was amplified from pNL4-3 and placed between the RRE and WPRE sequences in pHIV6 to generate pHIV-7. This parental plasmid, pHIV7-GFP (GFP, green fluorescent protein), was used to package the parental vector using a four-plasmid system.

[0095] The packaging signal psiΨ is ​​required for efficient packaging of the viral genome into the vector. The RRE and WPRE enhance the transport of RNA transcripts and expression of transgenes. It has been demonstrated that the flap sequence, in combination with the WPRE, enhances the transduction efficiency of lentiviral vectors in mammalian cells.

[0096] To reduce the possibility of generating replication-competent lentivirus by recombination, the helper functions required for viral vector production were separated into three separate plasmids: 1) pCgp encodes the gag / pol proteins required for viral vector construction; 2) pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence to help transport the viral genome for efficient packaging; 3) pCMV-G encodes the glycoprotein of vesiculo-stomatitis virus (VSV), which is required for infectivity of the viral vector.

[0097] There is minimal DNA sequence homology between the pHIV7-encoded vector genome and the helper plasmid. The regions of homology are: Approximately 600 nucleotides located in the gag / pol sequence of the pCgp helper plasmid the packaging signal region of ; The CMV promoter sequence in all three helper plasmids; and the RRE sequence in the helper plasmid pCgp; Since multiple recombination events are required, it is highly unlikely that homology in these regions would result in the generation of replication-competent recombinant virus. Furthermore, any resulting recombinants would lack functional LTR and tat sequences required for lentiviral replication.

[0098] The CMV promoter was replaced with EF1α-HTLV promoter (EF1p), and the new plasmid was named epHIV7 (Figure 4). EF1p has a length of 563 bp, and was introduced into epHIV7 using NruI and NheI after excising the CMV promoter.

[0099] The lentiviral genome, except for gag / pol and rev, which are necessary for wild-type virus virulence and productive infection of target cells, is removed from this system. Furthermore, since the IL13(EQ)BBZ-T2ACD19t_epHIV7 vector construct does not contain an intact 3'LTR promoter, the resulting expressed and reverse transcribed DNA proviral genome in target cells will have an inactive LTR. As a result of this design, HIV-1 derived sequences are not transcribed from the provirus and only therapeutic sequences are expressed from their respective promoters. Removal of LTR promoter activity in SIN vectors is expected to significantly reduce the possibility of unintended activation of host genes. Table 4 summarizes the various regulator elements present in IL13(EQ)BBZ-T2ACD19t_epHIV7.

[0100] Table 4: Functional elements of IL13(EQ)41BBZ-T2A-CD19t_epHIV7 [Table 4]

[0101] Example 3: Generation of vectors for transduction of patient T cells For each plasmid (IL13(EQ)BBZ-T2A-CD19t_epHIV7; pCgp; pCMV-G; and pCMV-Rev2), a seed bank is generated that is used to inoculate fermenters to produce sufficient amounts of plasmid DNA. Plasmid DNA is tested for identity, sterility, and endotoxins before use in lentiviral vector production.

[0102] Briefly, cells were grown from 293T working cell bulk (WCB) that were tested for sterility and the presence of viral contamination. A vial of 293T cells from the 293T WCB was The cells were grown and expanded until there were sufficient cells to plate an appropriate number of 10-layer cell factories (CF) for vector production and cell train maintenance. A single train of cells can be used for production.

[0103] Lentiviral vectors were produced in sub-batches of up to 10 CFs. Two sub-batches could be made in the same week, resulting in production of approximately 20 L of lentiviral supernatant per week. To produce one lot of product, the material produced from all sub-batches was pooled at downstream processing steps. 293T cells were seeded into the CFs in 293T medium (DMEM with 10% FBS). The factories were placed in a 37°C incubator and leveled to distribute the cells evenly across the entire layer of the CFs. After two days, the cells were transfected with the four lentiviral plasmids listed above using the CaPO4 method, which contains Tris:EDTA, 2M CaCl2, 2X HBS, and a mixture of the four DNA plasmids. Three days after transfection, the supernatant containing the secreted lentiviral vectors was harvested, purified, and concentrated. After the supernatant was removed from the CFs, the finished cells were harvested from each CF. The cells were trypsinized from each factory and harvested by centrifugation. The cells were resuspended in freezing medium and cryopreserved, and were subsequently used for replication-competent lentivirus (RCL) assays.

[0104] For vector purification and formulation, the crude supernatant was clarified by membrane filtration to remove cell debris. Host cell DNA and residual plasmid DNA were degraded by endonuclease digestion (Benzonase®). The viral supernatant was cleared from cell debris using a 0.45 μm filter. The clarified supernatant was collected in a pre-weighed container into which Benzonase® was added (final concentration 50 U / mL). Endonuclease digestion of residual plasmid DNA and host genomic DNA was carried out for 6 hours at 37°C. An initial tangential flow ultrafiltration (TFF) concentration of the endonuclease-treated supernatant was used to remove residual low molecular weight components from the crude supernatant while concentrating the virus by about 20 times. The clarified endonuclease-treated viral supernatant was subjected to a shear rate of about 4,000 s at maximum flow rate. -1 The supernatant was circulated through a hollow fiber cartridge with a NMWCO of 500 kD at a flow rate designed to maintain the performance of the cartridge. Diafiltration of the nuclease-treated supernatant was initiated during the concentration process to maintain the performance of the cartridge. An 80% permeate replacement rate was established using 4% lactose in PBS as the diafiltration buffer. The viral supernatant was brought to the target volume, which corresponds to a 20-fold concentration of the crude supernatant. Diafiltration was continued for four additional exchange volumes with a dialysate exchange rate of 100%.

[0105] Further concentration of the viral product was achieved by using high speed centrifugation techniques. Each sub-batch of lentivirus was pelleted using a Sorvall RC-26 plus centrifugation at 6000 RPM (6,088 RCF) at 6°C for 16-20 hours. The viral pellet from each sub-batch was then reconstituted in a 50 mL volume with 4% lactose in PBS. The reconstituted pellet in this buffer represents the final formulation for virus preparation. The entire vector concentration process resulted in approximately a 200-fold volume reduction. After completion of all sub-batches, the material was then placed at -80°C while samples from each partial batch were tested for sterility. After confirming sample sterility, the sub-batches were rapidly thawed at 37°C with frequent agitation. This material was then pooled and manually aliquoted into the viral vector suite in a Class II Type A / B3 biosafety cabinet. A fill configuration of 1 mL of concentrated lentivirus in sterile USP Class 6, externally threaded O-ring cryovials was used. The Quality System (QS) at COH's Center for Applied Technology Development (CATS) follows policies and standard operating procedures for CBG and releases all materials in accordance with current Good Manufacturing Practices (cGMPs).

[0106] To ensure the purity of lentiviral vector preparations, they were tested for residual host DNA contamination and for transfer of residual host and plasmid DNA. Among other tests, vector identity was assessed by RT-PCR to ensure that the correct vector was present. All release criteria were met for the vectors intended for use in this study.

[0107] Example 4: Preparation of T cells suitable for use in ACT T lymphocytes are obtained from the patient by leukopheresis. Appropriate allogeneic or autologous T cell subsets, such as central memory T cells (T CM) are genetically modified to express a CAR and then administered back to the patient by any clinically acceptable means to achieve anti-cancer treatment.

[0108] Appropriate T CM can be prepared as follows: Apheresis products obtained from consented study participants are ficolled, washed and incubated overnight. Monocytes, regulatory T cells and naive T cell populations are depleted from cells using GMP grade anti-CD14, anti-CD25 and anti-CD45RA reagents (Miltenyi Biotec) and the CliniMACS™ Separator. After depletion, CD62L+ T cells are isolated using DREG56-biotin (COH clinical grade) and anti-biotin microbeads (Miltenyi Biotec) on the CliniMACS™ Separator. CM The negative fraction cells are enriched for cells.

[0109] After enrichment, T CM Cells are formulated in complete X-Vivo15 plus 50 IU / mL IL-2 and 0.5 ng / mL IL-15 and transferred to Teflon® cell culture bags. CM The cells are stimulated with Dynal ClinEx® Vivo CD3 / CD28 beads. Five days after stimulation, the cells are transduced with the IL13(EQ)BBZ-T2A-CD19t_epHIV7 lentiviral vector at a multiplicity of infection (MOI) of 1.0 to 0.3. Cultures are maintained for up to 42 days by adding complete X-Vivo15 and IL-2 and IL-15 cytokines required for cell proliferation (cell density of 3 x 10 5 ~2×10 6 (Maintain viable cells / mL and cytokine supplementation every Monday, Wednesday, and Friday of culture). Cells typically grow to approximately 10 cells / mL under these conditions within 21 days. 9 At the end of the culture period, cells are harvested, washed twice, and formulated in clinical grade cryopreservation medium (Cryostore CS5, BioLife Solutions).

[0110] On the day(s) of T cell infusion, the cryopreserved and released product is thawed, washed, and formulated for reinfusion. The cryopreserved vials containing the released cell product are removed from liquid nitrogen storage, thawed, cooled, and washed with PBS / 2% human serum albumin (HSA) wash buffer. After centrifugation, the supernatant is removed and the cells are resuspended in preservative free saline (PFNS) / 2% HSA infusion diluent. A sample is removed for quality control testing.

[0111] Two qualification runs on cells sourced from healthy donors were performed using the manufacturing platform described above. Each preclinical qualification run product was assigned a human donor (HD) number, HD006.5 and HD187.1. Importantly, as shown in Table 5, these qualification runs expanded >80-fold within 28 days and the expanded cells expressed the IL13(EQ)BBγ / CD19t transgene.

[0112] Table 5: Summary of expression data from preclinical qualification test products [Table 5]

[0113] Example 5: IL13(EQ)BBγ / CD19t+ T CM Flow cytometric analysis of surface transgene and T cell marker expression in The two preclinical qualification test products described in Example 4 were used in preclinical studies as described below. Figures 6A-C show the results of flow cytometry analysis of surface transgene and T cell marker expression. IL-13(EQ)BBγ / CD19t+ T CMHD006.5 and HD187.1 were co-stained with anti-IL-13-PE and anti-CD8-FITC to detect CD8+ CAR+ and CD4+ (i.e., CD8 negative) CAR+ cells (Figure 6A), or with anti-CD19-PE and anti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4 negative) CAR+ cells (Figure 6B). CM HD006.5 and HD187.1 were stained with fluorochrome-conjugated anti-CD3, TCR, CD4, CD8, CD62L, and CD28 (grey histograms) or isotype controls (black histograms) (Figure 6C). In each of Figures 6A-C, the percentages shown are based on viable lymphocytes (DAPI negative) stained for isotype.

[0114] Example 6: Comparison of CAR T cell delivery routes for the treatment of large TS-initiated PBT tumors The following is an investigation comparing the route of delivery, intravenous (iv) or intracranial (ic), for antitumor activity against an aggressive primary PBT line. In a pilot study (data not shown), unexpectedly, iv-administered IL13(EQ)BBζ+ T CM We observed that i.c. delivery of IL13(EQ)BBZ+ T cells did not confer a therapeutic benefit compared to PBS for the treatment of small (day 5) PBT030-2 EGFP:ffLuc tumors. This is in contrast to the robust therapeutic effect observed with i.c. administered CAR+ T cells. We speculated that PBT030-2 tumors may be too small to mobilize therapeutic T cells from the periphery on day 5, and performed a comparison of i.c. delivery to large, day 19 PBT030-2 EGFP:ffLuc tumors. For these studies, PBT030-2-implanted mice were treated with i.c. delivery of IL13(EQ)BBZ+ T cells. CM or sham TCM (without CAR), two iv injections (5 × 10 6 CAR+T CM ; days 19 and 26) or four i.c. injections (1 × 10 6 CAR+T CMCAR+ T cells were treated with either i.v. or i.c. (days 19, 22, 26, and 29). Again, no therapeutic benefit was monitored by Xenogen imaging or Kaplan-Meier survival analysis for iv administered CAR+ T cells (Figures 7A and 7B). In contrast, potent antitumor activity was observed in ic administered IL13(EQ)BBζ+ T cells. CM Next, brains from the mouse cohort 7 days after T cell injection were harvested and assessed for CD3+ human T cells by IHC. Surprisingly, Mock T CM or IL13(EQ)BBζ T CM In mice treated i.v. with either IgG4 or IgG5, there were no detectable CD3+ human T cells in the tumor or in other mouse brain regions where human T cells are typically present (i.e., the leptomeninges) (Figure 7C), suggesting a defect in tumor tropism. This is in contrast to the significant numbers of T cells detected in i.c.-treated mice (Figure 7D).

[0115] Tumor-derived cytokines, especially MCP-1 / CCL2, are important in recruiting T cells to tumors. Accordingly, we evaluated PBT030-2 tumor cells and found that this line produces high levels of MCP-1 / CCL2 comparable to U251T cells, a glioma line previously shown to attract effector CD8+ T cells administered iv to ic-implanted tumors (data not shown). Malignant gliomas are highly invasive tumors and are often multifocal in presentation. The above studies suggest that IL13BBZ T CMWe have established that CAR T cells can clear infiltrated tumors, such as PBT030-2, and mediate long-term durable antitumor activity. The ability of intracranially delivered CAR T cells to traffic to multifocal disease was also examined. For this study, PBT030-2 EGFP:ffLuc TS was implanted in both the left and right hemispheres (Figure 8A) and CAR+ T cells were injected only into the right tumor site. Encouragingly, we detected T cells by CD3 IHC 7 days after T cell infusion both at the site of injection (i.e., right tumor) and also within the tumor in the left hemisphere for all mice evaluated (n=3) (Figure 8B). These findings provide evidence that CAR+ T cells can reach and infiltrate tumor foci at distant sites. Similar findings were observed in a second tumor model using the U251T glioma cell line (data not shown).

[0116] Example 7: Amino acid sequence of IL13(EQ)BBζ / CD19t The complete amino acid sequence of IL13(EQ)BBζ / CD19t is shown in Figure 9. The full sequence (SEQ ID NO:1) is: the 22 amino acid GMCSF signal peptide (SEQ ID NO:2); The 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); IgG4 sequence of 229 amino acids (SEQ ID NO: 4 with bolded amino acid substitutions L235E and N297Q); the 22 amino acid CD4 transmembrane sequence (SEQ ID NO:5); the 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Gly linker; the 112 amino acid CD3ζ sequence (SEQ ID NO:7); The 24 amino acid T2A sequence (SEQ ID NO:8); and CD19t sequence of 323 amino acids (SEQ ID NO:9); Includes:

[0117] The mature chimeric antigen receptor sequence (SEQ ID NO: 10) is The 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); IgG4 sequence of 229 amino acids (SEQ ID NO: 4 with amino acid substitutions L235E and N297Q shown in bold); The 22 amino acid CD4 sequence (SEQ ID NO:5); the 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Gly linker; and the 112 amino acid CD3ζ sequence (SEQ ID NO:7); Within this CAR sequence (SEQ ID NO: 10) is the IL-13 / IgG4 / CD4t / 41-BB sequence (SEQ ID NO: 11), which is The 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitutions shown in bold) E13Y); IgG4 sequence of 229 amino acids (SEQ ID NO: 4 with bolded amino acid substitutions L235E and N297Q); The 22 amino acid CD4 sequence (SEQ ID NO:5); and the 42 amino acid 4-1BB sequence (SEQ ID NO:6); The IL13 / IgG4 / CD4t / 4-1BB sequence (SEQ ID NO:11) can be linked to the 112 amino acid CD3ζ sequence (SEQ ID NO:7) by a linker, such as a Gly Gly Gly linker. The CAR sequence (SEQ ID NO:10) can be preceded by a 22 amino acid GMCSF signal peptide (SEQ ID NO:2).

[0118] FIG. 10 shows a comparison of the sequences of IL13(EQ)41BBζ [IL13(EQ)41BBζ T2A-CD19t_epHIV7;pF02630] (SEQ ID NO: 12) and CD19Rop_epHIV7 (pJ01683) (SEQ ID NO: 13).

[0119] Example 8: Amino acid sequences of additional CARs targeting IL13Rα2 Figures 11-18 show the amino acid sequences of additional CARs directed against IL13Rα2. In each case, the various domains are labeled, except for the GlyGlyGly spacer located between the specific intracellular domains. Each encompasses human IL13 and Glu through Tyr (SEQ ID NO:3; amino acid substitution E13Y highlighted). In the expression vectors used to express these CARs, the expressed amino acid sequences are: The 24 amino acid T2A sequence (SEQ ID NO:8); and the 323 amino acid CD19t sequence (SEQ ID NO:9) that allows coordinated expression of a truncated CD19 sequence on the surface of CAR-expressing cells; can be included.

[0120] A panel of CARs containing the human IL13 (E13Y) domain, CD28tm domain, CD28gg costimulatory domain, 4-1BB costimulatory domain, and CD3ζ domain CAR scaffold and encompassing either the HL (22 amino acids), CD8 hinge (48 amino acids), IgG4-HL-CH3 (129 amino acids) or IgG4(EQ) (229 amino acids) spacers were tested for their ability to mediate IL13Ra2-specific killing assessed in a 72-hour co-culture assay. All were active except for HL (22 amino acids), which appears to have low CAR expression in this system.

[0121] Example 9: Structure of two HER2-CARs One CAR comprising the HER2 scFv described herein is called Her2scFv-IgG4(L235E,N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t. This CAR comprises the following: HER2-targeting scFv; an IgG4 Fc region mutated at two sites within the CH2 region (L235E; N297Q) in a manner that reduces binding by the Fc receptor (FcR); CD28 transmembrane domain, CD28 costimulatory domain, and CD3ζ activation domain; Figure 20 shows the amino acid sequence of this CAR, including the sequence of the truncated CD19 sequence used to monitor CAR expression and the T2A ribosomal skip sequence that allows the CAR to be produced without fusion of the truncated CD19 sequence. As shown in Figure 21, the immature CAR contains a GMCSFR signal peptide, a HER2 scFv, an IgG4 that acts as a spacer, a CD8 transmembrane domain, a 4-1BB costimulatory domain that includes a LL-GG sequence modification, three G The transcript also encodes a T2A ribosomal sequence and a truncated CD19 sequence that are not part of the CAR protein sequence. The mature CAR is identical to the immature CAR but lacks the GMCSF signal peptide.

[0122] Example 10: Expression of HER2-targeted CAR FIG. 22A is a schematic diagram of the two HER2-specific CAR constructs shown in FIG. 20 and FIG. 21. In HER2(EQ)28ζ, the scFv is tethered to the membrane by a modified IgG4 Fc linker (double mutant, L235E;N297Q) containing the CD28 transmembrane domain, the intracellular CD28 costimulatory domain, and the cytolytic CD3ζ domain. A T2A skip sequence separates the CAR from the truncated CD19 (CD19t) protein used for cell tracking. HER2(EQ)BBζ is similar, except that the costimulatory domain is 4-1BB instead of CD28, and the transmembrane domain is the CD8 transmembrane domain instead of the CD28 transmembrane domain. Human central memory (T CM ) cells were transfected with lentiviral vectors expressing either HER2(EQ)28ζ or HER2(EQ)BBζ. CM Representative FACS data of surface phenotype are shown. Figure 22C shows the surface phenotype of T cells transfected with lentiviral vectors expressing either HER2(EQ)28ζ or HER2(EQ)BBζ. CMFigure 22 shows the results of assays for CD19 and protein L expression in HER2(EQ)BBζ. As can be seen from these results, transfection efficiency, as assessed by CD19 expression, was similar for both CARs. However, protein L expression was lower for HER2(EQ)BBζ than for HER2(EQ)BBζ, suggesting that the HER2(EQ)BBζ CAR is less stable than HER2(EQ)BBζ. Analysis of cell proliferation (Figure 22D) shows that neither CAR interferes with T cell proliferation.

[0123] Example 11: In vitro characterization of HER2-targeted CAR A variety of breast cancer cell lines were used to characterize HER2(EQ)28ζ and HER2(EQ)BBζ, including HER2-negative lines (LCL lymphoma, MDA-MB-468, U87 glioma), low HER2 expressing lines (MDA-MB-361,231BR) and high HER2 expressing lines (SKBR3, BT474, BBM1). Figure 23A shows the HER2 expression levels of each of these lines. Flow cytometry (gated on CAR+ T cells) was used to characterize CD107a degranulation and IFNγ production in Mock (non-transduced), HER2(EQ)28ζ or HER2(EQ)BBζ CAR T cells after 5 hours of co-culture with either MDA-MB-361 tumor cells (low HER2 expression) or BBM1 tumor cells (high HER2 expression). The results of this analysis are shown in Figure 23B. Similar studies were performed with other breast cancer cell lines and the results are summarized in Figure 23C. IFNγ production by HER2-CAR T cells after 24 h of culture with recombinant HER2 protein or tumor targets was measured by ELISA and the results of this analysis are shown in Figure 23D.

[0124] Example 12: In vitro antitumor activity of HER2-targeted CAR Flow cytometry was used to assess tumor cell killing after 72 hours of co-culture of Mock (non-transduced), HER2(EQ)28ζ or HER2(EQ)BBζ CAR T cells with tumor targets. The results of this analysis are shown in Figure 24A. PD-1 and LAG-3 induction was measured in total CAR T cells after 72 hours of co-culture with HER2-negative MDA-MB-468 or HER2-positive BBM1 cells. The results of this analysis are shown in Figure 24B. PD-1 induction was measured in CD8+ CAR T cells after 72 hours of co-culture with tumor targets that were HER2-negative (LCL lymphoma, MDA-MB-468, U87 glioma), low HER2 expressing (MDA-MB-361,231BR) or high HER2 expressing (SKBR3, BT474, BBM1). The results of this analysis are shown in Figure 24C. These studies have demonstrated that HER2 ( These results suggest that HER2(EQ)BBζ results in lower PD-1 induction than HER2(EQ)28ζ. Tumor cell killing at effector:tumor (E:T) ratios ranging from 0.25:1 to 2:1 was measured for HER2(EQ)28ζ or HER2(EQ)BBζ CAR T cells. The results of this analysis are shown in Figure 24D. Figure 24D shows that both HER2(EQ)28ζ and HER2(EQ)BBζ are effective in tumor cell killing in vitro. CFSE proliferation of HER2-CAR T cells after 72 hours of co-culture with MDA-MB-468 or BBM1 cells was measured by flow cytometry. The results of this analysis are shown in Figure 24E. Figure 24E shows that HER2(EQ)BBζ CAR T cells proliferate more than HER2(EQ)28ζ CAR T cells.

[0125] Example 13: In vivo antitumor activity of HER2-targeted CAR The activity of intratumoral delivered HER2 CAR T cells was evaluated in a patient-derived breast-to-brain metastasis model. Figures 25A-25C are H&E staining of tumors. Mice were treated with Mock (non-transduced) or HER2(EQ)BBζ CAR T cells by direct injection into the tumor. Figures 25D-25F show the results of optical imaging of the tumors, and Figures 25G-25I are Kaplan-Meier survival curves of mice treated locally either 3, 8 or 14 days after tumor injection. These studies show that HER2(EQ)BBζ CAR T cells have a strong antitumor effect in vivo when directly injected into the tumor.

[0126] To evaluate the antitumor effect in a human xenograft model of breast-brain metastasis, BBM1 cells (0.2M) or BT474 (0.15M) were injected intracranially into NSG mice. Eight days after tumor injection, HER2(EQ)28ζ or HER2(EQ)BBζ, or Mock (non-transduced) T cells (1M) were injected intratumorally. BBM1 (Figure 26A) and BT474 (Figure 26B) tumors were monitored by luciferase-based optical imaging. Kaplan Meier curves are shown in Figure 26C and Figure 26D.

[0127] A human patient-derived orthotopic xenograft model of breast-brain metastasis was also used to evaluate HER2(EQ)28ζ and HER2(EQ)BBζ CAR T cells. Figure 27A shows the area of ​​tumor implantation by stereotactic injection of BBM1 cells (0.2M) and intraventricular T cell delivery. Tumor staining is shown in Figure 27B. 14 days after tumor injection, HER2(EQ)28ζ, HER2(EQ)BBζ, or Mock (non-transduced) T cells (0.5M) were injected intratumorally. Tumor growth was monitored by luciferase-based optical imaging. Figure 27C shows the flux mean for each treatment group, and Figure 27D shows the Kaplan Meier survival curve for each treatment group.

[0128] Example 14: T cells expressing a CAR targeting IL13Ra2 CM Comparison of intracranial and intratumoral administration of Two different intracranial (ic) delivery routes, i.e., intratumoral (ict) and intracerebroventricular (icv), were used to deliver T cells transduced with the IL13(EQ)BBZ-T2A-CD19t_epHIV7 lentiviral vector and propagated in vitro as proposed for the clinical treatment of glioblastoma (GBM). CM CAR T cells generated from the enriched cell line were evaluated in a murine model of glioblastoma for in vivo safety, trafficking, and efficacy. The in vivo safety and functional efficacy of these cells administered either ict or icv was examined in immunodeficient NSG mice using the IL13Rα2+ primary low-passage GBM tumor sphere line PBT030-2 engineered to express a firefly luciferase (ffLuc) reporter gene.

[0129] T enriched from PBMCs by CliniMACS™ / AutoMACS selection CM The cell line was IL13(EQ)BBZ-T2A-CD19t_epHIV7 lentivirus. The CAR T cells were lenti-transduced with IL13Rα2+ and expanded, then cryopreserved using the same method as above. Freshly thawed CAR T cells administered either ict or icv were then evaluated for potential toxicity, their ability to transport to multifocal GBM tumors, and their ability to control the in vivo growth of ic-implanted IL13Rα2+ GBM line PBT030-2 cells. To assess general toxicity, mice were observed daily for general health, including weight and alertness. Tumor burden, measured by Xenogen imaging, was examined. Immunohistochemistry (IHC) to detect T cell recruitment / infiltration of tumors was also performed on a subset of mice.

[0130] Male NSG mice (10–12 weeks old) were injected with 1 × 10 IgG in both the left and right contralateral hemispheres on day 0. 5ffLuc+ PBT030-2 cells were stereotactically injected i.c. and allowed to engraft for 6 days. Mice were then grouped based on tumor size as determined by Xenogen imaging for equal tumor size distribution per mouse. Mice groups were then left untreated (n=4) or injected with 1×10 6 CAR+ IL13(EQ)BBζ / CD19t+ T CM PBT030-2 tumor growth was monitored over time by Xenogen imaging and quantification of ffLuc flux (photons / sec). At different time points, mice from each group were euthanized, their brains were harvested, embedded in paraffin, and immunohistochemistry (IHC) was performed to assess the presence of human CD3-expressing cells (i.e., human T cells). Specifically, one week after T cell administration, three mice were euthanized from each CAR T cell treatment group (day 13 of the experiment), so these mice were not included in the Xenogen imaging analysis in Figure 29. Then, two weeks after T cell administration, two mice were euthanized in each of the mouse groups (day 21 of the experiment).

[0131] As this was not a survival study, and therefore to assess brain T cell trafficking, all mice were euthanized at specific time points (see below). The mice were monitored daily for overt signs of distress or general toxicity. Mice treated with either the ICT or ICV regimen did not exhibit weight loss and were bright, alert, and responsive throughout the experiment. Thus, there were no signs of treatment-related side effects, regardless of the route of T cell administration. Thus, there were no signs of any treatment-related side effects, regardless of the route of T cell administration.

[0132] As shown in Figure 29A-C, IL13(EQ)BBζ / CD19t+ T CM As expected, ict delivery of IL13(EQ)BBζ / CD19t+ T cells showed potent antitumor activity against PBT030-2 tumors. CMICV delivery of appeared to provide greater therapeutic efficacy against i.c.-implanted PBT030-2 tumors than that observed with i.c.t. administration, particularly against tumor lesions in the contralateral (left) hemisphere.

[0133] To determine whether the route of administration affected the ability of T cells to migrate to tumor sites, IHC analysis of CD3+ T cells was performed on the brains of mice from each group 1 and 2 weeks after T cell administration. As shown in Figure 30, human CD3+ T cells were found in both the left and right tumor sites in mice that received either ict or icv administered T cells. These data are representative of 3 mice from each group 1 week after T cell administration and 2 mice from each group 2 weeks after T cell administration.

[0134] This study demonstrates that both intratumoral and intracerebroventricular administration of T cells were well-tolerated in this NSG mouse model. Furthermore, T cells transduced with the IL13(EQ)BBZ-T2A-CD19t_epHIV7 vector were well-tolerated in this NSG mouse model. CM With both ict and icv delivery of the qualification test cells, in vivo multifocal antitumor effects and IHC detection of T cells at the tumor site can be observed. This study further demonstrated that icv delivery This suggests that delivered T cells may have greater efficacy than ict-delivered T cells.

[0135] Example 15: Phase 1 clinical trial evaluating IL-13Rα2CAR T cells for the treatment of glioblastoma This example demonstrates that autologous IL13BBζ T in patients with recurrent IL13Rα2+ GBM cm This paper describes initial findings from a clinical trial evaluating the safety, feasibility, and bioactivity of weekly intracranial infusions of IL13BBζ+ T cells. As described in more detail below, enrolled patients underwent leukapheresis to harvest autologous PBMCs and were randomized to receive IL13BBζ+ T cells. cmConcurrent with the preparation of the tumor, a reservoir / catheter device is placed to perform tumor biopsy or resection. Following baseline MR and PET imaging and recovery from surgery, patients will be randomized to receive IL13BBζ+ T cm These patients will be treated with a 4-week treatment regimen consisting of 3 weeks of intracranial infusion of IL13BBζ T followed by a 1-week break for toxicity and disease evaluation. To date, results for this first low-dose cohort of 3 resected patients have shown that IL13BBζ T cm These results suggest that local delivery of IL13BBζ T cells following surgical resection is safe and well tolerated with no observed treatment-related grade 3 or higher toxicity, and importantly, show early evidence of antitumor activity following CAR T cell administration. For all patients for whom samples were available, CAR T cells were detected in tumor cyst fluid or cerebrospinal fluid (CSF) by flow cytometry for at least 7 days following treatment. One patient of particular interest presented with recurrent multifocal GBM, including one metastatic site in the spine and extensive leptomeningeal disease. This patient was initially administered IL13BBζ T cells per protocol into the resection cavity of the largest recurrent tumor focus in the occipitotemporal-occipital region. cm The patient was treated with six local infusions of IL13BBζ T. Encouragingly, the CAR T cell injection site remained stable for over seven weeks with no evidence of disease recurrence, although other lesions distant from the CAR T cell injection site continued to progress. The patient was then treated with IL13BBζ T cm The patient was treated with a special-use protocol using five weekly intraventricular infusions of 100 mg of CAR T-cells each day without any other therapeutic intervention. One week after the final intraventricular CAR T-cell infusion, all intracranial and spinal tumors had regressed, with the majority decreasing by more than 75% by volume, and the patient remained clinically stable 4 months after initiation of CAR T-cell therapy.

[0136] The CAR used in this study, IL13(EQ)BBζ, is described above. The sequence of the immature CAR, including the CD19t marker, is shown in Figure 9. The entire immature sequence (SEQ ID NO: 1) is: the 22 amino acid GMCSF signal peptide (SEQ ID NO:2), the 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); IgG4 sequence of 229 amino acids (SEQ ID NO: 4 with amino acid substitutions L235E and N297Q shown in bold); the 22 amino acid CD4 transmembrane sequence (SEQ ID NO:5); the 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Gly linker; the 112 amino acid CD3ζ sequence (SEQ ID NO:7); The 24 amino acid T2A sequence (SEQ ID NO:8); and CD19t sequence of 323 amino acids (SEQ ID NO:9); Includes:

[0137] CD8+ CD4+ T CM To prepare the cells, autologous cells from each patient were used. They were then transfected with the lentiviral vector expressing IL13(EQ)BBζ. CM , CD45RO+ / CD62L+ T CM To immunoselect the IgG antibodies, they were enriched from peripheral blood mononuclear cells (PBMCs) using a CliniMACS® instrument. These cells were activated with anti-CD3 / CD28 Dynal beads. Activated / genetically modified IL13(EQ)BBζ / CD19t+ T cells were transduced with a SIN lentiviral vector directing expression of the IL13(EQ)BBζ CAR. CM Cells were expanded in vitro with IL-2 / IL-15 and then cryopreserved.

[0138] The treatment of two patients suffering from recurrent / refractory GBM with either therapy is described below. Intracavitary administration of CAR T cells was performed manually over approximately 5-10 minutes through a Rickham catheter with up to 1.0 mL of preservative-free normal saline (PFNS) flush delivered at 0.5 ml / hr by convection-enhanced delivery (CED). Intraventricular administration of CAR T cells was performed manually over approximately 5-10 minutes through a Rickham catheter placed in the lateral ventricle. This was followed by up to 0.5 mL of preservative-free normal saline (PFNS) flush delivered over 5-10 minutes by manual push technique. The PFNS flush is meant to clear the administration line and push the remaining CAR T cells through the catheter.

[0139] The time course of CAR T cell preparation and treatment is shown in Figure 31. Concurrent with the manufacturing process, study participants underwent resection of their tumors, followed by placement of a Rickham catheter and baseline imaging.

[0140] Patient UPN097 underwent tumor resection and received 2 × 10 6 cells in the second cycle, and 10 × 10 6 The patient was treated with 100x10 cells. For both cycles 1 and 2, the cells were administered into the resection cavity. After cycle 2, patient UPN097 was removed from the study due to rapid tumor progression.

[0141] Patient UPN109 received 2 × 10 6 cells, 10 × 10 in the second and third cycles 6 After a rest period, patient UPN109 was treated with 10 × 10 cells in cycles 4, 5, and 6. 6 In cycles 1-6, cells were administered. In cycle 7, patients were treated with 2 × 10 6 In cycles 8 and 9, patients were treated with 10 × 10 6 In cycles 7-10, administration was intracerebroventricularly.

[0142] Figure 32A shows the analysis of CAR T cell persistence monitored by CD19. This analysis shows good T cell persistence 8 days after the second cycle. Figure 32B shows the reduction in the presence of GBM cells monitored by IL13Rα2 expression on the cells.

[0143] Figures 33A and 33B show imaging results for patient UPN097 in the area of ​​the catheter used for intratumoral administration. Figure 33A shows the almost complete absence of CD3+ or CD8+ T cells before treatment. Figure 33B, a sample taken from the left frontal tumor cavity wall 16 days after treatment, shows a large area of ​​necrotic tumor and the significant presence of CD3+ and CD8+ cells.

[0144] As shown in Figures 34A-D, levels of IL-13 did not change significantly (a Th2 cytokine), but there was an increase in IFN-γ (a Th1 cytokine) over the two cycles of treatment (Figures 34A and 34B). IL-6, a tumor-associated cytokine, decreased during the first cycle and was at lower levels during the second cycle (Figure 34C). IL-8, another tumor-associated cytokine, decreased during the first cycle but increased during the second cycle toward its pre-first cycle levels (Figure 34D).

[0145] Patient UPN109 presented with recurrent multifocal GBM, including one metastatic site in the spinal cord and extensive leptomeningeal disease. As mentioned above, this patient received IL13BBζ T cm The patient was treated with six local infusions of IL13BBζ T cells. The CAR T cell injection site remained stable for over 7 weeks without evidence of disease recurrence, but other lesions distant from the CAR T cell injection site continued to progress (data not shown). The patient was then treated with IL13BBζ T cells as described above. cmPatients received intraventricular infusions of 100 mg / kg / day (five weekly). Figures 35A-B show MRI and / or PET images of transverse (Figure 35A) and sagittal (Figure 35B) brain sections. Figure 35C shows transverse (top) and frontal (bottom) sections of the spinal column before (left) and one week after (right) intraventricular treatment. The tumor lesion sites are indicated by red arrows in each image.

[0146] Example 16: Case report of intraventricular administration A 50-year-old man was diagnosed with a low-grade brain tumor in the right temporal lobe after initially presenting with grand mal seizures. After 4 months of monitoring, the right temporal tumor showed MRI enhancement. The patient underwent tumor resection, confirming the diagnosis of WHO grade IV GBM. The patient then received standard of care adjuvant proton beam radiation to a total dose of 59.4 cobalt Gy equivalent with concurrent temozolomide (140 mg daily), followed by 4 cycles of temozolomide with the Novocure device (NovoTTF-100A) for 3 months. Six months after primary tumor resection, PET and MRI images showed evidence of disease progression.

[0147] Next, after confirmation of IL13Rα2 expression in the primary right temporal lobe tumor by IHC, the patient was treated with autologous IL13Rα2-targeted CAR T cells ( FIG. 36 ), resulting in an H-score of 100 ( FIG. 37 ). The patient was then treated with leukapheresis, followed by CD4+ CD8+ T cell selection via a two-step depletion selection method as previously described. CM Peripheral blood mononuclear cells (PBMCs) were collected by enrichment of IL13BBζ T CM During the development of the T cell injection, the patient was treated with another clinical protocol evaluating a fibroblast growth factor inhibitor (NCT01975701) and progressed through treatment with symptoms of headache, confusion and increasing disorientation. Additionally, the patient was tapered off steroids prior to T cell injection.

[0148] Ten months after primary tumor resection, the patient underwent another surgical resection of three of the five identified progressive GBM lesions (Figure 38), including the largest lesion in the right occipital-temporal region (T1) where the reservoir / catheter device was placed, and two lesions in the right frontal lobe (T2, T3). Two additional tumors in the left temporal region were not surgically removed (T4, T5). Six days after surgical resection, the patient underwent two 4-week treatment regimens. Each regimen consisted of IL13BBζ T CM This patient received a low dose of 2 × 10 6 Starting with CAR+ T cells, followed by 10 × 10 6 The patient was treated with five infusions of CAR+ T cells (Figure 39). After these six ICT infusions, the patient was treated with IL13BBζ T cells under a special use protocol. CM The patient received five additional intracerebroventricular (ICV) treatment cycles, again with a 2 × 10 6 Start with a low dose of CAR T cells, 10 × 10 6 A second catheter was placed into the right lateral ventricle, followed by four infusions of CAR T cells (Figure 40).

[0149] Due to limited therapeutic product, only five ICV infusion cycles were feasible. Overall, 94 × 10 patients 6 The patient received 11 cell infusions for a total dose of 1000 IL13BBζ T cells. The treatment course encompassed 15 weeks, including evaluation weeks for toxicity and disease assessment (i.e., MRI and PET imaging) after every third cycle and after the last two ICV infusions. The patient received no other therapeutic interventions during this CAR T cell treatment course. Findings up to a 190-day evaluation period encompassing 11 infusion cycles are reported here. Subsequently, a second IL13BBζ T CM Beginning on day 192, the patient continued to receive ICV infusions of this second product approximately every 3 weeks.

[0150] Example 17: Study Design These studies, which encompassed exception-use protocols, were approved by the Institutional Review Board. Patients provided written informed consent. Eligibility included a histologically confirmed diagnosis of currently recurrent IL13Rα2+ grade IV glioma, age >18 years with a Karnofsky performance status (KPS) >60, adequate cardiopulmonary function, and expected survival >4 weeks. Patients must have completed initial radiation therapy at least 12 weeks prior to enrollment and must not have any other active malignancies, infections, or indirect diseases, or be receiving other investigational drugs or require dexamethasone >2mg TID (3x / day) during T-cell therapy.

[0151] This patient was treated with autologous IL13Rα2-targeted CAR T cells (IL13BBζ T) in a patient diagnosed with relapsed / refractory IL13Rα2+ high-grade glioma (WHO grades III and IV). CM This patient was initially treated under our ongoing Phase I trial (NCT02208362) to evaluate the safety and feasibility of weekly intracranial infusions of IL13BBζ T cells. This is a two-arm study in which T cells were administered either directly into the tumor (stratum 1 = intratumoral) or into the tumor resection cavity (stratum 2 = intracavitary). After completing six intracavitary (ICT) infusion cycles, this patient was then treated with IL13BBζ T cells. CM Patients were treated under a separate special-use protocol allowing for intracerebroventricular (ICV) delivery of

[0152] Example 18: Cellular Product Production and Infusion A lentiviral vector encoding the 4-1BB costimulatory, IL13Rα2-targeted CAR, IL13BBζ, is detailed herein. Briefly, the codon-optimized CAR sequence contains IL13Rα1, a human IgG4 Fc spacer containing two mutations (L235E; N297Q) that prevent Fc receptor-mediated recognition, a human CD4 transmembrane domain, a human costimulatory 4-1BB cytoplasmic signaling domain, and a membrane-tethered human IL-13 ligand mutated at a single site (E13Y) to reduce potential binding to human CD3ζ cytoplasmic signaling. A T2A ribosomal skip sequence then separates this IL13BBζ CAR sequence from a truncated human CD19 sequence (CD19t), an inactive, non-immunogenic cell surface marker.

[0153] On the day of leukapheresis, IL13BBζ T CMTo prepare the PBMCs, PBMCs were isolated by density gradient centrifugation on Ficoll-Paque (GE Healthcare) followed by two washes in PBS / EDTA. PBMCs were then washed once in PBS, resuspended in X Vivo15 medium (Bio Whittaker) containing 10% fetal calf serum (FCS) (Hyclone), transferred to a 300 cc transfer bag and stored on a 3-D rotator overnight at room temperature (RT). The next day, 5x109 PBMCs were incubated in X Vivo15 containing 10% FCS in a 300 cc transfer bag containing clinical grade anti-CD14 (1.25 mL), anti-CD25 (2.5 mL) and anti-CD45RA (2.5 mL) microbeads (Miltenyi Biotec) for 30 min at RT. CD14+, CD25+, and CD45RA+ cells were immediately depleted using CliniMACS™ depletion according to the manufacturer's instructions (Miltenyi Biotec). After centrifugation, the unlabeled negative fraction of cells was resuspended in CliniMACS™ PBS / EDTA buffer (Miltenyi Biotec) containing 0.5% human serum albumin (HSA) (CSL Behring) and then labeled with clinical grade biotinylated DREG56mAb (COHNMC CBG) in 0.6 mL for 30 min at RT. Cells were then washed and resuspended in a final volume of 100 mL CliniMACS™ PBS / EDTA containing 0.5% HSA and transferred to a new 300 cc transfer bag. Incubated with 1.25 mL of anti-biotin microbeads (Miltenyi Biotec) for 30 min. After immunization, the CD62L+ fraction (T CM ) were purified by positive selection on a CliniMACS™ according to the manufacturer's instructions and resuspended in X Vivo15 containing 10% FCS.

[0154] Within 2 hours of enrichment, 26.9 × 10 6TCM were stimulated and transduced with clinical grade IL13BBζ-T2A-CD19t_epHIV7 at an MOI of 0.3 in 5.5 mL of X Vivo15 containing 10% FCS with 5 μg / mL protamine sulfate (APP Pharmaceutical), 50 U / mL rhIL-2 and 0.5 ng / mL rhIL-15 in 32 Vuelife tissue culture bags (AFCs) placed in a horizontal position on a culture rack at 37 °C and 5% CO2. The cell density was then reduced to 4 × 10 5 ~2×10 6 Cultures were maintained with cytokine supplementation (final concentrations of 50U / mL rhIL-2 and 0.5ng / mL rhIL-15) every Monday, Wednesday, and Friday of culture by adding X-Vivo15 10% FCS as needed to maintain viable cells / mL. T cells were transferred into 730 Vuelife bags (AFC) based on culture volume. Seven days after lentiviral transduction, CD3 / CD28 Dynabeads were removed using a Dynal ClinEx Vivo magnetic particle concentrator bag magnet. Bead-free T cells were expelled into a new 730 Vuelife bag. Cultures were cultured at approximately 4.53×10 8 Cultures were grown until they generated approximately 1.3 × 10 cells, at which point the cultures were harvested, washed in Isolyte (Braun) with 2% HSA, and then frozen in a Cryostor CS5 (BioLife Solutions) for cryopreservation using a Mr. Frosty (Nalgene) and portable controlled rate freezer system (Custom Biogenics). 7 Cells were resuspended at 1000 cells / mL. Quality control tests included viability, potency (CD19t expression), identity (CD3 expression), transgene copy number (WPRE qPCR), replication-competent virus testing (VSV-G qPCR and formal RCL testing at Indiana University), residual bead count, and sterility.

[0155] As described above, the T2A ribosomal skip sequence 12 separates this IL13BBζ sequence from CD19t, an inactive, non-immunogenic cell surface marker that marks cell transduction (Figure 39A). This T2A binding results in the synchronous expression of both IL13BBζ and CD19t from a single transcript.

[0156] CD62L+ CD45RA-CD4+ CD8+ central memory T cells (T CM ), manufacturing methods for immunogenic enrichment of lentiviral transduction and ex vivo propagation are also detailed herein. CM The product underwent quality control release testing according to clinical protocols. For each injection, T cells were thawed, washed, and reformulated in pharmaceutical preservative-free saline (PFNS) containing 2% human serum albumin (HSA) to a final volume of 0.5 mL. Cells were manually injected into a Rickham reservoir using a 21-gauge butterfly needle to deliver a 0.5 mL volume over 5-10 minutes, followed by a maximum of 1 mL of PFNS flush delivered by convection-enhanced delivery (CED) of 0.5 mL per hour.

[0157] Example 19: Clinical Imaging Post-gadolinium T1-weighted MRI sequences of the brain and spine were acquired on a Siemens Viro 3 Tesla scanner. Lesions were measured on axial T1 MPR-weighted images obtained after administration of Multihance®. 18-F-fluorodeoxyglucose (18-F-FDG) was measured using a GE Discovery DST HP60 PET-CT scanner (70 cm axial field of view, slice thickness 3.75 mm). ) imaging. Maximum standard uptake values ​​(SUV) were obtained using Vital Images Vitrea version 6.7.2 software. The area of ​​contrast-enhancing tumor lesions was calculated as the maximum tumor area (mm 2 The CT scan was reviewed by a radiologist for measurement of tumor volume (cm) and tumor volume (cm 3 ) was calculated.

[0158] Quality control released autologous IL13BBζ T CM A cryopreserved cell bank of 1000 cells was thawed and reconstituted by washing twice with phosphate-buffered saline (PBS) containing 2% HSA and resuspending in pharmaceutical preservative-free saline containing 2% HSA. Delivery of therapeutic CAR T cells to either the glioma resection cavity (ICT) or lateral ventricle (ICV) was achieved using a Holter™ Rickham Ventriculostomy Reservoir (Codman) equipped with an intraventricular catheter (Integra Pudenz) and stylet. For ICT delivery, the reservoir / catheter device was inserted at the time of tumor resection. The tip of the catheter was partially embedded in the resection wall to allow cell delivery both within and into the peritumoral brain tissue. Postoperative imaging (CT and MRI) was obtained to confirm catheter location and extent of tumor resection.

[0159] Example 20: IL13BBζ T CM exhibit a central memory-like T cell phenotype Concentrated T CM (36×10 6 ) were stimulated ex vivo, transduced with lentivirus, and cultured for 17 days at 638 × 10 6 The CAR T cell product was expanded to obtain total cells. The final T cell product (CD3+ and TCR+) consisted of CD4 (74%) and CD8 (16%) T cell subsets expressing IL13BBζ and CD19t with genetic modification co-staining for both cell surface proteins (Figure 39A). The CAR T cell product exhibited a central memory T cell phenotype, expressing CD45RO (97%), CD62L (57%), CCR7 (28%), CD28 (97%) and CD27 (59%) (Figure 39A). This product also exhibited several exhaustion markers, including TIM-3 (65%) and LAG-3 (49%), but not significant levels of PD-1 and KLRG1 (Figure 39A).

[0160] Example 21: IL13BBζ T CM Safety and Tolerability of Repeated Intracranial Infustions of Patients received IL13BBζ T administered via a reservoir / catheter device via two different intracranial delivery routes: intracavitary (ICT) delivery after tumor resection (cycles 1-6) and intracerebroventricular (ICV) delivery to the cerebrospinal fluid (CSF) (cycles 7-11). CM The patients were treated with weekly injections of 10 × 10 6 Eleven intracranial infusions of CAR+ T cells at the maximum cell dose were well tolerated with no grade 3 or higher toxicities (NCI Common Toxicity Criteria) with possible or higher attribution to treatment observed. Minor events observed after CAR T cell infusion included the following:

[0161] Table 6: Safety and Tolerability [Table 6]

[0162] *Only events with possible or higher attribution to T cell administration are reported; all occurred once and were grade 1-2 according to NCI Common Toxicity Criteria, and no grade 3 or higher events were observed.

[0163] Example 22: Clinical Response At the time of treatment, the patient's tumor displayed features of highly aggressive recurrent GBM with poor prognostic features. This included evidence of recurrence from primary diagnosis within 6 months of treatment following standard of care, presentation of multifocal tumor disease including spinal cord involvement and extensive soft tissue tumor disease (Figure 38), histological features of dedifferentiated GBM, and high tumor proliferation rate with over 60% of cells staining positive for Ki67 (Figure 37B). Tumor expression of IL13Rα2 assessed by IHC on FFPE of resected tumor tissue was similar between primary and recurrent tumors with H-scores of 100 and 80, respectively (Figure 37). Intratumoral IL13Rα2 in recurrent tumors α2 expression was heterogeneous: 10% of cells showed high staining intensity (2-3+), 60% showed low expression (1+), and 30% of cells stained no more than background (0+). Of potential concern, the highest levels of IL13Rα2 expression were often observed in tumor areas of pseudopalisading necrosis, an expression pattern that has been noted for other GBM tumors (Figure 37A).

[0164] After enrollment in the clinical protocol, the patient underwent surgical resection of three of the five recurrent lesions (Figure 38). The reservoir / catheter device was placed in the resected cavity of the largest recurrent lesion (T1) in the right occipital-temporo-occipital region. The patient was initially treated with CAR+ T cells (2 × 10 6 1st cycle: 10×10 6 The patients were treated per protocol with intracavitary thoracic (ICT) infusions for six weeks (cycles 2–6) (Figure 1B). During this period, the temporo-occipital tumor lesion (T1) remained stable for more than 45 days after surgery without evidence of progressive disease (Figure 39C). However, MRI revealed that other unresected tumor lesions in the left temporal lobe (T4 and T5) as well as a new recurrent lesion adjacent to tumor 3 (T6) and a lesion in the olfactory sulcus (T7) had progressed over this same period (Figure 39C). In addition, one large tumor (270 mm 2 ) and one or more small tumor foci (<1 cm 2 Metastatic lesions in the spine were also detected, including IL13BBζ T CM These findings were encouraging, as they suggested that local ICT delivery may have prevented disease recurrence in the resected occipitotemporal-occipital region, but they also suggested that local ICT delivery was insufficient to effectively control tumor progression in distant areas away from the injection site.

[0165] Based on these findings, and supported by preclinical studies showing that ICV delivery of CAR T cells can be delivered to multifocal GBM in the NSG mouse model (data not shown), this patient was enrolled in a special use protocol and was treated with IL13BBζ T cells without any other therapeutic intervention. CM The patients were treated with ICV infusions of 2 × 10 6 7th cycle: 10×10 6 (Cycles 8-11) (Figure 40A). One week after three ICV infusions (Cycle 9; Day 133), all tumor lesions showed dramatic regression. After the final ICV infusion (Cycle 11; Day 156), the majority of intracranial and spinal tumors had regressed by more than 70% by both maximum area and volume measurements (Figures 40B-E, Table 7 below, and Figure 41). Follow-up MR and PET imaging 6 weeks after the last ICV infusion (Day 190), when the patient received no other therapeutic intervention, continued to show disease regression. Tumors were reduced by ≧78% by both maximum area and volume measurements (Figures 40B-E, Table 7 below, and Figure 41). At this Day 190 time point, it was not possible to distinguish between disease versus residual radiographic evidence of inflammation, scarring, and / or dural enhancement. CM ICV delivery of IL13BBζ T induced a near complete elimination of all metastatic tumors within the spine. The largest area of ​​the largest lesions was reduced by 97%. Only one small tumor focus was visible out of more than 10 present before ICV treatment. Concurrent with the time course and tumor regression of ICV treatment, the system dexamethosome could be reduced from 2 mg bid to 0.5 mg qd. The patient remained clinically stable and returned to normal life and work activities, thus supporting the durability of this CAR T cell-mediated antitumor response. These results support the efficacy and safety of IL13BBζ T CAR T cell-mediated antitumor response. CM These results show that treatment with mediated a near complete response based on stringent RANO criteria.

[0166] Table 7: MRI assessment of non-resected lesions (volume cm 3 , area mm 2 ) [Table 7]

[0167] *New lesions occurring during cycles 1-6 Bold, values ​​compared to maximum % reduction NA, lesion not identified 0, the lesion may be visually identified, but the value was lower than the value of the analysis software parameter ND, no imaging performed

[0168] Example 23: CAR T cell persistence and CNS inflammatory responses IL3BBζ T CM To elucidate the immunological changes associated with the antitumor response observed after ICV infusions (cycles 6-11), CSF was evaluated for cellular infiltration, CAR+ T cell persistence, and cytokine levels. Immediately after each ICV infusion (i.e., days 1-2 of cycles 6-11), the number of cells per mL of CSF increased 7.0±3.6-fold compared to pre-infusion levels (day 0 of each cycle) and 153±128-fold compared to pre-ICV (C7D0) levels (Figure 42). Total cell numbers in the CSF typically decreased over the 7-day treatment cycle. When evaluated for C9D2, the cellular infiltrate encompassed the majority of CD3+ T cells, both endogenous and CAR-expressing, as well as CD14+ CD11b+ HLA-DR+ mature myeloid populations (Figure 42A). Only rare CD19+ B cells and CD11b+ CD15+ granulocytes were detected (Figure 42A). Consistent with the flow cytometry data, CSF cytopathology for C11D1 reported the presence of reactive lymphocytes, monocytes and macrophages.

[0169] CAR-T persistence was also monitored over the course of ICV treatment. Due to low cell recovery in the CSF of cycles 7 and 8, analysis focused on evaluating time points immediately following cycles 9, as well as cycles 10 and 11. Importantly, CAR+ T cells were detected at all time points evaluated, including C9D0, which corresponds to 7 days after cycle 8 (Figure 42B), thus demonstrating persistence of the therapeutic cells for at least 7-8 days after infusion. However, the number of CAR T in the CSF after infusion decreased in later cycles when tumor burden significantly decreased (C10D1 and C11D1) (Figure 40B). Of note, no significant expansion of CAR T cells in the CSF at cycle 9 was detected. The number of CAR+ cells increased 1.6-fold from pre-infusion (C9D0) to 2 days after (C9D2), then decreased 2.3-fold at day 8 (C9D8).

[0170] The presence of immune cells, including CAR+ T cells, after each infusion corresponded to a significant increase in cytokine levels in the CSF. The measured levels and calculated fold-change above baseline for the 30 cytokines measured are shown below in Tables 9 and 10. Notably, 11 cytokines increased 10-fold or more from pre-ICV baseline (C7D0) immediately after IL3BBζ TCM infusion, including cytokines IFNγ, TNF, IL-2, IL-10, IL-5, IL-6, and IL-8, and chemokines CXCL9 / MIG, CXCL10 / IP-10, and CCR2 / MCP-1, and soluble cytokine receptor IL-1Rα (FIG. 3C). Seven other cytokines showed a 5-fold or greater increase from baseline (C7D0), including G-CSF, IL-12, IL-2-R, IL-4, IL-7, and MIP-1b. IL3BBζ TCM increased 10-fold or more from pre-ICV baseline (C7D0) immediately after infusion compared to pre-ICV (C7D0). CMThe inflammatory cytokines that showed the highest fold increase immediately after infusion were IL-2 (>90-fold for C9D2) and the IFN-γ-inducible chemokines CXCL9 and CXCL10 (>40-fold for C8D1, C9D2, C10D1 and C11D2). These cytokines returned to near baseline levels within 7 days between treatment cycles. Cytokines that did not show a significant increase after CAR T cell infusion included IL-13, RANTES and VEGF (Tables 9 and 10).

[0171] Table 9: UPN 109 CSF cytokine analysis (pg / mL), ICV cycles 7-11 [Table 8]

[0172] OOR<, Out of Range (less than) *, Value extrapolated beyond normal range

[0173] Table 10: UPN 109 CSF cytokine fold change analysis, ICV cycles 7-11 [Table 9]

[0174] Bolded values ​​from Table 10, "OOR<" values ​​were replaced with the lowest measurable value for that cytokine to allow for calculation of fold change. *, cytokines for which a >10-fold increase was observed at least once.

[0175] These immunological changes in the CSF were local, as there were no significant changes in cytokine levels (Table 11) and no detectable CAR+ T cells were observed by qPCR and flow cytometry in peripheral blood (data not shown). The changes in the CSF could not be compared to those in the resected cavity of tumor lesion 1 (T1) due to the inability to obtain cyst fluid from the cavity during the ICT treatment course.

[0176] Table 11: UPN 109 serum cytokine analysis (pg / mL), ICV cycles 7-11 [Table 10]

[0177] OOR<, Out of Range (less than) *, Value extrapolated beyond normal range

[0178] Example 24: Patient Sample Processing and Analysis Tumor resection material was collected through the COH Department of Pathology according to clinical protocols.

[0179] IL-13Rα2 immunohistochemistry (IHC) was performed on 5 μm sections of formalin-fixed paraffin-embedded specimens as described above. Ki67 IHC was performed similarly except for antigen retrieval by heating at pH 8.0 and a 1:75 dilution of anti-K167 (Dako Corp). IL-13Rα2 immunoreactivity was scored by a clinical neuropathologist and quantified based on the percentage of tumor cells showing cytoplasmic and Golgi-like staining of weak (1+), moderate (2+), or strong (3+) intensity. The H score was calculated using the formula: (3 x percentage of strongly staining cells) + (2 x percentage of moderately staining cells) + percentage of weakly staining cells The H score was obtained by , giving a range of 0-300. The H score can be translated into the intensity scoring system described in the enrollment criteria as follows: 0 represents negative (H score 0), 1+ represents low (H score 1-100), 2+ represents moderate (H score 101-200), and 3+ represents high (H score 201-300). Inclusion criteria were at least 20% of cells scoring 1+ staining intensity (>20%, 1+), representing an H score of 20. Appropriate positive (testis) and negative (prostate) controls were used for IL-13Rα2 IHC staining. The "+" symbol reflects the presence of membranous staining. The study was performed in the Department of Pathology at City of Hope National Medical Center and is considered a clinical trial for research purposes. The laboratory is Clinical Laboratory Improvement Amendments of 1988 (CLIA) certified as qualified to perform complex clinical laboratory tests.

[0180] Peripheral blood samples were collected in vacutainer tubes ± EDTA. Samples with EDTA were ficolled immediately upon receipt. Peripheral blood mononuclear cells (PBMCs) were frozen in a Crystor CS5 at -80°C and then transferred to liquid nitrogen for long-term storage. Samples without EDTA were allowed to clot for 2-3 hours at room temperature. Serum was collected by centrifugation, divided into single-use aliquots of 100-200 μL, and stored at -80°C. Cerebrospinal fluid (CSF) was collected from the ICV reservoir with a 3 cc syringe, spun down, and the supernatant was divided into aliquots and stored at -80°C. CSF cells were immediately resuspended in HBSS- / - with 2% FCS and sodium azide (Corning CellGro) for flow cytometry analysis. The remaining cells were resuspended, frozen in a Cryostor CS4 at -80°C, and then transferred to liquid nitrogen for long-term storage.

[0181] Cell surface phenotyping of immune cells was performed by flow cytometry using fluorochrome conjugates specific for CD3, CD4, CD11b, CD14, CD19, CD27, CD28, CD62L, CD45RA, CD45RO, IL-13, TCR-α / β (BD Biosciences), KLRG1, CD15 (BioLegend), HLA-DR, PD1 (eBiosciences), CD8 (Fisher Scientific), LAG-3 (Lifespan Biosciences), CCR7, or TIM-3 (R&D Systems) and their respective isotype controls.

[0182] Study participant serum and CSF samples were analyzed by cytokine bead array. Assays were performed using the Human Cytokine 30-Plex Panel Kit (Invitrogen) and FLEXMAP 3D® (Luminex).

Claims

1. 1. A pharmaceutical composition for treating a patient diagnosed with a tumor of the central nervous system, comprising an effective amount of T cells, said T cells comprising a chimeric antigen receptor that targets a tumor antigen, for administration intraventricularly to said patient, wherein said tumor of the central nervous system expresses said tumor antigen.

2. 1. A pharmaceutical composition for treating a patient diagnosed with a tumor of the central nervous system, comprising an effective amount of T cells, said T cells comprising a chimeric antigen receptor that targets a tumor antigen, said chimeric antigen receptor comprising: A tumor targeting domain, A spacer selected from SEQ ID NO: 4, 14-20, 50-52, or each variant thereof having 1-5 amino acid modifications; a transmembrane domain selected from the CD4 transmembrane domain or a variant thereof having from 1 to 5 amino acid modifications, the CD8 transmembrane domain or a variant thereof having from 1 to 5 amino acid modifications, the CD28 transmembrane domain or a variant thereof having from 1 to 5 amino acid modifications, and the CD3 zeta transmembrane domain or a variant thereof having from 1 to 5 amino acid modifications, At least one costimulatory domain selected from a CD28 costimulatory domain or a variant thereof having 1 to 5 amino acid modifications, and a 4-1BB costimulatory domain or a variant thereof having 1 to 5 amino acid modifications, and CD3zeta domain, and said central nervous system tumor expresses said tumor antigen.

3. The pharmaceutical composition described in claim 1 or 2, wherein the central nervous system tumor is glioblastoma.

4. The pharmaceutical composition of claim 1 or 2, wherein the T cells are autologous or allogeneic T cells.

5. At least 1 x 10 ６ 3. The pharmaceutical composition of claim 1 or 2, comprising cells.

6. 3. The pharmaceutical composition of claim 1 or 2, which is administered at least twice, optionally with different total numbers of T cells administered or with stepwise escalating or de-escalating doses.

7. The pharmaceutical composition described in claim 1 or 2, wherein the tumor of the central nervous system is caused by a diffuse infiltrating tumor, a primary brain tumor, a secondary brain tumor, or a primary cancer selected from breast cancer, lung cancer, head and neck cancer, and melanoma.

8. 3. The pharmaceutical composition of claim 1 or 2, wherein the chimeric antigen receptor binds to a protein expressed on the surface of a glioblastoma cell.

9. The pharmaceutical composition according to claim 1 or 2, wherein the T cells comprise both CD4+ cells and CD8+ cells, and / or the T cells comprise at least 10% Tcm cells.

10. The pharmaceutical composition of claim 2 , wherein the tumor targeting domain comprises an scFv targeted to the tumor antigen.

11. The pharmaceutical composition of claim 10, wherein the scFv is represented by SEQ ID NO:49 or comprises a variant thereof having 1 to 5 amino acid modifications of SEQ ID NO:

49.

12. 3. The pharmaceutical composition of claim 2, wherein the tumor targeting domain comprises human IL-13 or a variant thereof, wherein the human IL-13 variant comprises the amino acid sequence of SEQ ID NO:3 with 1 to 5 amino acid modifications, with the proviso that the amino acid at position 11 of SEQ ID NO:3 is other than E.

13. The pharmaceutical composition of claim 2 , wherein the transmembrane domain is selected from a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a CD3ζ transmembrane domain.

14. The pharmaceutical composition of claim 2, wherein the spacer comprises an amino acid sequence represented by an amino acid sequence selected from SEQ ID NOs: 4, 14-20, 50, 51, and 52.

15. The pharmaceutical composition of claim 2, wherein the at least one costimulatory domain is selected from a CD28 costimulatory domain and a 4-1BB costimulatory domain.

16. Tumors include the following: primary CNS malignancies and secondary malignancies resulting from cancer elsewhere, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratopoietic / rhabdoid tumor, central nervous system, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, bronchial tumor, Burkitt's lymphoma, carcinoma id tumors, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, fibrous histiocytoma of bone, malignant and osteosarcoma, gallbladder cancer, gastric,stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, hepatocellular carcinoma (primary), pancreatic cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, bone cancer fibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with primary midline carcinoma associated with the NUT gene, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumor / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer of the oropharynx, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiated pineal parenchymal tumor, pineoblastoma (pineoblastoma), cerebellar tentorial epithelial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell The pharmaceutical composition according to claim 1 or 2, which is derived from a cancer selected from the group consisting of lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell cervical cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, ureteral cancer, renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

17. The pharmaceutical composition described in claim 1 or 2, wherein the tumor of the central nervous system is neuroblastoma (neuroblastoma).

18. The pharmaceutical composition of claim 1 or 2, wherein the central nervous system tumor is a tumor expressing IL13Rα2, HER2, PSCA, EGFR, EGFRvIII, EphA2, NY-ESO-1, or CD19.

19. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is HER2.

20. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is IL13Rα2.

21. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is PSCA.

22. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is EGFR.

23. The pharmaceutical composition of claim 1 or 2, wherein the tumor antigen is EGFRvIII.

24. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is EphA2.

25. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is NY-ESO-1.

26. The pharmaceutical composition described in claim 1 or 2, wherein the tumor antigen is CD19.

27. A pharmaceutical composition for administration intracerebroventricularly to treat a patient diagnosed with a tumor of the central nervous system, comprising an effective amount of T cells, the T cells comprising a chimeric antigen receptor that targets CD19.

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