Chimeric antigen receptor containing a chlorotoxin domain

Abstract

To provide chimeric transmembrane immunoreceptors (CARs).SOLUTION: The chimeric transmembrane immunoreceptors (CAR) include an extracellular domain that includes chlorotoxin or a related toxin, or a variant of chlorotoxin or a related toxin, that binds to human glioma or other human tumor cells, a transmembrane domain, a costimulatory domain and an intracellular signaling domain.SELECTED DRAWING: None

Description

[Background technology]

[0001] Tumor-specific T cell-based immunotherapy, including therapy with recombinant T cells, has been investigated for antitumor treatment. Chimeric antigen receptors (CARs) are composed of an extracellular tumor recognition / targeting region, an extracellular linker / spacer, a transmembrane region, and an intracellular T cell activation and costimulatory signaling region. The design of the recognition / targeting region is crucial to avoid deleterious off-target effects. The majority of CAR tumor targeting regions are single-chain variable fragments (scFvs) derived from antibody sequences, exploiting the specificity of antibodies to bind to specific antigens. There are also examples of CAR tumor targeting regions derived from conventional receptor ligands, such as the IL-13 cytokine CAR, which targets cells expressing the IL-13 receptor, IL13Rα2. Despite some notable successes, the identification and validation of novel CAR tumor targeting regions remains a major challenge in the field.

[0002] Malignant gliomas (MG), including anaplastic astrocytoma (AA-WHO grade III) and glioblastoma (GBM-WHO grade IV), are diagnosed in the United States with approximately 20,000 new cases annually. According to the American Brain Tumor Association, the total prevalence of individuals with malignant brain tumors is approximately 140,000 in the United States based on 2010 census data. MG is a rare disease, highly aggressive and heterogeneous in its malignant behavior and generally fatal. The current standard treatment for high-grade MG provides short-term benefits, and these brain tumors are virtually incurable. In fact, even with modern surgical and radiotherapy techniques, the location within the central nervous system (CNS) often exacerbates the already severe morbidity, and the 5-year survival rate is quite low. Furthermore, there are few treatment options for the majority of patients whose disease recurs. Therefore, more effective treatments are highly desirable, especially for patients who relapse / progress after initial treatment.

[0003] Adoptive T cell therapy (ACT), which utilizes recombinant T cells expressing CAR, can provide a safe and effective method to reduce the recurrence rate of MG, in which CAR T cells are engineered to specifically recognize antigenically distinct tumor populations (Non-Patent Documents 1-5). T cells can also migrate through the brain parenchyma to target and kill invasive malignant cells (Non-Patent Documents 6-8). 8 ). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cartellieri et al.2010 J Biomed Biotechnol 2010:956304 [Non-Patent Document 2] Ahmed et al.2010 Clin Cancer Res 16:474 [Non-Patent Document 3] Sampson et al.2014 Clin Cancer Res 20:972 [Non-Patent Document 4] Brown et al.2013 Clin Cancer Res 2012 18:2199 [Non-Patent Document 5] Chow et al.2013 Mo / Ther 21 :629 [Non-Patent Document 6] Hong et al.2010 Clin Cancer Res 16:4892 [Non-Patent Document 7] Brown et al.2007 J Immunol 179:3332 [Non-Patent Document 8] Yaghoubi 2009 Nat Clin Pract Oncol 6:53 Summary of the Invention [Means for solving the problem]

[0005] Described herein are chimeric transmembrane immune receptors (chimeric antigen receptors or CARs) that include an extracellular region, a transmembrane region, and an intracellular signaling region. The extracellular region includes chlorotoxin (a 36 amino acid peptide toxin found in the venom of the scorpion Leiurus quinquestriatus), or a related toxin, or a variant of chlorotoxin or a related toxin, and optionally a spacer, e.g., including a portion of a human Fc region. The transmembrane portion includes, e.g., a CD4 transmembrane region, a CD8 transmembrane region, a CD28 transmembrane region, or a CD3 transmembrane region. The intracellular signaling region includes a signaling region from the zeta chain of the human CD3 complex (CD3ζ) and one or more costimulatory domains, e.g., a 4-1BB costimulatory domain. When the extracellular region is expressed on the surface of a T cell, the CAR can direct T cell activity to the cell expressing a receptor for chlorotoxin, such as a glioblastoma cell. The inclusion of a costimulatory domain, such as the 4-1BB (CD137) costimulatory domain, in tandem with CD3ζ within the intracellular domain allows the T cells to receive costimulatory signals. T cells, e.g., patient-specific autologous T cells, may be engineered to express the CARs described herein, and the engineered cells may be expanded and used in ACT. Various T cell subsets may be used, including both alpha beta (αβ) T cells and gamma delta (γδ) T cells. Additionally, CARs can also be expressed in other immune cells, such as NK cells. When a patient is treated with immune cells expressing the CARs described herein, the cells may be autologous or allogenic T cells. In some cases, the cells used are both CD4+ and CD8+ central memory T cells (T cells), which are CD62L+, CCR7+, CD45RO+, and CD45RA-. CM ) or the cells used are a cell population comprising CD4+ and CD8+ T CM A cell population that includes T cells, stem central memory T cells, and naive T cells (i.e., T CM / SCM / NA population of cells. CM / SCM / N The population of cells is CD62L+, CCR7+, and contains both CD45RA+ and CD45RO+ cells, as well as both CD4+ and CD8+ cells. The use of such cells can improve long-term persistence of cells following adoptive transfer compared to the use of other types of patient-specific T cells.

[0006] Described herein is a nucleic acid molecule encoding a CAR: Chlorotoxin (MCMPCFTTDHQMA R.K. CDDCCGGKGRGKCYGPQCLCR; SEQ ID NO: 1), or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted) with the cysteine ​​residues remaining unmodified; transmembrane domains: CD4 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), CD8 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), CD28 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), and CD3 zeta transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted). a transmembrane region selected from; a costimulatory region (e.g., a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3 zeta signaling region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications.

[0007] In one embodiment, the CAR comprises a toxin related to chlorotoxin instead of chlorotoxin. Thus, the CAR is GaTx2, a toxin from the scorpion (Leiurus quinquestriatus hebraeus) (VSCEDCPDHCSTQKARAKCDNDKCVCEPI; SEQ ID NO: 56), or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), with the proviso that the cysteine ​​residues are not modified; transmembrane domains: CD4 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), CD8 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), CD28 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), and CD3 zeta transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted). a transmembrane region selected from a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory region (e.g., a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3 zeta signaling region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0008] In some embodiments, a CAR may contain more than one chlorotoxin sequence (e.g., two or three or more copies of SEQ ID NO:1, either contiguous or separated by 1-10 amino acids), or more than one toxin related to chlorotoxin. Thus, a CAR may contain two or more chlorotoxin sequences (e.g., SEQ ID NO:1 followed by SEQ ID NO:1 followed by the remainder of the molecule), or a CAR may contain a chlorotoxin sequence followed by a sequence of a toxin related to chlorotoxin (e.g., SEQ ID NO:57 or other toxins shown in FIG. 25).

[0009] The CAR is GaTx1, a toxin from the scorpion (Leiurus quinquestriatus hebraeus) (CGPCFTTDHQMEQKCAECCGGIGKCYGPQCLCNR; SEQ ID NO: 57), or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), with the proviso that the cysteine ​​residues are not modified; transmembrane domains: the CD4 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), the CD8 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), the CD28 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), and the CD3 zeta transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted). a transmembrane region selected from a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory region (e.g., a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3 zeta signaling region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0010] The CAR is AaCtx, a toxin from the scorpion Androctonus australis (MCIPCFTTNPNMAAKCNACCGSRRGSCRGPQCIC; SEQ ID NO: 58), or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), with the proviso that the cysteine ​​residues are not modified; transmembrane domains: CD4 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), CD8 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), CD28 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), and CD3 zeta transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted). a transmembrane region selected from a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory region (e.g., a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3 zeta signaling region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0011] The CAR is BmKCT, a toxin from the scorpion Buthus martensii (CGPCFTTDANMARKCRECCGGIGKCFGPQCLCNRI; SEQ ID NO: 59), or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), with the proviso that the cysteine ​​residues are not modified; a transmembrane domain: a transmembrane domain shown in Table 2 or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), a CD4 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), a CD8 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), a CD28 transmembrane domain or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted), and a CD3 zeta transmembrane domain or a transmembrane region selected from a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory region (e.g., a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3 zeta signaling region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0012] In one embodiment, the CAR comprises the amino acid sequence of any of SEQ ID NOs: 26-55, in which the chlorotoxin sequence (SEQ ID NO: 1) is replaced by any of SEQ ID NOs: 56-59 or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted).

[0013] In some embodiments, the costimulatory region is selected from the group consisting of: a costimulatory region shown in Table 3 or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory region or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory region or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications, and an OX40 costimulatory region or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, there are 4-1BB costimulatory regions or variants thereof with 1-5 (e.g., 1 or 2) amino acid modifications. In some embodiments, there are two costimulatory regions, such as a CD28 costimulatory region or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a 4-1BB costimulatory region or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In one embodiment, 1-5 (eg, 1 or 2) amino acid modifications are substitutions.

[0014] In some cases, there is a short sequence of 1-6 amino acids (eg, GGG) between the costimulatory domain and the CD3 zeta signaling domain and / or between two costimulatory domains.

[0015] Additional embodiments of CARs include: variants of chlorotoxin with 1-5 amino acid modifications that enhance binding specificity or immunogenicity for the chlorotoxin receptor (Cltx-R); the chlorotoxin variants are variants that include the amino acid sequence of SEQ ID NO: 1 with 1-5 (e.g., 1 or 2) amino acid modifications; two different costimulatory domains: the CD28 costimulatory domain or variants thereof with 1-5 (e.g., 1 or 2) amino acid modifications, the 4-1BB costimulatory domain or variants thereof with 1-5 (e.g., 1 or 2) amino acid modifications, two different costimulatory regions selected from the group consisting of: a CD28 costimulatory region or a variant thereof with 1-2 amino acid modifications, a 4-1BB costimulatory region or a variant thereof with 1-2 amino acid modifications, and an OX40 costimulatory region or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications; two different costimulatory regions selected from the group consisting of: a CD28 costimulatory region or a variant thereof with 1-2 amino acid modifications, a 4-1BB costimulatory region or a variant thereof with 1-2 amino acid modifications, and an OX40 costimulatory region or a variant thereof with 1-2 amino acid modifications. a stimulatory domain; a chlorotoxin or a variant thereof modified by 1-2 amino acids; a transmembrane domain selected from: a CD4 transmembrane domain or a variant thereof modified by 1-2 amino acids, a CD8 transmembrane domain or a variant thereof modified by 1-2 amino acids, a CD28 transmembrane domain or a variant thereof modified by 1-2 amino acids, and a CD3 zeta transmembrane domain or a variant thereof modified by 1-2 amino acids; a costimulatory domain (e.g., a CD28 costimulatory domain or a variant thereof modified by 1-5 (e.g., 1 or 2) amino acids); or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3 zeta signaling region or a variant thereof having 1-2 amino acid modifications;a spacer region located between the chlorotoxin or variant thereof and the transmembrane region (e.g., a spacer region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2-12 (Table 3) or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications); the spacer comprises an IgG hinge region; the spacer region comprises 1-150 amino acids; there is no spacer; the 4-1BB signaling region comprises the amino acid sequence of SEQ ID NO:24, the CD3ζ signaling region comprises the amino acid sequence of SEQ ID NO:21, and a linker of 3 to 15 amino acids is located between the costimulatory region and the CD3ζ signaling region or variant thereof. In particular embodiments with two costimulatory regions, one is a 4-1BB costimulatory region and the other is a costimulatory region selected from: CD28 and CD28gg. In certain embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions;

[0016] In one embodiment, the nucleic acid molecule expresses a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 26-55; the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NOs: 26-55.

[0017] Also disclosed is a population of human T cells transduced with a vector comprising an expression cassette encoding a chimeric antigen receptor, the chimeric antigen receptor being either: chlorotoxin or a variant thereof having 1-5 amino acid modifications (e.g., 1 or 2), a chlorotoxin-related toxin or a variant thereof having 1-5 amino acid modifications (e.g., 1 or 2); a transmembrane region selected from: a CD4 transmembrane region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., 1 or 2) thereof, a CD8 transmembrane region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., 1 or 2) thereof, a CD28 transmembrane region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., 1 or 2) thereof, and a CD3 zeta transmembrane region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., 1 or 2) thereof; or a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both a CD28 costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a CD3ζ signaling region or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In one embodiment, the population of human T cells comprises a vector expressing a chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NOs: 26-55 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); the population of human T cells comprises a central memory T cell (T CM ), e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T CM Alternatively, the population of human T cells may be a combination of central memory T cells, naive T cells and stem central memory T cells (T CM / SCM / Ncells), e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T CM / SCM / N In either case, the population of T cells includes both CD4+ and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+, at least 3% of the CD3+ T cells are CD8+, and at least 70%, 80%, or 90% are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the cells that are CD3+ cells are CD4+, and at least 4%, 5%, 8%, 10%, 20% of the CD3+ cells are CD8+ cells).

[0018] Also provided is a method of treating cancer in a patient, comprising administering to the patient a population of autologous or allogeneic human T cells (e.g., central memory T cells (T CM cells) or a combination of central memory T cells, naive T cells, and stem central memory T cells (i.e., T cells are T CM / SCM / N cells, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells being T CM / SCM / NAlso described are methods comprising administering a population of T cells (autologous or allogeneic T cells) to a patient in need of treatment with a glioma. In either case, the population of T cells comprises both CD4+ and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+, at least 3% of the CD3+ T cells are CD8+, and at least 70%, 80% or 90% are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the cells that are CD3+ cells are CD4+, and at least 4%, 5%, 8%, 10%, 20% of the CD3+ cells are CD8+ cells), introduced by a vector comprising an expression cassette encoding a chimeric antigen receptor, the chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NOs: 26-55 or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted). In one embodiment: the cancer is glioblastoma; the transduced human T cells were prepared by a method comprising the steps of: obtaining T cells from the patient, treating the T cells to isolate central memory T cells, and transducing into at least a portion of the central memory T cells with a viral vector comprising an expression cassette encoding a chimeric antigen receptor, the chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NOs: 26-55, or a variant thereof in which 1-5 (e.g., 1 or 2) amino acids have been modified (e.g., substituted).

[0019] Also described are: nucleic acid molecules, which encode a polypeptide comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NO:26-55; nucleic acid molecules, which encode a polypeptide comprising an amino acid sequence which corresponds to an amino acid sequence selected from SEQ ID NO:26-55 except for the presence of not more than five amino acid substitutions, deletions or insertions; nucleic acid molecules, which encode a polypeptide comprising an amino acid sequence which corresponds to an amino acid sequence selected from SEQ ID NO:26-55 except for the presence of not more than five amino acid substitutions; and nucleic acid molecules, which encode a polypeptide comprising an amino acid sequence which corresponds to an amino acid sequence selected from SEQ ID NO:26-55 except for the presence of not more than two amino acid substitutions.

[0020] T cells expressing a CAR containing chlorotoxin or a variant thereof may be useful in the treatment of cancers, such as glioblastoma and other cancers that express the receptor for chlorotoxin. Such cancers include, but are not limited to: primary brain tumors and gliomas (glioblastoma multiforme WHO grade IV, anaplastic astrocytoma WHO grade III, low-grade astrocytoma WHO grade II, pilocytic astrocytoma WHO grade I, other ungraded gliomas, oligodendroglioma, gliosarcoma, ganglioglioma, meningioma, ependymoma), neuroectodermal tumors (medulloblastoma, neuroblastoma, ganglioneuroma, melanoma (metastatic), melanoma (primary), pheochromocytoma, Ewing's sarcoma, primary neuroectodermal tumor, small cell lung carcinoma, schwannoma), other brain tumors (epidermoid cyst (epidermoid), brain tumor of unknown pathology, glioblastoma (pt.) of the pituitary gland), metastatic tumors to the brain of unknown tissue origin), and other cancers (breast cancer, breast cancer metastases, kidney cancer, liver cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer).

[0021] The disclosure also includes nucleic acid molecules encoding any of the CARs described herein (e.g., a vector comprising a nucleic acid sequence encoding one of the CARs) and isolated T lymphocytes expressing any of the CARs described herein.

[0022] The CARs described herein may include a spacer region located between the chlorotoxin region (i.e., chlorotoxin or a variant thereof) and the transmembrane region. A variety of different spacers may be used. Some of them include at least a portion of a human Fc region, such as a hinge portion or a CH3 region of a human Fc region or a variant thereof. Table 1 below provides various spacers that may be used in the CARs described herein. Table 1: Examples of spacers [Table 1]

[0023] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence interposed between the immunoglobulin CH1 and CH2 regions, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 region or both the CH3 and CH2 regions. The immunoglobulin-derived sequence may include one or more amino acid modifications, e.g., 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0024] "Amino acid modification" refers to amino acid substitutions, insertions and / or deletions in a protein or peptide sequence. "Amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in an original peptide or protein sequence with another amino acid. Substitutions may be made to change the amino acids in the resulting protein in a non-conservative manner (i.e., by changing a codon from an amino acid belonging to a class of amino acids having a particular size or characteristics to an amino acid belonging to another class) or in a conservative manner (i.e., by changing a codon from an amino acid belonging to a class of amino acids having a particular size or characteristics to an amino acid belonging to the same class). Such conservative changes generally lead to smaller changes in the structure and function of the resulting protein. The following are examples of different classifications of amino acids: 1) Amino acids with non-polar 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): lysine, arginine, histidine (pH 6.0). Another classification could be amino acids with phenyl groups: phenylalanine, tryptophan and tyrosine.

[0025] In certain embodiments, the spacer is derived from an IgG1, IgG2, IgG3, or IgG4 that comprises one or more amino acid residues substituted with an amino acid residue different from that present in the unmodified spacer. The one or more substituted amino acid residues are selected from, but are not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, or combinations thereof. In this numbering system, which is described in more detail below, the first amino acid of the IgG4(L235E, N297Q) spacer in Table 1 is 219, just as the first amino acid of the IgG hinge sequences and the IgG4 hinge linker (HL) sequences in Table 1 is 219, and the first amino acid of the IgG4(HL-CH3) spacer in Table 1 is 219.

[0026] In certain embodiments, the modified spacer is derived from IgG1, IgG2, IgG3, or IgG4, including, but not limited to, one or more of the following amino acid residue substitutions: C220S, C226S, S228P, C229S, P230S, E233P, V234A, L234V, L234F, L234A, L235A, L235E, G236A, G237A, P238S, S239D, F243L, P247I, S267E, H268Q, S280H, K290S, K290E, K290N, R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I, V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S, P331S, I332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L or combinations thereof.

[0027] In certain embodiments, the modified spacer is derived from an IgG4 region that comprises one or more amino acid residues replaced by an amino acid residue different from that present in the unmodified spacer. The one or more replaced amino acid residues are selected from, but are not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, or combinations thereof.

[0028] In one embodiment, the modified spacer is derived from an IgG4 region that includes, but is not limited to, one or more of the following amino acid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E, 268Q, 249I, 250I, 251I, 252I, 253I, 254I, 255I, 256I, 257I, 258I, 259I, 260I, 261I, 262I, 263I, 264I, 2 80H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L or combinations thereof, where the amino acids in the unmodified spacer are replaced at the indicated positions by the amino acids identified above.

[0029] For amino acid positions in immunoglobulins discussed herein, numbering is according to the EU index or EU numbering system (Kabat et al., 1991 Sequences of Proteins of Immunological Interest, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, incorporated herein by reference in its entirety). The EU index or EU numbering system as in Kabat refers to the EU antibody numbering (Edelman et al., 1969 Proc Natl Acad Sci USA 63: 78-85).

[0030] A variety of transmembrane regions may be used. Table 2 includes examples of suitable transmembrane regions. If a spacer region is present, the transmembrane region is located carboxy-terminal to the spacer region. Table 2: Examples of transmembrane domains [Table 2]

[0031] Many of the CARs described herein include one or more (e.g., two) costimulatory domains. The costimulatory domain(s) are located between the transmembrane domain and the CD3 zeta signaling domain. Table 3 includes examples of suitable costimulatory domains, along with the CD3 zeta signaling domain. Table 3: Examples of CD3 ζ and costimulatory regions [Table 3]

[0032] Among the chlorotoxin-containing CARs described herein are summarized in Table 4, which shows the spacer region, transmembrane region and costimulatory region(s) for each CAR. Table 4: Examples of CARs containing chlorotoxin [Table 4] *SEQ ID NO for sequence with signal sequence / SEQ ID NO for sequence excluding signal sequence [Brief description of the drawings]

[0033] [Figure 1] Generation of CLTX-CAR-expressing T cells. (A) Schematic of lentiviral construct encoding chlorotoxin (CLTX)-redirected chimeric antigen receptor (CAR) cassette, where transcription of CLTX-CAR and T2A ribosomal skip and truncated CD19 (CD19t) sequences is driven by the EF1 promoter (EF1p). (B) Diagram of CLTX-CAR, including the extracellular 36 amino acid chlorotoxin peptide and IgG4 Fc (EQ) spacer region, CD28 transmembrane domain, and intracellular CD28 and CD3 zeta cytoplasmic signaling domain sequences. (C) Flow cytometry analysis of healthy donor T cells (HD187.2 TCM / SCM / N) engineered to express CLTX-CAR. Shown are anti-CD19 anti-Fc and anti-CD8 stainings depicting co-expression of CLTX-CAR and CD19t transgenes in both CD8+ and CD4+ (CD8-) T cell subsets. Percentages of immunoreactive cells for transduced (CLTX-CAR) and non-transduced cells (mock) 18 days after CD3 / CD28 bead stimulation are shown, demonstrating the ability to transduce human T cells with CLTX-CAR.

[0034] [Diagram 2]Figure 1 shows that CLTX-CAR T cells specifically recognize the glioblastoma cell line U251T. (A-F) CLTX binds to GBM cells and shows minimal binding to non-GBM cells. Shown is evaluation of chlorotoxin-conjugated Cy5.5 (CLTX-Cy5.5) binding to A, human peripheral blood mononuclear cells (PBMCs) from a healthy donor; B, human EBV-transformed lymphoblastoid cell line, LCL; C, large T antigen-transformed human embryonic kidney cell line 293T; D, human astrocytes differentiated from induced pluripotent stem cells (iPSCs) from a healthy donor; and E, human glioblastoma cell line U251T. Cell lines were cultured in medium (untreated) or medium containing 1 μM CLTX-Cy5.5 for 1 hour at 37°C and then evaluated by flow cytometry. (F) Specific killing of glioma tumor cell line U251T, but not LCL, 293T or primary human astrocytes, by CLTX-CAR T cells. Plotted are the numbers of viable target cells (LCL, 293T, astrocytes and U251T) co-cultured with CLTX-CAR T cells for 72 hours at an effector:target ratio = 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. **: p<0.01; ns: non-specific, Student's t-test was performed between groups as indicated in the figure.

[0035] [Diagram 3] Figure 1: CLTX binding to multiple low-passage, human primary brain tumor (PBT) lines is independent of IL13Rα2 expression. Flow cytometry analysis of (A) four IL13Rα2-low and (B) four IL13Rα2-high cell lines cultured in medium containing 1 μM CLTX-Cy5.5 for 1 h and then stained with a PE-conjugated IL13Rα2 antibody.

[0036] [Figure 4]Figure 1: Low passage PBT human glioblastoma cell line CLTX-CAR T cell recognition and killing is independent of IL13Rα2 expression. (A) CLTX-CAR T cells show statistically significant killing of a panel of primary GBM lines versus the embryonic kidney cell line 293T. Plotted are the number of viable target cells co-cultured with CLTX-CAR T cells for 24, 48 and 72 hours at an effector:target ratio = 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. ***: p<0.001, Student's t-test performed between PBT cell viability and 293T cells. (B) Elimination of PBT003-4 and PBT009 tumor cells by CLTX-CAR T cells compared to mock control observed using live cell imaging. Representative images of PBT003-4 and PBT009 cells co-cultured with mock or CLTX-CAR T cells at an effector:target ratio of 1:4 (4,000 T cells, 16,000 target cells) taken by brightfield microscopy immediately after co-culture (0 h) and 3 days after co-culture (72 h).

[0037] [Diagram 5] Figure 1: CLTX-CAR T cell activation after stimulation with GBM cells. T cells were stimulated with target cells for 5 hours at an effector:target ratio of 1:1 (25,000 T cells, 25,000 target cells) in the presence of a protein transport inhibitor. The percentage of degranulating CAR-T cells was determined by flow cytometry (A) CD107a immunoreactivity and (B) cytokine production as determined by intracellular staining. **: p<0.01; ***: p<0.001, one-way ANOVA with Sidak-Bonferroni correction comparing degranulation / cytokine secretion in 293T cell-stimulated T cells and PBT-stimulated T cells, respectively.

[0038] [Figure 6] Figure 1 shows the antitumor effect of CLTX-CAR T cells with different linker designs. (A) Schematic of CLTX-CAR constructs with different linkers (transmembrane domain not shown) including IgG4Fc (EQ), IgG4 (HL-CH3), CD8h and short linker (L). (B) CLTX-CAR T cells with different linkers are able to kill U251T GBM cells. Plotted are the numbers of viable U251T cells co-cultured with T cells harboring different CLTX-redirected constructs for 24, 48 and 72 hours at an effector:target ratio = 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. (C) CLTX-CAR T cells with different linkers show different cytokine production levels following antigen challenge. T cells engineered with different LTX-redirected constructs were stimulated with U251 T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFNγ secretion was detected by ELISA assay of supernatants. *: p<0.05; **: p<0.01; ***: p<0.001, one-way ANOVA with Sidak-Bonferroni correction comparing the indicated CAR T cells with mock T cells.

[0039] [Figure 7]Antitumor efficacy of CLTX-CAR T cells with different intracellular signaling regions. (A) Schematic of CLTX-CAR constructs with different intracellular costimulatory regions (CD28 and 41BB). (B) CLTX-CAR T cells with different costimulatory regions are able to kill U251T GBM cells. Plotted are the number of viable U251T cells co-cultured with T cells harboring different CLTX-redirected constructs for 24, 48 and 72 hours at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. (C) CLTX-CAR T cells with different costimulatory regions produce different levels of cytokines following antigen challenge. T cells recombined with different LTX-redirected constructs were stimulated with U251T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFNγ secretion was detected by ELISA assay of the supernatants. **: p<0.01; ***: p<0.001, one-way ANOVA with Sidak-Bonferroni correction comparing the indicated CAR T cells with mock T cells.

[0040] [Figure 8] Figure 1: CLTX-CAR T cells reduce the growth of established U251T GBM tumors in vivo. (A) Schematic showing U251T xenograft growth and T cell treatment in NSG mice. Mice with subcutaneously implanted U251T cells (days -14 to 0) were treated with PBS (tumor only), mock T cells, or CLTX-CAR T cells. (B) Tumor progression is inhibited by CLTX-CAR T cell treatment. Tumor growth was measured by caliper measurement over a 20-day period (days 0 to 20) from the time of T cell injection. ***: p<0.001, one-way ANOVA with Sidak-Bonferroni correction performed on data at day 20 after T cell injection comparing tumor volumes in tumor only or mock treated groups and CLTX-CAR T cell treated mice.

[0041] [Figure 9] FIG. 2 shows the amino acid sequence of CLTX-IgG4(L235E, N297Q)-CD28tm-CD28-zeta (SEQ ID NO: 26).

[0042] [Figure 10] FIG. 27 shows the amino acid sequence of CLTX-IgG4(HL-CH3)-CD28tm-CD28gg-zeta (SEQ ID NO:27).

[0043] [Figure 11] FIG. 2 shows the amino acid sequence of CLTX-CD8h-CD28tm-CD28gg-zeta (SEQ ID NO:28).

[0044] [Figure 12] FIG. 2 shows the amino acid sequence of CLTX-IgG4(hinge)-CD28tm-CD28gg-zeta (SEQ ID NO:29).

[0045] [Figure 13] FIG. 3 shows the amino acid sequence of CLTX-L-CD28tm-CD28gg-zeta (SEQ ID NO:30).

[0046] [Figure 14] FIG. 3 shows the amino acid sequence of CLTX-IgG4(L235E, N297Q)-CD28tm-4gg-1BB-zeta (SEQ ID NO:31).

[0047] [Figure 15] FIG. 3 shows the amino acid sequence of CLTX-IgG4(HL-CH3)-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO: 32).

[0048] [Figure 16] FIG. 2 shows the amino acid sequence of CLTX-CD8h-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO: 33).

[0049] [Figure 17] FIG. 3 shows the amino acid sequence of CLTX-IgG4(hinge)-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO:34).

[0050] [Figure 18] FIG. 3 shows the amino acid sequence of CLTX-L-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO:35).

[0051] [Figure 19] FIG. 3 shows the amino acid sequence of CLTX-IgG4(L235E, N297Q)-CD4tm-CD28tm-4-1BB-zeta (SEQ ID NO:36).

[0052] [Figure 20] FIG. 3 shows the amino acid sequence of CLTX-IgG4(HL-CH3)-CD4tm-4-1BB-zeta (SEQ ID NO:37).

[0053] [Figure 21] FIG. 3 shows the amino acid sequence of CLTX-CD8h-CD28tm-4-1BB-zeta (SEQ ID NO:38).

[0054] [Figure 22] FIG. 3 shows the amino acid sequence of CLTX-IgG4(hinge)-CD28tm-4-1BB-zeta (SEQ ID NO:39).

[0055] [Figure 23] FIG. 4 shows the amino acid sequence of CLTX-L-CD28tm-4-1BB-zeta (SEQ ID NO: 40).

[0056] [Figure 24] FIG. 22 shows the CAR of FIG. 21 equipped with T2A (ribosomal skip sequence and truncated CD19; SEQ ID NO: 60). The truncated CD19 is co-expressed with the CAR, allowing a simple method to identify and quantify transfected cells.

[0057] [Diagram 25] FIG. 1 shows various chlorotoxin-related toxins (SEQ ID NOs: 1, 56-59, and 61-73) and an alignment of their amino acid sequences (Dardevet et al., 2015 Toxins (Basel) 7:1079). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Described below are the structures, constructions and characterizations of various chimeric antigen receptors, including chlorotoxin (CLTX). Chimeric antigen receptors (CARs) are recombinant biological molecules that, at a minimum, include an extracellular recognition region, a transmembrane region, and an intracellular signaling region. Thus, the term "antigen" is not limited to molecules that bind to antibodies, but any molecule that can specifically bind to a target. For example, a CAR may include a ligand that specifically binds to a cell surface receptor. The extracellular recognition region (sometimes referred to as the extracellular region or simply the recognition element it contains) includes a recognition element that specifically binds to a molecule present on the cell surface of a target cell. The transmembrane region tethers the CAR in the membrane. The intracellular signaling region includes a signaling region derived from the zeta chain of the human CD3 complex, and optionally includes one or more co-stimulatory signaling regions. CARs are capable of binding to antigens and inducing T cell activation independent of MHC restriction. Thus, CARs are "universal" immune receptors that can treat a population of patients 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 the treatment of cancer.

[0059] One CAR containing chlorotoxin described herein is called CLTX-IgG4(EQ)-CD28gg-Zeta. This CAR contains several key features including: chlorotoxin; an IgG4 Fc region mutated in a manner that reduces binding by Fc receptors (FcRs) at two sites within the CH2 domain (L235E; N297Q); a CD28 costimulatory domain, and a CD3ζ activation domain.

[0060] In some cases, the CARs described herein may be produced using vectors in which the CAR open reading frame is followed by a T2A ribosomal skip sequence and a truncated CD19 (CD19t) that lacks the cytoplasmic signaling tail (truncated at amino acid 323). In this configuration, co-expression of CD19t provides an inert, non-immunogenic surface marker that allows accurate measurement of genetically modified cells, positive selection of genetically modified cells, and efficient cell tracking in vivo following adoptive transfer and / or imaging of therapeutic T cells. Co-expression of CD19t provides a marker for immunological targeting of introduced cells in vivo using clinically available antibodies and / or immunotoxin reagents to selectively eliminate therapeutic cells, thereby acting as a suicide switch.

[0061] The CAR described herein may be produced by any means known in the art, but preferably it is produced using recombinant DNA technology.The nucleic acid encoding several regions of the chimeric receptor can be conveniently prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (such as 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.

[0062] Various T cell subsets or T cells isolated from patients, including unselected PBMCs or enriched CD3 T cells or enriched CD3 or memory T cell subsets. CM Or T CM / SCM / N can be introduced with a vector for CAR expression. Central memory T cells are one useful T cell subset. Central memory T cells may be isolated from peripheral blood mononuclear cells (PBMCs) by enrichment for CD45RO+ / CD62L+ cells, for example using a CliniMACS® device to immunomagnetically select cells expressing the desired receptor. Cells enriched for central memory T cells may be activated with anti-CD3 / CD28, for example, introduced with a SIN lentiviral vector directing expression of the CAR and truncated human CD19 (CD19t), a non-immunogenic surface marker for in vivo detection and potential ex vivo selection. Activated / genetically modified central memory T cells may be expanded in vitro with IL-2 / IL-15 and then cryopreserved. EXAMPLES

[0063] Example 1: Construction and structure of CLTX-IgG4Fc(EQ)-CD28-zeta CAR A useful CAR structure containing chlorotoxin, CLTX-IgG4Fc(EQ)-CD28-zeta, is described below. The codon-optimized CAR sequence contains: chlorotoxin, an IgG4 Fc spacer containing mutations (S228P, L235E) that greatly reduce Fc receptor-mediated recognition, a CD28 transmembrane domain, a costimulatory CD28 cytoplasmic signaling domain, and a CD3 zeta cytoplasmic signaling domain. A T2A ribosomal skip sequence separates this CAR sequence from CD19t, an inactive and non-immunogenic cell surface detection / selection marker. This T2A linkage results in coordinate expression of CAR and CD19t from a single transcript. Figure 1A shows the structure of the CLTX-IgG4Fc(EQ)-CD28-zeta codon-optimized CAR sequence. TSchematic diagram of the translated region of X-IgG4Fc(EQ)-CD28-zeta-T2ACD19t. In this figure, the CLTX-IgG4Fc(EQ)-CD28-zeta CAR, as well as the T2A ribosomal skip and truncated CD19 sequences, are all shown. Expression of the CAR and CD19t cassette is driven by the human EF1 promoter (EF1p). Figure 1B shows a schematic of the expressed, mature CAR.

[0064] The CLTX-IgG4Fc(EQ)-CD28-zeta sequence was generated by fusion of the human GM-CSF receptor alpha leader peptide chlorotoxin, the S228P / L235E / N297Q modified IgG4 Fc hinge (where the double mutation L235E / N297Q prevents FcR recognition), the CD28 transmembrane, the CD28 cytoplasmic signaling region, and the CD3 zeta cytoplasmic signaling region 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 up to the transmembrane component of a CD19-containing plasmid (i.e., base pairs 1-972). All three fragments, 1) CLTX-IgG4Fc(EQ)-CD28-zeta, 2) T2A, and 3) CD19t, were cloned into the multiple cloning site of the epHIV7 lentiviral vector. When transfected into appropriate cells, the vector is integrated into the host cell genome. The amino acid sequence of CLTX-IgG4Fc(EQ)-CD28-zeta is presented in Figure 9 with the various regions indicated. EXAMPLES

[0065] Example 2: Construction and structure of epHIV7 used for expression of CLTX-IgG4Fc(EQ)-CD28-zeta The pHIV7 plasmid is a clinical vector. CLTX-IgG4Fc(EQ)-CD28-zeta-T2A-CD19t_epHIV7 is the original plasmid provided by the T Cell Therapeutic Research Laboratory (TCTRL) at City of Hope (COH). The epHIV7 vector used to express the CAR was generated from the pHIV7 vector. Importantly, this vector uses the human EF1 promoter to drive expression of the CAR. Both the 5' and 3' sequences of the vector were obtained from pv653RSN, as previously obtained from the HXBc2 provirus. The polypurine tract DNA flap sequence (cPPT) was derived from the HIV-1 strain pNL4-3 from the NIH AIDS Reagent Repository. The woodchuck posttranscriptional regulatory element (WPRE) sequence has been described previously.

[0066] The construction of pHIV7 was carried out as follows. Briefly, pv653RSN, containing 653 bp from the 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, sequences from the 5'LTR to the rev-responsive element (RRE) were removed to create p5'HIV-151, then the 5'LTR was modified to remove sequences upstream of the TATA box and linked 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, then 400 bp was deleted in the 3'LTR enhancer / promoter to remove cis-regulatory elements in HIV U3 to form p3'HIV-2. In step 3, fragments isolated from p5'HIV-3 and p3'HIV-2 were ligated to generate pHIV-3. In step 4, p3'HIV-2 was further modified to remove extra upstream HIV sequences to generate p3'HIV-3, and a 600 bp BamHI-SalI fragment containing the WPRE was added to p3'HIV-3 to generate 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 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 of pHIV6 to generate pHIV-7. This original plasmid, pHIV7-GFP (GFP, green fluorescent protein), was used to package the original vector using a four-plasmid system.

[0067] The packaging signal, psi (Ψ), 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.

[0068] Helper functions (necessary for viral vector production) were split into three separate plasmids to reduce the possibility of generating replication-competent lentivirus by recombination: 1) pCgp encodes the gag / pol proteins required for viral vector assembly; 2) pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence to help transport the viral genome for efficient packaging; and 3) pCMV-G encodes the glycoprotein of vesiculo-stomatitis virus (VSV), required for viral vector infectivity.

[0069] There is minimal DNA sequence homology between the pHIV7-encoded vector genome and the helper plasmids. The regions of homology include the approximately 600 nucleotide packaging signal region located in the gag / pol sequence of the pCgp helper plasmid; the CMV promoter sequence in all three helper plasmids; and the RRE sequence in the helper plasmid pCgp. Because of the need for multiple recombinations, it is highly unlikely that the homology in this region would result in the generation of a replication-competent recombinant virus. In addition, any resulting recombinants would lack functional LTR and tat sequences required for lentiviral replication.

[0070] The CMV promoter was replaced with the EF1α-HTLV promoter (EF1p) and the new plasmid was named epHIV7. EF1p has 563 bp and was introduced into epHIV7 using NruI and NheI after the CMV promoter was excised.

[0071] The lentiviral genome, except for gag / pol and rev, which are necessary for wild-type virus virulence and productive infection of target cells, was removed from this system. CLTX -IgG4Fc(EQ)-CD28-Zeta-T2ACD19t_epHIV7 vector construct does not contain an intact 3'LTR promoter, therefore the LTRs of the resulting expressed and reverse transcribed DNA proviral genome in target cells will be inactive. As a result of this design, all HIV-1 derived sequences will not be transcribed from the provirus and only the therapeutic sequences will be expressed from each promoter. Removal of LTR promoter activity in SIN vectors is expected to significantly reduce the chance of unintended activation of host genes. EXAMPLES

[0072] Example 3: Production of vectors for transduction of patient T cells Vectors for transfer of patient T cells may be prepared as follows: For each plasmid (i.e., plasmids expressing 1) CAR and optionally a marker such as truncated CD19; 2) pCgp; 3) pCMV-G; and 4) pCMV-Rev2), a seed bank was generated that was used to inoculate fermenters to produce sufficient amounts of plasmid DNA. Plasmid DNA was tested for identity, sterility, and endotoxins before use in lentiviral vector production.

[0073] Briefly, cells are expanded from 293T working cells (WCB) that have been tested to ensure sterility and absence of viral contamination. A vial of 293T cells from the 293T WCB is thawed. The cells are grown and expanded until there are sufficient cell numbers to seed an appropriate number of 10-layer cell factories (CFs) for vector production and cell train maintenance. A single cell train may be used for production.

[0074] Lentiviral vectors are produced in sub-batches of up to 10 CFs. Two sub-batches can be produced in the same week, leading to production of approximately 20 L of lentiviral supernatant / week. To produce one lot of product, the material produced from all sub-batches is pooled during downstream processing steps. 293T cells are plated in CFs in 293T medium (DMEM with 10% FBS). Factories are placed in a 37°C incubator and leveled to obtain an even distribution of cells over the entire layer of the CF. After 2 days, cells are cultured in Tris:EDTA, 2M CaCl 2 , 2X HBS, and CaPO with a mixture of four DNA plasmids 4 The cells are transfected with the four lentiviral plasmids described above using the ELISA method. Three days after transfection, the supernatant containing the secreted lentiviral vectors is collected, purified, and concentrated. After the supernatant is removed from the CFs, production end cells are harvested from each CF. Cells are trypsinized from each factory and harvested by centrifugation. The cells are resuspended in freezing medium and cryopreserved. These cells are later used for replication-competent lentivirus (RCL) testing.

[0075] To purify and formulate the vector, the crude supernatant is clarified by membrane filtration to remove cell debris. Host cell DNA and residual plasmid DNA are degraded by endonuclease digestion (Benzonase®). The viral supernatant is cleared from cell debris using a 0.45 μm filter. The clarified supernatant is collected in a pre-weighed container into which Benzonase® is added (final concentration 50 U / mL). Endonuclease digestion of residual plasmid DNA and host genomic DNA is carried out for 6 hours at 37° C. A first tangential flow ultrafiltration (TFF) concentration of the endonuclease-treated supernatant is 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 is circulated through a hollow fiber cartridge with a NMWCO of 500 kD at a flow rate designed to maintain a shear rate of approximately 4,000 sec-1 or less while maximizing the flux rate. Dialysis of the nuclease-treated supernatant is initiated during the concentration process to maintain cartridge performance. An 80% permeate replacement rate is established using 4% lactose in PBS as the dialysis buffer. The viral supernatant is brought to a target volume to represent 20-fold concentration of the crude supernatant, and dialysis is continued for four additional exchange volumes at a dialysate replacement rate of 100%.

[0076] Further concentration of the viral product is achieved using high speed centrifugation techniques. Each sub-batch of lentivirus is pelleted using a Sorvall RC-26 plus centrifuge at 6000 RPM (6,088 RCF) at 6°C for 16-20 hours. The viral pellet from each sub-batch is then reconstituted to 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 results in approximately a 200-fold volume reduction. Following completion of all sub-batches, the material is then placed at -80°C while samples from each sub-batch are tested for sterility. Following confirmation of sample sterility, the sub-batches are rapidly thawed at 37°C with frequent agitation. The material is then pooled and manually dispensed in a Class II Type A / B3 biosafety cabinet. A fill configuration of 1 mL of concentrated lentivirus in sterile USP Class 6, male thread O-ring cryovials is used.

[0077] To ensure the purity of the lentiviral vector preparation, it is tested for residual host DNA contaminants, as well as for transfer of residual host and plasmid DNA. Among other tests, vector identity is assessed by RT-PCR to ensure that the correct vector is present. EXAMPLES

[0078] Example 4: Preparation of T cells suitable for use in ACT T to express CAR CM When used, suitable patient cells may be prepared as follows: First, T lymphocytes are obtained from the patient by leukopheresis and a suitable allogeneic or autologous T cell subset, such as central memory T cells (T CM ) are genetically altered to express CAR and then administered back to the patient by any clinically acceptable means to achieve anti-cancer treatment.

[0079] Appropriate T CMmay be generated as follows: Apheresis products obtained from consented study participants are ficolled, washed and incubated overnight. Cells are then depleted of monocyte, regulatory T cell and naive T cell populations using GMP grade anti-CD14, anti-CD25 and anti-CD45RA reagents (Miltenyi Biotec) and a CliniMACS™ separation device. Following depletion, negative fraction cells are purified using DREG56-biotin (COH clinical grade) and anti-biotin microbeads (Miltenyi Biotec) on a CliniMACS™ separation device to identify CD62L+T CM Enriched for cells.

[0080] Following enrichment, T CM Cells are formulated in complete X-Vivo15 plus 50 IU / mL IL-2 and 0.5 ng / mL IL-15, transferred to Teflon cell culture bags, and stimulated with Dynal Clin™ Vivo CD3 / CD28 beads. Up to 5 days post-stimulation, cells are transduced with lentiviral vectors expressing the desired CAR at a multiplicity of infection (MOI) of 1.0 to 0.3. Culturing is continued with the addition of complete X-Vivo15 and IL-2 and IL-15 cytokines as required for cell growth (cell density of 3×10 5 and 2×10 6 Cells are maintained at between 10 and 200 cells / mL and cytokine supplementation is continued every Monday, Wednesday and Friday of culture for up to 42 days. 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).

[0081] On the day of T cell infusion, the cryopreserved and released product is thawed, washed, and formulated for reinfusion. 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 normal saline (PFNS) / 2% HSA infusion diluent. A sample is removed for quality control testing. EXAMPLES

[0082] Example 5: Expression of Cltx-IgG4(EQ)-CD28gg-Zeta FIG. 1C shows healthy donor T cells (HD187.2 T cells) engineered to express CLTX-CAR. CM / SCM / N ) are shown. Shown are anti-CD19 anti-Fc and anti-CD8 stainings depicting the co-expression of CLTX-CAR and CD19t transgene in both CD8+ and CD4+ (CD8-) T cell subsets. The percentage of immunoreactive cells for transduced (CLTX-CAR) and non-transduced (mock) cells 18 days after CD3 / CD28 bead stimulation is shown, demonstrating the ability to transduce human T cells with CLTX-CAR. EXAMPLES

[0083] Example 6: Chlorotoxin and Cltx-IgG4(EQ)-CD28gg-Zeta T cells specifically recognize the glioma cell line U251T The fluorescent label, chlorotoxin conjugated to Cy5.5 (CLTX-Cy5.5), was used to assess chlorotoxin binding to various cell types. The results of this study are depicted in Figure 2A-E (A, human peripheral blood mononuclear cells (PBMCs) from a healthy donor; B, human EBV-transformed lymphoblastoid cell line, LCL; C, large T antigen-transformed human embryonic kidney cell line 293T; D, human astrocytes differentiated from induced pluripotent stem cells (iPSCs) from a healthy donor; and E, human glioblastoma cell line U251T). Cell lines were cultured in medium (untreated) or in medium containing 1 μM CLTX-Cy5.5 for 1 h at 37°C and then assessed by flow cytometry.

[0084] As shown in Figure 2F, CLTX-CAR T cells specifically killed the glioma tumor cell line U251T, but not LCL, 293T, or primary human astrocytes. Plotted are the numbers of viable target cells (LCL, 293T, astrocytes, and U251T) co-cultured with CLTX-CAR T cells for 72 hours at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. EXAMPLES

[0085] Example 7: Chlorotoxin binds to low passage PBT, a human primary glioblastoma cell line, independently of IL13Rα2 expression To examine whether chlorotoxin binding is independent of IL13Rα2 expression, flow cytometry analysis was performed on IL13Rα2-low and IL13Rα2-high cell lines that were cultured in medium containing 1 μM CLTX-Cy5.5 for 1 h and then stained with PE-conjugated IL13Rα2 antibody. As can be seen in Figure 3A-B, chlorotoxin binds to low passage PBT, a human primary glioblastoma cell line, independently of IL13Rα2 expression. EXAMPLES

[0086] Example 8: CLTX-IgG4(EQ)-CD28gg-Zeta T cells recognize and kill low passage PBT human glioblastoma cell lines independent of IL13Rα2 expression and TCGA molecular subtype As shown in Figure 4A, CLTX-CAR T cells show statistically significant killing of a panel of primary GBM lines versus the embryonic kidney cell line 293T. Plotted are the numbers of viable target cells co-cultured with CLTX-CAR T cells at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells) for 24, 48 and 72 hours, after normalization to those co-cultured with mock T cells for the same period.

[0087] Figure 4B shows the elimination of PBT003-4 and PBT009 tumor cells by CLTX-CAR T cells compared to mock controls, observed using live cell imaging. Representative images of PBT003-4 and PBT009 cells co-cultured with mock or CLTX-CAR T cells at an effector:target ratio of 1:4 (4,000 T cells, 16,000 target cells) captured by brightfield microscopy immediately after co-culture (0 h) and after 3 days (72 h) of co-culture. EXAMPLES

[0088] Example 9: CLTX-IgG4(EQ)-CD28gg-Zeta T cells are activated by stimulation with GBM cells T cells (expressing mock or CLTX CAR) were stimulated with target cells at an effector:target ratio of 1:1 (25,000 T cells, 25,000 target cells) for 5 hours in the presence of protein transport inhibitors. The percentage of degranulated CAR-T cells was determined by flow cytometry for CD107a immunoreactivity (Figure 5A) and cytokine production as determined by intracellular staining (Figure 5B). EXAMPLES

[0089] Example 10: CLTX-CAR T cells with different linker designs are effective against tumor cells Figure 6A is a schematic diagram of CLTX-CAR constructs with different spacers (linkers), including IgG4Fc (EQ), IgG4 (HL-CH3), CD8h, and a short linker (L). All have the CD28 transmembrane domain (not shown). As shown in Figure 6B, CLTX-CAR T cells with different linkers are able to kill U251T GBM cells. Plotted are the numbers of viable U251T cells co-cultured with T cells harboring different CLTX-redirected constructs for 24, 48, and 72 hours at an effector:target ratio = 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. As shown in Figure 6C, CLTX-CAR T cells with different linkers show different cytokine production levels following antigen challenge. T cells engineered with different LTX-redirected constructs were stimulated with U251 T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFNγ secretion was detected by ELISA assay of the supernatants. EXAMPLES

[0090] Example 11: Antitumor effects of CLTX-CAR T cells with different intracellular signaling domains Figure 7A is a schematic diagram of CLTX-CAR constructs with different intracellular costimulatory domains CD28 and 41BB. As shown in Figure 7B, CLTX-CAR T cells with different costimulatory domains are able to kill U251T GBM cells. Plotted are the numbers of viable U251T cells co-cultured with T cells harboring different CLTX-redirected constructs for 24, 48 and 72 hours at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells) after normalization to those co-cultured with mock T cells for the same period. As shown in Figure 7C, CLTX-CAR T cells with different costimulatory domains produce different levels of cytokines following antigen challenge. T cells recombined with different LTX-redirected constructs were stimulated with U251T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFNγ secretion was detected by ELISA assay of the supernatants. EXAMPLES

[0091] Example 12: CLTX-CAR T cells reduce the growth of established U251T GBM tumors in vivo. Figure 8A is a schematic depiction of U251T xenograft growth and T cell treatment studies in NSG mice. Mice with subcutaneously implanted U251T cells (days -14 to 0) were treated with PBS (tumor only), mock T cells, or CLTX-CAR T cells. Figure 8B, Tumor progression is inhibited by CLTX-CAR T cell treatment. Tumor growth was measured by caliper measurement over a 20 day period (days 0 to 20) from the time of T cell injection. EXAMPLES

[0092] Example 13: Additional CLTX CARs Figures 9-24 present the amino acid sequences of various additional CLTX-CARs that can be constructed and expressed as described above for the CLTX-IgG4(EQ)-CD28gg-Zeta CAR. In Figures 8-24, various regions (listed under the sequence in each figure from amino terminus to carboxy terminus) are indicated by alternating underlined and non-underlined portions. Thus, in Figure 9, the GMCSFRa signal peptide is underlined, the chlorotoxin sequence is not underlined, the spacer (IgG4(SmP)(L235E, N297Q)) is underlined, the CD28 transmembrane sequence is not underlined, the CD28cyto(LLmGG) costimulatory region is underlined, the (Gly)3 sequence that separates the costimulatory region from the CD3ζ sequence is not underlined, and the CD3ζ sequence is underlined. In Figures 9-23, the T2A and T19t sequences co-expressed with the CAR are not shown. Figure 24 shows the CAR of Figure 23 with T2A (ribosomal skipping) and truncated CD19. The truncated CD19 is co-expressed with the CAR, allowing for a convenient method of identification and quantification of transfected cells. EXAMPLES

[0093] Example 14: Additional Toxin Sequences Figure 25 shows a sequence alignment of chlorotoxin and various chlorotoxin-related toxins (Dardevet et al., 2015 Toxins (Basel) 7:1079). These toxins may, in some cases, be substituted for chlorotoxin in the CARs described herein.

Claims

1. A pharmaceutical composition for the treatment of glioblastoma, comprising a T cell expressing a chimeric antigen receptor comprising the amino acid sequence of any one of SEQ ID NOs: 26, 28, 31, and 33.

2. The pharmaceutical composition of claim 1, wherein the T cell expresses a chimeric antigen receptor consisting of the amino acid sequence of any one of SEQ ID NOs: 26, 28, 31 and 33.

3. 2. The pharmaceutical composition of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

26.

4. The pharmaceutical composition of claim 1 , wherein at least 50% of the T cells are central memory T cells, naive T cells or stem central memory cells.

5. The pharmaceutical composition of claim 1 , wherein at least 60% of the T cells are central memory T cells, naive T cells or stem central memory cells.

6. The pharmaceutical composition of claim 1 , wherein at least 70% of the T cells are central memory T cells, naive T cells or stem central memory cells.

7. The pharmaceutical composition of claim 1 , wherein at least 80% of the T cells are central memory T cells, naive T cells or stem central memory cells.

8. The pharmaceutical composition of claim 1 , wherein at least 50% of the T cells are central memory T cells.

9. The pharmaceutical composition of claim 1 , wherein at least 60% of the T cells are central memory T cells.

10. The pharmaceutical composition of claim 1 , wherein at least 70% of the T cells are central memory T cells.

11. The pharmaceutical composition of claim 1 , wherein at least 80% of the T cells are central memory T cells.

12. 5. The pharmaceutical composition of claim 4, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

26.

13. 7. The pharmaceutical composition of claim 6, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:26.

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