Chimeric antigen receptors containing a chlorotoxin domain

Abstract

Chimeric transmembrane immunoreceptors (CARs) are described that include an extracellular domain comprising a chlorotoxin or related toxin, or a variant of a chlorotoxin or related toxin, a transmembrane region, a costimulatory domain, and an intracellular signaling domain that bind to human glioma or other human tumor cells.

Description

Background of the Invention

[0002] Immunotherapy based on tumor-specific T cells has been investigated, including the use of engineered T cells for anti-tumor treatment. Chimeric antigen receptors (CARs) consist of an extracellular tumor recognition / targeting domain, an extracellular linker / spacer, a transmembrane domain, and an intracellular T cell activation and co-stimulatory signaling domain. The design of the recognition / targeting domain is crucial to avoiding undesirable off-target effects. Most CAR tumor-targeting domains are single-chain variable fragments (scFvs) derived from antibody sequences, utilizing the specificity of antibody binding to specific antigens. There are also examples of CAR tumor-targeting domains derived from normal receptor ligands, such as IL-13 cytokine CARs that target cells expressing the IL-13 receptor IL-13Rα2. Despite some significant successes, identifying and validating novel CAR tumor-targeting domains remains a major challenge in this field.

[0003] Malignant gliomas (MG), including glioblastoma multiforme (AA-WHO grade III) and glioblastoma glioblastoma (GBM-WHO grade IV), have an incidence of approximately 20,000 new cases diagnosed annually in the United States. According to the American Brain Tumor Association, based on data from the 2010 U.S. Census, the overall prevalence of MG among surviving individuals is roughly 140,000. Although MG is a rare disease, it is highly aggressive and heterogeneous in its malignant behavior and is almost always fatal. Current standard of care for advanced MG provides only short-term benefit, and these brain tumors are, in fact, incurable. Indeed, even with modern surgical and radiation therapy techniques (which often exacerbate an already severe morbidity imposed by targeting the central nervous system (CNS), 5-year survival rates are very low. Furthermore, for the majority of patients with recurrent disease, there are very few treatment options. Therefore, there is a clear need for more effective treatments, especially for those patients who have relapsed / progressed after first-line therapy.

[0004] Adoptive T-cell therapy (ACT) using engineered T cells expressing chimeric antigen receptors (CARs) can provide a safe and effective way to reduce the relapse rate of mycoplasma leukemia (MG) because CAR T cells can be engineered to specifically recognize antigenically unique tumor populations (Cartellieri et al. 2010 J Biomed Biotechnol 2010:956304; Ahmed et al. 2010 Clin Cancer Res 16:474; Sampson et al. 2014 Clin Cancer Res 20:972; Brown et al. 2013 Clin Cancer Res 2012 18:2199; Chow et al. 2013 Mol Ther 21:629), and T cells can migrate through the brain parenchyma to target and kill invasive malignant cells (Hong et al. 2010 Clin Cancer Res 16:4892; Brown et al. 2007 J Immunol 179:3332; Hong et al. 2007 J Immunol 179:3332; Hong et al. 2010 Clin Cancer Res 16:4892; Brown ... Hong et al. 2010 J Immunol etal.2010 Clin Cancer Res16:4892; Yaghoubi 2009 Nat Clin Pract Oncol 6:53). Invention Overview

[0006] This article describes a chimeric transmembrane immune receptor (chimeric antigen receptor, or "CAR") comprising an extracellular domain, a transmembrane region, and an intracellular signaling domain. The extracellular domain comprises chlorhexidine (a 36-amino acid peptide toxin found in the venom of the scorpion *Leiurus quinquestriatus*) or a related toxin, or a variant of chlorhexidine or a related toxin, and optionally a spacer region containing, for example, a portion of the human Fc domain. The transmembrane portion comprises, for example, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3 transmembrane domain. The intracellular signaling domain comprises a signaling domain derived from the ζ chain (CD3ζ) of the human CD3 complex and one or more co-stimulatory domains, such as a 4-1BB co-stimulatory domain. When expressed on the surface of T cells, the extracellular domain enables the CAR to direct T cell activity to cells expressing the chlorhexidine receptor. Such cells include glioblastoma cells. The intracellular region contains a co-stimulatory domain tandem with CD3ζ, such as the 4-1BB (CD137) co-stimulatory domain, enabling T cells to receive co-stimulatory signals. T cells can be engineered, for example, with patient-specific autologous T cells to express the CAR described herein, and engineered cells can be expanded for use in ACT. Various T cell subsets can be used, including both αβ T cells and γδ T cells. Additionally, the CAR can be expressed in other immune cells, such as NK cells. In cases where patients are treated with immune cells expressing the CAR described herein, the cells can be autologous T cells or allogeneic T cells. In some cases, the cells used include CD4+ and CD8+ central memory T cells (T cells). CM Both cell populations consist of CD62L+, CCR7+, CD45RO+, and CD45RA-, or the cells used include CD4+ and CD8+ T cells. CM Cells, stem cells, central memory T cells, and naive T cells (i.e., T cells) CM / SCM / N (Cell population). T CM / SCM / N The cell population consisted of CD62L+, CCR7+, and included both CD45RA+ and CD45RO+ cells, as well as both CD4+ and CD8+ cells. The use of these cells improved long-term cell persistence after adoptive transfer compared to other types of patient-specific T cells.

[0007] This document describes a nucleic acid molecule encoding a CAR, which comprises: chloramphenicol (MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR; SEQ ID NO: 1) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), provided that no cysteine ​​residues are modified; a transmembrane domain selected from: a CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a co-stimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a co-stimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions)). or a variant with 2 amino acid modifications (e.g., substitution); or a 4-1BB costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or a CD28 costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and a 4-1BB costimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and a CD3ζ signaling domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications.

[0008] In some embodiments, the CAR includes a toxin associated with chlorine rather than chlorine. Therefore, the CAR may include GaTx2, a toxin from *Leiurus quinquestriatus hebraeus* (VSCEDCPDHCSTQKARAKCDNDKCVCEPI; SEQ ID NO: 56) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), provided that cysteine ​​residues are not modified; a transmembrane domain selected from: the CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and the CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). Variants of the following: co-stimulatory domains (e.g., CD28 co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or 4-1BB co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or both CD28 co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and 4-1BB co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and CD3ζ signaling domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications.

[0009] In some cases, a CAR may include more than one chlortoxin sequence (e.g., two or three or more copies of SEQ ID NO:1, which are consecutive or separated by 1-10 amino acids) or more than one chlortoxin-associated toxin. Therefore, a CAR may include two or more chlortoxin sequences (e.g., SEQ ID NO:1 followed by SEQ ID NO:1 followed by the remainder of the molecule), or a CAR may include a chlortoxin sequence followed by a sequence of a chlortoxin-associated toxin (e.g., SEQ ID NO:57 or...). Figure 25 (Another toxin described in the text).

[0010] CARs may include GaTx1, which is a toxin derived from Leiurus quinquestriatus hebraeus (CGPCFTTDHQMEQKCAECCGGIGKCYGPQCLCNR; SEQ ID). NO:57) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), provided that cysteine ​​residues are not modified; a transmembrane domain selected from: the CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and the CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory domain (e.g., the CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or the 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or the CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or 2) amino acid modifications (e.g., substitutions) variants and 4-1BB co-stimulatory domains or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and CD3ζ signaling domains or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications.

[0011] The CAR may include AaCtx, which is a toxin from the yellow fattail scorpion (Androctonus australis) (MCIPCFTTNPNMAAKCNACCGSRRGSCRGPQCIC; SEQ ID NO:58) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), provided that cysteine ​​residues are not modified; a transmembrane domain selected from: the CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and the CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and a co-stimulatory domain (e.g., the CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications). Variants with 1-5 amino acid modifications (e.g., substitution); or a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and a CD3ζ signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications.

[0012] The CAR may include BmKCT, which is a toxin from the East Asian scorpion (Buthus martensii) (CGPCFTTDANMARKCRECCGGIGKCFGPQCLCNRI; SEQ ID NO:59) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), provided that no cysteine ​​residues are modified; a transmembrane domain selected from: a CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory domain (e.g., a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a variant with 2 amino acid modifications (e.g., substitution); or a CD28 co-stimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and a 4-1BB co-stimulatory domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and a CD3ζ signaling domain or a variant thereof with 1-5 (e.g., 1 or 2) amino acid modifications.

[0013] In several embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NO:26-55, wherein the chloramphenicol sequence (SEQ ID NO:1) is replaced by an amino acid sequence selected from SEQ ID NO:56-59 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0014] In several embodiments, the co-stimulatory domain is selected from the group consisting of: the co-stimulatory domains described in Table 3 or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the CD28 co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), the 4-1BB co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and the OX40 co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In some embodiments, a 4-1BB co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) are present. In some embodiments, two co-stimulatory domains are present, such as the CD28 co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and the 4-1BB co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In several embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions.

[0015] In some cases, there are short sequences of 1-6 amino acids (e.g., GGG) between the costimulatory domain and the CD3ζ signaling domain and / or between the two costimulatory domains.

[0016] In another embodiment, the CAR includes: a variant of chlortoxin having 1-5 amino acid modifications, said amino acid modifications increasing binding specificity or immunogenicity to the chlortoxin receptor (Cltx-R); the chlortoxin variant is a variant comprising the amino acid sequence of SEQ ID NO:1 having 1-5 (e.g., 1 or 2) amino acid modifications; two different co-stimulatory domains selected from the group consisting of: a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, and an OX40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications; and two different co-stimulatory domains selected from the group consisting of: a CD28 co-stimulatory domain or a variant thereof having 1-2 amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-2 amino acid modifications, and an OX40 co-stimulatory domain. The co-stimulatory domain or a variant thereof having 1-2 amino acid modifications; chlortoxin or a variant thereof having 1-2 amino acid modifications; a transmembrane region selected from: a CD4 transmembrane domain or a variant thereof having 1-2 amino acid modifications, a CD8 transmembrane domain or a variant thereof having 1-2 amino acid modifications, a CD28 transmembrane domain or a variant thereof having 1-2 amino acid modifications, and a CD3ζ transmembrane domain or a variant thereof having 1-2 amino acid modifications; a co-stimulatory domain (e.g., a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or both a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and a CD3ζ signaling domain or a variant thereof having 1-2 amino acid modifications; A spacer region located between chloramphenicol or a variant thereof and a transmembrane domain (e.g., the spacer region contains an amino acid sequence selected from SEQ ID NO:2-12 (Table 3) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications); a spacer region containing an IgG hinge region; a spacer region containing 1-150 amino acids; no spacer region; a 4-1BB signaling domain containing the amino acid sequence of SEQ ID NO:24; a CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO:21 and a 3-15 amino acid linker located between the costimulatory domain and the CD3ζ signaling domain, and a CD3ζ signaling domain or a variant thereof. In some embodiments, where two costimulatory domains are present, one is a 4-1BB costimulatory domain and the other costimulatory domain is selected from CD28 and CD28gg. In many embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions.

[0017] In some embodiments: the nucleic acid molecule expresses a polypeptide comprising an amino acid sequence selected from the following: SEQ ID NO: 26-55; the chimeric antigen receptor comprises an amino acid sequence selected from the following: SEQ ID NO: 26-55.

[0018] Also disclosed is a population of human T cells transduced using a vector containing an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: chloramphenicol or a variant thereof having 1-5 amino acid modifications (e.g., 1 or 2) amino acid modifications (e.g., substitution), or a chloramphenicol-associated toxin or a variant thereof having 1-5 amino acid modifications (e.g., 1 or 2) amino acid modifications (e.g., substitution); and a transmembrane domain selected from: a CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution), a CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution). The variants of the CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and the CD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); co-stimulatory domains (e.g., CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or both the CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and the 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and the CD3ζ signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In several embodiments, the human T cell population comprises a vector expressing a chimeric antigen receptor, the chimeric antigen receptor comprising a subset selected from SEQ ID NO. The amino acid sequence of NO:26-55 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); the human T cell population includes central memory T cells (T cells). CM (cells), for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T cells. CM Cells, or the human T cell population, comprise a combination of central memory T cells, naive T cells, and stem central memory cells (T cells). CM / SCM / N (cells), for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T cells. CM / SCM / NT cells. In any case, the T cell population includes both CD4+ cells and CD8+ cells (e.g., at least 20% of CD3+ T cells are CD4+ and at least 3% of CD3+ T cells are CD8+, and at least 70, 80, or 90% are CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, or 60% of CD3+ cells are CD4+ and at least 4%, 5%, 8%, 10%, or 20% of CD3+ cells are CD8+).

[0019] It also describes methods for treating cancer in patients, which include administering autologous or allogeneic human T cells (e.g., containing central memory T cells (T cells)). CM T cells (or central memory T cells), a combination of naive T cells and stem central memory cells (i.e., T cells are T cells that are T cells in the central memory T cells). CM / SCM / N (cells), at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T cells. CM / SCM / N A population of autologous or allogeneic human T cells. In either case, the T cell population comprises both CD4+ and CD8+ cells (e.g., at least 20% of CD3+ T cells are CD4+ and at least 3% of CD3+ T cells are CD8+, and at least 70, 80, or 90% are CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, or 60% of CD3+ cells are CD4+ and at least 4%, 5%, 8%, 10%, or 20% of CD3+ cells are CD8+), transduced by a vector containing an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NO:26-55 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In several embodiments: the cancer is glioblastoma; and transduced human T cells are prepared by means of obtaining T cells from a patient, processing the T cells to isolate central memory T cells, and transducing at least a portion of the central memory cells using a viral vector containing an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor contains an amino acid sequence selected from SEQ ID NO:26-55 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).

[0020] Also described are: nucleic acid molecules encoding polypeptides comprising an amino acid sequence that is at least 95% identical to the amino acid sequence selected from SEQ ID NO:26-55; nucleic acid molecules encoding polypeptides comprising an amino acid sequence identical to the amino acid sequence selected from SEQ ID NO:26-55, except for the presence of no more than 5 amino acid substitutions, deletions, or insertions; nucleic acid molecules encoding polypeptides comprising an amino acid sequence identical to the amino acid sequence selected from SEQ ID NO:26-55, except for the presence of no more than 5 amino acid substitutions; and nucleic acid molecules encoding polypeptides comprising an amino acid sequence identical to the amino acid sequence selected from SEQ ID NO:26-55, except for the presence of no more than 2 amino acid substitutions.

[0021] T cells expressing CARs containing chlortoxin or its variants can be used to treat cancers such as glioblastoma, and other cancers expressing chlortoxin receptors, including but not limited to: primary brain tumors and gliomas (WHO grade IV glioblastoma multiforme, WHO grade III anaplastic astrocytoma, WHO grade II low-grade astrocytoma, WHO grade I pilocytic astrocytoma, other ungraded gliomas, oligodendrogliomas, gliosarcomas, gangliogliomas, meningiomas, etc.). Ependymoma, neuroectodermal tumors (medulloblastoma, neuroblastoma, ganglioneuroma, melanoma (metastatic), melanoma (primary), pheochromocytoma, Ewing's sarcoma, primitive neuroectodermal tumor, small cell lung cancer, schwannoma), other brain tumors (epidermoid cysts, brain tumors of unknown pathology, pituitary pt. of glioblastoma multiforme, metastatic brain tumors of unknown tissue origin), and other tumors (breast cancer, metastatic breast cancer, kidney cancer, liver cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer).

[0022] This disclosure also includes nucleic acid molecules encoding any of the CARs described herein (e.g., vectors including nucleic acid sequences encoding one of the CARs) and isolated human T lymphocytes expressing any of the CARs described herein.

[0023] The CAR described herein may include a spacer region positioned between the chlortoxin domain (i.e., chlortoxin or a variant thereof) and the transmembrane domain. A variety of different spacers may be used. Some of them include at least a portion of the human Fc region, such as the hinge portion of the human Fc region or the CH3 domain or a variant thereof. Table 1 below provides several spacers that may be used in the CAR described herein.

[0024] Table 1: Examples of spacers

[0025]

[0026]

[0027]

[0028] Some spacer regions comprise whole or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., a sequence falling between the CH1 and CH2 domains of an immunoglobulin, such as the IgG4 Fc hinge or the CD8 hinge. Some spacer regions contain the immunoglobulin CH3 domain or both the CH3 and CH2 domains. Immunoglobulin-derived sequences may contain one or more amino acid modifications, such as 1, 2, 3, 4, or 5 substitutions, for example, substitutions that reduce off-target binding.

[0029] "Amino acid modification" refers to the substitution, insertion, and / or deletion of amino acids in a protein or peptide sequence. "Amino acid substitution" or "replacement" refers to the replacement of an amino acid at a specific position in a parent peptide or protein sequence with another amino acid. Substitution can be performed to change the amino acids in the resulting protein in a non-conservative manner (i.e., by changing the codon belonging to an amino acid group of a specific size or characteristic to an amino acid belonging to another group) or a conserved manner (i.e., by changing the codon belonging to an amino acid group of a specific size or characteristic to an amino acid belonging to the same group). This conserved change generally results in minimal alteration to the structure and function of the resulting protein. Here are examples of several 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): lysine, arginine, histidine (at pH 6.0). Another group might be those amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0030] In some embodiments, the spacer is derived from IgG1, IgG2, IgG3, or IgG4 and comprises one or more amino acid residues substituted with amino acid residues different from those present in the unmodified spacer. The one or more substituted amino acid residues are selected from, but 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, and 339, or combinations thereof. In this numbering system, as described in more detail below, the first amino acid in the IgG4 (L235E, N297Q) spacer in Table 1 is 219, and the first amino acid in the IgG4 (HL-CH3) spacer in Table 1 is 219. The first amino acid in the IgG hinge sequence and the IgG4 hinge joint (HL) sequence in Table 1 is also 219.

[0031] In some embodiments, the modified spacer is derived from IgG1, IgG2, IgG3, or IgG4 and comprises, but is not limited to, substitutions for one or more of the following amino acid residues: 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.

[0032] In some embodiments, the modified spacer is derived from the IgG4 region and comprises one or more amino acid residues substituted with amino acid residues different from those present in the unmodified region. The one or more substituted amino acid residues are selected from, but not limited to, one or more amino acid residues or combinations thereof 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, and 339.

[0033] In some embodiments, the modified spacer is derived from the IgG4 region and comprises, but is not limited to, one or more of the following amino acid residues substituted: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E, 268Q, 280H, 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, wherein the amino acid in the unmodified spacer is replaced by the amino acid identified above at the indicated position.

[0034] For the amino acid positions in the immunoglobulins discussed in this article, they were numbered according to the EU index or the EU numbering system (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, thus the whole is incorporated here). The EU index in the EU index or Kabat or EU numbering system refers to the number of EU antibodies (Edelman et al. 1969 Proc Natl Acad Sci USA 63:78-85).

[0035] Various transmembrane domains can be used. Table 2 contains examples of suitable transmembrane domains. In the presence of spacer domains, the transmembrane domain is located at the carboxyl terminus of the spacer domain.

[0036] Table 2: Examples of transmembrane domains

[0037]

[0038] Many of the CARs described herein contain one or more (e.g., two) co-stimulatory domains. These co-stimulatory domains are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable co-stimulatory domains that accompany the sequence of the CD3ζ signaling domain.

[0039] Table 3: Examples of CD3ζ domain and co-stimulatory domain

[0040]

[0041]

[0042] The chloramphenicol-containing CARs described herein are those summarized in Table 4, where the spacer domain, transmembrane domain, and co-stimulatory domain are indicated for each CAR.

[0043] Table 4: Examples of CARs containing chlorine toxin

[0044]

[0045]

[0046] *SEQ ID NO of the sequence including the signal sequence / SEQ ID NO of the sequence excluding the signal sequence Brief description of the attached diagram

[0048] Figure 1 AC: Generation of T cells expressing CLTX-CAR. (A) Schematic diagram of a lentiviral construct encoding a chloramphenicol (CLTX)-redirected chimeric antigen receptor (CAR) cassette, wherein transcription of CLTX-CAR, along with T2A ribosomal jumping and truncated CD19 (CD19t) sequences, is driven by the EF1 promoter (EF1p). (B) Diagram of CLTX-CAR containing an extracellular 36-amino acid chloramphenicol peptide and an IgG4Fc (EQ) spacer domain, a CD28 transmembrane domain, and intracellular CD28 and CD3ζ cytoplasmic signaling domain sequences. (C) Engineered healthy donor T cells (HD187.2T) to express CLTX-CAR. CM / SCM / N Flow cytometry analysis of the cells showed anti-CD19, anti-Fc, and anti-CD8 staining, representing CLTX-CAR and CD19t transgenes on CD8. + and CD4+ (CD8 - Co-expression in T cell subsets. The percentage of immunoreactive cells in transduced cells (CLTX-CAR) and untransduced cells (mimics) 18 days after CD3 / CD28 bead stimulation is shown to demonstrate the ability to transduce human T cells with CLTX-CAR.

[0049] Figure 2 AF: CLTX-CAR T cell-specific recognition of the glioblastoma cell line U251T. (AE) CLTX binds to GBM cells and shows minimal binding with non-GBM cells. The following evaluations are shown for chlortoxin-conjugated Cy5.5 (CLTX-Cy5.5): A, peripheral blood mononuclear cells (PBMCs) derived from healthy donors; B, human EBV-transformed lymphoblastoid cell line LCL; C, large T antigen-transformed human embryonic kidney line 293T; D, human astrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from healthy donors; and E, human glioblastoma cell line U251T. Cell lines were cultured in untreated medium or medium containing 1 μM CLTX-Cy5.5 at 37°C for 1 h and then evaluated by flow cytometry. (F) Specific killing of glioma tumor lines U251T by CLTX-CAR T cells, but not LCL, 293T, or primary human astrocytes. The number 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) was plotted relative to the number of target cells co-cultured with simulated T cells for the same duration. **: p < 0.01; ns: nonspecific, Student's t-test performed between groups as shown in the figure.

[0050] Figure 3 AB: The binding of CLTX to multiple low-passage human primary brain tumor (PBT) cell lines is independent of IL13Rα2 expression. Flow cytometry analysis of (A) four IL13Rα2-low and (B) four IL13Rα2-high cell lines cultured for 1 hour in medium containing 1 μM CLTX-Cy5.5 and then stained with PE-conjugated IL13Rα2 antibody.

[0051] Figure 4AB: CLTX-CAR T cell recognition and killing of low-passage PBT human glioblastoma lines independent of IL13Rα2 expression. (A) CLTX-CAR T cells showed statistically significant killing of a primary GBM lineage relative to the embryonic kidney lineage 293T. The plot shows the number of live 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, normalized to the number of live target cells co-cultured with simulated T cells for the same duration. ***: p < 0.001, Student's t-test performed between PBT cell viability and 293T cell viability. (B) Clearance of PBT003-4 and PBT009 tumor cells by CLTX-CAR T cells compared to simulated controls, observed using live-cell imaging. Representative images of PBT003-4 and PBT009 cells co-cultured with mimics or CTLX-CAR T cells at an effector:target ratio of 1:4 (4,000 T cells, 16,000 target cells) and immediately after co-culture (0 h) and 3 days after co-culture (72 h) using bright-field microscopy.

[0052] Figure 5 AB: CLTX-CAR T cell activation after GBM cell stimulation. T cells were stimulated for 5 hours with target cells at an effector:target ratio of 1:1 (25,000 T cells, 25,000 target cells) in the presence of protein transport inhibitors. The percentage of CAR-T cells undergoing degranulation was determined by CD107a immunoreactivity (A) using flow cytometry, and cytokine production was detected by intracellular staining (B). **: p < 0.01; ***: p < 0.001. One-way ANOVA, adjusted for Sidak-Bonferroni, was used to compare degranulation / cytokine secretion in each type of T cells stimulated by PBT and those stimulated by 293 T cells.

[0053] Figure 6AC: Antitumor activity of CLTX-CAR T cells with different adapter designs. (A) Schematic diagram of CLTX-CAR constructs with different adapters, including IgG4Fc(EQ), IgG4 (HL-CH3), CD8h, and a short adapter (L) (transmembrane domain not shown). (B) CLTX-CAR T cells with different adapters were able to kill U251T GBM cells. The plot shows the number of live U251T cells co-cultured with T cells containing different CLTX retargeting constructs for 24, 48, and 72 hours, normalized relative to the number of live U251T cells co-cultured with simulated T cells for the same length of time, at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells). (C) CLTX-CAR T cells with different adapters showed different levels of cytokine production after antigen challenge. T cells engineered with different CTLX-redirected constructs were stimulated with U251 T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFN-γ secretion was detected by ELISA of the supernatant. *: p < 0.05; **: p < 0.01; ***: p < 0.001. Univariate ANOVA analysis corrected for Sidak-Bonferroni was used to compare CAR-T cells and simulated T cells.

[0054] Figure 7 AC: Antitumor activity of CLTX-CAR T cells with different intracellular signaling domains. (A) Schematic diagram of different CLTX-CAR constructs in the intracellular co-stimulatory domains CD28 and 41BB. (B) CLTX-CAR T cells with different co-stimulatory domains are able to kill U251T GBM cells. The plot shows the number of live U251T cells co-cultured with T cells containing 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) for the same duration of co-culture with simulated T cells, after normalization relative to the number of live U251T cells co-cultured with simulated T cells. (C) CLTX-CAR T cells with different co-stimulatory domains produce various levels of cytokines after tumor attack. T cells engineered with different CLTX-redirected constructs were stimulated with U251T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFN-γ secretion was detected by ELISA of the supernatant. **: p < 0.01; ***: p < 0.001. Univariate ANOVA analysis with Sidak-Bonferroni correction was used to compare CAR-T cells and simulated T cells.

[0055] Figure 8 AB: CLTX-CAR T cells reduce the growth of established U251T GBM tumors in vivo. (A) Schematic diagram showing U251T xenograft growth and T cell therapy in NSG mice. Mice with subcutaneously implanted U251T cells were treated with PBS (tumor only), mimic T cells, or CLTX-CAR T cells (days -14 to 0). (B) CLTX-CAR T cell therapy inhibits tumor progression. Tumor growth was determined by caliper measurement over 20 days from T cell injection (day 0 to 20). ***: p < 0.001. Data were analyzed by one-way ANOVA with Sidak-Bonferroni correction on day 20 after T cell injection, comparing tumor volume in CLTX-CAR-treated mice with tumor-only or mimic-treated mice.

[0056] Figure 9 The amino acid sequence of CLTX-IgG4(L235E,N297Q)-CD28tm-CD28gg-zeta was depicted (SEQ ID NO:26).

[0057] Figure 10 The amino acid sequence of CLTX-IgG4(HL-CH3)-CD28tm-CD28ggzeta was described (SEQ ID NO:27).

[0058] Figure 11 The amino acid sequence of CLTX-CD8h-CD28tm-CD28gg-zeta was described (SEQ ID NO:28).

[0059] Figure 12 The amino acid sequence of CLTX-IgG4 (hinge)-CD28tm-CD28gg-zeta was described (SEQ ID NO:29).

[0060] Figure 13 The amino acid sequence of CLTX-L--CD28tm-CD28gg-zeta was described (SEQ ID NO:30).

[0061] Figure 14 The amino acid sequence of CLTX-IgG4(L235E,N297Q)-CD28tm-CD28gg-4-1BB-zeta was depicted (SEQ ID NO:31).

[0062] Figure 15The amino acid sequence of CLTX-IgG4(HL-CH3)-CD28tm-CD28gg-4-1BB-zeta was described (SEQ ID NO:32).

[0063] Figure 16 The amino acid sequence of CLTX-CD8h-CD28tm-CD28gg-4-1BB-zeta was described (SEQ ID NO:33).

[0064] Figure 17 The amino acid sequence of CLTX-IgG4(hinge)-CD28tm-CD28gg-4-1BB-zeta was described (SEQ ID NO:34).

[0065] Figure 18 The amino acid sequence of CLTX-L-CD28tm-CD28gg-4-1BB-zeta was described (SEQ ID NO:35).

[0066] Figure 19 The amino acid sequence of CLTX-IgG4(L235E,N297Q)-CD28tm-4-1BB-zeta was depicted (SEQ ID NO:36).

[0067] Figure 20 The amino acid sequence of CLTX-IgG4(HL-CH3)-CD4tm-4-1BB-zeta was described (SEQ ID NO:37).

[0068] Figure 21 The amino acid sequence of CLTX-CD8h-CD28tm-4-1BB-zeta was described (SEQ ID NO: 38).

[0069] Figure 22 The amino acid sequence of CLTX-IgG4(hinge)-CD28tm-4-1BB-zeta was described (SEQ ID NO:39).

[0070] Figure 23 The amino acid sequence of CLTX-L-CD28tm-4-1BB-zeta was described (SEQ ID NO:40).

[0071] Figure 24 The image depicts a sequence with T2A (ribosomal skipping sequence and truncated CD19; SEQ ID NO:60). Figure 21 The CAR. Truncated CD19 is co-expressed with the CAR, allowing for easy identification and quantification of transfected cells.

[0072] Figure 25The sequence of chloramphenicol (SEQ ID NO:1) was described. Invention Details

[0074] The following describes the structure, construction, and characterization of several chimeric antigen receptors containing chloramphenicol (CLTX). Chimeric antigen receptors (CARs) are recombinant biomolecules containing at least an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain. Therefore, the term "antigen" is not limited to molecules that bind antibodies, but also refers to any molecule that can specifically bind a target. For example, a CAR may include a ligand that specifically binds to a cell surface receptor. The extracellular recognition domain (also called the extracellular domain or simply by the recognition element it contains) contains a recognition element that specifically binds to molecules present on the cell surface of the target cell. The transmembrane region anchors the CAR to the membrane. The intracellular signaling domain contains a signaling domain derived from the zeta chain of the human CD3 complex and optionally includes one or more co-stimulatory signaling domains. In an MHC-independent manner, CARs can both bind antigens and induce T cell activation. Therefore, 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 treating cancer.

[0075] The CAR containing chloramphenicol described in this article is called CLTX-IgG4(EQ)-CD28gg-Zeta. This CAR includes several important features, including: chloramphenicol; IgG4 Fc region mutated at two sites in the CH2 region (L235E; N297Q) in a manner that reduces Fc receptor (FcR) binding; CD28 co-stimulatory domain; and CD3ζ activation domain.

[0076] In some cases, a vector can be used to generate the CAR described herein, wherein the CAR open reading frame is followed by a T2A ribosomal skipping sequence and a truncated CD19 (CD19t) lacking the cytoplasmic signaling tail (truncated at amino acid 323). In this arrangement, co-expression of CD19t provides an inert, non-immunogenic surface marker that allows for precise assay of genetically modified cells and enables positive selection of genetically modified cells, as well as efficient cell transport and / or in vivo therapeutic T-cell imaging following adoptive transfer. Co-expression of CD19t provides a marker for the selective deletion of therapeutic cells by immunotargeting transduction of cells in vivo using clinically available antibodies and / or immunotoxins, thereby functioning as a suicide switch.

[0077] The CAR described herein can be generated by any means known in the art, although recombinant DNA technology is preferred. Nucleic acids encoding several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence using standard molecular cloning techniques known in the art (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding regions are preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte cell line, and most preferably selected from in vivo T lymphocyte cell lines.

[0078] Multiple T cell subsets isolated from patients can be transduced using vectors for CAR expression, including unselected PBMCs or enriched CD3 T cells or enriched CD3 or memory T cell subsets or T cells. CM or T CM / SCM / N Central memory T cells are a useful subset of T cells. They can be utilized by, for example... The device uses immunomagnetic selection to enrich CD45RO+ / CD62L+ cells expressing the desired receptor to isolate central memory T cells from peripheral blood mononuclear cells (PBMCs). Cells enriched for central memory T cells can be activated using anti-CD3 / CD28 and transduced using a SIN lentiviral vector, for example, that directs CAR expression as well as truncated human CD19 (CD19t), a non-immunogenic surface marker for both in vivo detection and possible in vitro selection. Activated / genetically modified central memory T cells can be expanded in vitro using IL-2 / IL-15 and then cryopreserved. Example

[0079] Example 1: Construction and structure of CLTX-IgG4Fc(EQ)-CD28-zeta CAR

[0080] The structure of a useful CAR containing chlortoxin, CLTX-IgG4Fc(EQ)-CD28-zeta, is described below. The codon-optimized CAR sequence comprises: chlortoxin, an IgG4 Fc spacer containing mutations (S228P, L235E) that significantly reduce Fc receptor-mediated recognition, a CD28 transmembrane domain, a co-stimulatory CD28 cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain. A T2A ribosomal skipping sequence separates this CAR sequence from the inert, non-immunogenic cell surface detection / selection marker CD19t. This T2A linkage results in the coordinated expression of both the CAR and CD19t from a single transcript. Figure 1A is a schematic diagram of the open reading frame of CLRX-IgG4Fc(EQ)-CD28-zeta-T2ACD19t. The diagram indicates the CLRX-IgG4Fc(EQ)-CD28-zetaCAR and the T2A ribosomal skipping and truncated CD19 sequence. Expression of the CAR and CD19t cassette is driven by the human EF1 promoter (EF1p). Figure 1 B schematically depicts the mature CAR expression.

[0081] The CLTX-IgG4Fc(EQ)-CD28-zeta sequence was generated by fusing the human GM-CSF receptor α leader peptide chlorotoxin, the S228P / L235E / N297Q modified IgG4Fc hinge (where the double mutation L235E / N297Q interferes with FcR recognition), the CD28 transmembrane domain, the CD28 cytoplasmic signaling domain, and the CD3ζ cytoplasmic signaling domain sequence. This sequence was synthesized de novo after codon optimization. The T2A sequence was obtained by digesting plasmids containing T2A. The CD19t sequence was obtained by transmembrane component (i.e., base pairs 1-972) sequence from the leader peptide sequence of plasmids containing CD19. 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 suitable cells, the vector integrated into the host cell genome. Figure 9 The amino acid sequence of CLTX-IgG4Fc(EQ)-CD28-zeta was presented, indicating multiple domains.

[0082] Example 2: Construction and structure of epHIV7 for expressing CLTX-IgG4Fc(EQ)-CD28-zeta

[0083] The pHIV7 plasmid is a parent plasmid from which the clinical plasmid CLTX-IgG4Fc(EQ)-CD28-zeta-T2A-CD19t_epHIV7 was derived at the T cell Therapeutics Research Laboratory (TCTRL) in City of Hope (COH). The epHIV7 vector for CAR expression was generated from the pHIV7 vector. Importantly, this vector uses the human EF1 promoter to drive CAR expression. Both the 5' and 3' sequences of the vector are derived from pv653RSN, as previously derived from the HXBc2 provirus. The polypurine fragment DNA flap sequence (cPPT) is derived from the HIV-1 strain pNL4-3 in the NIH Reagent Repository for AIDS. The prairie dog posttranscriptional regulatory element (WPRE) sequence has been previously described.

[0084] pHIV7 was constructed as follows. In short, the pv653RSN containing a 653bp sequence from gag-pol, plus 5' and 3' long terminal repeats (LTRs) and the intermediate SL3-neomycin phosphotransferase gene (Neo), was subcloned into pBluescript: In step 1, p5'HIV-1 51 was prepared from the sequence from the 5'LTR to the rev-response element (RRE), then the 5'LTR was modified by removing the sequence upstream of the TATA box and first ligated to the CMV enhancer, then ligated to the SV40 origin of replication (p5'HIV-2). In step 2, after cloning the 3'LTR into pBluescript to prepare p3'HIV-1, a 400-bp deletion was performed in the 3'LTR enhancer / promoter to remove the cis-regulatory element in HIV U3 and form p3'HIV-2. In step 3, fragments isolated from p5'HIV-3 and p3'HIV-2 were ligated to prepare pHIV-3. In step 4, p3'HIV-2 was further modified by removing additional upstream HIV sequences to generate p3'HIV-3, and a 600-bp BamHI-SalI fragment containing WPRE was added to p3'HIV-3 to prepare p3'HIV-4. In step 5, the size of the pHIV-3 RRE was reduced by PCR and ligated to the 5' fragment (not shown) from pHIV-3 and ligated to p3'HIV-4 to prepare pHIV-6. In step 6, a 190-bp BglII-BamHI fragment containing a cPPT DNA flap sequence from HIV-1 pNL4-3 (55) was amplified from pNL4-3 and placed between the RRE and WPRE sequences in pHIV-6 to prepare pHIV-7. This parental plasmid pHIV7-GFP (GFP, green fluorescent protein) was used to package the parental vector using a four-plasmid system.

[0085] The packaging signal psi(ψ) is required for the efficient packaging of the viral genome into the vector. RRE and WPRE enhance RNA transcript transport and transgene expression. Valve sequences combined with WPRE have been shown to enhance the transduction efficiency of lentiviral vectors in mammalian cells.

[0086] The auxiliary functions required for generating the viral vector were divided into three separate plasmids to reduce the likelihood of generating replicative lentiviruses via recombination: 1) pCgp encodes the gag / pol protein 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 vesicular stomatitis virus (VSV), which is required for the infectivity of the viral vector.

[0087] Minimal DNA sequence homology exists between the pHIV7-encoded vector genome and the helper plasmid. Homologous regions include a packaging signal region of approximately 600 nucleotides located in the gag / pol sequence of the pCgp helper plasmid; the CMV promoter sequences in all three helper plasmids; and the RRE sequence in the pCgp helper plasmid. It is highly unlikely that replicative recombinant viruses could be generated due to homology in these regions, as this would require multiple recombination events. Furthermore, any resulting recombinants would lack the functional LTR and tat sequences required for lentiviral replication.

[0088] The CMV promoter was replaced by the EF1α-HTLV promoter (EF1p), and the new plasmid was named epHIV7. EF1p has a length of 563 bp and was introduced into epHIV7 using NruI and NheI after the CMV promoter was removed.

[0089] The lentiviral genome has been removed from this system, excluding the gag / pol and rev, which are essential for the pathogenicity of the wild-type virus and for productive infection of target cells. Furthermore, the CLTX-IgG4Fc(EQ)-CD28-zeta-T2ACD19t_epHIV7 vector construct does not contain a complete 3' LTR promoter; therefore, the expressed and reverse-transcribed proviral DNA genome obtained in target cells will have an inactive LTR. Due to this design, no HIV-1-derived sequences will be transcribed from the provirus, and only therapeutic sequences will be expressed from their respective promoters. The removal of LTR promoter activity in the SIN vector will significantly reduce the likelihood of unintentional activation of the host gene.

[0090] Example 3: Generation of a vector for transducing patient T cells

[0091] Vectors for transducing patient T cells can be prepared as follows: For each plasmid (i.e., 1) a plasmid expressing CAR and optional markers such as truncated CD19; 2) pCgp; 3) pCMV-G; and 4) pCMV-Rev2), a seed library is generated, which is used to inoculate a fermenter to produce a sufficient quantity of plasmid DNA. The identity, sterility, and endotoxin content of the plasmid DNA are tested before use to generate lentiviral vectors.

[0092] In short, cells are expanded from 293T working cells (WCBs) that have been tested to confirm their identity and are free from viral contamination. A vial of 293T cells from the 293T WCBs is thawed. The cells are cultured and expanded until a sufficient number of cells are available for plating into an appropriate number of 10-layer cell factory (CF) plates for vector production and cell line maintenance. Single-cell lines can be used for production.

[0093] Lentiviral vectors are produced in sub-batches of up to 10 CFs. Two sub-batches can be produced within the same week, resulting in approximately 20 L of lentiviral supernatant per week. During downstream processing, material from all batches is combined to produce a single batch of product. 293T cells are seeded in CFs in 293T medium (DMEM containing 10% FBS). The plants are placed in a 37°C incubator and leveled to achieve uniform cell distribution across all layers of the CF. After two days, cells are transfected using the CaPO4 method with the four lentiviral plasmids described above, which involves a mixture of Tris:EDTA, 2M CaCl2, 2X HBS, and the four DNA plasmids. On day 3 post-transfection, the supernatant containing the secreted lentiviral vector is collected, purified, and concentrated. Cells are collected from each CF after removing the supernatant. Cells are digested with trypsin from each plant and collected by centrifugation. Cells are resuspended in freezing medium and cryopreserved. These cells are then used for replication-capable lentivirus (RCL) assays.

[0094] To purify and prepare the vector, the crude supernatant was clarified by membrane filtration to remove cell debris. Digestion with endonuclease was then performed. Degrade host cell DNA and residual plasmid DNA. Clarify cell debris in the viral supernatant using a 0.45 μm filter. Collect the clarified supernatant and add [a specific ingredient]. Pre-weighed containers (final concentration 50 U / mL) were used. Endonuclease digestion of residual plasmid DNA and host genomic DNA was performed at 37°C for 6 hours. 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 approximately 20-fold. The clarified endonuclease-treated viral supernatant was circulated through a hollow fiber cartridge with 500 kD NMWCO at a flow rate designed to maintain a shear rate of approximately 4000 / s or lower while maximizing throughput. Perfiltration of the nuclease-treated supernatant was initiated during the concentration process to maintain cartridge performance. An 80% permeate replacement rate was established using 4% lactose in PBS as the perfiltration buffer. The viral supernatant was brought to the target volume, representing approximately 20-fold concentration of the crude supernatant, and further perfiltration was continued for 4 exchange volumes, where the permeate replacement rate was 100%.

[0095] Further concentration of the viral products was achieved using high-speed centrifugation. Individual sub-batches of lentivirus were precipitated using a Sorvall RC-26plus centrifuge at 6000 RPM (6,088 RCF) at 6°C for 16–20 hours. Viral pellets from each sub-batch were then reconstituted in 50 mL of PBS with 4% lactose. The reconstituted pellets in this buffer represent the final formulation of the viral preparation. The entire vector concentration process resulted in approximately a 200-fold volume reduction. After all sub-batches were completed, the material was placed at -80°C while the sterility of each sub-batch sample was tested. Once sterility was confirmed, the sub-batches were rapidly thawed with agitation at 37°C. The material was then pooled and manually aliquoted in a Class II A / B3 biosafety cabinet. 1 mL of concentrated lentivirus was packed into sterile USP Class 6, externally threaded O-ring cryovials.

[0096] To ensure the purity of the lentiviral vector preparation, it was tested for residual host DNA contamination and for the transfer of residual host and plasmid DNA. In other tests, the vector identity was assessed by RT-PCR to ensure the presence of the correct vector.

[0097] Example 4: Preparation of T cells suitable for ACT

[0098] If using T CM To express CAR, suitable patient cells can be prepared as follows: First, T lymphocytes are obtained from the patient via leukocyte ablation, and suitable allogeneic or autologous T cell subsets, such as central memory T cells (T cells), are genetically modified. CM The CAR is expressed and then administered back to the patient by any clinically acceptable means to achieve anti-cancer treatment.

[0099] Suitable T-shirt CM The following steps can be taken: Apheresis products obtained from consenting study participants are treated with Ficoll, washed, and incubated overnight. Then, GMP-grade anti-CD14, anti-CD25, and anti-CD45RA reagents (Miltenyi Biotec) and CliniMACS are used. TM The separation device depleted populations of monocytes, regulatory T cells, and naive T cells. After depletion, the cells were processed in CliniMACS. TM The isolation device used DREG56-Biotin (COH clinical grade) and anti-biotin microbeads (Miltenyi Biotec) to enrich CD62L+ T cells in the negative fraction. CM cell.

[0100] After enrichment, T was prepared in complete X-Vivo15 with 50 IU / mL IL-2 and 0.5 ng / mL IL-15. CM The cells were then transferred to Teflon cell culture bags, where they were cultured using Dynal ClinEx. TM Cells were stimulated with Vivo CD3 / CD28 beads. Five days post-stimulation, cells were transduced with a lentiviral vector expressing the desired CAR at a multiplicity of infection (MOI) of 1.0 to 0.3. Cultures were maintained for 42 days, during which complete X-Vivo15 and IL-2 and IL-15 cytokines were added as needed for cell expansion (maintaining a cell density of 3 x 10⁻⁶ cells / year). 5 and 2x10 6 Live cells / mL, and cytokine supplementation is given every Monday, Wednesday, and Friday during culture. Under these conditions, cells typically expand to approximately 10⁻⁶ cells / mL within 21 days. 9 Cells were harvested at the end of the culture cycle, washed twice, and prepared in clinical-grade cryopreservation medium (Cryostore CS5, BioLife Solutions).

[0101] On the day of T-cell infusion, the cryopreserved and released product was thawed, washed, and prepared for re-infusion. The cryopreserved vials containing the released cell product were removed from liquid nitrogen storage, thawed, cooled, and washed with PBS / 2% human serum albumin (HSA) wash buffer. After centrifugation, the supernatant was removed, and the cells were resuspended in preservative-free normal saline (PFNS) / 2% HSA infusion diluent. Samples were then used for quality control testing.

[0102] Example 5: Expression of Cltx-IgG4(EQ)-CD28gg-Zeta

[0103] Figure 1 C shows engineered healthy donor T cells (HD187.2 T cells) expressing CLTX-CAR. CM / SCM / N Flow cytometry analysis results. The images show anti-CD19, anti-Fc, and anti-CD8 staining, representing co-expression of CLTX-CAR and CD19t transgenes in both CD8+ and CD4+ (CD8-) T cell subsets. The percentage of immunoreactive cells in transduced (CLTX-CAR) and untransduced (mimic) cells 18 days after CD3 / CD28 bead stimulation is shown to demonstrate the ability to transduce human T cells with CLTX-CAR.

[0104] Example 6: Chlorine toxin and Cltx-IgG4(EQ)-CD28gg-Zeta T cells specifically recognize glioma cell lines U251

[0105] Chlorine toxin conjugated with the fluorescently labeled Cy5.5 (CLTX-Cy5.5) was used to evaluate the binding of chlorine toxins to various cell types. The results of this study were presented in... Figure 2 The cells in the AE array are shown as follows: (A) peripheral blood mononuclear cells (PBMCs) derived from healthy donors; (B) human EBV-transformed lymphoblastic cell line LCL; (C) human embryonic kidney line 293T transformed with large T antigen; (D) human astrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from healthy donors; and (E) human glioblastoma cell line U251T). Cell lines were cultured in untreated medium or medium containing 1 μM CLTX-Cy5.5 at 37°C for 1 hour and then evaluated by flow cytometry.

[0106] like Figure 2 As shown in F, CLTX-CAR T cells specifically kill the glioma tumor cell line U251T without killing LCL, 293T, or primary human astrocytes. The number of live 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) was plotted relative to the number of live target cells co-cultured with simulated T cells for the same duration, at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells).

[0107] Example 7: Chlorine toxin binds to low-passage PBT human glial cells in a manner independent of IL13Rα2 expression. Tumor

[0108] To examine whether chloramphenicol binding is independent of IL13Rα2 expression, cell lines were cultured for 1 hour in medium containing 1 μM CLTX-Cy5.5, followed by flow cytometry analysis of IL13RA2-low and IL13RA2-high cell lines stained with PE-conjugated IL13Rα2 antibody. Figure 3 As can be seen from AB, chloramphenicol binds to low-passage PBT human glioma lineage in a manner independent of IL13Rα2 expression.

[0109] Example 8: CLTX-IgG4(EQ)-CD28gg-ZetaT cells expressing IL13Rα2 and TGGA molecules independently. The subtype method identifies and kills low-passage PBT human glioblastoma lines.

[0110] like Figure 4 As shown in Figure A, CLTX-CAR T cells demonstrated statistically significant killing of a primary GBM cell line compared to the embryonic kidney cell line 293T. The plot shows the number of live target cells co-cultured with CLTX-CAR T cells for 24, 48, and 72 hours at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells) after normalization relative to the number of live target cells co-cultured with simulated T cells for the same duration.

[0111] Figure 4 Image B shows the clearance of PBT003-4 and PBT009 tumor cells by CLTX-CAR T cells compared to the mimic control, as observed using live-cell imaging. Representative images of PBT003-4 and PBT009 cells co-cultured with mimic or CLTX-CAR T cells at an effector:target ratio of 1:4 (4,000 T cells, 16,000 target cells) were captured by bright-field microscopy immediately after co-culture (0 h) and 3 days after co-culture (72 h) at the mimic or CLTX-CAR T cell ratio.

[0112] Example 9: Activation of CLTX-IgG4(EQ)-CD28gg-ZetaT cells by stimulation with GBM cells

[0113] In the presence of protein transport inhibitors, T cells (mimicry or CLTX-expressing CARs) were stimulated for 5 hours via target cell stimulation at an effector:target ratio of 1:1 (25,000 T cells, 25,000 target cells). The percentage of CAR-T cells undergoing degranulation was determined by flow cytometry based on CD107a immunoreactivity. Figure 5 A), and detect cytokine production by intracellular staining ( Figure 5 B).

[0114] Example 10: CLTX-CAR T cells with different spacer designs effectively target tumor cells

[0115] Figure 6 A is a schematic diagram of a CLTX-CAR construct with different spacers (connectors), said spacers comprising IgG4Fc(EQ), IgG4(HL-CH3), CD8h, and a short connector (L). All have a CD28 transmembrane domain (not shown). Figure 6 As shown in B, CLTX-CAR T cells with different connectors were able to kill U251T GBM cells. The plot shows the number of live U251T cells co-cultured with T cells containing different CLTX retargeting constructs for 24, 48, and 72 hours, normalized relative to the number of live U251T cells co-cultured with simulated T cells for the same duration, at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells). Figure 6 As shown in Figure C, CLTX-CAR T cells with different connectors exhibited varying levels of cytokine production after antigen challenge. T cells engineered with different CTLX-redirected constructs were stimulated with U251 T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFN-γ secretion was detected by ELISA of the supernatant.

[0116] Example 11: Antitumor effects of CLTX-CAR T cells with different intracellular signal transduction domains

[0117] Figure 7 A is a schematic diagram of CLTX-CAR constructs with different intracellular co-stimulatory domains CD28 and 41BB. (See diagram for reference.) Figure 7 As shown in B, CLTX-CAR T cells with different co-stimulatory domains were able to kill U251T GBM cells. The plot shows the number of live U251T cells co-cultured with T cells containing different CLTX-redirected constructs, at an effector:target ratio of 1:1 (15,000 T cells, 15,000 target cells), after normalization relative to those co-cultured with Mock T cells for the same length of time. Figure 7 As shown in Figure C, CLTX-CAR T cells with different co-stimulatory domains produced various levels of cytokines after tumor attack. T cells engineered with constructs retargeted with different CLTX were stimulated with U251 T cells at an effector:target ratio of 1:1 (20,000 T cells, 20,000 target cells). IFN-γ secretion was detected by ELISA assay of the supernatant.

[0118] Example 12: CLTX-CAR T cells reduce the growth of established U251T GBM tumors in vivo.

[0119] Figure 8 A is a schematic diagram of U251T xenograft growth and T-cell therapy in NSG mice. Mice with subcutaneously implanted U251T cells were treated with PBS (tumor only), mimic T cells, or CLTX-CAR T cells (days -14 to 0). Figure 8 B. CLTX-CAR T-cell therapy inhibits tumor progression. Tumor growth was measured using calipers within 20 days from the time of T-cell injection (day 0 to day 20).

[0120] Example 13: Another CLTX CAR

[0121] Figure 9-24 Various additional CLTX-CAR amino acid sequences were presented, which can be constructed and expressed as described above for the CLTX-IgG4(EQ)-CD28gg-Zeta CAR. Figure 8-24 In the diagrams, the multiple regions listed below the sequence, from the amino to the carboxyl terminus, are indicated by alternating underlined and ununderlined portions. Therefore, in... Figure 9In the diagram, the GMCSFRa signal peptide is underlined, the chloramphenicol sequence is not underlined, the spacer (IgG4(SmP)(L235E,N297Q)) is underlined, the CD28 transmembrane sequence is not underlined, the CD28cyto(LLmGG) co-stimulatory domain is underlined, the (Gly)3 sequence separating the co-stimulatory domain from the CD3zeta sequence is not underlined, and the CD3zeta sequence is underlined. Figure 9-23 The T2A and CD19t sequences co-expressed with CAR were not shown in the data. Figure 24 Depicting Figure 23 The CAR contains T2A (a ribosomal skipping sequence) and a truncated CD19. The truncated CD19 is co-expressed with the CAR, allowing for easy identification and quantification of transfected cells.

[0122] Example 14: Additional toxin sequences

[0123] Figure 25 The sequence of chloramphenicol (SEQ ID NO:1) is depicted. In some cases, these toxins can replace the chloramphenicol in the CAR described herein. sequence list <110> City of Hope <120> Chimeric antigen receptors containing chlortoxin domains <130> 40056-0024WO1 <140> PCT / US2016 / 056901 <141> 2016-10-13 <150> US 62 / 241,021 <151> 2015-10-13 <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> PRT <213> Leiurus quinquestriatus <400> 1 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg 35 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220> <223> connector <400> 2 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Mutant IgG4 hinge <400> 3 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 4 <211> 12 <212> PRT <213> Homo sapiens <400> 4 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> 5 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Mutant IgG4 hinge + adapter <400> 5 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly 20 <210> 6 <211> 39 <212> PRT <213> Homo sapiens <400> 6 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> 7 <211> 48<着 <212> PRT <着213> Homo sapiens <400> 7 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 8 <211> 45 <212> PRT <213> Homo sapiens <400> 8 It should be noted that there may be some inaccuracies in the original text, especially in the tags like "<着213>" which seem to be incorrect notations. This translation is based on the best understanding of the provided content.Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 9 <211> 129 <212> PRT <213> Artificial sequence <220> <223> Human chimeric IgG4 HL-Ch3 with mutation <400> 9 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 20 25 30 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 35 40 45 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 50 55 60 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 65 70 75 80 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 85 90 95 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 100 105 110 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 115 120 125 Lys <210> 10 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Mutated IgG4 <400> 10 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Gln Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 11 <211> 229 <212> PRT <213> Artificial sequence <220> <223> Mutated IgG4 <400> 11 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Gln Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 12 <211> 107 <212> PRT <213> Homo sapiens <400> 12 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100 105 <210> 13 <211> 21 <212> PRT <213> Homo sapiens <400> 13 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu 20 <210> 14 <211> 27 <212> PRT <213> Homo sapiens <400> 14 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 15 <211> 28 <212> PRT <213> Synthetic Sequence <220> <223> Mutant human CD28 <400> 15 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 16 <211> 22 <212> PRT <213> Homo sapiens <400> 16 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 1 5 10 15 Gly Leu Gly Ile Phe Phe 20 <210> 17 <211> 21 <212> PRT <213> Homo sapiens <400> 17 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 18 <211> 23 <212> PRT <213> Homo sapiens <400> 18 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr 20 <210> 19 <211> 24 <212> PRT <213> Homo sapiens <400> 19 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 20 <211> 27 <212> PRT <213> Homo sapiens <400> 20 Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu 1 5 10 15 Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val 20 25 <210> 21 <211> 112 <212> PRT <213> Homo sapiens <400> 21 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 22 <211> 41 <212> PRT <213> Homo sapiens <400> 22 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 [[ID=G30]]Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 23 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Mutated CD28 <400> 23 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 24 <211> 42 <212> PRT <213> Homo sapiens <400> 24 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 25 <211> 42 <212> PRT <213> Homo sapiens <400> 25 Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His 1 5 10 15 Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln 20 25 30 Ala Asp Ala His Ser Thr Leu Ala Lys Ile 35 40 <210> 26 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Comprising the chimeric CLTX-IgG4(EQ)-CD28tm-CD28-zeta of the signal <400> 26 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val 65 70 75 80 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 100 105 110 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 165 170 175 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185 190 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 210 215 220 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 225 230 235 240 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 245 250 255 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met 275 280 285 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 290 295 300 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 305 310 315 320 Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 325 330 335 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 340 345 350 Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp 355 360 365 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 370 375 380 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 385 390 395 400 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 405 410 415 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 420 425 430 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 435 440 445 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 450 455 460 Met Gln Ala Leu Pro Pro Arg 465 470 <210> 27 <211> 371 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(HL-CH3)-CD28tm-CD28-zeta including a signal <400> 27 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 65 70 75 80 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 85 90 95 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 100 105 110 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 115 120 125 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 130 135 140 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 145 150 155 160 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 165 170 175 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu 180 185 190 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 195 200 205 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His 210 215 220 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 225 230 235 240 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 245 250 255 Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 260 265 270 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 275 280 285 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 290 295 300 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 305 310 315 320 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 325 330 335 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 340 345 350 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 355 360 365 Pro Pro Arg 370 <210> 28 <211> 287 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-CD8h-CD28tm-CD28-zeta including a signal <400> 28 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Thr Thr Thr Pro Ala Pro 50 55 60 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 65 70 75 80 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 85 90 95 Gly Leu Asp Phe Ala Cys Asp Met Phe Trp Val Leu Val Val Val Gly 100 105 110 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 115 120 125 Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met 130 135 140 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 145 150 155 160 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Arg 165 170 175 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 180 185 190 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 195 200 205 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 210 215 220 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 225 230 235 240 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 245 250 255 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 260 265 270 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 275 280 285 <210> 29 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(hinge)-CD28tm-CD28-zeta including signal <400> 29 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly 65 70 75 80 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 85 90 95 Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn 100 105 110 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 115 120 125 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val 130 135 140 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 145 150 155 160 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 165 170 175 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 180 185 190 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 195 200 205 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 210 215 220 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 225 230 235 240 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 245 250 [[ID=XX]]<210> 30 <211> 252 <212> PRT <2XX> Synthetic sequence <220> <223> Chimeric CLTX-L-CD28tm-CD28-zeta including a signal <400> 30 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly 50 55 60 It should be noted that in the original text, there is an unclear "人工序列" which is tentatively translated as "Synthetic sequence" here. And "<210>" and "<211>" etc. are likely some kind of specific identifiers in the original context and are directly retained as they are. If there is more specific information about these tags, a more accurate translation can be made. Also, "<2XX>" is a placeholder for the correct tag number in the "人工序列" translation part.Gly Gly Ser Gly Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu 65 70 75 80 Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 85 90 95 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 100 105 110 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 115 120 125 Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe 130 135 140 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 145 150 155 160 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 165 170 175 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 180 185 190 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 195 200 205 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 210 215 220 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 225 230 235 240 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 245 250 <210> 31 <211> 516 <212> PRT <213> Artificial Sequence <220> <223> Chimeric CLTX-IgG4(EQ)-CD28tm-CD28-4-1BBzeta including a signal <400> 31 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val 65 70 75 80 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 100 105 110 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 165 170 175 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185 190 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 210 215 220 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 225 230 235 240 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 245 250 255 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met 275 280 285 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 290 295 300 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 305 310 315 320 Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 325 330 335 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 340 345 350 Ala Tyr Arg Ser Gly Gly Gly Lys Arg Gly Arg Lys Lys Leu Leu Tyr 355 360 365 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 370 375 380 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 385 390 395 400 Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 405 410 415 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 420 425 430 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 435 440 445 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 450 455 460 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 465 470 475 480 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 485 490 495 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 500 505 510 Leu Pro Pro Arg 515 <210> 32 <211> 416 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(HL-CH3)-CD28tm-CD28-4-1BBzeta including the sequence <400> 32 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 65 70 75 80 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 85 90 95 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 100 105 110 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 115 120 125 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 130 135 140 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 145 150 155 160 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 165 170 175 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu 180 185 190 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 195 200 205 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His 210 215 220 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 225 230 235 240 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 245 250 255 Gly Gly Gly Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 260 265 270 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 275 280 285 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly 290 295 300 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 305 310 315 320 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 325 330 335 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 340 345 350 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 355 360 365 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 370 375 380 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 385 390 395 400 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 405 410 415 <210> 33 <211> 332 <212> PRT <213> Synthetic Sequence <220> <223> Comprising the chimeric CLTX-CD8h-CD28tm-CD28-4-1BB-zeta sequence <400> 33 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Thr Thr Thr Pro Ala Pro 50 55 60 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 65 70 75 80 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 85 90 95 Gly Leu Asp Phe Ala Cys Asp Met Phe Trp Val Leu Val Val Val Gly 100 105 110 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 115 120 125 Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met 130 135 140 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 145 150 155 160 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Lys 165 170 175 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 180 185 190 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 195 200 205 Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe 210 215 220 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 225 230 235 240 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 245 250 255 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 260 265 270 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 275 280 285 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 290 295 300 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 305 310 315 320 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 325 330 <210> 34 <211> 299 <212> PRT <213> Artificial sequence <220> <223> Chimeric LTX-IgG4(hinge)-CD28tm-CD28-4-1BBzeta including the sequence <400> 34 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly 65 70 75 80 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 85 90 95 Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn 100 105 110 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 115 120 125 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Lys Arg 130 135 140 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 145 150 155 160 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 165 170 175 Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser 180 185 190 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 195 200 205 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 210 215 220 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 225 230 235 240 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 245 250 255 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 260 265 270 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 275 280 285 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 290 295 <210> 35 <211> 297 <212> PRT <213> Synthetic sequence <220> <223> Chimeric CLTX-L-CD28tm-CD28-4-1BB-zeta including a sequence <400> 35 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly 50 55 60 Gly Gly Ser Gly Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu 65 70 75 80 Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 85 90 95 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 100 105 110 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 115 120 125 Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Lys Arg Gly Arg 130 135 140 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 145 150 155 160 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 165 170 175 Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser 180 185 190 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 195 200 205 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 210 215 220 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 225 230 235 240 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 245 250 255 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 260 265 270 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 275 280 285 Leu His Met Gln Ala Leu Pro Pro Arg 290 295 <210> 36 <211> 466 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(EQ)-CD4tm-4-1BB-zeta including the sequence <400> 36 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val 65 70 75 80 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 100 105 110 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 165 170 175 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185 190 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 210 215 220 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 225 230 235 240 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 245 250 255 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met 275 280 285 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 290 295 300 Leu Gly Ile Phe Phe Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 305 310 315 320 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 325 330 335 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly 340 345 350 Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 355 360 365 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 370 375 380 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 385 390 395 400 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 405 410 415 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 420 425 430 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 435 440 445 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 450 455 460 Pro Arg 465 <210> 37 <211> 366 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(HL-CH3)-CD4tm-4-1BB-zeta including the sequence <400> 37 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 65 70 75 80 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 85 90 95 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 100 105 110 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 115 120 125 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 130 135 140 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 145 150 155 160 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 165 170 175 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Ala Leu Ile Val 180 185 190 Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe 195 200 205 Phe Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 210 215 220 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 225 230 235 240 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val 245 250 255 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 260 265 270 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 275 280 285 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 290 295 300 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 305 310 315 320 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 325 330 335 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 340 345 350 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 38 <211> 288 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-CD8h-CD28tm-4-1BB-zeta including the sequence <400> 38 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Thr Thr Thr Pro Ala Pro 50 55 60 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 65 70 75 80 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 85 90 95 Gly Leu Asp Phe Ala Cys Asp Met Phe Trp Val Leu Val Val Val Gly 100 105 110 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 115 120 125 Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 130 135 140 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 145 150 155 160 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly 165 170 175 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 180 185 190 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 195 200 205 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 210 215 220 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 225 230 235 240 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 245 250 255 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 260 265 270 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 275 280 285 <210> 39 <211> 255 <212> PRT <213> Synthetic Sequence <220> <223> Chimeric CLTX-IgG4(hinge)-CD28tm-4-1BB-zeta including the sequence <400> 39 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro 50 55 60 Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly 65 70 75 80 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 85 90 95 Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 100 105 110 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 115 120 125 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg 130 135 140 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 145 150 155 160 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 165 170 175 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 180 185 190 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 195 200 205 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 210 215 220 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 225 230 235 240 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 245 250 255 <210> 40 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> Comprising the chimeric CLTX-L-CD28tm-4-1BB-zeta of the sequence <400> 40 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly 50 55 60 Gly Gly Ser Gly Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu 65 70 75 80 Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 85 90 95 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 100 105 110 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 115 120 125 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys 130 135 140 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 145 150 155 160 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 165 170 175 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 180 185 190 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 195 200 205 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 210 215 220 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 225 230 235 240 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 245 250 <210> 41 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(EQ)-CD28tm-CD28-zeta excluding signal <400> 41 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 50 55 60 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 65 70 75 80 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 85 90 95 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 100 105 110 Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 115 120 125 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 130 135 140 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 145 150 155 160 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 165 170 175 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 180 185 190 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 195 200 205 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 210 215 220 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 225 230 235 240 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 245 250 255 Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu Val Val 260 265 270 Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 275 280 285 Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp 290 295 300 Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 305 310 315 320 Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly 325 330 335 Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 340 345 350 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 355 360 365 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 370 375 380 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 385 390 395 400 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 405 410 415 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 420 425 430 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 435 440 445 Arg <210> 42 <211> 349 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(HL-CH3)-CD28tm-CD28-zeta excluding signal <400> 42 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro 50 55 60 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 65 70 75 80 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 85 90 95 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 100 105 110 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 115 120 125 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 130 135 140 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 145 150 155 160 Leu Ser Leu Gly Lys Met Phe Trp Val Leu Val Val Val Gly Gly Val 165 170 175 Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp 180 185 190 Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met 195 200 205 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 210 215 220 Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys 225 230 235 240 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 245 250 255 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 260 265 270 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 275 280 285 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 290 295 300 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 305 310 315 320 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 325 330 335 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 340 345 <210> 43 <211> 265 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-CD8h-CD28tm-CD28-zeta excluding signal <400> 43 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 35 40 45 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 50 55 60 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 65 70 75 80 Asp Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 85 90 95 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 100 105 110 Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 115 120 125 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 130 135 140 Phe Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser 145 150 155 160 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 165 170 175 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 180 185 190 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 195 200 205 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 210 215 220 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 225 230 235 240 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 245 250 255 Leu His Met Gln Ala Leu Pro Pro Arg 260 265 <210> 44 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(hinge)-CD28tm-CD28-zeta excluding signal <400> 44 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 50 55 60 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 65 70 75 80 Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 85 90 95 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 100 105 110 Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala 115 120 125 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 130 135 140 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 145 150 155 160 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 165 170 175 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 180 185 190 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 195 200 205 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 210 215 220 His Met Gln Ala Leu Pro Pro Arg 225 230 <210> 45 <211> 230 <212> PRT <213> Artificial Sequence <220> <223> Chimeric CLTX-L-CD28tm-CD28-zeta excluding signal <400> 45 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe 35 40 45 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 50 55 60 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 65 70 75 80 Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 85 90 95 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 100 105 110 Tyr Arg Ser Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 115 120 125 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 130 135 140 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 145 150 155 160 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 165 170 175 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 180 185 190 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 195 200 205 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 210 215 220 Gln Ala Leu Pro Pro Arg 225 230 <210> 46 <211> 494 <212> PRT <213> Artificial Sequence <220> Chimeric CLTX-IgG4(EQ)-CD28tm-CD28-4-1BBzeta for exclusion of signals <400> 46 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 50 55 60 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 65 70 75 80 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 85 90 95 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 100 105 110 Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 115 120 125 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 130 135 140 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 145 150 155 160 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 165 170 175 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 180 185 190 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 195 200 205 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 210 215 220 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 225 230 235 240 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 245 250 255 Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu Val Val 260 265 270 Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 275 280 285 Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp 290 295 300 Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 305 310 315 320 Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly 325 330 335 Gly Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val 370 375 380 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 385 390 395 400 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 405 410 415 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 420 425 430 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 435 440 445 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 450 455 460 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 465 470 475 480 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 47 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> Chimeric CLTX-IgG4(HL-CH3)-CD28tm-CD28-4-1BBzeta excluding signal <400> 47 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro 50 55 60 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 65 70 75 80 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 85 90 95 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 100 105 110 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 115 120 125 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 130 135 140 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 145 150 155 160 Leu Ser Leu Gly Lys Met Phe Trp Val Leu Val Val Val Gly Gly Val 165 170 175 Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp 180 185 190 Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met 195 200 205 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 210 215 220 Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Lys Arg Gly 225 230 235 240 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 245 250 255 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 260 265 270 Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg 275 280 285 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 290 295 300 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 305 310 315 320 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 325 330 335 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 340 345 350 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 355 360 365 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 370 375 380 Ala Leu His Met Gln Ala Leu Pro Pro Arg 385 390 <210> 48 <211> 310 <212> PRT <213> Artificial Sequence <220><000181�>Chimeric CLTX-CD8h-CD28tm-CD28-4-1BB-zeta for exclusion signal <400> 48 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 35 40 45 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 50 55 60 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 65 70 75 80 Asp Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 85 90 95 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys[[ID=2⑨]] 100 105 110 Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 115 120 125 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 130 135 140 Phe Ala Ala Tyr Arg Ser Gly Gly Gly Lys Arg Gly Arg Lys Lys Leu 145 150 155 160 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 165 170 175 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 180 185 190 Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 195 200 205 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 210 215 220 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 225 230 235 240 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 245 250 255 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 260 265 270 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 275 280 285 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 290 295 300 Gln Ala Leu Pro Pro Arg 305 310 <210> 49 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Chimeric LTX-IgG4(hinge)-CD28tm-CD28-4-1BBzeta excluding signal <400> 49 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 50 55 60 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 65 70 75 80 Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 85 90 9​​​​​Ala Ala Tyr Arg Ser Gly Gly Gly Lys Arg Gly Arg Lys Lys Leu Leu 115 120 125 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 130 135 140 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 145 150 155 160 Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 165 170 175 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 180 185 190 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 195 200 205 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 210 215 220 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 225 230 235 240 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 245 250 255 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 260 265 270 Ala Leu Pro Pro Arg 275 <210> 50 <211> 275 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-L-CD28tm-CD28-4-1BB-zeta excluding signal <400> 50 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe 35 40 45 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 50 55 60 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 65 70 75 80 Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 85 90 95 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 100 105 110 Tyr Arg Ser Gly Gly Gly Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 115 120 125 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 130 135 140 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 145 150 155 160 Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 165 170 175 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 180 185 190 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 195 200 205 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 210 215 220 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 225 230 235 240 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 245 250 255 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 260 265 270 Pro Pro Arg 275 <210> 51 <211> 444 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(EQ)-CD4tm-4-1BB-zeta excluding signal <400> 51 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 50 55 60 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 65 70 75 80 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 85 90 95 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 100 105 110 Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 115 120 125 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 130 135 140 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 145 150 155 160 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 165 170 175 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 180 185 190 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 195 200 205 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 210 215 220 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 225 230 235 240 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 245 250 255 Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Ala Leu Ile Val Leu Gly 260 265 270 Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys 275 280 285 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 290 295 300 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 305 310 315 320 Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe 325 330 335 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 340 345 350 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 355 360 365 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 370 375 380 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 385 390 395 400 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 405 410 415 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 420 425 430 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 435 440 <210> 52 <211> 344 <212> PRT <213> Artificial Sequence <220> <223> Chimeric CLTX-IgG4(HL-CH3)-CD4tm-4-1BB-zeta excluding signal <400> 52 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro 50 55 60 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 65 70 75 80 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 85 90 95 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 100 105 110 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 115 120 125 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 130 135 140 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 145 150 155 160 Leu Ser Leu Gly Lys Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 165 170 175 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Arg Gly Arg Lys 180 185 190 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 195 200 205 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 210 215 220 Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala 225 230 235 240 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 245 250 255 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 260 265 270 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 275 280 285 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 290 295 300 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 305 310 315 320 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 325 330 335 His Met Gln Ala Leu Pro Pro Arg 340 <210> 53 <211> 266 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-CD8h-CD28tm-4-1BB-zeta signal <400> 53 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 35 40 45 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 50 55 60 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 65 70 75 80 Asp Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 85 90 95 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 100 105 110 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 115 120 125 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 130 135 140 Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg 145 150 155 160 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 165 170 175 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 180 185 190 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 195 200 205 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 210 215 220 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 225 230 235 240 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 245 250 255 Ala Leu His Met Gln Ala Leu Pro Pro Arg 260 265 <210> 54 <211> 233 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-IgG4(hinge)-CD28tm-4-1BB-zeta excluding signal <400> 54 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 35 40 45 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 50 55 60 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg 65 70 75 80 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 85 90 95 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 100 105 110 Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser 115 120 125 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 130 135 140 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 145 150 155 160 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 165 170 175 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 180 185 190 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 195 200 205 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 210 215 220 Leu His Met Gln Ala Leu Pro Pro Arg 225 230 <210> 55 <211> 231 <212> PRT <213> Artificial sequence <220> <223> Chimeric CLTX-L-CD28tm-4-1BB-zeta excluding signal <400> 55 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln 20 25 30 Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe 35 40 45 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 50 55 60 Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys 65 70 75 80 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 85 90 95 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 100 105 110 Gly Cys Glu Leu Gly Gly Gly Arg Val Lys Phe Ser Arg Ser Ala Asp 115 120 125 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 130 135 140 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 145 150 155 160 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 165 170 175 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 180 185 190 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 195 200 205 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 210 215 220 Met Gln Ala Leu Pro Pro Arg 225 230 <210> 56 <211> 29 <212> PRT <213> Leiurus quinquestriatus hebraeus <400> 56 Val Ser Cys Glu Asp Cys Pro Asp His Cys Ser Thr Gln Lys Ala Arg 1 5 10 15 Ala Lys Cys Asp Asn Asp Lys Cys Val Cys Glu Pro Ile 20 25 <210> 57 <211> 34 <212> PRT <213> Leiurus quinquestriatus hebraeus <400> 57 Cys Gly Pro Cys Phe Thr Thr Asp His Gln Met Glu Gln Lys Cys Ala 1 5 10 15 Glu Cys Cys Gly Gly Ile Gly Lys Cys Tyr Gly Pro Gln Cys Leu Cys 20 25 30 Asn Arg <210> 58 <211> 34 <212> PRT <213> Androctonus australis <400> 58 Met Cys Ile Pro Cys Phe Thr Thr Asn Pro Asn Met Ala Ala Lys Cys 1 5 10 15 Asn Ala Cys Cys Gly Ser Arg Arg Gly Ser Cys Arg Gly Pro Gln Cys 20 25 30 Ile Cys <210> 59 <211> 35 <212> PRT <213> Buthus martensii <400> 59 Cys Gly Pro Cys Phe Thr Thr Asp Ala Asn Met Ala Arg Lys Cys Arg 1 5 10 15 Glu Cys Cys Gly Gly Ile Gly Lys Cys Phe Gly Pro Gln Cys Leu Cys 20 25 30 Asn Arg Ile 35 <210> 60 <211> 600 <212> PRT <213> Artificial sequence <220> <223> Chimeric CTLX-L-CD28tm-41BB-zeta-T2A-CD19 <400> 60 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Cys Met Pro Cys Phe Thr Thr Asp His 20 25 30 Gln Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly 35 40 45 Lys Cys Tyr Gly Pro Gln Cys Leu Cys Arg Gly Gly Gly Ser Ser Gly 50 55 60 Gly Gly Ser Gly Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu 65 70 75 80 Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 85 90 95 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 100 105 110 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 115 120 125 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Arg Val Lys 130 135 140 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 145 150 155 160 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 165 170 175 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 180 185 190 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 195 200 205 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 210 215 220 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 225 230 235 240 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu Glu Gly 245 250 255 Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu 260 265 270 Asn Pro Gly Pro Arg Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu 275 280 285 Phe Leu Thr Pro Met Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys 290 295 300 Val Glu Glu Gly Asp Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser 305 310 315 320 Asp Gly Pro Thr Gln Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys 325 330 335 Pro Phe Leu Lys Leu Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met 340 345 350 Arg Pro Leu Ala Ile Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met 355 360 365 Gly Gly Phe Tyr Leu Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp 370 375 380 Gln Pro Gly Trp Thr Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg 385 390 395 400 Trp Asn Val Ser Asp Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg 405 410 415 Ser Ser Glu Gly Pro Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys 420 425 430 Leu Tyr Val Trp Ala Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro 435 440 445 Pro Cys Val Pro Pro Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp 450 455 460 Leu Thr Met Ala Pro Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro 465 470 475 480 Pro Asp Ser Val Ser Arg Gly Pro Leu Ser Trp Thr His Val His Pro 485 490 495 Lys Gly Pro Lys Ser Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro 500 505 510 Ala Arg Asp Met Trp Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala 515 520 525 Thr Ala Gln Asp Ala Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr 530 535 540 Met Ser Phe His Leu Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp 545 550 555 560 Leu Leu Arg Thr Gly Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr 565 570 575 Leu Ile Phe Cys Leu Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg 580 585 590 Ala Leo Val Leo Arg Arg Lys Arg 595 600

Claims

1. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the following: (i) Chlorine toxins as shown in SEQ ID NO: 1; (ii) Spacer area; (iii) Select transmembrane domains from the following groups: CD4 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane domain, and CD3ζ transmembrane domain; (iv) Costimulatory domains; (v) GGG spacers, and (vi) CD3ζ signal propagation domain; The spacer region therein consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2-12.

2. The nucleic acid molecule of claim 1, wherein the spacer region is located between the chlortoxin and the transmembrane domain.

3. The nucleic acid molecule of claim 1, wherein the chimeric antigen receptor is composed of an amino acid sequence selected from SEQ ID NO: 26-55.

4. A vector comprising the nucleic acid molecule of claim 1.

5. A vector comprising the nucleic acid molecule of claim 3.

6. A carrier comprising an expression cassette, said expression cassette comprising the nucleic acid molecule of claim 1.

7. A population of T cells or NK cells comprising the nucleic acid molecule of claim 1 or claim 3.

8. A population of T cells or NK cells comprising the vector of claim 4 or claim 5.

9. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: chlorine toxin as shown in SEQ ID NO:1; a spacer composed of sequences selected from the group consisting of SEQ ID NO:2-12; a transmembrane domain composed of sequences selected from the group consisting of SEQ ID NO:13-20; a co-stimulatory domain composed of sequences selected from the group consisting of SEQ ID NO:22-25; a GGG spacer; and a CD3ζ signaling domain as shown in SEQ ID NO:

21.

10. The nucleic acid molecule of claim 9, wherein the spacer is composed of sequences selected from the group consisting of: SEQ ID NO: 2, 3, 5, 8 and 9-12; a transmembrane domain composed of sequences selected from SEQ ID NO: 15 and 16; and a co-stimulatory domain composed of sequences selected from the group consisting of: SEQ ID NO: 22-24.

11. The nucleic acid molecule of claim 9, wherein the GGG spacer is located between the co-stimulatory domain and the CD3ζ signaling domain.

12. The nucleic acid molecule of claim 9, wherein the GGG spacer is located between the first and second co-stimulatory domains.

13. A vector comprising a nucleic acid molecule of any one of claims 9-12.

14. A carrier comprising an expression cassette, the expression cassette comprising a nucleic acid molecule of any one of claims 9-12.

15. A population of T cells or NK cells comprising a nucleic acid molecule of any one of claims 9-12.

16. A population of T cells or NK cells comprising the vector of claim 13.

17. A population of T cells or NK cells comprising the vector of claim 14.

18. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the following: (i) the chloramphenicol shown in SEQ ID NO: 1; (ii) a spacer consisting of 5-300 amino acids; (iii) a transmembrane domain selected from the group consisting of: CD4 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane domain, and CD3ζ transmembrane domain; (iv) co-stimulatory domain; (v) CD3ζ signaling domain; and optionally a spacer of 1-6 amino acids between the co-stimulatory domain and the CD3ζ signaling domain.

19. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: chlorine toxin as shown in SEQ ID NO:1; a spacer composed of sequences selected from the group consisting of SEQ ID NO:2-12; a transmembrane domain composed of sequences selected from the group consisting of SEQ ID NO:13-20; a co-stimulatory domain composed of sequences selected from the group consisting of SEQ ID NO:22-25; a CD3ζ signaling domain as shown in SEQ ID NO:21; and optionally a spacer of 1-6 amino acids between the co-stimulatory domain and the CD3ζ signaling domain.

20. A vector comprising the nucleic acid molecule of claim 18 or 19.

21. A carrier comprising an expression cassette containing the nucleic acid molecule of claim 18 or 19.

22. A population of T cells or NK cells comprising the nucleic acid molecule of claim 18 or 19.

23. A population of T cells or NK cells comprising the vector of claim 20 or 21.

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