MUC16-specific chimeric antigen receptors and their uses

Abstract

To provide an isolated nucleic acid encoding a chimeric antigen receptor (CAR) containing a scFv from anti-MUC16 monoclonal antibody for cancer treatment.SOLUTION: Provided is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising: (a) a MUC16 antigen binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a CD3 zeta signaling domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is incorporated herein by reference in its entirety. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 680,297, filed on 2004 / 01 / 11.

[0003] Sequence Listing This application has been filed electronically in ASCII format and is incorporated by reference in its entirety. Contains a sequence listing incorporated herein at The ASCII copy is named 50471_715_601_SL.txt and is located at 3 It is 31,880 bytes in size. [Background technology]

[0004] Recombinant polypeptides, including chimeric polypeptides, are useful in research, diagnostic, manufacturing, and therapeutic applications. Engineered effector cells that express antigen-binding polypeptides, such as CARs are useful in the treatment of diseases and disorders such as infectious diseases, autoimmune disorders, and cancer.

[0005] Incorporation by Reference All publications, patents, and patent applications mentioned herein are to be construed as separate entities each individually identified as such. Where any patent or patent application is specifically and individually indicated to be incorporated by reference, to the same extent as if each of the preceding claims were incorporated by reference herein. Summary of the Invention

[0006] Provided herein is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR (a) MUC16 antigen-binding domain; (b) stalk domain; (c) transmembrane domain (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) an isolated nucleic acid comprising a CD3 zeta signaling domain. In this embodiment, the MUC16 antigen binding domain is: (a) SEQ ID NOs: 1, 3, 5, 7, 9 12, and 14. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or a polypeptide having 100% identity; (b) a polypeptide having SEQ ID NOs: 2, 4, 6, 8, 10, 1 1, 13, and 15. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a polypeptide having 100% identity thereto; and (c) a polypeptide set forth in SEQ ID NOs: 27 to 57. and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity The polypeptides include at least one of

[0007] In some embodiments, the MUC16 antigen binding domain is any of the sequences set forth in SEQ ID NOs: 27-57. and at least 90%, 91%, 92%, 93%, or 100% identity with at least one of the amino acid sequences 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the stalk domain is a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In embodiments, the costimulatory signaling domain comprises 4-1BB. The costimulatory signaling domain of 4-1BB has at least one amino acid sequence with the amino acid sequence of SEQ ID NO:22. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 In some embodiments, the co-stimulatory polypeptides include polypeptides having 100% or 100% identity. The signaling domain comprises CD28. In some embodiments, the costimulatory signal of CD28 The transduction domain has at least 90%, 91%, 92%, or 100% identity with the amino acid sequence of SEQ ID NO: 23. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical It includes polypeptides having the following properties.

[0008] In some embodiments, the CD3 zeta signaling domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the isolated nucleic acids provided herein are truncated forms. epithelium In some embodiments, the truncated form may further comprise a growth factor receptor. epithelium The growth factor receptor is HER1t and comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the truncated form epithelium The growth factor receptor is HER1t-1 and includes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:66.

[0009] In some embodiments, the CAR is a CAR having at least one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 27-57. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It includes polypeptides with 99% or 100% identity.

[0010] As used herein, a scaffold and (1) a truncated form comprising at least one of HER1t, HER1t-1, or a functional variant thereof. epithelium Provided is a vector comprising a nucleic acid sequence encoding a growth factor receptor; (2) a cytokine; and (3) a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a CD3 zeta signaling domain.

[0011] In some embodiments, the cytokine is IL-15 or IL-12. In some embodiments, the vector is a lentiviral vector, a retroviral vector, or a non-viral vector. In some embodiments, the truncated epithelium The growth factor receptor comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the IL-15 is membrane-bound IL-15. In some embodiments, the membrane-bound IL-15 comprises a nucleotide sequence encoding SEQ ID NO: 161.

[0012] In some embodiments, any of the vectors presented herein is a self-cleaving Nucleotide encoding the Thosea asigna virus (T2A) peptide In some embodiments, the backbone may further comprise a Sleeping Beaut In some embodiments, the plasmid is a pFUGW transposon DNA plasmid. Any of the vectors presented in the detailed description may further include a promoter. In some embodiments, the promoter is hEF1a1. In some embodiments, the MUC16 antigen-binding domain is: (a) at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14; a polypeptide having; (b) at least 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 11, 13, and 15; and (c) at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% identical to at least one of the amino acid sequences shown in SEQ ID NOs: 27-57, including at least one of the polypeptides having. In some embodiments, the MUC16 antigen-binding domain is at least 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 0% identical to at least one of the amino acid sequences shown in SEQ ID NOs: 27-57. In some embodiments, the stalk domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, including a polypeptide having. In some embodiments, the co-stimulatory signaling domain includes 4-1BB. In one embodiment, the co-stimulatory signaling domain of 4-1BB is the amino acid sequence of SEQ ID NO: 22, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, including a polypeptide domain having. In some embodiments, the co-stimulatory signaling domain includes 4-1BB. In some embodiments, the co-stimulatory signaling domain of 4-1BB is the amino acid sequence of SEQ ID NO: 22. Acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% identity to the nucleic acid sequence.

[0013] In some embodiments, the costimulatory signaling domain comprises CD28. In this embodiment, the costimulatory signaling domain of CD28 is a nucleotide sequence having the amino acid sequence of SEQ ID NO: 23. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identity. The 3 zeta signaling domain has at least 90% identity with the amino acid sequence of SEQ ID NO:26. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Includes nucleic acid sequences with 100% identity.

[0014] In some embodiments, the vector comprises a plasmid. In some embodiments, each vector In some embodiments, the non-viral vector comprises an expression plasmid. ng Beauty transposon.

[0015] As used herein, immunological effects of nucleotides containing any of the nucleotides presented herein are The present invention relates to the use of (1) cell tagging (2) IL-15 and (3) An immune effector cell comprising a chimeric antigen receptor (CAR), wherein the CAR is (a) MU C16 antigen-binding domain; (b) stalk domain; (c) transmembrane domain; (d) 4 -1BB or CD28, or both containing costimulatory signaling domains; and (e ) Immune effector cells containing the CD3 zeta signaling domain are presented.

[0016] In some embodiments, the IL-15 is membrane-bound IL-15. The membrane-bound IL-15 comprises the polypeptide sequence of SEQ ID NO: 161. The cell tag comprises HER1t, wherein HER1t comprises the polypeptide sequence of SEQ ID NO: 65. In some embodiments, the cell tag comprises HER1t-1, wherein HER1t-1 is No. 66.

[0017] As used herein, immune effectors comprising any of the vectors described herein are In some embodiments, the cells are selected from the group consisting of T cells, natural killer (NK) cells, and the like. In some embodiments, the target cell is a T cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell. The CAR comprises at least one of the amino acid sequences of SEQ ID NOs: 27 to 57.

[0018] Provided herein is a method for stimulating a T cell-mediated immune response in a target cell population or tissue in a human subject in need thereof, comprising administering to the human subject an effective amount of cells genetically modified to express a CAR, wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; (e) a CD3 zeta signaling domain; and (f) a truncated epithelium The present invention provides a method for detecting ovarian cancer or breast cancer, comprising growth factor receptor (HER1t).In some embodiments, the human is diagnosed with at least one of ovarian cancer and breast cancer.In some embodiments, the ovarian cancer or breast cancer is recurrent or refractory ovarian cancer or breast cancer.

[0019] Provided herein is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR (a) at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 15 or 27 to 57; (b) a MUC16 antigen-binding domain with one of the amino acid sequences of SEQ ID NO: 16; (c) a costimulatory signaling domain comprising CD28 with the amino acid sequence of SEQ ID NO: 23; (d) HER1t with the amino acid sequence of SEQ ID NO: 65 and SEQ ID NO: 6 a HER1 tag comprising at least one of HER1t-1 with an amino acid sequence of 6; and and (e) a CD3 zeta signaling domain having the amino acid sequence of SEQ ID NO: 26. , an isolated nucleic acid is presented.

[0020] Provided herein is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR (a) at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 15 or 27 to 57; (b) a MUC16 antigen-binding domain with one of the amino acid sequences of SEQ ID NO: 16; (c) a costimulatory signaling domain comprising 4-1BB with the amino acid sequence of SEQ ID NO: 23; (d) a HER1t having the amino acid sequence of SEQ ID NO: 65 and a nucleic acid sequence of SEQ ID NO: 66; a HER1 tag containing at least one of HER1t-1 with a 66 amino acid sequence; and (e) a CD3 zeta signaling domain having the amino acid sequence of SEQ ID NO: 26. Thus, an isolated nucleic acid is presented.

[0021] As used herein, a nucleic acid sequence comprising any one or more of the polynucleotides described herein is In some embodiments, the vector is a lentiviral vector, In some embodiments, the vector is a retroviral vector or a non-viral vector. The virus vector is the Sleeping Beauty transposon. In the embodiment, the vector is a plurality of vectors.

[0022] Provided herein is a system for expressing a CAR in immune effector cells, the system comprising: Systems are provided that include one or more vectors encoding the isolated nucleic acids provided herein. In some embodiments, the immune effector cells are T cells or NK cells. In embodiments, the systems presented herein include a gene encoding at least one additional gene. In some embodiments, the additional gene comprises a cytokine. In some embodiments, the cytokine is IL-2, IL-15, IL-12, IL-2 1, and at least one of a fusion of IL-15 and IL-15Rα. In some embodiments, the cytokine is in a secreted form. In some embodiments, the system comprises one vector. In embodiments, the one or more vectors are lentiviral vectors, retroviral vectors, or In some embodiments, the non-viral vector is , Sleeping Beauty transposon. The system presented herein further comprises a Sleeping Beauty transposase. In some embodiments, the Sleeping Beauty transposase is SB1 1, SB100X, or SB110. In some embodiments, the immune effector cells The cells are mammalian cells.

[0023] Provided herein are methods for expressing a CAR in immune effector cells, comprising: A method is presented that includes contacting an effector cell with the system described herein. .

[0024] Provided herein is a method for stimulating the proliferation and / or survival of engineered T cells, comprising: (a) Obtaining a cell sample from a subject, the sample comprising T cells or T cell precursors. (b) administering to the cell a gene encoding a gene that is present in any one of claims 1 to 34 and 47 to 52. one or more vectors encoding the isolated nucleic acid to be transduced, and a vector encoding the transposase. The mice were transfected with a vector that encodes the engineered MUC16 CAR, resulting in a population of engineered MUC16 CAR-expressing T cells. (c) and, optionally, allowing the population of MUC16 CAR T cells to incubate for 2 days. or less. .

[0025] In some embodiments, the method of stimulating the proliferation and / or survival of engineered T cells comprises administering to the cells The method may further comprise the step of transfecting a vector encoding a cytokine. In some embodiments, the cytokine is a fusion protein comprising IL-15 and IL-15Rα. In some embodiments, the one or more vectors are lentiviral vectors. In some embodiments, the vector is a viral vector, a retroviral vector, or a non-viral vector. The non-viral vector is the Sleeping Beauty transposon. In this embodiment, the method further comprises a Sleeping Beauty transposase. In some embodiments, the Sleeping Beauty transposase is S B11, SB100X or SB110.

[0026] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering an effective amount of administering one or more doses of engineered T cells to a subject, wherein the engineered T cells In some embodiments, methods are presented that include administering IL-16 CAR and membrane-bound IL-15. A first dose of an effective amount of engineered T cells is administered intraperitoneally. In some embodiments, the cancer is ovarian. In some embodiments, the cancer is breast cancer. In some embodiments, MUC16 CAR is represented by SEQ ID NO: 95-107, 119-149, or 194-195 In some embodiments, the membrane-bound IL-1 is encoded by any one of the sequences. 5 is encoded by SEQ ID NO: 161. In some embodiments, an effective amount of the engineered T cells is at least 10 per kg 2 In some embodiments, the engineered T cells The effective dose is at least 10 per kg. 4 In some embodiments, the engineered T The effective amount of cells is at least 10 per kg. 5 Each cell is an individual cell.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the present invention and its advantages may be obtained by reference to exemplary embodiments in which the principles of the present disclosure are employed. This can be had by reference to the following detailed description and accompanying drawings, which set forth: [Brief explanation of the drawings]

[0028] [Figure 1]1 shows a schematic representation of the Sleeping Beauty transposon system. Sleeping Beauty-derived DNA plasmids encoding cytokines such as MUC16 CAR and membrane-bound IL15 (mbIL15) transposon, and DNA plasmids encoding SB transposase are delivered to immune effector cells, for example, by electroporation. Engineered immune effector cells can be produced under the point-of-care (POC) method described herein. In certain cases, MUC16 CAR and / or cytokines can be co-expressed with cell tags or kill tags for conditional ablation in vivo. [Figure 2] FIG. 1 depicts exemplary gene expression cassettes for MUC16 CAR and cytokines in different configurations with gene switch components. [Figure 3] FIG. 1 depicts examples of MUC16 CARs with different stalk lengths. [Figure 4] Figure 1 shows the expression of CAR, mbIL15, and HER1t1 transgenes. T cells were nucleofected with MUC16-3 CAR-HER1t1 or MUC16-3 CAR-mbIL15-HER1t1 transposon and SB transposase. Transgene expression was measured one day after nucleofection. Cells were gated on live CD3+ cells. Data shown are the mean ± SEM from three healthy donors. [Figure 5] Figure 1 shows the expression of MUC16 CAR with different spacer lengths derived from the CD8 alpha hinge region in CAR-T cells. MUC16 CAR-T cells were generated by electroporation of SB-based plasmids in healthy donor T cells (donor number D133098). CAR-T cells were numerically expanded ex vivo by co-culture with MUC16t-expressing AaPCs with weekly stimulation. CAR expression was measured by multiparameter flow cytometry on days 1 and 8 after nucleofection. Test articles are listed in Table 6. [Figure 6] Figure 7 shows CAR-T cells expressing MUC16 CAR with different spacer lengths derived from the CD8 alpha hinge region. CAR-T cells were generated by electroporation of SB-based plasmids in T cells from a healthy donor (donor number D132552). CAR-T cells were numerically expanded ex vivo by co-culture with MUC16t-expressing AaPCs with weekly stimulation. CAR expression was measured by multiparameter flow cytometry on days 1 and 8 post-nucleofection. Test articles are listed in Table 7. [Figure 7] Figure 1 shows Western blot analysis of MUC16 CAR expression. MUC16 CAR expression was measured by staining with mouse anti-human CD3ζ antibody. Lane 1: protein marker; lane 2: MUC16-3 CAR-HER1t1 T cells; and lane 3: MUC16-3 CAR-mbIL15-HER1t1 T cells. [Figure 8] Figure 1 shows Western blot analysis of mbIL15 expression. mbIL15 expression was measured by staining with anti-IL-15 antibody. Lane 1: protein marker; Lane 2: MUC16-3 CAR-HER1t1 T cells and Lane 3: MUC16-3 CAR-mbIL15-HER1t1 T cells. [Figure 9] Figure 1 shows Western blot analysis of HER1t1 expression. HER1t1 expression was measured by Western blot after immunoprecipitation. Lane 1: MUC16-3 CAR-HER1t1 T cells; Lane 2: MUC16-3 CAR-mbIL15-HER1t1 T cells; Lane 3: immunoprecipitation control without cell lysate; Lane 4: A431 cell line lysate; and Lane 5: protein marker. [Figure 10A] FIG. 1 shows flow cytometry analysis of MUC16 expression in various tumor cell lines tested. [Figure 10B]Figure 1 shows the specific cytotoxicity of MUC16 CAR-T cells. Cytotoxicity of MUC16-3 CAR-HER1t1 T cells and MUC16-3 CAR-mbIL15-HER1t1 T cells against various tumor cell lines at varying E:T ratios. Control CAR-mbIL15-HER1t1 T cells served as a negative control. Mean ± SEM of triplicate wells from three donors is shown. [Figure 11] Figure 1 shows cytokine production by MUC16-CAR T cells upon co-culture with various tumor cell lines. CAR-T cells generated using T cells from three healthy donors were co-cultured with the indicated tumor cell lines at an E:T ratio of 1:1 for 7 days. Shown is the mean ± SEM of triplicate wells from three donors. [Figure 12] Figure 8 shows the cytotoxicity of MUC16-CAR-T cells in a luciferase assay. CAR-T cells generated from T cells of two healthy donors were cultured with tumor cell lines. Test articles are listed in Table 8. [Figure 13A] Figure 1 shows the biological function of mbIL15 in MUC16 CAR-T cells. CAR-T cells were generated from T cells from three healthy donors. Persistence of MUC16-3 CAR-HER1t1 and MUC16-3 CAR-mbIL15-HER1t1 T cells in in vitro culture in the absence of antigen and exogenous cytokines. Each symbol represents CAR-T cells from an individual donor. Mean ± SEM from three donors is shown. [Figure 13B]

[0023] Figure 1 shows the biological function of mbIL15 in MUC16 CAR-T cells. CAR-T cells were generated from T cells of three healthy donors. CAR-T cell proliferation is shown. Each symbol represents an individual donor CAR-T cell. Average ± SEM from three donors is shown. [Figure 13C]

[0023] Figure 1 shows the biological function of mbIL15 in MUC16 CAR-T cells. CAR-T cells were generated from T cells from three healthy donors. Memory marker analysis of MUC16-3 CAR-mbIL15-HER1t1 T cells is shown. Each symbol represents an individual donor CAR-T cell. Mean ± SEM from three donors is shown. [Figure 14]

[0023] Figure 1 shows MUC16-CAR-T cell survival in vitro in the absence of extracellular cytokines. MUC16-2 CAR-HER1t1 and MUC16-2 CAR-mbIL15-HER1t1 T cells derived from T cells of three healthy donors were cultured in the presence or absence of extracellular cytokines for two weeks. Surviving T cells cultured without cytokines were graphed as a fraction of T cells cultured with cytokines. Data shown are the mean ± SEM from three donors. [Figure 15] Figure 1 shows cetuximab-mediated ADCC activity (expressed as % cytotoxicity; y-axis) of MUC16 CAR-T cells co-expressing HER1t1. Allogeneic NK cells were used as effector cells at an E:T ratio of 10:1. Cetuximab demonstrated specific cytotoxicity against HER1t1-expressing MUC16 CAR-T cells. Data shown are mean ± SEM. [Figure 16] Figure 1 shows quantitative analysis of SKOV3-fLUC-MUC16 tumor burden measured by in vivo bioluminescence (IVIS) imaging. NSG mice (N = 5-7 mice per group) were administered the SKOV3-fLUC-MUC16 tumor cell line by i.p. injection on day 0. Tumor-bearing mice were treated with different MUC16 CAR-T treatments by single i.p. or i.v. injection, and tumor burden was quantified by IVIS throughout the treatment series. Data shown are mean ± SEM. Arrows indicate the day of CAR-T administration. [Figure 17]Figure 1 shows quantitative analysis of SKOV3-fLUC-MUC16 tumor burden measured by in vivo bioluminescence (IVIS) imaging. NSG mice (N = 5-7 mice per group) were administered the SKOV3-fLUC-MUC16 tumor cell line by i.p. injection on day 0. Tumor-bearing mice were treated with different MUC16 CAR-T treatments by single i.p. injection on day 5 after tumor cell administration, and tumor burden was quantified by IVIS throughout the treatment series. Data shown are mean ± SEM. [Figure 18A] Figure 18 shows quantification of MUC16 CAR-T cells in the blood of treated tumor-bearing NSG mice. NSG mice (N = 5-7 mice per group) were administered the SKOV3-fLUC-MUC16 tumor cell line by i.p. injection. Tumor-bearing mice were treated with different MUC16 CAR-T treatments by single i.p. injection 5 days after tumor cell administration, and CAR-T cells were quantified via measurement of CD8+ HER1t1+ (Figure 18A) and CD4+ HER1t1+ (Figure 18B) T cells in mouse blood samples (number of CAR-T cells per mL of blood; y-axis). Data shown are mean ± SEM; n = 5-7 mice per time point. [Figure 18B] Same as above. [Figure 19] Figure 9 shows quantitative analysis of SKOV3-fLUC-MUC16 tumor burden measured by in vivo bioluminescence (IVIS) imaging. NSG mice (N=5 mice per group) were administered the SKOV3-fLUC-MUC16 tumor cell line by ip injection on day 0. Tumor-bearing mice were treated with one of three different doses of MUC16-2 CAR-T cells by single ip injection on day 5 after tumor cell administration, and tumor burden was quantified by IVIS throughout the treatment course. Test articles are listed in Table 9. Data shown are mean ± SEM. [Figure 20]Figure 9 shows quantitative analysis of SKOV3-fLUC-MUC16 tumor burden measured by in vivo bioluminescence (IVIS) imaging. NSG mice (N=5 mice per group) were administered the SKOV3-fLUC-MUC16 tumor cell line by ip injection on day 0. Tumor-bearing mice were treated with one of three different doses of MUC16-3 CAR-T cells by single ip injection on day 5 after tumor cell administration, and tumor burden was quantified by IVIS throughout the treatment course. Test articles are listed in Table 9. Data shown are mean ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of the Invention The following description and examples further illustrate embodiments of the present invention. It is not limited to the specific embodiments described herein and, as such, may vary. It will be understood that numerous variations and modifications to the present invention will occur to those skilled in the art. will recognize that the scope of

[0030] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise specified, the technical and scientific terms used herein Terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. do.

[0031] The section headings used herein are for organizational purposes only and do not limit the scope of the subject matter described. It should not be considered as limiting the subject matter.

[0032] While various features of the invention may be described in the context of a single embodiment, the features may also be provided separately. Conversely, the terms "common" and "commonly used" may be used interchangeably herein, and may be presented in any suitable combination. Although, for clarity, the present invention may be described in the context of individual embodiments, the present invention also encompasses a single It can be implemented in one embodiment.

[0033] The following definitions supplement those in the art and are intended to orient the present application and any Related or unrelated matters, e.g., commonly owned, belonging to any patent or application Any methods and materials similar or equivalent to those described herein are not intended to be limiting. Although various materials and methods may be used in carrying out the tests of this disclosure, preferred materials and methods are described herein. Accordingly, the terminology used herein refers to specific embodiments and methods. are for descriptive purposes only and are not intended to be limiting. There is no.

[0034] In this application, unless otherwise stated, the use of the singular refers to the plural. As used herein, unless the context clearly dictates otherwise, Unless otherwise specified, the singular forms "a", "an" and "the" are used in conjunction with the plural It should be noted that the referent includes the Unless otherwise stated, the use of "or" means "and / or." In addition to the term "including," "include," "includes" The use of other forms, such as "included" and "included" is not limiting.

[0035] As used herein, "some embodiments," "an embodiment," "one embodiment," or References to "other embodiments" may include modifications to the particular features, structures, or aspects described in connection with the embodiments. The features are included in at least some embodiments of the present invention, but in all embodiments, It means that it is not necessarily included.

[0036] As used in this specification and claims, "comprising" means "comprise" and "comprises" are examples of "comprising" g)), "having" ("have" and "having any form of "has," "including," ("include" and "includes" are examples of "including" any form of "containing") or "containing" ("contain " and "contains," or any form of "containing" The term "list" is inclusive or open-ended and may include additional, unlisted elements or methods. Any embodiment discussed herein does not exclude any method steps of the present invention. Alternatively, it is contemplated that the present invention may be practiced with respect to compositions, and vice versa. Additionally, the compositions of the present invention can be used to achieve the methods of the present invention.

[0037] As used herein, the term "about" in relation to reference numerical values ​​and its grammatical equivalents Values ​​can include the number itself and values ​​within a range of ±10% from the number. For example, "about 10 The quantity " includes 10 and any quantity between 9 and 11. For example, " in relation to a base number The term "about" also means a value plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 6% of this value. This includes ranges of 3%, 2%, or 1%.

[0038] By "isolated" is meant the removal of a nucleic acid from its natural environment. Nucleic acids may be derived from nature (including genomic DNA and mRNA) or may be synthesized. whether derived (including cDNA) and / or amplified under laboratory conditions. The purity is increased, and in this case "purity" is a relative term, not "absolute purity." On the other hand, nucleic acids and proteins are prepared with a diluent or adjuvant. It is understood that although the compounds may be formulated, for practical purposes they may be isolated. For example, nucleic acids, when used for introduction into cells, may be used in an acceptable carrier or diluent. Typically, it is mixed with a solvent.

[0039] As used herein, a "polynucleotide" or "oligonucleotide" refers to a ribonucleotide. A polynucleotide of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Double-stranded and single-stranded DNA, triplex DNA, as well as double-stranded and single-stranded RNA The term also includes, for example, methylation and / or capping of polynucleotides. The term may also include modified forms of polynucleotides, such as those modified by cleavage, as well as unmodified forms of polynucleotides. Unnatural or synthetic nucleotides, as well as nucleotide analogs It is also intended to include molecules comprising

[0040] "Polypeptide" refers to "polypeptides" and " Used interchangeably with the term "protein(s)" and refers to polymers of amino acid residues "Mature protein" refers to a full-length protein, optionally glycosylated or unglycosylated. or proteins containing other modifications typical of proteins in a given intracellular environment.

[0041] Nucleic acids and / or nucleic acid sequences may be derived from natural or artificial common ancestral nucleic acids or ancestral nucleic acid sequences. A protein and / or protein sequence is "homologous" if it is derived from that sequence. their coding DNA is derived, natural or artificial, from a common ancestral nucleic acid or ancestral nucleic acid sequence Homologous molecules are sometimes referred to as homologs. For example, Any naturally occurring protein may be modified by any available mutagenesis method. When expressed, this mutant nucleic acid produces the protein encoded by the original nucleic acid. Homology generally refers to the sequence of two or more nucleic acids or is deduced from the sequence identity between proteins (or their sequences). The exact percentage of identity between sequences useful for identifying nucleic acids and proteins of interest is Although varying, at least 25% sequence identity is used to establish homology. Sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% %, or 99% or greater sequence identity may also be used to establish homology. It is possible.

[0042] In the context of two nucleic acid or amino acid sequences of a polypeptide, "identical" or The term "sequence identity" refers to the alignment of sequences over a specified comparison window for maximum similarity. refers to residues in two sequences that are the same when aligned. In the present specification, the polypeptides are implemented using, for example, BLAS TP (or CLUSTAL, or any other available alignment software) ) at least 80%, 85% identical to the reference polypeptide or fragment thereof, as measured by , 90%, 98%, 99%, or 100% identical to the starting nucleic acid. They can also be described in terms of, for example, Use BLASTN (or CLUSTAL, or any other available alignment) The reference nucleic acid or this fragment is 50%, 60%, or 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical A molecule may have a certain percentage of sequence identity to a larger molecule. If the two molecules are optimally aligned, this means that the percentage of Residues are ordered by the optimal alignment of the two molecules, with matching residues in the larger molecule. This means finding out.

[0043] A "transposon" or "transposable element" (TE) is a gene that determines its location in the genome. altering, possibly creating or storing mutations and altering the genome size of the cell , the vector DNA sequence. Transposition often results in duplication of the TE. Class I TEs are copied in two steps: first, class I TEs are copied from DNA to RNA; The resulting RNA is then reverse transcribed into DNA. The reverse transcription step is carried out by the TE itself. It is catalyzed by reverse transcriptase, which can be encoded by the host. , similar to retroviruses such as HIV. Cut-and-paste by class II TEs The transposition mechanism does not involve an RNA intermediate. Transposition is mediated by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA. whereas other transposases bind to specific DNA sequence targets. The enzymes produce staggered cleavages at the target site, resulting in 5 This results in single-stranded DNA overhangs (sticky ends) at the 1' or 3' ends. The first step is to excise the DNA transposon, and then the DNA transposon is inserted into the new The new target site is ligated to the target site, a process that involves DNA polymerase It involves the enzyme activity and the DNA ligase activity that joins the sugar-phosphate backbone. The insertion site of a DNA transposon is located at the target DNA site. A can be created by staggered cuts in A and filling in the gaps with DNA polymerase. short direct repeats, followed by excision of the TE by transposase. During the S phase of the cell cycle, the donor site Transposition occurs when the target site has not yet replicated, but the target site has already replicated. The metastasis is classified into two types: Class I TE and Class II TE. Both can be classified as "autonomous" or "non-autonomous." While E can move on its own, non-autonomous TEs require the presence of another TE to move. This means that non-autonomous TEs require a transposase (in the case of class II) or This is often due to a lack of reverse transcriptase (in the case of class I).

[0044] "Transposase" refers to a gene that binds to the ends of transposons and functions as a cut-and-paste enzyme. It catalyzes the movement of transposons to other parts of the genome by either the mitotic or replicative transfer machinery. In some embodiments, the catalytic activity of a transposase is used to transduce genes It can be transferred from a vector to the genome.

[0045] The nucleic acid sequences and vectors disclosed or contemplated herein can be "transfected" into cells. introduced by "transfection," "transformation," "nucleofection," or "transduction" As used herein, "transfection," "transformation," or or "transduction" means the transfer of one or more genes to a cell by using physical or chemical methods. The term "transfection" refers to the introduction of multiple exogenous polynucleotides into a host cell. Infection methods are known, for example, calcium phosphate DNA co-precipitation (e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expr. See Session Protocols, Humana Press (1991); DEAE-dextran method ;Electroporation;Cationic liposome-mediated transfection;Tungsten particles Enhanced gene gun method (Johnston, Nature, 346: 776-777 (1990)); strontium phosphate co-transfection precipitation method (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)); and nucleofection method (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). (Trompeter et al., J. Immunol. Methods 274:245-256 (2003)). The vectors or viral vectors are available in suitable packaging, many of which are commercially available. The infectious particles can be propagated in the growing cells and then introduced into host cells.

[0046] "Promoter" refers to a region of a polynucleotide that induces transcription of a coding sequence A promoter is a gene that is located near the transcription start site of a gene, on the same strand of DNA, and A promoter is located upstream (towards the 5' region of the sense strand). Constitutive promoters are active under all conditions, whereas other promoters, e.g. Inducible promoters are regulated and active in response to a specific stimulus.

[0047] The term "promoter activity" refers to the operably linked promoter whose activity is being measured. It refers to the degree of expression of the linked nucleotide sequence. It can be measured directly by determining the amount of RNA transcript produced by immunoblot analysis. The promoter may be linked to a reporter nucleic acid sequence, for example. It can also be measured indirectly by determining the amount of product encoded by the sequence. .

[0048] As used herein, an "inducible promoter" refers to a promoter that is regulated by a transcriptional regulator, e.g., a biological factor. Or refers to a promoter that is induced to activity by the presence or absence of an abiotic factor. Inducible promoters are promoters that allow expression of genes operably linked to them to be expressed in an organism or in a particular They are useful because they can be turned on or off at certain stages of tissue development. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, and promoter, steroid-regulated promoter, metal-regulated promoter, pathogenesis-regulated promoter promoters, temperature-regulated promoters, and light-regulated promoters. In some embodiments, the inducible promoter is part of a gene switch.

[0049] As used herein, the term "enhancer" refers to, for example, a gene that is operably linked to An enhancer refers to a DNA sequence that increases the transcription of a nucleic acid sequence. They can be located many kilobases away from the target region, allowing for regulatory factor binding, DNA These may mediate changes in the methylation patterns of the target gene or in the DNA structure. Numerous enhancers are known and have been cloned from various sources. available as a peptide or within a cloned polynucleotide (e.g., (e.g., from depositories such as ATCC, as well as other commercial or private sources). Many polynucleotides, including promoters (such as the commonly used CMV promoter), It also includes enhancer sequences. Enhancers may be located upstream of a coding sequence. It may be located within this or downstream of it. The term "enhancer" refers to an enhancer region that maps within the immunoglobulin (Ig) locus. Enhancer elements derived from (such enhancers include, for example, heavy chain (mu) 5' enhancer, light chain (kappa) 5' enhancer, kappa intron enhancer This refers to enhancers that are intronic and mu intronic enhancers, as well as 3' enhancers. In general, see Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New Y See Work (1993), pages 353-363, and U.S. Pat. No. 5,885,827. sea ​​bream).

[0050] As used herein, a "coding sequence" refers to a polynucleotide that encodes a polypeptide. A region or sequence is a sequence that begins near the 5' end and ends with an initiation codon. Near the 3' end, a stop codon is bounded. It can also be called a gframe.

[0051] As used herein, "operably linked" refers to the ability of a DNA segment to interact with another DNA segment. a physical and / or functional linkage to the A segment, wherein the segment is It refers to a linkage that allows the gene products to function in their intended manner. The DNA sequence to be loaded may be, for example, a promoter, enhancer, and / or Regulatory sequences, such as silencers, can modulate the transcription of DNA sequences either directly or operably linked to a regulatory sequence when linked in an indirectly enabling manner For example, a DNA sequence may be selected if it is located downstream of the transcription start site of a promoter. ligated to the promoter in the correct reading frame relative to the transcription start site. When a DNA sequence is inserted into a target region, allowing transcription elongation to proceed through the DNA sequence, An enhancer or silencer is operably linked to a promoter. are ligated to a DNA sequence to increase or decrease transcription of the DNA sequence, respectively. When inserted, it is operably linked to a DNA sequence encoding a gene product. Enhancers and silencers are often located upstream of the coding region of a DNA sequence. It may be located downstream of this or embedded within it. Null sequences are signal transduction pathways when expressed as a preprotein that participates in the secretion of the polypeptide. The DNA for the polypeptide sequence is operably linked to the DNA encoding the polypeptide. Linkage of the A sequence to the regulatory sequence is typically by ligation at appropriate restriction sites. or by using restriction endonucleases known to those skilled in the art to insert This is achieved via an adapter or linker.

[0052] The term "transcriptional regulator" refers to a promoter-driven DNA Biochemical elements that act to prevent or inhibit transcription of a sequence (e.g., repressors) promoter proteins or nuclear inhibitory proteins), or under certain environmental conditions, A biosynthetic molecule that acts to enable or stimulate the transcription of a motor-driven DNA sequence. "Enhanced expression" refers to a genetic element (e.g., inducer or enhancer) that mediates a specific target gene expression.

[0053] The term "induction" refers to the transcription, promotion, or transcription of a nucleic acid sequence brought about by a transcriptional regulatory factor. activity of the target gene and / or promoter expression relative to some basal level of transcription. Refers to rising.

[0054] A "target" gene or "heterologous" gene or "gene of interest (GOI)" is a gene Refers to a gene that is introduced into a host cell by transduction.

[0055] As used herein, a "recombinase" refers to a gene that induces site-specific recombination between defined sites. A group of enzymes that can facilitate the synthesis of DNA fragments, where the sites are physically separated on a single DNA molecule. Defined recombination refers to a group of enzymes in which the target gene is a nucleotide or site on a separate DNA molecule. The DNA sequences at the sites are not necessarily identical. Recombination is initiated by protein-DNA interaction. Within the group of enzymes, there are enzymes involved in phage integration and excision (e.g., λ Integra). enzyme, ΦC31), resolution of circular plasmids (e.g., Tn3, gamma delta, Cre, Flp), DNA inversion for expression of alternative genes (e.g. Hin, Gin, Pin), genes during development (e.g., in the Anabaena genus) assembly of nitrogen fixation genes) and transposition (e.g., IS607 transposon) There are many proteins that catalyze the recombination of DNA. Most site-specific recombinases have evolved over time. Based on affinity and mechanistic similarity, they fall into one of two families: , λ integrase family or tyrosine recombinases (e.g., Cre, Flp , XerD), and the resolvase / integrase family, or serine recombinase enzyme family (e.g., ΦC31, TP901-1, Tn3, gamma delta) .

[0056] "Recombination junction sites" refer to sites recognized by the recombinase enzymes described herein. Typically, one site is a specific polynucleotide sequence. a site within the target region (e.g., a chromosome or episome in a prokaryotic or nucleotidic organism) that allows targeted recombination Two different sites (called "complementary sites") present on the nucleic acid to be integrated at the site ) are involved. Herein, the terms "conjugation" and "conjugation" refer to the bacterial target and phage donor, respectively. The terms "attB" and "attP" are used to refer to the fusion (or recombination) sites. However, recombination sites for specific enzymes may have different names. The core region includes a left arm and a right arm separated by a core region or spacer region. Therefore, the attB recombination site is BOB' [where B and B' are , the left arm and the right arm, and O is the core region. tP is POP' [in the sequence, P and P' are arms, and O is again a codon] The attB site is the target region. The recombination sites flanking the DNA to be integrated during simultaneous integration of nucleic acids are designated as "attL" and and "attR." Thus, using the terminology above, the attL site and The attR site consists of BOP' and POB', respectively. In the designation, the "O" is omitted and attB and attP are designated, for example, as BB' and BB', respectively. It's called PP'.

[0057] An "expression vector" or "vector" is an autonomous vector that mediates replication of a polynucleotide within a cell. behaves as a unit (i.e., is capable of replicating under its own control), or is any genetic element that can replicate upon insertion into a host cell chromosome, e.g. , plasmids, chromosomes, viruses, transposons, and the like, which are complexes of joined segments. and / or by incorporating another polynucleotide segment into it to effect its production and / or expression. Suitable vectors include plasmids, transposons, Vectors include, but are not limited to, bacteriophages and cosmids. Ligation or insertion of the vector into a host cell of the joined segments is carried out. The host may contain polynucleotide sequences necessary to effect expression of the host. Varies depending on the host organism: promoter sequences that effect transcription, enhancers that increase transcription nucleotide sequence, a ribosome binding site sequence, and a transcription / translation termination sequence. The vector involves ligation or integration of the vector into the DNA sequence of the host cell. It may be possible to directly express the product of a nucleic acid sequence encoded therein without the need for transfection.

[0058] A vector may also contain a "selectable marker gene." The term "selectable marker gene" refers to a gene that identifies cells expressing a nucleic acid sequence in the presence of a corresponding selectable agent. It refers to a nucleic acid sequence that allows specific selection in the forward or reverse direction of the nucleic acid sequence in the presence of the nucleic acid sequence. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publication No. Publication No. 1992 / 08796 and No. 1994 / 28143 Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980), O'Hare et al. , Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981), Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981 ), Santerre et al., Gene, 30: 147 (1984), Kent et al., Science, 237: 901-903 (19 87), Wigler et al., Cell, 11: 223 (1977), Szybalska & Szybalski, Proc. Natl. Aca d. Sci. USA, 48: 2026 (1962), Lowy et al., Cell, 22: 817 (1980), and U.S. Pat. The invention described in U.S. Pat. Nos. 5,122,464 and 5,770,359 It is being done.

[0059] In some embodiments, the vector is capable of replicating in a host cell and can be selected appropriately. In the presence of selective pressure, "episodes" persist as extrachromosomal segments of DNA within the host cell. These vectors are "episomes" or "episomes" (see, e.g., Conese et al., Gene Therapy (See, for example, J. Immunol., 11:1735-1742 (2004)). Representative, commercially available episomal expression vectors include: , Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EB V) Episomal plasmids, including but not limited to, those that use an origin of replication (oriP) The vectors pREP4, pCEP4, and pREP7, as well as the Invitrogen pcDNA3.1 from Stratagen (Carlsbad, Calif.) pBK-CMV from e (LaJolla, Calif.) contains EBNA1 and oriP Non-limiting examples of episomal vectors include those that use the T antigen and SV40 origin of replication instead of the This shows a typical example.

[0060] "Cancer cells" refer to cells in the early, middle, or late stages of multistage tumor progression, as previously described. This refers to cells that undergo the process (Pitot et al., Fundamentals of Oncology, 15-28 (1978)). cells in the early, middle, and late stages of tumor progression, including "pre-cancerous" cells (i.e., "hyper- These include tumor cells at the end of tumor progression (e.g., dysplastic and metaplastic cells), and tumor cells at the end of tumor progression (e.g., dysplastic cells). nothing.

[0061] "Metastatic" cancer cells are cells that have spread from the primary site of the cancer (i.e., normal, hyperplastic, or dysplastic cells). Cancer cells that have migrated from the area where they first formed to a site other than the primary site This refers to the area where the migrated cancer cells remain and grow.

[0062] "Cancer" refers to ovarian cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, and colon cancer. Cancer, stomach cancer, esophageal cancer, oral cancer, tongue cancer, gum cancer, skin cancer (e.g., melanoma) , basal cell carcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer , bone cancer, testicular cancer, kidney cancer, endometrial cancer, uterine cancer, bladder cancer, bone marrow cancer, lymphatic cancer tumors, spleen cancer, thymus cancer, thyroid cancer, brain cancer, nerve cancer, mesothelioma, gallbladder cancer, eye cancer (e.g., corneal cancer, uveal cancer, choroidal cancer, macula cancer, vitreous humor cancer, etc.) metastatic cancers such as joint cancer (e.g., synovial cancer), glioblastoma, lymphoma, and leukemia It refers to multiple cancer cells that may or may not exist.

[0063] MUC16 CA-125 (cancer antigen 125, tumor antigen 125, or carbohydrate antigen 125) or MUC16, also known as mucin 16, is a member of the mucin family of glycoproteins. MUC16 mediates tumorigenesis and tumorigenesis by several different mechanisms. MUC16 has been shown to play a role in the progression of proliferation. However, this has had very little success. Therefore, other treatment strategies are needed.

[0064] MUC16 is a large carbohydrate antigen, also known as CA125. It is encoded by the MUC16 gene located on chromosome 19. MUC16 is a It is a highly glycosylated multidomain type I transmembrane protein containing the C-terminal domain. Insulin is a multi-protein complex consisting of multiple extracellular SEA (sea urchin testis protein, E. coli) proteins with autoproteolytic activity. SEA contains a transmembrane (TM) domain. It has two proteolytic sites near the main one, the larger one called CA-125. The oxidized domain is released into the circulation at acidic pH. CA-125 is a disease-causing agent of ovarian cancer. The highly conserved truncated outer membrane MUC1 is commonly used as a marker. MUC16 antibodies target a protein domain called the ectodomain. It was identified as specifically binding to the ectodomain of MUC16 that is retained on the cell surface. "MUC16 overexpression" by target cells (e.g., cancer cells) is observed in control cells (e.g., non- MUC16 expressed by cells of interest at high levels compared to cancer cells, normal cells, etc. Refers to protein and / or mRNA.

[0065] Chimeric Antigen Receptor In the embodiments described herein, the CAR comprises a single extracellular antibody-derived target recognition molecule. The scFv may comprise a transmembrane domain and / or a single chain variable domain (scFv). For example, the intracellular signaling domains containing CD3ζ for T cell activation are flexible. Typically, when T cells are activated in vivo, These studies have demonstrated that primary antigen-induced TCR signaling is mediated by cytokines (i.e., IL-2 and I L-21), which then activates the signal transduction pathway in an autocrine / paracrine manner. This is accompanied by a secondary costimulatory signal from CD28, which feeds back to the target group. Thus, CARs can be engineered to contain signaling domains, such as CD28 cytoplasmic signaling domain, or other costimulatory molecule signaling domains such as the 4-1BB signaling domain Chimeric CD28 costimulation may induce upregulation of anti-apoptotic molecules and IL-1. T-2 production and expansion of peripheral blood mononuclear cell (PBMC)-derived T cells Improves cell survival.

[0066] In one embodiment, monoclonal antibodies specific for various epitopes of MUC16 are A fusion of a single chain variable fragment (scFv), e.g., a transmembrane domain and a CD Zeta Such molecules respond to recognition by the scFv of their target. This results in the transmission of the Zeta signal.

[0067] In one embodiment, a CAR comprises an ectodomain (extracellular), a transmembrane domain, and an endodomain. In one embodiment of the CAR ectodomain, the signal peptide The tides direct the nascent protein to the endoplasmic reticulum, where receptors, e.g., glycosylate The signal peptide sequence of any eukaryotic organism is the signal peptide that initiates the functioning of the protein. Generally, the amino acid sequence that is originally attached to the amino terminus of most components is Signal peptides are used (e.g., scFv with light chain-linker-heavy chain orientation). In some embodiments, the signal peptide is , GM-CSFRa (SEQ ID NO: 58) or IgK (SEQ ID NO: 59). Other signal peptides that may be involved include those derived from CD8 alpha and CD28. nothing.

[0068] The antigen recognition domain may be an scFv, but may also be an alternative. The antigen recognition domains derived from the alpha and beta chains of the TCR are a single ectodomain domain. domains (e.g., CD4 ectodomains) as well as linked, e.g., cytokines (e.g., It is thought that the ATPase inhibitors possess other recognition components, such as ATP, which result in the recognition of cells bearing cytokine receptors. For example, most antibodies that bind a given target, such as a virus-associated antigen, with high affinity are expected to bind the target. Any of these can be used as the antigen recognition region.

[0069] Generally, CARs exist in a dimeric form, consisting of an extracellular scFv (VH linked to VL) region. , a stalk domain, a transmembrane domain, and a fusion domain linking to intracellular signaling motifs. The endodomain of the first generation CAR is expressed as a CD3-ζ signaling protein. Second-generation CARs induce T cell activation through CD3-ζ and CD3-ζ receptors alone. Signaling via CD28 or other endodomains such as 4-1BB or OX40 Third-generation CARs target CD28, 4-1BB, or OX4 Activating T cells through a CD3ζ-containing combination of three signaling motifs, such as 0 Sexualize.

[0070] In some embodiments, the present invention provides a method for detecting a dendritic cell comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a chimeric antigen receptor (CAR) comprising a null transduction domain is provided. The extracellular domain is otherwise the antigen-binding portion or scFv and stalk domain. In some embodiments, the target-specific binding element is a target-specific binding element that binds to an intracellular signaling pathway. The reaching domain or otherwise cytoplasmic signaling domain is a co-stimulatory signaling domain. It contains the zeta chain and zeta chain segments.

[0071] The costimulatory signaling region of the CAR contains the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are molecules other than antigen receptors that are required for the effective response of lymphocytes to antigens. extracellular cell surface molecules or their ligands.

[0072] In some embodiments, a stalk is located between the extracellular domain and the transmembrane domain of the CAR. The main or stalk region is incorporated. As used herein, "stalk domain" Alternatively, the term "stalk region" generally refers to the transmembrane domain of an scFv or polypeptide. Any oligonucleotide that functions to link the peptide chain to either of the cytoplasmic domains of the peptide chain. The stalk domain refers to a peptide or polypeptide. In some cases, the antibody may comprise a range of Fc and optionally one or two constant domains of Fc. In the alternative, the stalk region comprises a hinge region derived from IgG1. , comprising the CH2CH3 region of an immunoglobulin and optionally a portion of CD3. The stalk region is described in WO 2016 / 073755. The CD8α hinge region, IgG4-Fc12 amino acid hinge region (ESKYG PPCPPCP) (SEQ ID NO: 196), or an IgG4 hinge region.

[0073] In other embodiments, a spacer is present between the extracellular domain of the CAR and the transmembrane domain. The spacer is incorporated into the stalk region and stalk extension region depicted in FIG. In one embodiment, the spacer may comprise a single stalk region. In this embodiment, the spacer comprises a stalk region (denoted as "s") and a spacer region (denoted as "s" herein). For example, the spacer may include a stalk extension region designated as "-n". The area of ​​the network, and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, including s'-n In a further embodiment, the stalk region may be linked to the stalk extension region s via a linker. Can be concatenated to '-n'.

[0074] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be any membrane-bound or transmembrane protein. Suitable transmembrane domains include the alpha and beta chains of the T cell receptor. , or may contain the transmembrane domain of the zeta chain, and may be involved in CD28, CD3 epsilon, CD3 Zeta, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD3 3, CD37, CD64, CD80, CD86, CD134, CD137, or CD1 Alternatively, the transmembrane domain may be synthetic. and may contain hydrophobic residues such as leucine and valine. At one or both ends of the transmembrane domain, phenylalanine, tryptophan, and and valine triplets are found. Optionally, in some embodiments, 2 to 10 amino acids Short oligonucleotide or polypeptide linkers, the length of which is between amino acids may form a link between the transmembrane domain and the cytoplasmic signaling domain of CAR. In some embodiments, the linker is a glycine-serine linker. In In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In this embodiment, the transmembrane domain comprises a CD3ζ transmembrane domain.

[0075] The intracellular domain may include one or more costimulatory domains. Exemplary costimulatory domains include: The markers are CD8, CD27, CD28, 4-1BB (CD137), ICOS, and DAP10. , DAP12, OX40 (CD134), CD3-zeta, or fragments or combinations thereof In some cases, the CARs described herein may be a combination of C D8, CD27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP 12, OX40 (CD134), or a fragment or combination thereof. It may contain one or more, or two or more, of the following domains: Therefore, the CARs described herein bind to CD27, CD28, 4-1BB (CD13 7), ICOS, OX40 (CD134), or a fragment or combination thereof one or more, or two or more, of the co-stimulatory domains Optionally, the CARs described herein include CD8, CD28, 4-1BB ( CD137), DAP10, DAP12, or a fragment or combination thereof. and one or more, or two or more, of the costimulatory domains Optionally, the CARs described herein are capable of targeting CD28, 4-1BB (CD137), or a fragment or combination thereof. Plural, including two or more. The CARs used in this study contain the costimulatory domains CD28 and 4-1BB (CD137), or Optionally, the CARs described herein comprise fragments of each of these. The main CD28 and OX40 (CD134) or their respective fragments Optionally, the CARs described herein comprise a co-stimulatory domain, CD8 and and CD28, or fragments of each of these. The CAR comprises the costimulatory domain of CD28, or a fragment thereof. The CARs described herein may contain a co-stimulatory domain, 4-1BB (CD137), or In some cases, the CARs described herein comprise a co-stimulatory domain. OX40 (CD134), or a fragment thereof. The CAR comprises the costimulatory domain CD8, or a fragment thereof. The CARs described herein contain at least one co-stimulatory domain, DAP10, and In some cases, the CARs described herein comprise at least one , the costimulatory domain of DAP12, or a fragment thereof.

[0076] The intracellular signaling domain, also known as the cytoplasmic domain, of the CAR of the present disclosure comprises: Contributes to the activation of at least one of the normal effector functions of CAR-loaded immune cells The term "effector function" refers to the specialized function of a cell. The target function may be, for example, cytolytic activity, or helper activity including secretion of cytokines. Therefore, the term "intracellular signaling domain" is used to describe Proteins that transmit target functional signals and direct cells to carry out specialized functions Usually, the entire intracellular signaling domain can be used, but in many cases It is not necessary to use the entire chain. To the extent that the modified portion is used to transmit the effector function signal, such a The linked portion can be used in place of the intact strand. The term signal transduction domain refers to a domain that is sufficient to transmit an effector function signal. It is intended to include any truncated portion of the intracellular signaling domain. In some embodiments, the intracellular domain is a signaling domain for T cell activation. Optionally, the signaling domain for T cell activation further comprises a TCR enzyme. FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, Contains domains derived from CD5, CD22, CD79a, CD79b, or CD66d In some cases, the signaling domain for T cell activation is derived from CD3ζ. Includes the domain.

[0077]

[0013] In embodiments, provided herein is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; (e) a CD3 zeta signaling domain; and, optionally, (f) a truncated human epithelium An isolated nucleic acid is presented that includes a growth factor receptor (HER1t or HER1t1).

[0078] Nucleic acid sequences encoding functional portions of the CARs described herein are within the scope of the present invention. A functional portion includes, for example, a portion that is functional to the same extent as, comparable to, or better than, the parent CAR. To a greater extent, they recognize target cells or detect, treat, or prevent disease. Regarding the nucleic acid sequence encoding the parent CAR, the portion of the CAR that retains the ability to bind to the parent CAR is also included. The nucleic acid sequence encoding the functional portion of the CAR may be, for example, about 10% of the parent CAR. 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CA It may encode a protein containing R.

[0079] In embodiments, the CAR is located at the amino or carboxyl terminus of the moiety, or It contains additional amino acids at both ends, which are identical to the amino acid sequence of the parent CAR. Desirably, the additional amino acids enhance the biological activity of the functional moiety, e.g., It does not interfere with target cell recognition, cancer detection, cancer treatment or prevention, etc. More preferably, Compared to the biological activity of parent CAR, the additional amino acids enhance the biological activity of CAR .

[0080] As used herein, the term "functional variant" refers to a functional variant of a reference polypeptide. Polypeptides or proteins with qualitative or significant sequence identity or sequence similarity A functional variant refers to a polypeptide that retains the biological activity of the reference polypeptide of which it is a variant. A variant is, for example, a mutant of a CAR described herein (parent CAR), which is capable of inducing target cell retain the ability to recognize the parent CAR to a similar, comparable, or higher degree The functional variants of the CAR, relative to the nucleic acid sequence encoding the parent CAR, are included. The nucleic acid sequence encoding the variant is, for example, about 10% identical to the nucleic acid sequence encoding the parent CAR. , about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% They may be identical, about 95% identical, or about 99% identical.

[0081] The CARs described herein (including functional portions and functional variants thereof) are glycosylation, amidation, carboxylation, phosphorylation, esterification, N-acylation, e.g. , cyclization via disulfide bridges or conversion to acid addition salts and / or optionally This includes dimerization or multimerization, or conjugation.

[0082] antigen binding portion In embodiments, the CARs described herein do not otherwise comprise an antigen-binding portion. In some embodiments, the antibody comprises a target-specific binding element, called a molecule, that binds specifically to an antigen on a cell. Targeting the antigen of interest can be achieved by engineering a desired antigen-binding moiety that differentially binds to the target antigen. The CARs described herein are engineered to interact with the target gene.

[0083] In embodiments, the antigen-binding portion of the CAR described herein binds to MUC16. MUC16-specific CARs are expressed on the cell surface. In some embodiments, the specificity of T cells is redirected to human MUC16. The antigen-binding domain is a variable domain of an anti-MUC16 monoclonal antibody specific for the target antigen. A main light chain (VL) and a variable domain heavy chain (VH) comprising a glycine-serine linker. VL and VH connected by a flexible linker such as a β-linker or Whitlow linker In some embodiments, the scFv comprises a single chain antibody fragment (scFv) comprising MUC16- 1 scFv, MUC16-2 scFv, MUC16-3 scFv, MUC16-4 scFv, MUC16-5 scFv, MUC16-6 scFv, or MUC16 In some embodiments, the scFv is humanized. In this form, the antigen-binding portion may be, for example, N-terminal to C-terminal, VH-linker-VL or Alternatively, it may comprise a VH and a VL directionally linked together as VL-linker-VH.

[0084] In embodiments, the CARs described herein are selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14 (MUC16 VL) of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 an antigen-binding portion comprising a VL polypeptide having 8%, 99%, or 100% identity; Includes minutes.

[0085] In embodiments, the CARs described herein are selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10 , 11, 13, and 15 (MUC16 VH) or one with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 Antigens comprising VH polypeptides with 7%, 98%, 99%, or 100% identity Contains a binding moiety.

[0086] In embodiments, the CARs described herein comprise a Gly-Ser linker (sequence SEQ ID NO: 83 or SEQ ID NO: 197) or a functional variant of a linker. VL (SEQ ID NO: 1, 3, 5, 7, 9, 12, or 14) and VH (SEQ ID NO: 2, 4, 6, 8, 10, 11, 13, or 15).

[0087] In embodiments, the CARs described herein are selected from the group consisting of SEQ ID NOS: 25-57 (VH, V L, and a linker) 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or polypeptides having 100% identity thereto.

[0088] In embodiments, the CAR described herein is SEQ ID NO: 2 (MUC16-1 VH) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide The antigen-binding portion comprises an agonist.

[0089] In embodiments, the CAR described herein is SEQ ID NO: 1 (MUC16-1 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with a VL polypeptide The antigen-binding portion comprises an agonist.

[0090] In embodiments, the CAR described herein is SEQ ID NO: 4 (MUC16-2 VH) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide The antigen-binding portion comprises an agonist.

[0091] In embodiments, the CAR described herein is SEQ ID NO: 3 (MUC16-2 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with a VL polypeptide The antigen-binding portion comprises an agonist.

[0092] In embodiments, the CAR described herein is SEQ ID NO: 6 (MUC16-3 VH) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide The antigen-binding portion comprises an agonist.

[0093] In embodiments, the CAR described herein is SEQ ID NO: 5 (MUC16-3 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with a VL polypeptide The antigen-binding portion comprises an agonist.

[0094] In embodiments, the CAR described herein is SEQ ID NO: 8 (MUC16-4 VH) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide The antigen-binding portion comprises an agonist.

[0095] In embodiments, the CAR described herein is SEQ ID NO: 7 (MUC16-4 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with a VL polypeptide The antigen-binding portion comprises an agonist.

[0096] In embodiments, the CAR described herein is SEQ ID NO: 10 (MUC16-5 At least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of the VH-L 95%, 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide. The antigen-binding portion comprises a polypeptide.

[0097] In embodiments, the CAR described herein is SEQ ID NO: 11 (MUC16-5 At least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of the VH-F 95%, 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide. The antigen-binding portion comprises a polypeptide.

[0098] In embodiments, the CAR described herein is SEQ ID NO: 9 (MUC16-5 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with a VL polypeptide The antigen-binding portion comprises an agonist.

[0099] In embodiments, the CAR described herein is SEQ ID NO: 13 (MUC16-6 At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, 11 VH polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises a peptide.

[0100] In embodiments, the CAR described herein is SEQ ID NO: 12 (MUC16-6 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, VL polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises a peptide.

[0101] In embodiments, the CAR described herein is SEQ ID NO: 15 (MUC16-7 At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, 11 VH polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises a peptide.

[0102] In embodiments, the CAR described herein is SEQ ID NO: 14 (MUC16-7 VL) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, VL polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises a peptide.

[0103] In some embodiments, the antigen-binding portion has at least one amino acid sequence identical to that of SEQ ID NO:58. Also 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or has 100% identity to the GM-CSFRa signal peptide.

[0104] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 27. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 28. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0105] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 30. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 The present invention includes polypeptides having 8%, 99%, or 100% identity to the polypeptide.

[0106] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 31. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 32. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0107] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 34. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 35. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0108] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 37. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 38. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0109] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 40. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 41. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0110] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 43. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein is a CAR having at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 44. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0111] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 46. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 47. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0112] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 49. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 50. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0113] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 52. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 53. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0114] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 55. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the polypeptide. The CAR described herein has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 56. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the polypeptides described herein include polypeptides having 100% identity to the polypeptides described herein. The described CARs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention also includes polypeptides having the same identity.

[0115] Stork Domain In embodiments, the MUC16 CAR of the invention comprises an antigen-binding portion and a T cell membrane In some embodiments, the stalk domain provides a separation between the establishes the optimal effector-target membrane distance. In some embodiments, the stalk domain The amine provides flexibility to the antigen-binding domain to reach its target. In this case, the stalk domain is the CD8 alpha hinge domain.

[0116] In embodiments, the CD8 alpha hinge domain has the amino acid sequence of SEQ ID NO: 16. With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, This includes polypeptides with 98%, 99%, or 100% identity.

[0117] Spacer In other embodiments, a spacer is provided between the extracellular domain of the CAR and the transmembrane domain. As described herein, the spacer is incorporated into the storage medium depicted in FIG. In one embodiment, the spacer comprises a single stalk region and a stalk extension region. In another embodiment, the spacer may comprise a stalk region (denoted as "s"). (wherein the fragment is a fragment of a nucleotide sequence) and a stalk extension region, designated herein as "s-n'". For example, the spacer may have one stalk region and n may be 0, 1, 2, 3, 4, 5, 6, or 7. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or In a further embodiment, the stalk region may comprise s'-n, which may be 20. The stalk extension regions s'-n can be linked via the CAR. Examples of the linker include a GSG linker (SEQ ID NO: 85), a SGSG linker (SEQ ID NO: No. 86), (G4S)3 linker (SEQ ID NO: 83), (G4S)4 linker (SEQ ID NO: 2 00), and / or Whitlow linkers.

[0118] In one embodiment, the stalk region and stalk extension region are present in a naturally occurring polypeptide. It may be derived from or designated as a polypeptide of synthetic origin. The stalk region and / or stalk extension region may be a cell surface protein or a In some embodiments, the hinge domain may be derived from a derivative or variant of these. , the stalk region and / or stalk extension region may be CD28 or CD8 alpha (C In some embodiments, each stalk region and The CD8 alpha hinge domain, CD28 hinge domain, and stalk extension region are TLA-4 hinge domain, LNGFR extracellular domain, IgG1 hinge, IgG4 hinge The stalk region may be derived from at least one of the ribosomal domains, the ribosomal domain, the ribosomal domain, and the CH2-CH3 domain. and the stalk extension region contains the CD8 alpha hinge domain, the CD28 hinge domain, CTLA-4, LNGFR extracellular domain, IgG1 hinge, IgG4 hinge, or C It can be derived individually from any combination of the H2-CH3 domains. The region may be derived from the CD8 alpha hinge domain and include at least one stalk extension. The long region may be derived from the CD28 hinge domain, thus forming a hybrid spacer In another example, the stalk region is attached to an IgG1 hinge or an IgG4 hinge. At least one stalk extension region may be derived from the CH2-CH3 domain of an IgG. It may also be derived from

[0119] In certain embodiments, the stalk region comprises one or more dimerization sites and is homodimerizable. Alternatively, a heterodimerized chimeric polypeptide may be formed. The region or one or more stalk extension regions contain a mutation that completely abolishes the dimerization site. In some embodiments, the stalk extension region may have at least For example, if the stalk region contains two dimerization sites, the stalk region may contain one less dimerization site. If included, the stalk extension region may contain one or zero dimerization sites. If the stalk region contains one dimerization site, the stalk extension region contains zero dimerization sites. In some instances, the stalk extension region lacks a dimerization site.

[0120] In some aspects of the embodiments disclosed herein, the storage of a subject antigen-binding polypeptide is The CD8 alpha hinge domain is at least about 65%, 70%, 75%, or 80% homologous to the CD8 alpha hinge domain. %, 80%, 85%, 90%, 95%, 99% or more identity The CD8 alpha hinge domain may be a fragment of at least one amino acid sequence corresponding to the sequence shown in SEQ ID NO: 16. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more Optionally, the stalk extension region may comprise a polypeptide sequence with a sequence At least 65%, 70%, 75%, 80%, 85% of the sequence shown in number 16 , 90%, 95% or more identity to a polypeptide sequence. Thus, the stalk extension region has at least 65% of the sequence shown in SEQ ID NO: 108. , with 70%, 75%, 80%, 85%, 90%, 95% or more identity In some instances, the stalk region and stalk extension region comprise a nucleotide sequence of At least 65%, 70%, 75%, 80%, 85% of the sequence shown in number 16 , 90%, 95% or more identity to a polynucleotide sequence do.

[0121] Transmembrane domain In some embodiments, the CAR is fused to the extracellular domain of the CAR stalk domain. In one embodiment, the CAR comprises a transmembrane domain that is naturally associated with one of the domains of the CAR. In some embodiments, a transmembrane domain is used. In some embodiments, a transmembrane domain is a loosely coupled molecule that spans the membrane. It is an aqueous alpha helix.

[0122] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be any membrane-bound or transmembrane protein. The transmembrane region of particular use in the present invention may be derived from the alpha domain of the T cell receptor. chain, beta chain, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD 5, CD8 alpha, CD9, CD16, CD22, CD33, CD37, CD64, C CD80, CD86, CD134, CD137, and CD154 (i.e., these Alternatively, the transmembrane domain may be synthetic. There are also other methods, which preferentially contain hydrophobic residues such as leucine and valine. In this form, at each end of the synthetic transmembrane domain, there are phenylalanine, tryptophan, and Optionally, in some embodiments, 2 to 10 amino acids are found in the valine triplet. A short oligonucleotide or polypeptide linker, the length of which is between A link between the transmembrane domain of the CAR and the cytoplasmic signaling domain may be formed. In some embodiments, the linker is a glycine-serine linker.

[0123] In some embodiments, the transmembrane domain of the CAR described herein is CD8 alpha. In some embodiments, the CD8 alpha transmembrane domain is At least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of No. 20 %, 96%, 97%, 98%, 99%, or 100% identity to Includes:

[0124] In embodiments, the transmembrane domain of the CAR described herein is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain is the amino acid sequence of SEQ ID NO: 21. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence , 97%, 98%, 99%, or 100% identity to the sequence.

[0125] Cytoplasmic domain (costimulatory and signaling domains) The cytoplasmic domain, also known as the intracellular signaling domain of the CAR described herein. The main goal is to activate at least one of the normal effector functions of CAR-infused immune cells. The term "effector function" refers to the specialized function of a cell. T cells The effector functions of the IL-1 receptor may include, for example, cytolytic activity and cytokine secretion. It may also have helper activity. Hence the term "intracellular signaling domain" , transducing effector function signals and directing cells to carry out specialized functions; It refers to a portion of a protein. Usually, the entire intracellular signaling domain can be used, but In many cases, it is not necessary to use the entire chain. To the extent that the truncated portion is used to transmit the effector function signal, A truncated portion such as Therefore, the term intracellular signaling domain refers to the domain that transmits the effector function signal. and any truncated portion of the intracellular signaling domain sufficient to is intended.

[0126] Examples of intracellular signaling domains for use in the CARs described herein include antibody- T cell receptors (TCRs) and receptors that act together to initiate signal transduction after receptor binding. The cytoplasmic sequences of the receptors and co-receptors, as well as any derivatives or variants of these sequences and the same It may include any synthetic sequence that has functional capability.

[0127] Signals generated through the TCR alone are usually insufficient for full T cell activation. A secondary or costimulatory signal is also required. can be mediated by two different classes of cytoplasmic signaling sequences: via the TCR Those that initiate antigen-dependent primary activation (primary cytoplasmic signaling sequences) and those that do not Acting to provide a secondary or costimulatory signal in a stimulatory-dependent manner (secondary cytoplasmic signaling sequence).

[0128] Primary cytoplasmic signaling sequences mediate TCs in either a stimulatory or inhibitory manner. Controls primary activation of the R complex. Primary cytoplasmic signaling sequence that acts in a stimulatory manner These proteins contain signal transduction motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. It may contain a nucleotide sequence.

[0129] For particular use in the present invention, ITAMs containing primary cytoplasmic signaling sequences Examples are TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD 3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d-derived In embodiments, the cytoplasmic signaling molecule of a CAR described herein. contains a cytoplasmic signaling sequence derived from CD3 zeta.

[0130] In embodiments, the cytoplasmic domain of the CAR, by itself or as described herein, in combination with any other desired cytoplasmic domain useful in the context of the described CAR For example, the CAR may be designed to contain a CD3-zeta signaling domain. The cytoplasmic domain may include a CD3 zeta chain portion and a costimulatory signaling region. The signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. The molecules are antigen receptors or their ligands, which are necessary for an adequate response of lymphocytes to antigens. Examples of such molecules are CD27, CD28, 4-1BB, and the like. (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function Continuous antigen-1 (FA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, DA These include ligands that specifically bind to P10, DAP12, and CD83. In some embodiments, costimulatory molecules may be used together, for example, CD28 and 4-1BB or CD2 8 and OX40. Therefore, the present invention primarily utilizes 4-1 as a costimulatory signaling element. Although exemplified by BB and CD28, other costimulatory elements are within the scope of the present invention.

[0131] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CARs described herein include They can be linked together in a random order or in a specific order. Optionally, 2 to 10 amino acids Short oligo- or polypeptide linkers, between 1 and 2 in length, can form the linkage. The glycine-serine pair provides a particularly suitable linker.

[0132] In one embodiment, the cytoplasmic domain comprises the signaling domain of CD3 zeta and In another embodiment, the cytoplasmic domain comprises a CD3 signaling domain. The signaling domains of 4-1BB and 4-1K are also included. In embodiments, the cytoplasmic domain comprises the signaling domain of CD3 zeta as well as the signaling domain of CD2 8 and 4-1BB signaling domains.

[0133] In one embodiment, the cytoplasmic domain of a CAR described herein comprises the 4-1BB signaling domain. It contains the nucleotide transduction domain and the signal transduction domain of CD3 zeta, and has the signal of 4-1BB. The null transduction domain has at least 90%, 91%, or more homology with the polypeptide sequence of SEQ ID NO: 22. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and the signaling domain of CD3 zeta comprises a polypeptide sequence having an identity of At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, 11 Polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% identity Contains code arrays.

[0134] In one embodiment, the cytoplasmic domain of a CAR described herein is capable of activating the CD28 signaling pathway. The antibody was designed to contain the CD3 transduction domain and the signal transduction domain of CD3 zeta, The signaling domain of CD28 is a polypeptide having the sequence of SEQ ID NO: 23. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a polypeptide sequence having 100% identity thereto, which is involved in signal transduction of CD3 zeta. The target domain has at least 90%, 91%, 92%, or 100% identity with the polypeptide sequence of SEQ ID NO: 26. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The polypeptide sequence includes a polypeptide sequence having the following properties:

[0135] In one embodiment, the cytoplasmic domain of a CAR described herein comprises the 4-1BB signaling domain. It contains the nucleotide transduction domain and the signal transduction domain of CD3 zeta, and has the signal of 4-1BB. The null transduction domain comprises the amino acid sequence set forth in SEQ ID NO: 22 and is a signaling domain of CD3 zeta. The loop transduction domain comprises the amino acid sequence set forth in SEQ ID NO:26.

[0136] Further genetic elements Cell therapy holds great promise for the treatment of human diseases, but the cells themselves or their Significant toxicity from the transgene products has hindered clinical exploration. In some embodiments, the method described herein is infused into a mammalian subject, e.g., a human. Immune effector cells containing the described CARs may be used to treat inflammatory bowel diseases, such as rheumatoid arthritis, ... and rheumatoid arthritis. can be depleted to modulate the effects of such immune effector cells. Thus, in certain embodiments, the MUC16-specific chimeric antigen receptor agonists described herein In addition to the RA antigen receptor, a second gene may also be engineered to induce immune effector responses, as described herein. The second gene is effectively transduced into the target cell, either MUC16 CAR or MUC16 CAR. 16 A "kill switch" that allows for the depletion of cells containing CAR / mbIL-15 In certain embodiments, a "kill switch" refers to a low level of HER1 or CD20. at least one antibody binding epitope or functional fragment thereof and, optionally, a signal peptide a HER1 polypeptide or a CD20 polypeptide comprising a polypeptide sequence or a fragment thereof The antibody is a HER1 tag or CD20 tag containing the nucleotide sequence.

[0137] In certain embodiments, the second gene is epithelium In some embodiments, the second gene is a truncated human HER1 tag, which is a growth factor receptor (HER1) or a fragment or variant thereof. epithelium Optionally, the second gene is a human truncated human HER1 tag, which is growth factor receptor 1 (e.g., HER1t or HER1t1). epitheliumIt is a variant of growth factor receptor 1. In embodiments, at least one of HER1, HER1t, and HER1t1 provides a safety mechanism by allowing depletion of infused CAR-T cells via administration of FDA-approved cetuximab or any antibody that recognizes HER1, HER1t, and / or HER1t1. In embodiments, the HER1t gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 65. In embodiments, the HER1t1 gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 66. Truncated HER1 sequences, such as HER1t and HER1t1, abolish the potential for EGF ligand binding, EGFR homodimerization and heterodimerization, and EGFR-mediated signaling, while leaving cetuximab's receptor binding intact (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Volume 37, pp. 353-373).

[0138] In a further embodiment, the therapeutic chimeric antigen receptor specific for MUC16 of the present invention In addition, the second gene introduced is a CD20 tag. The CD20 polypeptide may be a full-length CD20 polypeptide or a truncated CD20 polypeptide (CD20 t-1). Optionally, a CD20 tag, e.g., CD20 or CD20t-1 also demonstrated the efficacy of infused CA via administration of FDA-approved rituximab therapy. Allowing for depletion of RT cells also allows for a safety mechanism. In some embodiments, the CD20 tag has at least 90%, 91%, 92%, or 93% homology to the sequence of SEQ ID NO: 36. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the CD20 tag has a polypeptide sequence comprising: t-1 tag, which has at least 90%, 91%, 92%, 93%, or 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the CD20 tag has the nucleic acid sequence of SEQ ID NO: 160. Sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identity to a nucleotide sequence of CD20 In some embodiments, the CD20 tag is encoded by the gene of SEQ ID NO: 160. Nucleic acid sequences and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 CD containing nucleotide sequences with 7%, 98%, 99%, or 100% identity It is encoded by the 20t-1 gene.

[0139] In embodiments, the CAR vector comprising the CAR described herein is selected from the group consisting of SEQ ID NO: 159 nucleic acid sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, A full length containing a nucleic acid sequence with 96%, 97%, 98%, 99%, or 100% identity It further contains a CD20 tag.

[0140] In some embodiments, a kill tag, e.g., HER1t, HER1t-1, CD20, or The gene encoding the CD20t-1 tag was inserted in-frame into the MUC16 CAR. At the 3' end of the The gene is fused via a self-cleaving peptide, such as, but not limited to, a peptide. In some embodiments, the T2A peptide has at least 9 amino acid sequences with the amino acid sequence of SEQ ID NO: 72. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or has an amino acid sequence with 100% identity.

[0141] In some embodiments, the kill tag gene is in-frame with the MUC16 CAR gene. , and cloned into the lentiviral plasmid backbone. In other embodiments, both genes are cloned into separate lentiviral vectors. The offspring are cloned into the Sleeping Beauty transposon vector. In yet other embodiments, HER1t, HER1t-1, CD20, or CD20t-1 Kill tags such as these were inserted into separate Sleeping Beauty transposon vectors. In certain embodiments, the kill tag is a signal peptide, e.g., In this case, the GM-CSFRα signal peptide The amino acid sequence of SEQ ID NO: 58 is identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 1 4%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In certain embodiments, the signal peptide comprises the nucleic acid sequence of SEQ ID NO: 59 and at least 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or 100% identity to the IgK sequence. In some cases, the signal peptide is , IgE, CD8α variants and fragments thereof.

[0142] Exemplary gene expression cassettes encoding CAR and kill tags described herein is shown in Figures 1 and 2.

[0143] Exemplary CAR open reading frames Exemplary CARs and Human Carcinomas Encompassed by the Methods and Compositions Described Herein The open reading frames of the MUC16 receptor are listed in Table 1:

[0144] [Table 1]

[0145] In embodiments, provided herein is an isolated nucleic acid encoding a CAR, wherein the CAR , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 26 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% An isolated nucleic acid comprising a polypeptide having the same identity as the nucleic acid is provided.

[0146] In each of the embodiments listed in Table 1 with "MUC16-2 scFv," the CAR The antigen-binding portion has at least 90%, 91%, 92%, or 93% affinity to the amino acid sequence of SEQ ID NOs: 3 to 4. 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, the MUC16-2 scFv comprises a polypeptide having the same identity as the MUC16-2 scFv. The MUC16-3 scFv has at least 90% identity with the amino acid sequence of SEQ ID NO: 58. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or It has a GM-CSFRa signal peptide with 100% identity.

[0147] In each of the embodiments of Table 1 with "CD8a," the transmembrane region of the CAR is SEQ ID NO: 20 At least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to the polypeptide. The stalk domain comprises the amino acid sequence of SEQ ID NO: 16. Columns with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identity to a CD8a polypeptide. .

[0148] In each of the embodiments of Table 1 involving "CD28m," the intracellular domain of the CAR is At least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of No. 21 %, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence. Contains CD28.

[0149] In each of the embodiments of Table 1 with "T2A," the CAR ORF is delivered from a single vector. A self-cleaving Thosea asigna gene allows for the production of multiple gene products ) viral (T2A) peptide. In some embodiments, the T2A peptide is At least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of No. 72 %, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence. Has.

[0150] In the embodiment of Table 1 with "HER1t," the ORF of the CAR is an FDA-approved This allows for depletion of infused CAR-T cells via administration of cetuximab therapy. This results in a safety mechanism due to the use of a truncated human epidermal growth factor receptor 1 (HER2). The HER1t gene described herein comprises the amino acid sequence of SEQ ID NO: 65. With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The polypeptide sequences may have 98%, 99%, or 100% identity. Unless otherwise noted, the HER1t tag is a GM-CSFRa signal. The nucleic acid sequence of the GM-CSFRsp gene is SEQ ID NO: 58. In some embodiments, HER1t may be replaced with another tag, for example, HER1t-1. The HER1t-1 gene described has at least 90% homology with the amino acid sequence of SEQ ID NO: 66. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and may contain polypeptide sequences with 100% identity. In embodiments, the signal peptide has at least 90 amino acid sequences with the amino acid sequence of SEQ ID NO:59. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and is an IgK with an amino acid sequence that is 100% identical.

[0151] In the embodiment of Table 1 with "4-1BB," the CAR ORF is located at amino acid sequence 22 of SEQ ID NO:22. With acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 Cosminexus having polypeptide sequences with 7%, 98%, 99%, or 100% identity Contains exciton molecules.

[0152] In the embodiment of Table 1 with "FL CD20", the ORF of the CAR is the amino acid sequence of SEQ ID NO: 67. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence , 97%, 98%, 99%, or 100% identity to a polypeptide sequence Contains a full-length CD20 tag. CD20 is the marker for FDA-approved cetuximab therapy. The safety mechanism is improved by allowing depletion of infused CAR-T cells via administration. In another embodiment, the FL CD20 is a nucleotide sequence selected from the group consisting of FL CD20, ... At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, CD20t-1 contains polypeptide sequences with 99% or 100% identity. can be substituted with

[0153] In certain embodiments of Table 1, the CAR ORF is an inducible promoter for gene transcription. In one embodiment, the inducible promoter is under the control of a gene switch ligator. Optionally, the inducible promoter is a RHEOS promoter. Small molecule ligand-inducible, two-polypeptide gene switches, such as the WITCH® gene switch In some embodiments, the gene switch may be an ecdysone receptor-based gene switch. The CAR ORF and gene switch components were constructed as depicted in Figures 1-2. It is possible.

[0154] cytokines In some embodiments, the CARs described herein include, but are not limited to, cytokines. The therapeutic agent is administered to a subject in combination with one or more additional therapeutic agents, which may be administered to the subject at the site of the therapy. A kinase is a protein that binds at least one chemokine, interferon, interleukin, or lymphokine. kinin, tumor necrosis factor, or variants or combinations thereof. In some cases, the interleukin is IL-1. , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 , IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL- 16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, I L-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29 , IL-30, IL-31, IL-32, IL-33, and their functional variants and fragments thereof. In some embodiments, the cytokine is membrane-bound. In some embodiments, cytokines are soluble or secreted. IL-15, soluble IL-15 / soluble IL-15Rα complex (e.g., ALT-803 In certain cases, the interleukin is membrane-bound IL-15 (mbIL- 15) or a fusion of IL-15 and IL-15Rα. bIL-15 can be co-expressed using the engineered immune effector cells described herein. In some embodiments, mbIL-15 is a membrane-bound chimeric IL-15. fused in frame to L-15Rα, or a functional fragment or variant thereof , full-length IL-15 (e.g., a native IL-15 polypeptide), or a fragment or fragments thereof. Optionally, IL-15 is linked to IL-15Rα via a linker. , indirectly linked. In some cases, mbIL-15 is linked to the IL-15 receptor as described in Hurton et al., "Tethered IL-15" -15 augments antitumor activity and promotes a stem-cell memory subset in tumor- specific T cells,” PNAS 2016. The cytokine is expressed on the same immune effector cells as the CAR.

[0155] In a further embodiment, an immune effector cell expressing a CAR described herein expresses membrane-bound IL-15 ("mIL-15 or mbIL-15"). In this embodiment, mbIL-15 is a fusion protein between IL-15 and IL-15Rα. In a further embodiment, mbIL-15 comprises a nucleotide sequence similar to that of SEQ ID NO: 69. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. The AR and cytokine are expressed on separate vectors. In certain cases, the vectors , lentiviral vectors, retroviral vectors, or Sleeping Bea It may be a uty transposon.

[0156] In some embodiments, mbIL-15 is a HER1t, HER1 It is expressed in conjunction with a cell tag such as mbIL-1, CD20t-1, or CD20. 15 is inflated with HER1t, HER1t-1, CD20t-1, or CD20 It can be expressed in a variety of ways.

[0157] In some embodiments, mbIL-15 inhibits the regulation of an inducible promoter for gene transcription. In one aspect, the inducible promoter controls the ligand induction of the gene switch. Optionally, the inducible promoter is RHEOSWITC H® gene switches, small molecule ligand-induced, two-polypeptide It may be an ecdysone receptor-based gene switch.

[0158] In another aspect, the interleukin can include IL-12. L-12 is a synthetic IL-12 derived from single-chain IL-12 (scIL-12), protease-sensitive IL-12, and anxiety-inducing IL-12. Immobilized IL-12, membrane-bound IL-12, and inserted IL-12. 12 variants are disclosed in the International Publication No. 2004 / 0100004, all of which are incorporated by reference in their entirety. International Publication No. 2015 / 095249, International Publication No. 2016 / 048903 FRET, as described in International Publication No. 2017 / 062953 It is.

[0159] In some embodiments, the cytokines described above act as inducible promoters for gene transcription. In one embodiment, the inducible promoter is under the control of a gene switch. Optionally, the inducible promoter is a RHEO promoter. Small molecule ligand-inducible, two-polypeptide gene switches, such as the SWITCH® gene switch It may be an ecdysone receptor-based gene switch by a peptide.

[0160] Genetic Switch As used herein, gene switch polypeptides, ligand-inducible gene switch polypeptides, Polynucleotides encoding these polypeptides and / or polypeptides Methods and systems for incorporating nucleotides are presented. The term "gene switch" refers to It refers to the combination of a promoter and associated response elements, e.g., one or more ligands In the presence of a nucleotide, the expression of a gene incorporating a response element and promoter is modulated. This refers to the ecdysone receptor (EcR)-based system that controls the expression of ecdysone. Molecular expression systems or gene switches are useful for gene therapy, intracellular, large-scale protein production, and cell-based high-throughput screening assays, functional genomics, etc. and the regulation of traits in transgenic plants and animals. Such an inducible gene expression system is useful for a variety of applications. It may comprise an expression system.

[0161] An early form of EcR-based gene switch was discovered in Drosophila melanogaster ( anogaster) EcR (DmEcR) polypeptide and mouse (Mus musculus) RXR ( MmRXR polypeptides, and these receptors bind to the steroid ponasterone. In the presence of A, reporter genes were expressed in mammalian cell lines and transgenic mice. It has been shown to transactivate genes (Christopherson et al., 1992; No et al., 1996). Subsequently, Suhr et al., 1998, investigated the nonsteroidal ecdysone agonist Tebufenozide inhibits the growth of Bombyx mori in the absence of exogenous heterodimer partners. ) EcR (BmEcR) for high-level transcription of reporter genes in mammalian cells. It was shown to induce lance activation.

[0162] PCT International Patent Application / US Application No. 97 / 05330 (International Publication No. 97 / 381 17) and PCT International Patent Application / U.S. Application No. 99 / 08381 (WO 99 / 58155) modulates the expression of exogenous genes a DNA construct comprising an exogenous gene and an ecdysone response element; The construct reacts with the ligand for the construct and, optionally, with a silent partner. In the presence of a receptor that can act as a Methods of activation by a second DNA construct containing an ecdysone receptor that induces gene expression In this example, the ecdysone receptor was expressed in Drosophila melanogaster (Dr. Typically, such systems have been isolated from Escherichia coli (Bombyx mori) for optimal activation. To achieve this, the presence of a silent partner, preferably the retinoid X receptor (RXR), is required. In mammalian cells, the insect ecdysone receptor (EcR) is required for the mammalian ecdysone receptor. Heterodimerizes with rhodopsin X receptor (RXR), thereby targeting target genes or heterologous genes. It can be used to regulate gene expression in a ligand-dependent manner. Patent application / US application No. 98 / 14215 specification (International Publication No. 99 / 02683 pamphlet The ecdysone receptor isolated from the silkworm moth, Bombyx mori, It has been shown to be functional in mammalian systems without the need for an exogenous dimer partner. It shows.

[0163] U.S. Patent No. 6,265,173 describes a steroid / thyroid superfamily of receptors. Various members of the Millipore Standards are incorporated herein by reference in their entirety, at least in accordance with the U.S.P. Drosophila melanogaster ultraconjugates containing dimerization domains It is disclosed that the spirulina receptor (USP) or a fragment thereof can be combined with the spirulina receptor (USP) or a fragment thereof. U.S. Patent No. 5,880,333 describes the use of cinnamon in plants. Drosophila melanogaster EcR and Ultraspiracle (USP) A heterodimer system according to the present invention, wherein the transactivation domain and the DNA binding domain are The present invention discloses a heterodimeric system, which is located on two different hybrid proteins. In each of these cases, a transactivation domain and a DNA-binding domain (P Native EcR as described in CT International Patent Application / US Application No. 98 / 14215. or as in PCT International Patent Application / US Application No. 97 / 05330 and (as modified EcR) into a single molecule, and other The heterodimer partners were used in their native state.

[0164] PCT International Patent Application / US Application No. 01 / 0905 discloses a transactivation domain The DNA-binding domain and the ATP-binding domain are separated from each other by placing them on two different proteins. In the absence of ligand, an ecdysone receptor-based inducible gene expression system was shown to produce background This results in a significant reduction in the activity of the ligand, which is above background This results in a significant increase in activity over that of the two high The hybrid line is described in PCT Application / US Application No. 97 / 05330 and PCT Application / US In comparison with the two systems disclosed in the application of the patent application No. 98 / 14215 The two-hybrid system is a significantly improved inducible gene expression modulation system. When the DNA-binding domain binds to the DNA-binding site on the gene, it binds to the transactivation domain. The main target is to increase the number of interacting proteins to activate the promoter more efficiently. but the ability to place the transcription activation domain in a more favorable position relative to the DNA binding domain. (See, for example, U.S. Pat. No. 5,283,173. The two-hybrid gene expression system uses a DNA fragment fused to a nuclear receptor polypeptide. A first gene expression cassette encoding a binding domain and a different nuclear receptor polypeptide. a second gene expression cassette encoding a transactivation domain fused to the peptide In the presence of a ligand, the first polypeptide This induces a conformational change in the polypeptide that facilitates its interaction with a second polypeptide, This results in dimerization of the DNA-binding domain with the transactivation domain. The DNA binding domain and the transactivation domain are thought to be Because the ligands are present on different molecules, in the absence of ligand, the background activity is is significantly reduced.

[0165] Certain modifications of the two-hybrid system also involve the use of steroidal ligands, e.g., ponasteroids. Non-steroidal anti-inflammatory drugs (NSAIDs) when compared with PonA or Muristerone A (MurA) It also resulted in improved sensitivity to steroid ligands, such as diacylhydrazines. That is, compared to steroids, nonsteroidal ligands have the following properties: Furthermore, the two-hybrid system demonstrated that unmodified RXR resulted in greater gene transcription activity. Some of the side effects resulting from overexpression of RXR may occur when used as a switching partner. In the preferred two-hybrid system, the natural DNA of EcR or RXR is The A-binding and transactivation domains were removed, resulting in the Hybrid molecules have the potential to interact with other steroid hormone receptors present in cells. This reduces the toxicity, thereby resulting in fewer side effects.

[0166] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily. Classified into subfamily 1, group H (referred to herein as "group H nuclear receptors") Members of each group share 40–60% amino acid identity within the E (ligand-binding) domain. (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor r Subfamily, 1999; Cell 97: 161-163). In addition to the ecdysone receptor, this nuclear receptor Other members of subfamily 1, group H, are ubiquitous receptors (URs), orphan receptors, and steroid hormone nuclear receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR interacting Liver protein 15 (RIP-15), liver x receptor beta (LXRβ), steroid hormone receptor receptor-like protein (RLD-1), liver x receptor (LXR), liver x receptor α (LXRα ), farnesoid x receptor (FXR), receptor-interacting protein 14 (RIP-1 4), and the farnesol receptor (HRR-1).

[0167] In some cases, the inducible promoter ("IP") is a promoter available from Intrexon Corp. Small molecule ligands, such as the RHEOSWITCH® gene switch from Biosciences Pharma An inducible, two-polypeptide ecdysone receptor-based gene switch In some cases, the gene switches may be any of the following, each of which is incorporated herein by reference in its entirety: Incorporated PCT / US Application No. 2001 / 009050 (International Publication No. WO 200 No. 1 / 070816; U.S. Pat. No. 7,091,038; U.S. Pat. No. 7, Specification No. 776,587; Specification No. 7,807,417; Specification No. 8,202,718 Specification; PCT / US Application No. 2001 / 030608 Specification (International Publication No. 2002 / 0 29075; U.S. Patent Nos. 8,105,825; 8,168 ,426; PCT / US Application No. 2002 / 005235 (International Publication No. 002 / 066613 brochure); U.S. Patent Application Publication No. 10 / 468,200 Details (U.S. Publication No. 20120167239); PCT / U.S. Application No. 2002 / 0 No. 05706 (International Publication No. WO 2002 / 066614); U.S. Pat. Specification No. 7,531,326; Specification No. 8,236,556; Specification No. 8,598,40 No. 9; PCT / US Application No. 2002 / 005090 (International Publication No. 2002 No. 8,715,959 (U.S. Patent Publication No. PCT / US Application No. 2002 / 005234 (International Publication No. WO 2003 / 027266); U.S. Patent No. 7,601,500 Specification No. 8; Specification No. 7,829,676; Specification No. 7,919,269; Specification No. 7,919,269; No. 8,030,067; PCT / US Application No. 2002 / 005708 (National International Publication No. 2002 / 066615; U.S. Patent Application Publication No. 10 / 468, 192 (U.S. Publication No. 20110212528); PCT / U.S. Application No. 2 002 / 005026 (International Publication No. WO 2003 / 027289); U.S. Patent Nos. 7,563,879; 8,021,878; 8,4 97,093; PCT / US Application No. 2005 / 015089 (International Publication No. 2005 / 108617; U.S. Patent No. 7,935,510; No. 8,076,454; PCT / US Application No. 2008 / 011270 (International Publication No. WO 2009 / 045370); U.S. Patent Application Publication No. 12 / 24 No. 1,018 (U.S. Publication No. 20090136465); PCT / U.S. Application No. 2008 / 011563 (International Publication No. 2009 / 048560 Pamphlet ); U.S. Patent Application Publication No. 12 / 247,738 (U.S. Publication No. 200901234 No. 41; PCT / US Application No. 2009 / 005510 (International Publication No. 20 10 / 042189 pamphlet); U.S. Patent Application Publication No. 13 / 123,129 specification Publication No. 20110268766; PCT / US Application No. 2011 / 02 9682 (International Publication No. WO 2011 / 119773); U.S. Patent Application Publication No. 13 / 636,473 (U.S. Publication No. 20130195800); PCT / US Application No. 2012 / 027515 (International Publication No. 2012 / 12202 No. 5); and U.S. Pat. No. 9,402,919. The ecdysone-based receptor component of any of the systems currently in use may be selected. , but not limited to these.

[0168] System for modulating CAR and / or cytokine expression in a host cell a polynucleotide encoding a gene switch polypeptide disclosed herein A system containing a ligand for ligand-induced control of gene expression is presented herein. A polynucleotide encoding a gene switch polypeptide, The peptide is (a) a DNA-binding domain fused to a nuclear receptor ligand-binding domain; (b) a first gene switch polypeptide comprising a nuclear receptor linker (DBD); and A second gene switch containing a transactivation domain fused to a nucleic acid binding domain. a first gene switch polypeptide and a second gene switch polypeptide; A polynucleotide in which the polypeptide is connected by a linker is presented. In some embodiments, the linker is a 2A linker, a GSG-2A linker, a GSG linker (SEQ ID NO: 85), SGSG linker (SEQ ID NO: 86), furin link mutant and A cleavable linker sequence or a ribosomal sequence selected from the group consisting of these derivatives. In certain embodiments, the 2A linker is a p2A linker. linker, T2A linker, F2A linker, or E2A linker.

[0169] In some embodiments, the DNA binding domain (DBD) is GAL4 (GAL4 DBD). D), LexA DBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor subunit The DBDs of the Parr family members, the bacterial LacZ DBD, and the yeast DBD Optionally, the transactivation domain comprises at least one VP16 transactivation domain. domain and at least one of the B42 acidic activator transactivation domain In other cases, the nuclear receptor ligand binding domain includes the ecdysone receptor (EcR ), ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor Body 1, retinoid X receptor interacting protein-15, liver X receptor β, steroid hormone luminal receptor-like protein, liver X receptor, liver X receptor α, farnesoid X receptor, Receptor-interacting protein 14, and a small fraction of the farnesol receptor In some embodiments, the nuclear receptor ligand binding domain comprises at least one of the sequences In some embodiments, the ecdysone receptor polypeptide sequences are derived from sequences of ecdysone receptor polypeptides having sequences numbered 91 and 92. The nuclear receptor ligand binding domain is the ecdysone receptor of SEQ ID NOs: 91 and 92. It is derived from a polypeptide sequence.

[0170] In yet another embodiment, the first gene switch polypeptide is an EcR nuclear receptor ligase. A second gene switch promoter was constructed containing the GAL4 DBD fused to the GAL4 binding domain. The peptide is fused to the retinoid receptor X (RXR) nuclear receptor ligand-binding domain. Optionally, the first gene switch comprises a VP16 transactivation domain linked to the first gene switch. The polypeptide and the second gene switch polypeptide are connected by a linker. The linker may be a 2A linker, a GSG-2A linker, a GSG linker (SEQ ID NO: 85 ), SGSG linker (SEQ ID NO: 86), furin link mutants and derivatives thereof is selected from one or more of:

[0171] In certain embodiments, the polypeptides encoded by the polynucleotides described herein are The two or more polypeptides may be separated by an intervening sequence encoding a linker polypeptide. In certain cases, the linker is a cleavable linker. In some embodiments, the polypeptide of interest is linked by a cleavable linker polypeptide. In certain embodiments, the cleavable linker is expressed as a fusion protein. The polypeptides are F / T2A, T2A, p2A, 2A, GSG-p2A, and GSG linker. (SEQ ID NO: 85), and any one or more of a furin link mutant In certain embodiments, the linker polypeptide is selected from the group consisting of SEQ ID NOs: 72-86 or 1. Includes 97 to 199.

[0172] In some cases, viral 2A sequences can be used. The 2A element is 5 to 100 bases long. In some cases, the 2A sequence may be 5, 10, 15, 2, or 3 amino acids shorter than the IRES with which it is paired. 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 The 2A-linked gene can have a length of 1, 90, or 100 nucleotides. The "self-cleaving" 2A polynucleotide can be expressed in the open reading frame of It can occur cotranslationally between the last two amino acids, GP, at the C-terminus of the peptide. This results in equal amounts of co-expressed proteins.

[0173] The viral 2A sequence can be about 20 amino acids. Optionally, the viral 2A sequence is , the consensus motif, Asp-Val / Ile-Glu-X-Asn-Pro The consensus motif sequence may contain -Gly-Pro (SEQ ID NO: 201). For example, the normal peptide bond between glycine and proline residues can be formed. This prevents ribosome skipping and cleavage of the nascent polypeptide. This effect can result in multiple genes being disrupted at equimolar levels. do.

[0174] 2A peptides are the sequences of multiple proteins within a single open reading frame. Afterwards, the polypeptides can be cleaved into individual polypeptides via the ribosomal skipping mechanism. This may allow translation into a polypeptide (Funston, Kallioinen et al. 2008). In the form, the 2A sequence is F / T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, and any combination thereof.

[0175] In some cases, the vector may include an IRES sequence and a 2A linker sequence. In this case, the expression of multiple genes linked to the 2A peptide is controlled by the This can be facilitated by a spacer sequence (GSG (SEQ ID NO: 85)). Therefore, the construct may contain spacers, linkers, adapters, promoters, or combinations thereof. For example, constructs may be constructed with different 2A peptides along with a spacer (SG SG (SEQ ID NO: 86) or GSG (SEQ ID NO: 85)) and a furin linker (R- The spacer may have a cleavage site for I-CeuI (CeuI). ) In some cases, the linker can be engineered. For example, the linker can be They can be designed to include chemical features such as hydrophobicity, At least two linker sequences may result in the same protein. A linker can be used in a vector. For example, the gene of interest can be The two linkers may be spaced apart.

[0176] In certain embodiments, the polypeptides encoded by the polynucleotides described herein are The two or more polypeptides may be separated by an intervening sequence encoding a linker polypeptide. In certain cases, the linker is a cleavable linker. In some embodiments, the polypeptide of interest is linked to the cleavable intervening linker polypeptide. In certain embodiments, the protein is expressed as a fusion protein in which the cleavable phosphorylase is linked to the cleavable phosphorylase. The car polypeptide may be a furin polypeptide set forth in SEQ ID NOs: 72-86 or 197-199. Any of the following: link, fmdv, p2a, GSG-p2a, and / or fp2a It could be one or two.

[0177] In some embodiments, the linker is a The linker may be a flexible linker, a non-flexible linker, or an in vivo cleavable linker. In some cases, the linker may be a functional linker. The target domains are linked together (as in the case of flexible and non-flexible linkers) ) and, as in the case of in vivo cleavable linkers, free functional domains. may also be released in vivo.

[0178] The linker improves biological activity and increases expression yield, thereby achieving desirable pharmacokinetics. The linker may also be a hydrazone, peptide, disulfide, or thioether. It may also include thioesther.

[0179] In some cases, the linker sequences described herein may include flexible linkers. Flexible linkers allow for some degree of movement or interaction between the joined domains. Flexible linkers can be small, non-polar (e.g., Gly), or can be composed of polar amino acids (e.g., Ser or Thr). , and may have a sequence consisting primarily of a string of Gly and Ser residues ("GS" linker). An example of a flexible linker is a linker having the sequence (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number "n", this exemplary G The length of the S linker can also be optimized to achieve proper separation of functional domains, if necessary. In addition to the GS linker, other flexible linkers can also be used to maintain the essential interdomain interactions. Linkers can also be used in recombinant fusion proteins. may also be rich in small or polar amino acids, such as Gly and Ser. , containing additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu are used to improve solubility. It can be improved.

[0180] The flexible linkers contained in the linker sequences described herein have good flexibility and Small or polar amino acids, such as Gly and Ser, provide solubility. The flexible linker may be used to allow certain movements or interactions to occur within the fusion protein. In addition, flexible linkers may be used to modify non-flexible structures. Although they may not have a specific structure, they are used as passive linkers that maintain the distance between functional domains. The length of the flexible linker can be adjusted to allow proper folding. This may allow for optimal biological activity of the fusion protein.

[0181] The linkers described herein can optionally further comprise a non-flexible linker. Using a non-flexible linker to maintain a constant distance between domains of a polypeptide Examples of non-flexible linkers include alpha-helix-forming linkers, to name a few. , Pro-rich sequence, (XP)n, X-Pro backbone, A(EAAAK)nA (n = 2-5 ) (SEQ ID NO: 202). The rigid linker may optionally have an alpha helix structure. Relatively rigid structure due to its structure or the presence of multiple Pro residues may present.

[0182] The linkers described herein may, in some cases, be cleavable. Non-cleavable linkers are used in in vivo or ex vivo processes. Functional domains are covalently linked together so that they act as one molecule throughout the process. The linker may also be cleaved in vivo. The anchor is introduced in a way that releases free functional domains in vivo. Cleavable linkers can be cleaved in the presence of reducing agents, proteases, to name a few. For example, reduction of a disulfide bond can be used to cleave the cleavable linker. In the case of disulfide linkers, thiols such as glutathione can be used. A cleavage event via disulfide exchange with could result in cleavage. In vivo cleavage of the linker in the recombinant fusion protein also In vivo, under pathological conditions (e.g., cancer or inflammation), within specific cells or tissues proteases that may be expressed within the cell or may be restricted to specific cellular compartments. In some cases, the cleavable linker allows for targeting of cleavage. For example, the specificity of many proteases is determined by the slow cleavage of linkers within the constraint compartment. Cleavable linkers may also be hydrazones, peptides, disulfides, or For example, hydrazones may confer serum stability. In this case, the hydrazone may allow cleavage within the acidic compartment. The acidic compartment may have up to 7 The linker may also include a thioether. The thioether is a non-reducing Thioethers can be designed for intracellular proteolysis. .

[0183] In certain embodiments, the fmdv linker polypeptide has at least one sequence selected from SEQ ID NO: 82 and SEQ ID NO: 83. All are approximately 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9% It includes sequences that may be 0%, 95%, 97%, 98%, 99%, or 100% identical. In certain embodiments, the fmdv linker polypeptide comprises two or more fusion One or more of the linkers that link the proteins are encoded in a single vector In certain instances, the fmdv linker polypeptide is It may be encoded by a nucleic acid sequence of an open reading frame (ORF). , the ORF encoding fmdv comprises or consists of the sequence of SEQ ID NO: 173 In certain embodiments, the polynucleotide encoding fmdv is SEQ ID NO: 173 and at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 5%, 90%, 95%, 97%, 98%, 99%, or 100% identical.

[0184] In certain cases, the linker polypeptide may be a "p2a" linker. In certain embodiments, the p2a polypeptide has at least about 45%, 5% or more of the amino acid sequence identical to SEQ ID NO:75. 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9 The sequences may be 7%, 98%, 99%, or 100% identical. In this embodiment, the p2a linker polypeptide links two or more fusion proteins. The linkers may be one or more of the linkers encoded within a single vector. By this combination, the p2a linker polypeptide is linked to the open reading frame of the polynucleotide. In certain embodiments, p2a may be encoded by an open reading frame (ORF) nucleic acid sequence. The ORF encoding comprises or consists of the sequence of SEQ ID NO: 167. In this case, the polynucleotide encoding p2a is a sequence identical to SEQ ID NO: 167 and at least 4 5%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9 5%, 97%, 98%, 99%, or 100% identical, or at least nearly identical These ratios may be the same.

[0185] Optionally, the linker polypeptide can be a "GSG-p2a" linker. In certain embodiments, the GSG-p2a linker polypeptide comprises SEQ ID NO: 76 and at least At most about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, It may contain sequences that may be 90%, 95%, 97%, 98%, 99%, or 100% identical. In certain embodiments, the GSG-p2a linker polypeptide comprises two or more of linkers encoded in a single vector linking more than Optionally, the GSG-p2a linker polypeptide may be one or more. The open reading frame (ORF) of nucleotides can be encoded by a nucleic acid sequence. The ORF encoding GSG p2a may comprise the sequence of SEQ ID NO: 168. Thus, the polynucleotide encoding GSG-p2a has the sequence of SEQ ID NO: 168 and at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical, or at least nearly so These ratios may be the same.

[0186] The linker polypeptide can be the "fp2a" linker provided herein. In certain embodiments, the fp2a linker polypeptide is selected from the group consisting of SEQ ID NO: 77 and at least Approximately 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 97%, 98%, 99%, or 100% identical. In certain instances, the fp2a linker polypeptide may comprise two or more fusion proteins. One or more of the linkers encoded within a single vector link the proteins. Optionally, the fp2a linker polypeptide may be used to It may be encoded by a nucleic acid sequence of an open reading frame (ORF). In the present invention, the polynucleotide encoding the fp2a linker is SEQ ID NO: 169 and at least Also 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 97%, 98%, 99%, or 100% identical, or at least nearly identical These ratios may be approximately the same.

[0187] In some cases, the linker can be engineered. For example, the linker can be hydrophobic, etc. In some cases, at least two The linker sequence may result in the same protein. The sequences are SEQ ID NO: 72 to SEQ ID NO: 86 , or the sequences of SEQ ID NO: 197 to SEQ ID NO: 199 and 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, It may be 99%, or 100% identical, or close to these percentages. In other cases, multiple linkers may be used in a vector. The gene of interest and one or more gene switch polypeptide sequences described in the document , can be separated by at least two linkers. , 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers It is possible.

[0188] The linker can be an engineered linker. Methods for designing linkers include computer-aided In some cases, the computational method may be a graphical method. Three-dimensional peptides derived from a database using computational methods. A library of structures can be searched for suitable peptides. Haven Protein Data Bank (PDB) was used to select linkers. The distance between selected amino acids can be measured.

[0189] In some embodiments, a polypeptide comprising a furin polypeptide and a 2A polypeptide A polynucleotide encoding a peptide construct is provided, in this case a furin polypeptide. and 2A polypeptides containing at least three hydrophobic amino acids. Optionally, the at least three hydrophobic amino acids are connected by a linker. Lysine (Gly) (G), Alanine (Ala) (A), Valine (Val) (V), Leucine Leucine (Leu) (L), isoleucine (Ile) (I), proline (Pro) (P), phenylalanine (P) Phe (F), Methionine (Met) (M), Tryptophan (Trp )(W). Optionally, the polypeptide linker is also selected from the list consisting of: One or more GS linker sequences, e.g., (GS)n (SEQ ID NO: 203), (SG)n (SEQ ID NO: 204), (GSG)n (SEQ ID NO: 198), and (SGSG)n (SEQ ID NO: No. 199) [where n can be any number from 0 to 15].

[0190] A method for obtaining improved expression of a polypeptide construct, comprising: and a polynucleotide encoding said polypeptide construct comprising a second functional polypeptide. providing a polypeptide comprising the first functional polypeptide and a second functional polypeptide; and a peptide with at least 60% identity with the sequence APVKQ (SEQ ID NO: 205). and wherein the linker polypeptide comprises a sequence selected from the group consisting of: Expressing the polynucleotide, wherein the sequence of the polypeptide construct is A linker comprising a sequence with at least 60% identity to APVKQ (SEQ ID NO: 205). - resulting in an improvement compared to a corresponding polypeptide construct without the polypeptide. and

[0191] In other cases, the linker may be an IRES. The term "internal ribosome entry site (IRES)" is intended to mean an internal ribosome entry site. In a vector containing an IRES sequence, the first gene is inserted into its own 5'-U Whereas translation can occur by cap-dependent ribosome scanning, a mechanism involving TR, Subsequent gene translation is cap-independent due to direct recruitment of ribosomes to the IRES. The IRES sequence allows eukaryotic ribosomes to bind and attach to the 5' cap end. The IRES sequence allows the initiation of translation from one transcript to the other without the need for a second transcript. It can allow the expression of multiple genes (Mountford and Smith 1995).

[0192] Exemplary IRES sequences can be found in SEQ ID NOs: 192 and 193. In this case, the polynucleotide encoding the 2xRbm3 IRES is SEQ ID NO: 192 and At least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical? or approximately these ratios. In certain cases, the EMCV IRES The polynucleotide encoding SEQ ID NO: 193 has at least 45%, 50%, 55%, or %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99%, or 100% identical, or nearly so It is possible.

[0193] In some embodiments, the expression of a CAR and a cytokine in a host cell is modulated. a system for generating a polypeptide comprising a first polynucleotide encoding a first polypeptide; a first gene expression cassette; a second polynucleotide encoding a second polypeptide; a second gene expression cassette comprising a ligand; and a ligand, wherein the host cell is transfected with the ligand. and contacting the first gene expression cassette and the second gene expression cassette in the presence of The first polypeptide and the second polypeptide are transfected so that the CAR and cytokine are expressed in the host cell. and a second polypeptide comprising: (i) a transactivation domain; (ii) a DNA-binding domain; (iii) a ligand-binding domain; (iv) a CAR; and (vi) a cytokine. and / or (vii) a cell tag. where the CAR is a MUC16 CAR and the cytokine is mbIL-15 In some cases, MUC16 CAR and mbIL-15 were co-expressed with one cell tag. MUC16 CAR and mbIL-15 are co-expressed with cell tags. In other cases, MUC16 CAR was expressed with a cell tag and mbIL-15 was expressed with a CAR tag. 2. The exemplary configuration of the gene expression cassette is shown in FIG. 1 and FIG. 2. In other cases, the CAR is a MUC16 CAR and the cytokine is IL-1 2. In some cases, MUC16 CAR, IL-12 are co-expressed with one cell tag. In some cases, MUC16 CAR and IL-12 are expressed as cell tags and In other cases, the MUC16 CAR is expressed with a cell tag and IL- 12 is expressed along with a second cell tag.

[0194] In some embodiments, the expression of a CAR and a cytokine in a host cell is modulated. a system for generating a polypeptide comprising a first polynucleotide encoding a first polypeptide; a first gene expression cassette; a second polynucleotide encoding a second polypeptide; a second gene expression cassette comprising a ligand; and a ligand, wherein the host cell is transfected with the ligand. and contacting the first gene expression cassette and the second gene expression cassette in the presence of The first polypeptide is transfected into the host cell so that the CAR and / or cytokine are expressed in the host cell. (ii) a DNA-binding domain; and (ii) i) a ligand-binding domain, and the second polypeptide comprises one or more of: (ii) a cytokine; and / or (iii) a cell tag; or Optionally, the CAR is a MUC16 CAR, and the CAR is a cytoplasmic CAR. The inhibitor is mbIL-15. Optionally, MUC16 CAR and mbIL- 15 are co-expressed with the respective cell tags. In other cases, MUC16 CAR is It is expressed with one cell tag and mbIL-15 is expressed with a second cell tag.

[0195] To modulate the expression of MUC16 CAR and cytokines in host cells Exemplary configurations of this system are depicted in Figures 1-2. In some embodiments, the gene expression cassette are introduced into immune effector cells using viral or virus-based systems. An example of a non-viral based delivery system described herein is the SB11 transposon system. , SB100X transposon system, SB110 transposon system, piggyBac transposon system In one embodiment, the gene expression cassette comprises one or more Slee It is introduced into immune effector cells using the ping Beauty transposon.

[0196] MUC16 CAR, cytokines (such as mbIL-15 or IL-12), and An exemplary embodiment of a gene expression cassette encoding constitutive expression of a cell tag is shown in FIG. 1a-b show various configurations of MUC16 CAR, mbIL-15, and 1 depicts an exemplary gene expression cassette for designing cell tags. In this embodiment, Gene expression cassettes were integrated into a single Sleeping Beauty transposon for immune expression. Figure 1c-d shows the MUC16 CAR transduced into vector cells. and the mbIL-15 and cell tag are in a second gene expression cassette. In this embodiment, the gene expression cassette is or multiple Sleeping Beauty transposons into immune effector cells and introduce it.

[0197] MUC16 CAR, cytokine (e.g., mbIL-15), and / or cell tag An exemplary embodiment of a gene expression cassette encoding inducible expression of is depicted in FIG. Figure 2a-d shows various configurations of MUC16 CAR under the control of an inducible promoter. 1 depicts exemplary gene expression cassettes for mbIL-15 and cell tag design. Figure 2e shows the expression of genes encoding the gene switch polypeptides described herein. 1 is an exemplary embodiment of a cassette. In this embodiment, the gene expression cassette can be one or more Multiple Sleeping Beauty transposons are used to direct the signaling pathway into immune effector cells. Enter.

[0198] Ligand In some embodiments, the ligand used to regulate the inducible gene switch is Bottom: N-[(1R)-1-(1,1-dimethylethyl)butyl]-N'-(2-ethyl- 3-Methoxybenzoyl)-3,5-dimethylbenzohydrazide (also known as berezimex) (2S, 3R, 5R, 9R, 10R, 13R, 14S, 17R)-17-[( 2S,3R)-3,6-dihydroxy-6-methylheptan-2-yl]-2,3,14 -trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,1 6,17-Decahydro-1H-cyclopenta[a]phenanthren-6-one; N'-( 3,5-dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3-hexanyl ]-2-Ethyl-3-methoxybenzohydrazide;5-Methyl-2,3-dihydrobenzohydrazide Zo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)-N '-(1-Ethyl-2,2-dimethyl-propyl)-hydrazide;5-Methyl-2,3- Dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy- 4-Methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydroxide 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)- Hydrazide; 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbohydrazide N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl)phosphoric acid 5-Ethyl-2,3-dihydro-benzo[1,4]dihydrobenzo[1,4]dihydrazide Xine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)-N'-(1-ethyl) -2,2-dimethyl-propyl)-hydrazide;5-ethyl-2,3-dihydro-benzo [1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl) 5-Ethyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide 1-(2,3-dihydrobenzo[1,4]dioxine-6-carboxylic acid N'-(1-te rt-Butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazide;5- Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1- tert-Butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl) -hydrazide;3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propionyl) (3-Methoxy-2-methyl-benzoyl)hydrazide;3,5-Dimethylbenzoyl N-(1-ethyl-2,2-dimethylpropyl)-4-methylbenzoic acid N' -(3-Methoxy-2-methyl-benzoyl)-hydrazide;3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoic acid 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-yl)-hydrazide Butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide;3 ,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-( 2-Ethyl-3-methoxy-benzoyl)-hydrazide;3,5-dimethoxy-4-methyl N-(1-ethyl-2,2-dimethylpropyl)-N'-(2-ethyl-)benzoic acid 3-Methoxy-benzoyl)-hydrazide;3,5-Dimethyl-benzoic acid N-(1-te rt-Butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazine N-(1-tert-butyl-3,5-dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl) ester -N'-(2-ethyl-3-methoxybenzoyl)hydrazide;2-Methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N'-(4-ethyl-benzoyl)- Hydrazide; 3,5-Dimethyl-benzoic acid N-(2,2-dimethyl-1-phenyl-propionyl) Pyr)-N'-(4-ethyl-benzoyl)-hydrazide;3,5-dimethyl-benzoic acid N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl) yl)-hydrazide; and 3,5-dimethoxy-4-methyl-benzoic acid N-(1-te rt-Butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydra The compound may be selected from, but is not limited to, any of the following:

[0199] Possibly, dose-regulated control of ecdysone receptor-based inducible gene switches. The ligands used for this purpose are ecdysone, 20-hydroxyecdysone, and ponasterone. A, ecdy steroids such as muristerone A, 9-cis-retinoic acid, retinoin Synthetic analogs of the acid, each of which is incorporated herein by reference, are disclosed in U.S. Pat. Specification No. 13,836; Specification No. 5,117,057; Specification No. 5,530,028 and US Patent No. 5,378,726, and US Patent Application Publication No. 2005 / 02 09283 and 2006 / 0020146 N,N'-diacylhydrazines such as N,N'-diacylhydrazines containing N,N'-diacylhydrazines; U.S. Patent Application Publication No. Oxazolines as described in European Patent Application No. 2004 / 0171651; Dibenzoylalkylcyanohydrazines such as those disclosed in US Pat. No. 61,809 Which dibenzoylalkylcyanohydrazine; U.S. Pat. No. 5,225,443 N-alkyl-N,N'-diaroylhydrazines and other N-alkyl-N,N'-diaroylhydrazines disclosed in N,N'-diaroylhydrazine; as disclosed in European Application No. 234,994 N-acyl-N-alkylcarbonylhydrazines, such as N-acyl-N-alkylcarbonylhydrazines, carbonylhydrazine; as described in U.S. Pat. No. 4,985,461 N-aroyl-N-alkyl-N'-aroylhydrazines Alkyl-N'-aroylhydrazines; U.S. Patent Application Publication No. 2004 / 0049037 Alnidoketones such as those described in the document; 3,5-di-tert -butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpage hydroxysteroids, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol Cholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6- Alpha-epoxycholesterol-3-sulfate (ECHS), 7- Ketocholesterol-3-sulfate, Flamezol, bile acid, 1,1- Other classes include biphosphonate esters, juvenile hormone III, etc. The material may be selected from, but is not limited to, any of a variety of similar materials. An example of a suitable diacylhydrazine ligand is RG-115819 (3,5-dimethyl-benzoyl) N-(1-ethyl-2,2-dimethylpropyl)-N'-(2-methyl-3-methoxy)- (R)-3,5-dimethyl-benzoyl)-hydrazide), RG-115932 ((R)-3,5-dimethyl- N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzyl)benzoate benzoyl)-hydrazide-), and RG-115830 (3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxybenzoyl) For example, any of which are incorporated by reference in their entirety. No. 12 / 155,111 and PCT Application No. 2004 / 010999, which are incorporated herein by reference. See Application / US Application No. 2008 / 006757.

[0200] Non-viral-based delivery systems Nucleic acids encoding CARs described in the present invention can also be prepared by incorporating DNA sequences into vertebrate chromosomes. "Sleeping Bea" refers to a synthetic DNA transposon system for introducing Non-viral-based delivery systems, such as the SB transposon system, are being used to deliver immune responses to An exemplary SB transposon system can be introduced into an effector cell, e.g., In U.S. Pat. Nos. 6,489,458 and 8,227,432 The Sleeping Beauty transposon system is described and depicted in Figure 3. , Sleeping Beauty (SB) transposase and SB transposon As used herein, the Sleeping Beauty transposon system includes S Sleeping Beauty transposase polypeptide and a derivative that retains its activity Conductors, mutants and / or fragments, and Sleeping Beauty transposons These may include polypeptides, derivatives, variants and / or fragments that retain activity. In certain embodiments, the Sleeping Beauty transposase is In some embodiments, the mRNA is provided as SB10, SB11, SB110x, or SB110 transposase. In some embodiments, the mRNA It contains a poly(A) tail and a poly(A) tail.

[0201] DNA transposons are simple cuts from one DNA site to another. Translocation occurs by a chain-and-paste method. Transposition involves the transfer of a defined DNA segment to a single DNA fragment. molecules and can be separated into the same DNA molecule or genome, or into different DNA molecules. This is a precise process that often moves the Tc1 / marine to another site in the genome. Like r-type transposases, SB transposases also transfer transposons to recipients. The insertion site is located at a TA dinucleotide base pair within the target DNA sequence. It may be at another site within the offspring, or it may be in another DNA molecule (or chromosome). There are approximately 200 million TA sites in the mammalian genome, including the genome. During the transposon integration process, the TA sequence is duplicated. This duplication is the most prominent site of transposition. It is a characteristic feature and is used in some experiments to confirm the mechanism. The transposase may be encoded within a transposon, and the transposase may be supplied by another source. In some cases, the transposon becomes a non-autonomous element. Transposons cannot excise and reinsert independently after insertion, so gene transcription is difficult. The SB transposon is most useful as a vector for introducing genes into the genome of vertebrates. It is envisioned that they can be used as non-viral vectors for gene therapy. Specifically, Sleeping Beau The ty(SB) system (Hackett et al., Mol Ther 18:674-83, (2010)) was adapted (Cooper et al., Blood 105:1622-31, (2005)). In one embodiment, two steps are performed: (i) T cell proliferation and differentiation; SB transposons to redirect vesicle specificity [i.e., chimeric antigen receptor Electrotransfer of DNA plasmids expressing CAR and SB transposase Hum Gene T (Jin et al., Gene Ther 18:849-56, (2011), Kebriaei et al. her 23:444-50, (2012)), and (ii) designer artificial Antigen presenting cells (AaPC) The integration sequence is stably attached to the host (also known as the integrating cell). In another embodiment, the second step (i i) is omitted, and the genetically modified T cells are either cryopreserved or immediately infused into the patient. do.

[0202] In one embodiment, the SB transposon system is described, for example, in its entirety by reference. See Hudecek et al., Critical Reviews in Biochemistry and Mol. ecular Biology, 52:4, 355-380 (2017), Singh et al., Cancer Res (8):68 (2008). Ap Ril 15, 2008 and Maiti et al., J Immunother. 36(2): 112-123 (2013) It has been done.

[0203] In certain embodiments, the MUC16 CAR and mbIL-15 are transposable. The SB transposase is encoded within a DNA plasmid vector, and the SB transposase is encoded within a separate vector. In certain embodiments, the MUC16 CAR described herein is encoded within is encoded within a transposon-based DNA plasmid vector, and mbIL-15 is encoded within a second The SB transposase is encoded within a transposon DNA plasmid vector. In some embodiments, the CAR is encoded within a third DNA plasmid vector. with a marker tag, e.g., HER1t, HER1t1, CD20, or CD20t-1. In some embodiments, mbIL-15 is encoded by a kill tag, e.g., HER1t, It is encoded together with HER1t1, CD20, or CD20t-1.

[0204] In embodiments, the MUC16 CAR is expressed as a transposon-based DNA plasmid vector. In a further embodiment, the IL-15 gene may be co-expressed with mbIL-15 and a cell tag. The MUC16 CAR can be under the control of an inducible promoter. The bIL-15 may be under the control of an inducible promoter. The promoter can be a ligand-inducible promoter of a gene switch. Inducible promoters are small molecule regulators, such as the RHEOSWITCH® gene switch. A Gand-induced, two-polypeptide, ecdysone receptor-based gene switch In certain embodiments, a MUC16 CAR, mbIL-15, and The tag may be constructed within one, two, or more transposons. Exemplary configurations of MUC16 CAR or mbIL-15 under the control of a CAR are shown in Figure 2. is depicted in.

[0205] In some embodiments, the SB11 transposon system, the SB100X transposon system, the S B110 transposon system, piggyBac transposon system (see, e.g., No. 9,228,180, which is incorporated herein in its entirety; Wils on et al, “PiggyBac Transposon-mediated Gene Transfer in Human Cells,” Molecule ar Therapy 15:139-145 (2007)), and / or piggyBat Tiger transposon system (e.g., Mitra et al., “Functional characterization of piggyBat f om the bat Myotis lucifugus unveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA 110:234-239 (2013)) to AR and other genetic elements into the cell. and transposon systems, each of which is incorporated herein by reference in its entirety. Nos. 7,148,203 and 8,227,432, incorporated herein by reference. Detailed description, US Patent Publication No. 2011 / 0117072, Mates et al., Nat Genet, 4 1(6):753-61 (2009). doi: 10.1038 / ng.343. Epub 2009 May 3, Gene Ther., 18(9):849- 56 (2011). doi: 10.1038 / gt.2011.40. Epub 2011 Mar 31 and Ivics et al., Cell. 9 1(4):501-10, (1997).

[0206] In other embodiments, a MUC16 CAR and cytokines, mbIL-15 and / or other gene tags such as HER1t / HER1t1 / CD20 / CD20t-1 tags The element is then transduced into immune effector cells via a recombinase and an expression vector for integration. Such vectors can be integrated randomly into the DNA of a host cell. It may be integrated into the chromosome of the expression vector, allowing specific recombination between the expression vector and the host cell chromosome. Such integrative expression vectors may contain recombination sites that allow the expression of the vector in a host cell. The endogenous expression control sequences of the chromosome may be used to direct expression of the desired protein. In embodiments, sequences on the donor polynucleotide that are homologous to sequences flanking the integration site are included. The presence of the sequence facilitates targeting of integration. For example, Targeting of integration using donor polynucleotides is a method similar to conventional transfection. Gene knockout or knockin can be created by homologous recombination techniques, e.g., homologous recombination. In other embodiments, the donor polynucleotides can be used in the preparation of the donor polynucleotides. The presence of sequences on the nucleotide that are homologous to the sequences flanking the integration site, and site-specific contacting the cells with the donor polynucleotide in the presence of a target recombinase site-specific recombinases, or simply A recombinase is an enzyme that catalyzes conservative site-specific recombination between compatible recombination sites. As used herein, site-specific recombinase refers to a polypeptide that mediates the transcription of a target gene. Natural polypeptides as well as derivatives, variants, and / or fragments that retain activity, and Natural polynucleotides, derivatives and mutants encoding recombinases that retain activity, and / or fragments.

[0207] The recombinase is introduced into the target cell prior to the introduction of the targeting vector. It can be introduced at the same time as this, or it can be introduced later. The enzyme can be delivered, for example, via liposomes, particle coatings, or microinjection. Alternatively, the protein can be directly introduced into cells using a suitable Expression vectors are used to express DNA or messenger RNA encoding the recombinase. A polynucleotide can also be introduced into a cell. The components of the recombination vector are expression vectors containing sequences encoding the recombinase of interest. However, expression of the recombinase can be achieved in other ways, e.g. For example, expression of the recombinase can be controlled by a regulatable promoter (i.e., the expression can be selectively It can also be regulated by placing it under the control of a promoter that can be induced or repressed by .

[0208] Recombinases come from the integrase or resolvase families. The integrase family of recombinases has over 100 members, Examples include FLP, Cre, and lambda integrase. - also called the tyrosine family or lambda integrase family, DNA The catalytic hydroxyl group of tyrosine is used for nucleophilic attack on the phosphodiester bond of Typically, members of the tyrosine family first nick the DNA, This then forms a double-strand break. Examples of tyrosine family integrases are: Cre integrase, FLP integrase, SSV1 integrase, and lambda (λ) integrases. In the solvase family, a conserved serine residue provides covalent attachment to the DNA target site. (Grindley, et al., (2006) Ann Rev Biochem 16:16).

[0209] In one embodiment, the recombinase is SPβc2 recombinase, SF370.1 recombinase, recombinase, Bxb1 recombinase, A118 recombinase, and ΦRv1 recombinase An isolated polymerase comprising a nucleic acid sequence encoding a recombinase selected from the group consisting of: Examples of serine recombinases are described in their entirety by reference. No. 9,034,652, which is incorporated herein by reference in its entirety. is described in.

[0210] Recombinases for use in the practice of the present invention can also be produced recombinantly. and purified as previously described. The polypeptides can be purified by ammonium sulfate precipitation of proteins using methods known in the art. methods, size fractionation, affinity chromatography, HPLC, ion exchange chromatography chromatography, heparin agarose affinity chromatography (e.g., Thor pe & Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998). The desired degree of purity can be achieved by a simple protein purification method.

[0211] In one embodiment, the recombinase is administered to the desired recombination site by any suitable method. The conjugation site can be introduced into a eukaryotic cell. Introducing functional proteins into cells by microinjection or other methods The introduction of purified recombinase protein results in the transient presence of the protein. , and ensures its functionality, which is often the preferred embodiment. The gene encoding the recombinase is located in the expression vector used to transform the cells. a polynucleotide encoding a recombinase is introduced into a polynucleotide in a eukaryotic cell The gene encoding the nucleotide sequence is contained within an expression vector operably linked to a promoter that mediates expression of the nucleotide sequence. The recombinase polypeptide also includes a message encoding the recombinase polypeptide. Recombinases can also be introduced into eukaryotic cells by means of transfectant RNA. The nucleic acid fragment is present for a period of time required for insertion into the genome to be modified. Generally preferred; therefore, the lack of persistence associated with most expression vectors is not detrimental. The recombinase gene is inserted into the target cell prior to introduction of the exogenous polynucleotide of interest. It can be installed at the same time as this, or at a later time. In one embodiment, the recombinase gene is a gene encoding a polynucleotide to be inserted. The recombinase gene is still present in the vector, and the recombinase gene is still present in the polynucleotide. In another embodiment, the recombinase gene can be expressed in a transgenic The recombinase is introduced into the nucleoprotein, either constitutively or via a cell-specific promoter. Tissue-specific promoters, development-specific promoters, organelle-specific promoters, or or small molecule inducible promoter or small molecule repressible promoter, Transgenic cells or animals can be generated. The enzyme also contains other peptides, proteins, nuclear localization signal peptides, signal peptides or organelle-specific signal peptides (e.g., mitochondrial or chloroplast signal peptides) Translocation in mitochondria or chloroplasts, facilitating recombination within the plasts It can also be expressed as a fusion protein with a nucleotide sequence (nucleotide sequence of interest).

[0212] In one embodiment, the method for site-specific recombination comprises a first recombination site and a second recombination site. providing a recombination site; and incorporating the first recombination site and the second recombination site into a prokaryotic cell. contacting the recombinant DNA fragment with a recombinase polypeptide of the present invention results in recombination between the recombination sites. and a step of transfecting the first recombination site with the second recombination site, wherein the recombinase polypeptide The first recombination site is capable of mediating recombination with an attP or The first recombination site is attB, the second recombination site is attB or attP, and the second recombination site is attB or attP. If the junction site is attB, the second recombination junction site is attP, and the first recombination junction site is attB. If the first recombination junction is attP, the second recombination junction is attB. In this case, the recombinase of Listeria monocytogenes Phage recombinase, phage recombinase of Streptococcus pyogenes enzymes, phage recombinases from Bacillus subtilis, Mycobacterium tuberculosis Phage recombinase from H. tuberculosis and Mycobacterium smegmatis atis phage recombinase.

[0213] Further embodiments involve the introduction of a site-specific recombinase into the cell whose genome is to be modified. One embodiment provides a method for obtaining site-specific recombination in a eukaryotic cell, comprising: providing a eukaryotic cell containing a first recombination junction site and a second recombination junction site; The first recombination junction site and the second recombination junction site are ligated with a prokaryotic recombinase polynucleotide. and contacting the recombinant DNA fragment with a peptide resulting in recombination between the recombination junction sites. the recombinase polypeptide comprises a first recombination junction site and a second recombination junction site. The first recombination junction site is capable of mediating recombination with the phage genome assembly. The recombination junction site (attP) or the recombination junction site (attB) of the bacterial genome, The second recombination junction is attB or attP, and the first recombination junction is at tB, the second recombination junction is attP, and the first recombination junction is If the second recombination junction is attP, the second recombination junction is attB. The enzyme binds to the phage recombinant protein of Listeria monocytogenes. Binase, phage recombinase of Streptococcus pyogenes, Bacillus subtilis Phage recombinase of Bacillus subtilis, Mycobacterium tuberculosis s) and the phage recombinase of Mycobacterium smegmatis In one embodiment, the recombinase is selected from the group consisting of: The recombinases are A118 recombinase, SF370.1 recombinase, and SPβc2 recombinase. recombinase, ΦRv1 recombinase, and Bxb1 recombinase. In one embodiment, the recombination results in integration.

[0214] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the exogenous nucleic acid may be expressed in a variety of ways within the host cell. A variety of assays can be performed to confirm the presence of the recombinant DNA sequence. Such assays include, for example, Southern and Northern blotting, RT-PC "Molecular biology" assays well known to those skilled in the art, such as R and PCR; immunological means, for example (ELISA and Western blot), or peptide or protein or Assays as described herein for identifying nucleic acids that fall within the scope of the present invention Assays that detect the presence or absence of specific peptides can be used in "biochemical" assays. Includes B.

[0215] Viral-based delivery systems Also provided herein are viral-based delivery systems incorporating the nucleic acids of the invention. Typical viral expression vectors are adenovirus-based vectors (e.g., Cruce Adenovirus, commercially available from Ill, Inc. (Leiden, Netherlands) virus-based Per.C6 system), lentivirus-based vectors (e.g., Lif Lentivirus, manufactured by e Technologies (Carlsbad, Calif.) vector-based pLPI), and retroviral vectors (e.g., pCFB-EGSH) Examples of such viruses include, but are not limited to, herpes viruses (e.g., pFB-ERV) and herpes viruses. In one embodiment, the viral vector is a lentiviral vector. Retrovirus-derived vectors allow the transgene to persist in daughter cells for long periods of time. This allows stable integration and propagation, thus achieving long-term gene transfer. Lentiviral vectors are suitable tools for transducing non-proliferating cells such as hepatocytes. In terms of the ability to introduce vectors, vectors derived from oncoretroviruses such as murine leukemia viruses are Lentiviral vectors also have the added advantage of being less immunogenic. Generally, and in some embodiments, suitable vectors include at least one Replication origins, promoter sequences, and favorable restriction endonucleases functional in one organism a nucleotide sequence encoding ... brochure, WO 01 / 29058 brochure, and U.S. Pat. No. 6,193).

[0216] In several embodiments, a lentiviral vector is provided that comprises a backbone and a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a CD3 zeta signaling domain. Optionally, the vector comprises a truncated form. epithelium Further included are nucleic acids encoding growth factor receptors (HER1t or HER1t), CD20t-1 or full-length CD20.

[0217] In some cases, the skeleton and (1) truncated epithelium Provided is a vector comprising a nucleic acid sequence encoding a growth factor receptor, e.g., HER1t or HER1t-1, or a functional variant thereof; and (2) a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a CD3 zeta signaling domain.

[0218] Optionally, the backbone and (1) full-length CD20, truncated CD20, or both and (2) a nucleic acid sequence encoding a chimeric antigen receptor (CAR). (a) a MUC16 antigen-binding domain; (b) a stoichiometric vector comprising a CAR; (c) the transmembrane domain; (d) 4-1BB or CD28, or both. (e) a co-stimulatory signaling domain containing a CD3 zeta signaling domain; and (f) a CD3 zeta signaling domain containing a CD3 zeta signaling domain. Hmm, a vector is presented.

[0219] In embodiments, the nucleic acid encoding the MUC16-specific CAR is cloned into a vector comprising a lentiviral backbone component. Exemplary backbone components include, but are not limited to, pFUGW and pSMPUW. The pFUGW lentiviral vector backbone is a self-inactivating (SIN) lentiviral vector backbone in which unnecessary HIV-1 viral sequences have been removed, resulting in a reduced potential for tumorigenesis, deleterious mutations, and replication of infectious particles. In embodiments, the vector encoding the MUC16 CAR also encodes mbIL-15 within a single construct. In embodiments, the MUC16 CAR and mbIL-15 are encoded on two separate lentiviral vectors. In some embodiments, mbIL-15 is a truncated form epithelium It is expressed together with a growth factor receptor tag. In several embodiments, the MUC16 CAR can be co-expressed with mbIL-15 and a cell tag from a single lentiviral vector. In a further embodiment, the MUC16 CAR can be under the control of an inducible promoter. In another embodiment, the mbIL-15 can be under the control of an inducible promoter. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible, two-polypeptide, ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch.

[0220] In one embodiment, a MUC16 CAR described herein comprises an anti-MUC16 scFv, a human CD8 hinge and transmembrane domain, and a human 4-1BB and CD3 zeta signaling domain. In another embodiment, a MUC16 CAR of the invention comprises an anti-MUC16 scFv, a human CD8 hinge and transmembrane domain, a human 4-1BB and CD3 zeta signaling domain, and, optionally, a truncated epithelium

[0013] The vectors include growth factor receptor (HER1t or HER1t-1) tags. Other suitable vectors include integrating expression vectors, which may integrate randomly into the DNA of a host cell or may contain recombination sites that allow specific recombination between the expression vector and the host cell chromosome. Such integrating expression vectors can use endogenous expression control sequences of the host cell chromosome to effect expression of the desired protein. Examples of site-specific integrating vectors include, for example, the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNA™5 / FRT) or components of the cre-lox system, such as the cre-lox system found in pExchange-6 Core Vectors from Stratagene (LaJolla, Calif.). Examples of vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T antigen) from Invitrogen (Carlsbad, Calif.) and pCI or pFN10A(ACT)FLEXI™ from Promega (Madison, Wis.). Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, promoter elements are located within the region 30 to 110 bp upstream of the start site, but many promoters have recently been shown to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is preserved even when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can increase to a distance of 50 bp before activity begins to decline. Individual elements may function cooperatively or independently to activate transcription, depending on the promoter.

[0221] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter. This promoter sequence is a promoter sequence that can be used to express any polynucleotide sequence operably linked to it. A strong constitutive promoter capable of driving high level expression of the nucleic acid sequence - It is an array.

[0222] Another example of a suitable promoter is human elongation growth factor 1 alpha 1 (hEF1a1). In embodiments, the vector constructs comprising the CAR described herein are derived from hEF Contains 1a1 functional variants.

[0223] However, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus ( MMTV), human immunodeficiency virus (HIV) LTR (long terminal repeat epeat promoter, MoMuLV promoter, avian leukosis virus promoter -, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter , as well as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, Other constitutive promoter sequences, including but not limited to the promoter, may also be used. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters, as previously described, are also contemplated as part of the present invention. The use of an inducible promoter allows for the expression of the gene to be operably linked when such expression is desired. to turn on expression of the ligated polynucleotide sequence or to turn off expression when expression is not desired. , which provides a molecular switch that can turn expression off. Examples of inducible promoters are: Metallothionein promoter, glucocorticoid promoter , progesterone promoter, and tetracycline promoter, In one embodiment, the inducible promoter is a ligand-inducible promoter of a gene switch. In some cases, the inducible promoter is RHEOSWITCH ( Small molecule ligand-induced, two-polypeptide gene switches, such as It may be an ecdysone receptor-based gene switch.

[0224] The expression vector introduced into cells to assess the expression of the CAR or portions thereof described herein may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells desired to be transfected or infected via a viral or non-viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as the neomycin resistance gene (neo) and the ampicillin resistance gene. In some embodiments, truncated forms epithelium A growth factor receptor (HER1t or HER1t-1) tag can be used as a selectable marker gene.

[0225] The reporter gene is used to identify potentially transfected cells and to identify the regulatory sequences. Reporter genes can be used to assess the functionality of a gene. not present in or expressed by the organism or tissue of the spirient, A polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. The DNA is then transferred to the recipient cell, where it is then inserted into the recipient cell. At each time point, the reporter gene expression is assayed. The enzymes used in the assay include luciferase, beta-galactosidase, and chloramphenicol acetyltransferase. a gene encoding secreted alkaline phosphatase, or green fluorescent protein Protein genes (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or purchased commercially. Generally, the smallest 5' fragment that gives the highest level of reporter gene expression is used. The construct with the linking region is identified as a promoter. The region can be linked to a reporter gene and the drug can be used to induce promoter-driven transcription. can be used to assess the ability to modulate

[0226] In embodiments, the viral vectors described herein are capable of directing the expression of a transgene. hEF1a1 promoter driving expression, bovine growth hormone poly(A) sequence enhancing transcription, In addition to the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), pFUGW plasmid It contains LTR sequences derived from the smid.

[0227] Methods for introducing and expressing genes into cells are well known. In the context of expression vectors, the vector The target can be cultured in a host cell, e.g., a mammalian cell, a bacterial cell, or the like, by any method known in the art. The vectors can be easily introduced into cells, yeast cells, or insect cells. The vector can be introduced into the host cell by physical means or by chemical means. It can also be introduced by biological means.

[0228] Physical methods for introducing polynucleotides into host cells, e.g., immune effector cells. The methods include calcium phosphate precipitation, lipofection, gene gun, and microinjection. These include vector and / or exogenous vector transfer techniques, electroporation, etc. Methods for generating cells containing nucleic acids are well known. See, for example, Sambrook et al. (Molecule ar Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)) In some embodiments, the polynucleotide is The methods include calcium phosphate transfection or polyethyleneimine (PEI) In some embodiments, the introduction of a polynucleotide into a host cell is The method for introduction is electroporation.

[0229] To introduce a polynucleotide of interest into a host cell, e.g., an immune effector cell, Biological methods for this purpose include the use of DNA and RNA vectors. Vectors, and especially retroviral vectors, are vectors that deliver genes to mammalian cells, e.g., human Other viral vectors have become the most widely used method for inserting the vector into mouse cells. - Lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated viruses, etc. See, e.g., U.S. Patent No. 5,350,674. See the specification and US Pat. No. 5,585,362.

[0230] Chemical means for introducing polynucleotides into host cells include polymer complexes, nanoparticles, and Capsules, microspheres, beads, as well as oil-in-water emulsions, micelles, and mixed emulsions cells, and colloidal dispersion systems such as lipid-based systems including liposomes. Exemplary colloids for use as delivery vehicles in vitro and in vivo The liquid-like system is a liposome (eg, an artificial membrane vesicle).

[0231] When a viral delivery system is used, an exemplary delivery vehicle is a liposome. Introduction into cells (in vitro, ex vivo, or in vivo) In another embodiment, the nucleic acid can be associated with a lipid. The lipid-associated nucleic acid is dispersed in the aqueous liposome, interspersed within the lipid bilayer of the liposome. It can also be encapsulated and associated with both liposomes and oligonucleotides. It can also be attached to liposomes via a linking molecule and incorporated into the liposomes. It can be complexed with liposomes or dispersed in a solution containing lipids. It can be mixed with lipids, can be combined with lipids, can be suspended in lipids It can be contained as a micelle or can be contained or complexed with a micelle. or otherwise associated with lipids. Compositions related to the present vectors are not limited to any particular structure in solution. For example, The composition may also exist in a bilayer structure as a micelle, forming a "collapsing" structure. The composition may also be simply dispersed in a solution. They may also form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic. In addition to lipid droplets generated within the cytoplasm, long-chain aliphatic hydrocarbons, as well as fatty acids and amino acids, alcohols, amines, amino alcohols, and aldehydes, as well as their derivatives. This includes classes of compounds.

[0232] Lipids suitable for use can be purchased from commercial sources. Dimethylpropional phospholipid ("DMPC") was purchased from Sigma, St. Louis, Mo. Dicetyl phosphate ("DCP") is available from K & K Laboratories Cholesterol ("Ch") can be purchased from Pharmacia (Plainview, NY). ol") is available from Calbiochem-Behring, and Jimilis Dimethylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Pharmaceuticals. It can be purchased from Color Lipids, Inc. (Birmingham, Ala.). Stock lipid solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol, so it is the only "Liposome" means an encapsulated lipid bilayer or lipid aggregate. In general, the present invention encompasses a variety of single lipid vehicles and multi-layer lipid vehicles formed by the creation of Liposomes are vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes can be characterized as having: Multilamellar liposomes have multiple lipid layers formed by suspending phospholipids in an excess amount of aqueous solution. The lipid components undergo a self-transition before forming the closed structure. , which traps water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, there are also compositions in solution that have structures different from the normal vesicle structure. For example, lipids may take on a micelle structure, and the lipid molecules may form heterogeneous structures. Lipofectamine-nucleic acid complexes are also envisioned. do.

[0233] Cells containing MUC16 CAR and vector This paper provides engineered cells that express the CAR described herein. In certain embodiments, the engineered cells described herein are immune effector cells. In some embodiments, this paper provides backbone and (1) truncated CAR. epithelium Immune effector cells are presented that comprise a vector comprising a nucleic acid sequence encoding a growth factor receptor (HER1t or HER1t1) and (2) a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a CD3 zeta signaling domain.

[0234] In certain embodiments, the present invention provides an immune effector cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; (e) a CD3 zeta signaling domain; and (f) a truncated epithelium Immune effector cells containing growth factor receptors (HER1t or HER1t1) are presented.

[0235] In embodiments, the present disclosure provides: (1) a cell tag for use as a kill switch; (2) a chimeric antigen receptor; an immune effector cell comprising a receptor (CAR), wherein the CAR (a) binds to a MUC16 antigen; (b) stalk domain; (c) transmembrane domain; (d) 4-1BB (e) a costimulatory signaling domain containing CD3 or CD28, or both; and In some embodiments, immune effector cells are presented that include a .alpha.-transduction domain. where the cell tag is HER1t, HER1t1, CD20t-1 or CD20.

[0236] In embodiments, immune effector cells include T cells, natural killer (NK) cells, and the like. In some embodiments, the target cells are T cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. The cells exhibit anti-tumor activity when the MUC16 antigen-binding domain binds to MUC16. vinegar.

[0237] Engineered immune effector cells The present invention relates to a method for producing a heterologous vector comprising: The immune effector cells modified with MUC16 C as described herein are presented. AR and a few of the HER1t, HER1t1, CD20, and CD20t-1 tags Immune effector cells engineered to express at least one of the following are presented. MUC16 CAR, mbIL-15m and HER1t, H disclosed herein The cells were designed to express at least one of the following tags: ER1t1, CD20, and CD20t-1. The modified immune effector cells are presented as follows.

[0238] As used herein, "T cell" or "T lymphocyte" refers to a cell-mediated immune response. T cells or T lymphocytes are a type of lymphocyte that play a central role in B cells and natural killer cells due to the presence of T cell receptors (TCR) on their cell surface They can be distinguished from other lymphocytes, such as NK cells.

[0239] In some embodiments, the engineered immune effector cells are T cells and / or natural T cells or T lymphocytes are modified immune cells, including killer cells. Exemplary T cells include helper T cells, cytotoxic T cells, and T H 17 cells, stem cell memory T cells (TSCM), naive T cells, memory T cells , effector T cells, regulatory T cells, or natural killer T cells.

[0240] "Helper T cells" (T H cells) regulate the development of B cells into plasma cells and memory B cells. in immune processes, including maturation, and activation of cytotoxic T cells and macrophages, Support other white blood cells. In some cases, T H The cells express the CD4 glycoprotein on their surface. Due to their expression, they are known as CD4+ T cells. Helper T cells act as antigen-presenting cells ( Peptide antigens are presented by MHC class II molecules expressed on the surface of APCs Once activated, helper T cells divide rapidly and mediate an active immune response. secrete small proteins called cytokines that inhibit or support the growth of These cells are called T H 1. T H 2. T H 3. TH 17, T H 9, or T FH Including There are several subtypes, including those that secrete different cytokines and induce different types of immune responses. Signaling from APCs facilitates differentiation into one of several subtypes. , directing T cells toward specific subtypes.

[0241] "Cytotoxic T cells" (TC cells or CTLs) are capable of targeting virus-infected cells and tumor cells. These cells also have a number of markers on their surface that destroy the nuclei of the nuclei and are involved in graft rejection. These cells express the CD8 glycoprotein and are therefore known as CD8+ T cells. These cells react with antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Regulatory T cells recognize their targets by binding to IL-1. Through ATP, adenosine, and other molecules, CD8+ cells are inducing a state of anergy. This may activate the immune system, thereby preventing autoimmune diseases.

[0242] "Memory T cells" are antigen-specific T cells that persist for a long time after the infection has cleared. Memory T cells are a subset of T cells that respond to their cognate antigen upon re-exposure. rapidly expand into large numbers of effector T cells, which provide the immune system with a means to Memory T cells are a subtype of stem cell memory T cells. (TSCM), central memory T cells (TCM cells) and two types of effector Memory cells include CD4+ cells and T cells (TEM cells and TEMRA cells). Memory T cells can be either CD8+ or CD8+ cells. They may express CD45RO, CD45RA, and / or CCR7.

[0243] "Regulatory T cells" (Treg cells), formerly known as suppressor T cells, ) play a role in maintaining immune tolerance. The primary role of regulatory T cells is to regulate T cell-mediated immune responses. By blocking the immune response, autoreactive T cells escape the negative selection process in the thymus. The aim is to suppress cells.

[0244] "Natural killer T cells" (NKT cells) act as a bridge between the adaptive immune system and the innate immune system. Conventional antibodies recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. Unlike normal T cells, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. Upon activation, these cells are attributed to both Th and Tc cells. functions (i.e., production of cytokines and release of cytolytic / cell-killing molecules) NKT cells also recognize some tumor cells and cells infected with herpes viruses. It is also possible to recognize and eliminate these.

[0245] Natural killer (NK) cells are a type of cytotoxic lymphocyte of the innate immune system. In some cases, NK cells act as a frontline defense against viral infection and / or tumor formation. NK cells detect MHC presented on infected or cancerous cells. It is possible to induce cytokine release, followed by lysis and apoptosis. NK cells also induce stress cells in the absence of antibody and / or MHC. It is possible to detect this, thereby enabling a rapid immune response.

[0246] Dose of engineered immune effector cells In some embodiments, a quantity of engineered immune effector cells is administered to a subject in need thereof. The dose is determined based on the efficacy and potential for inducing cytokine-associated damaging effects. In some cases, the amount of engineered immune effector cells is determined based on the potential for the cell to be administered per kg of Approximately 10 engineered immune effector cells 2 ~about 10 9 Optionally, modified immune fusion The amount of engineered immune effector cells is approximately 10 per kg. 3 ~about 10 9 Pieces In some cases, the amount of engineered immune effector cells is in units of 1 kg of engineered immune effector cells. Approximately 10 4 ~about 10 9 Optionally, optionally, modified immune effects The amount of engineered immune effector cells is approximately 10 per kg. 5 ~about 10 9 Includes In some cases, the amount of engineered immune effector cells is 100 mg / kg of engineered immune effector cells. Approximately 10 5 ~about 10 8 In some cases, the amount of engineered immune effector cells includes , approximately 10 engineered immune effector cells per kg 5 ~about 10 7 Including pieces. , the amount of engineered immune effector cells is approximately 10 engineered immune effector cells per kg. 6 ~about 10 9 In some cases, the amount of engineered immune effector cells may include Approximately 10 engineered immune effector cells 6 ~about 10 8 Optionally, modified immune effects The amount of engineered immune effector cells is approximately 10 per kg.7 ~about 10 9 Includes In some cases, the amount of engineered immune effector cells is 100 mg / kg of engineered immune effector cells. Approximately 10 5 ~about 10 6 In some cases, the amount of engineered immune effector cells includes , approximately 10 engineered immune effector cells per kg 6 ~about 10 7 Including pieces. , the amount of engineered immune effector cells is approximately 10 engineered immune effector cells per kg. 7 ~about 10 8 In some cases, the amount of engineered immune effector cells may include Approximately 10 engineered immune effector cells 8 ~about 10 9 Optionally, modified immune effects The amount of engineered immune effector cells is approximately 10 per kg. 9 Includes pieces. Therefore, the amount of engineered immune effector cells is approximately 1 kg of engineered immune effector cells. 10 8 In some cases, the amount of engineered immune effector cells may include 10 ... Immune effector cells approximately 10 7 Optionally, the amount of engineered immune effector cells Approximately 10 engineered immune effector cells per kg 6 In some cases, modified The amount of engineered immune effector cells is approximately 10 engineered immune effector cells per kg. 5 Contains .

[0247] In some embodiments, the CAR-T cells are MUC16-specific CAR-T cells. By combining these, the amount of MUC16-specific CAR-T cells was approximately 1000 CAR-T cells per kg. 10 2 ~about 109 In some cases, the amount of MUC16-specific CAR-T cells comprises 1 Approximately 10 CAR-T cells per kg 3 ~about 10 9 In some cases, MUC16 The amount of allogeneic CAR-T cells was approximately 10 CAR-T cells per kg. 4 ~about 10 9 Contains In some cases, the amount of MUC16-specific CAR-T cells is determined based on the number of CAR-T cells per kg of About 10 cells 5 ~about 10 9 In some cases, the amount of MUC16-specific CAR-T cells , approximately 10 CAR-T cells per kg 5 ~about 10 8 In some cases, MUC1 The amount of 6-specific CAR-T cells was approximately 10 CAR-T cells per kg. 5 ~about 10 7 Pieces In some cases, the amount of MUC16-specific CAR-T cells is 100 mg / kg of CAR-T cells. T cells approximately 10 6 ~about 10 9 Optionally, MUC16-specific CAR-T cells The amount is approximately 10 CAR-T cells per kg. 6 ~about 10 8 In some cases, MU The amount of C16-specific CAR-T cells was approximately 10 CAR-T cells per kg. 7 ~about 10 9 In some cases, the amount of MUC16-specific CAR-T cells includes 100 mg of CAR-T cells per kg of RT cells approx. 10 5 ~about 10 6 Optionally, MUC16-specific CAR-T cells The amount of cells is approximately 10 CAR-T cells per kg. 6 ~about 10 7 Including pieces. The amount of MUC16-specific CAR-T cells was approximately 10 CAR-T cells per kg. 7 ~about 1 0 8 In some cases, the amount of MUC16-specific CAR-T cells may include CAR-T cells approximately 10 8 ~about 10 9 In some cases, MUC16-specific CARs -T cell dose is approximately 10 CAR-T cells per kg 9 In some cases, M The amount of UC16-specific CAR-T cells was approximately 10 CAR-T cells per kg. 8 Contains In some cases, the amount of MUC16-specific CAR-T cells is 100 mg / kg of CAR-T cells. about 10 cells 7 In some cases, the amount of MUC16-specific CAR-T cells comprises 1 kJ Approximately 10 CAR-T cells per g 6 In some cases, MUC16-specific CARs -T cell dose is approximately 10 CAR-T cells per kg 5 In some cases, M The amount of UC16-specific CAR-T cells was approximately 10 CAR-T cells per kg. 4 Contains In some cases, the amount of MUC16-specific CAR-T cells is 100 mg / kg of CAR-T cells. about 10 cells 3 In some cases, the amount of MUC16-specific CAR-T cells comprises 1 kJ Approximately 10 CAR-T cells per g 2 Includes pieces.

[0248] Source of immune effector cells In certain aspects, the embodiments described herein provide redirected, antigen-specific allogeneic immune effector cells (e.g., T cells, Treg cells, NK cells, or NK T cells) A method for producing and / or expanding a cell (a CAR), the method comprising: Transfected with an expression vector containing a DNA (or RNA) construct encoding The cells are then optionally transfected with feeder cells, recombinant antigens, or receptors. In certain aspects, the method includes stimulating the cells with an antibody to cause cell proliferation. Cells (or cell populations) engineered to express CARs include hepatocytes, iPS cells, i T cells, immune effector cells, or precursor cells of these cells differentiated from PS cells be.

[0249] Sources of immune effector cells can include both allogeneic and autologous sources. By combining these cells, immune effector cells can be generated from stem cells or induced pluripotent stem cells (iPSCs). Thus, cells for manipulation according to embodiments may be derived from umbilical cord blood, They can be isolated from peripheral blood, human embryonic stem cells, or iPSCs. For example, allogeneic T cells The cells are engineered to contain a chimeric antigen receptor (and, optionally, to lack a functional TCR). In some embodiments, the immune effector cells can be engineered to be human peripheral blood mononuclear cells. Primary human T cells, such as T cells derived from PBMCs. PBMCs are derived from peripheral blood. They can also be harvested from the bone marrow after stimulation with G-CSF (granulocyte colony-stimulating factor). Alternatively, they can be collected from umbilical cord blood. In one embodiment, the immune effector cells are pan-T cells. After transfection or transduction (e.g., of a CAR expression construct), cells The cells can be immediately transfused or cryopreserved. After transfection or transduction, the cells are maintained in IL-2 and IL-1 until ready for infusion. The cells may be stored in a cytokine bath that may contain IL-1 and / or IL-21. So, after transfection, the cells are about 1, 2, 3, 4, Within 5 days or more, ex vivo, as a bulk population, The cells can be propagated over a period of days, weeks, or months. After transfection, the transfected cells are cloned and integrated or or the presence of a single expression cassette or plasmid maintained episomally, and Clones that support expression of the chimeric antigen receptor are expanded ex vivo. The clones selected for expansion specifically recognize target cells expressing the antigen. The recombinant T cells bind IL-2 or the common gamma chain, confirming their ability to lyse the T cells. Other cytokines (e.g., IL-7, IL-12, IL-15, IL-21, and others) Recombinant T cells can be expanded by stimulation with cytokines (such as They can be expanded by stimulation with antigen-presenting cells. It can also be expanded in contact with presenting cells and crosslinks CD3 on the surface of T cells. They can also be expanded with antibodies such as KT3. Further selection can be performed using nanobead-based isolation and / or fluorescence-activated cell sorting techniques. In a further embodiment, the genetic material can be selected from the AaPCs and further cultured with the AaPCs. The genetically modified cells can be cryopreserved.

[0250] T cells also reside in bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, and the peritoneal It can also be obtained from a number of sources, including water, pleural effusions, splenic tissue, and tumors. In certain embodiments, any number of T cell lines available in the art can be used. In certain embodiments of the present disclosure, T cells can be cultured using Ficoll (registered trademark). Blood collected from the subject using any number of techniques known to those skilled in the art, such as (i) ELISA (Immunoglobulin) or (ii) ELISA (Immunoglobulin) or (iii) ELISA (Immunoglobulin) or ... In some embodiments, cells derived from the circulating blood of an individual can be obtained from: The apheresis product typically contains T cells, monocytes, granulocytes, and The nucleated white blood cells (WBCs) contain lymphocytes, including B cells, other nucleated white blood cells, red blood cells, and platelets. In this embodiment, the cells collected by apheresis are washed to remove the plasma fraction, and the cells are For the processing steps in the term, in an appropriate buffer or medium. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In some cases, the cleaning solution is calcium-deficient and in some, but not all, magnesium-deficient. In some cases, the cells lack many divalent cations. As one skilled in the art will readily appreciate, washing steps can be used to enhance the activity of the product. Use a semi-automatic "flow-through" centrifuge (e.g., Cobe 29) according to the manufacturer's instructions. 91 Cell Processor, Baxter CytoMate, or Haemonetic This can be achieved by methods known to those skilled in the art, such as using Cell Saver 5). After washing, the cells can be resuspended in, for example, Ca2+-free PBS, Mg2+-free PB S, Plasmalyte A, or other saline solutions with or without buffers The cells can be resuspended in a variety of biocompatible buffers, such as ethanol solution. Remove unwanted components of the erythesis sample and resuspend the cells directly in culture medium. It is also possible to do so.

[0251] In another embodiment, the centrifugation may be performed using, for example, a PERCOLL® gradient, or By counterflow centrifugal elutriation, red blood cells are lysed and monocytes are depleted, thereby isolating T cells. Isolated from peripheral blood lymphocytes. CD3+, CD28+, CD4+, CD8+, CD45R Specific subpopulations of T cells, such as CD45A+ and CD45RO+ T cells, can also be identified using positive or negative selection methods. In another embodiment, CD14+ cells can be further isolated by sexual selection. For example, in one embodiment, T cells are depleted from the T cell population by injecting DYNABEADS ( anti-CD3 / anti-CD28 (i.e. , 3×28) with conjugated beads for a time sufficient for the desired T cell positive selection. Isolation is performed by incubation. In one embodiment, the time is about 30 minutes. In other embodiments, the time period is from 30 minutes to 36 hours, or longer, and In a further embodiment, the time is at least In yet another embodiment, the time is 10 to 20 minutes. In one embodiment, the incubation time is 4 hours. In another embodiment, the incubation time is 24 hours. Use of long incubation times, such as 24 hours, for isolation of T cells from patients with Tumor-infiltrating lymphocytes (TILs) can be isolated from tumor tissue or from immunocompromised individuals. In any situation where T cells are in low numbers compared to other cell types, such as in the isolation of ILs In this case, longer incubation times can be used to isolate T cells. Furthermore, the use of longer incubation times also increases the efficiency of CD8+ T cell capture. Therefore, simply by shortening or lengthening the time, T cells can be induced to differentiate into CD4+ T cells. 3 / CD28 beads and / or the beads can be directed against T cells. By increasing or decreasing the ratio (as further described herein) of T A subpopulation of cells may be preferentially screened for positive or negative results at the beginning of the culture or at other points during the process. In addition, antibodies against CD3 and CD4 on beads or other surfaces can be used to selectively or negatively select. T cell subpopulations were targeted by increasing or decreasing the ratio of anti-CD28 and / or anti-CD29 antibodies. Preferentially select positively or negatively at the initiation of culture, or at other desired time points. Those skilled in the art will appreciate that multiple rounds of selection may also be used in the context of the present invention. It will be appreciated that in certain embodiments, a selection procedure may be performed to identify "selected" It may also be desirable to use "uninfected" cells in the activation and expansion steps. The "unselected" cells can also be subjected to further rounds of selection. Cut.

[0252] Enrichment of T cell populations by negative selection directed against surface markers unique to negatively selected cells This can be achieved by combining antibodies that are present on negatively selected cells. A cocktail of monoclonal antibodies directed against specific cell surface markers was used to identify cell types. Selection via negative magnetic immunoadhesion or flow cytometry For example, monoclonal antibody citrates can be used to enrich for CD4+ cells by negative selection. The cells typically express CD14, CD20, CD11b, CD16, HLA-DR, and C In certain embodiments, the antibody is typically CD4+, CD25+, Regulatory T cells expressing CD62Lhi, GITR+, and FoxP3+ were enriched or isolated. Alternatively, in certain embodiments, it may be desirable to positively select for regulatory T cells. Cells can be depleted by anti-CD25 conjugated beads or other similar selection methods. can.

[0253] To isolate the desired cell population by positive or negative selection, the concentration and The surface (e.g., particles such as beads) can be varied. The beads and cells were mixed together in a significantly reduced volume (i.e., the cell concentration was It may be desirable to increase the number of beads to ensure maximum contact between the cells and the beads. For example, In one embodiment, a concentration of 2 billion cells per ml is used. A concentration of 1 billion cells per liter is used. In a further embodiment, 1 billion cells per ml In a further embodiment, more than 10 million cells per ml are used. , 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 450 In yet another embodiment, a cell concentration of 100,000,000, or 50 million cells per ml is used. 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells In a further embodiment, a cell concentration of 125 million or more cells per ml is used. A concentration of 150 million cells may be used. The use of high concentrations may improve cell yield, cell activation, Furthermore, the use of high cell concentrations can result in increased cell proliferation and expansion. CD28-negative T cells, or many tumor cells (i.e., leukemic blood, tumor tissue, etc.) The more cells that may express the target antigen of interest at a low level, such as cells from samples where This allows for more efficient capture of cells. Such cell populations may have therapeutic value. For example, the use of high cell concentrations may be beneficial in cases where CD28 expression is normally low. This allows for more efficient selection of CD8+ T cells.

[0254] In related embodiments, it may be desirable to use low concentrations of cells. By significantly diluting the mixture of particles (e.g., particles such as beads) This minimizes interactions between the cells expressing high amounts of the desired antigen that bind to the particles. For example, CD4+ T cells express high levels of CD28 and are selected for CD8 + T cells are captured more efficiently than + T cells. In one embodiment, the cell concentration used is In other embodiments, the concentration used is 5 x 10 cells per ml. Approximately 1 x 10 cells to 1 x 10 cells per ml, and any integer value between these It is possible.

[0255] In another embodiment, the cells are grown at varying speeds and lengths at 2-10°C or at room temperature. The mixture can be incubated on a rotator for a period of time to allow the mixture to separate.

[0256] The stimulated T cells can also be frozen after a washing step. After a washing step to remove platelets, the cells can be suspended in a freezing solution. Many freezing solutions and freezing parameters are known in the art and are also used in this context. One method that may be useful is to use a solution containing 20% ​​DMSO and 8% human serum albumin. PBS with 10% dextran 40 and 5% dextrose, or 20% of human serum albumin and 7.5% DMSO, or 0.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum Albumin and 7.5% DMSO, or, for example, Hespan and Plas The cells were then frozen in a suitable medium containing malyte A. , frozen to -80°C at a rate of 1°C per minute in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used, as well as storage at -20°C or in liquid nitrogen. Rapid, uncontrolled freezing may also be used. In certain embodiments, the cryopreserved cells are Thawed, washed, and then activated using the methods of the present invention as described herein. , and let rest at room temperature for 1 hour.

[0257] In certain embodiments, the time points at which expanded cells may be needed, as described herein, are also included. Also provided is the collection of a blood sample or apheresis product from a subject at an earlier time. Therefore, the source of cells to be expanded can be harvested at any desired time point. and the desired cells, such as T cells, can be transfected into T cells, such as in T cell therapy, as described herein. Subsequent T cell therapy for any number of diseases or conditions that would benefit from T cell therapy It can be isolated and frozen for use. In one embodiment, a blood sample or The eresis is generally taken from a healthy subject. In certain embodiments, a blood sample or Apheresis is generally used by people who are at risk of developing disease but have not yet developed the disease. The cells are collected from healthy subjects without any disease, and the cells of interest are isolated and frozen for subsequent use. In certain embodiments, the T cells are expanded, frozen, and used at a later time point. In certain embodiments, the sample can be obtained from a patient using the methods described herein. The patient is collected shortly after diagnosis of the particular disease in question, but before any treatment. In some embodiments, the cells are treated with natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, or the like. radiation, cyclosporine, azathioprine, methotrexate, mycophenolate, and and FK506, immunosuppressants, antibodies, or CAMPATH, anti-CD3 antibodies, Cytoxa cyclosporine, FK506, rapamycin, mycophenolate, Treatment with drugs such as steroids, FR901228, and other immunosuppressive agents such as irradiation Prior to any number of reasonable treatment modalities, including but not limited to, These drugs are isolated from blood samples or apheresis. Inhibits calcineurin (cyclosporine and FK506), a catalyzer or inhibiting p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin). (Liu et al., Cell 66:807-815, (1991), Henderson et al., Immun 73:316-32 1, (1991), Bierer et al., Curr. Opin. Immun 5:763-773, (1993). Further embodiments In this condition, cells are isolated for the patient and administered with other drugs such as bone marrow or stem cell transplants, fludarabine, etc. chemotherapy, external beam radiation therapy (XRT), cyclophosphamide, or OKT3 or T cell depletion therapy using antibodies such as CAMPATH (e.g., At the same time, or subsequently, the cells are frozen for later use. The cells are pre-isolated and treated with a drug that reacts with CD20, such as B12, B22, B32, B42, B52, B62, B72, B82, B9 ... They can be frozen for subsequent use for treatment after cell apheresis.

[0258] In a further embodiment of the invention, the T cells are obtained directly from the patient after treatment. In this regard, after certain cancer treatments, particularly treatments with drugs that impair the immune system, patients The T cells obtained after treatment, usually immediately after treatment, during the recovery period from treatment. Cell quality may be optimal or their ability to expand ex vivo may be compromised. It has been observed that improvements in strength can be achieved using the methods described herein. After ex vivo manipulation, these cells have demonstrated prolonged engraftment and in vivo This may be a favorable condition for expansion in vivo. During this recovery period, blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, are Furthermore, in certain embodiments, mobilization (e.g., GM-C In subjects using a SF mobilization regimen and conditioning regimen , specifically, repopulation, recirculation of specific cell types within a defined time window after treatment. Conditions can be created that favor cell proliferation, regeneration, and / or proliferation. include T cells, B cells, dendritic cells, and other cells of the immune system.

[0259] T cell activation and expansion T cells, whether before or after being engineered to express a CAR as described herein, Cells are generally cultured as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,000; Specification No. 55; Specification No. 6,905,680; Specification No. 6,692,964; Specification No. 6,692,964; Specification No. 5,858,358; Specification No. 6,887,466; Specification No. 6,905,6 Specification No. 81; Specification No. 7,144,575; Specification No. 7,067,318; Specification No. 7,172,869; Specification No. 7,232,566; Specification No. 7,175,8 Specification No. 43; Specification No. 5,883,223; Specification No. 6,905,874; Nos. 6,797,514; 6,867,041; and U.S. patent applications Activated using the method described in Publication No. 20060121005 This allows it to expand and multiply.

[0260] Generally, T cells as described herein are those that express signals associated with the CD3 / TCR complex. A surface conjugated with a stimulating agent and a ligand that stimulates costimulatory molecules on the surface of T cells In particular, the T cell population is expanded by contacting the T cell population with a T cell antigen as described herein. and an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD3 antibody immobilized on a surface. 2. Protein kinase C activation by contact with antibodies or calcium ionophores T cell surface can be stimulated by contact with factors (e.g., bryostatin). Use a ligand that binds to an accessory molecule to costimulate the accessory molecule above For example, a population of T cells may be stimulated with anti-CD40 antibodies under conditions appropriate to stimulate T cell proliferation. The CD4+ T cells or CD8+ T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody. To stimulate T cell proliferation, they can be contacted with anti-CD3 and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), as well as other methods commonly known in the art. can be used (Berg et al., Transplant Proc. 30(8):3975-3977, (1998), Haa nen et al., J. Exp. Med. 190(9):13191328, (1999), Garland et al., J. Immunol Met h. 227(1-2):53-63, (1999)).

[0261] In certain embodiments, the primary and costimulatory signals for the T cells are: Each signal is produced by a different protocol. For example, the agent producing each signal is In some cases, the surface is coupled to the substrate. If so, the agents can also be coupled to the same surface (i.e., in a "cis" configuration). ), or can be coupled to separate surfaces (i.e., a "trans" configuration). Alternatively, one agent can be coupled to a surface and the other agent can be in solution. In one embodiment, an agent that provides a costimulatory signal is bound to the cell surface and stimulates primary The agent that provides the activation signal is either in solution or coupled to a surface. In certain embodiments, both agents are in solution. It can be in a soluble form and then binds to an Fc receptor or antibody, or a drug. The antibody is crosslinked to the surface of a cell or the like that expresses another binding agent that binds to the antibody. , artificial antigen-presenting cells (a) envisioned for use in T cell activation and expansion APC) is described, for example, in U.S. Patent Application Publication No. 20040101519 and See also the specification of the same patent application, 20060034810.

[0262] In one embodiment, the two agents are on the same bead, i.e., "cis," or are immobilized on separate beads, i.e., "trans," on the beads. Specifically, the agent that provides the primary activation signal is an anti-CD3 antibody or its antigen. The binding fragment and agent that provides the costimulatory signal may be an anti-CD28 antibody or its antigen-binding fragment. In one embodiment, both drugs are co-immobilized to the same beads with equal molecular weights. In this form, CD4+ T cell expansion and T cell proliferation are achieved by binding to beads. In certain embodiments of the invention, a 1:1 ratio of each antibody bound to beads is used. The ratio of anti-CD3:CD28 antibodies used was compared to the expansion observed using a 1:1 ratio. In one particular embodiment, a 1:1 ratio is used to observe increased T cell expansion. An increase of about 1 to about 3 fold is observed compared to the expansion observed using the ratio of In an embodiment, the ratio of CD3:CD28 antibodies bound to the beads is between 100:1 and 1:10. 0, and all integer values ​​therebetween. In one embodiment of the invention, the anti-CD3 antibody The more anti-CD28 antibody bound to the particles, i.e., the ratio of CD3:CD28 is In certain embodiments of the invention, the binding of anti-CD28 antibodies to beads In one particular embodiment, the ratio of anti-CD3 to anti-CD3 bound to beads is greater than 2:1. A 1:100 ratio of CD3:CD28 is used. A ratio of 1:75 of combined CD3:CD28 is used. In another embodiment, a 1:50 ratio of CD3:CD28 bound to the antibody is used. A 1:30 ratio of CD3:CD28 bound to the antibody is used. In another embodiment, a 1:10 ratio of CD3:CD28 bound to beads is used. uses a 1:3 ratio of CD3:CD28 bound to beads. The format uses a 3:1 ratio of CD3:CD28 bound to beads.

[0263] Particle to cell ratios of 1:500 to 500:1, and any integer value between these. The ratio can be used to stimulate T cells or other target cells. As can be appreciated, the particle to cell ratio may depend on the particle size relative to the target cell. For example, small beads can only bind to a small number of cells, whereas large beads can bind to a small number of cells. In certain embodiments, the binding of the cells to the particle may be accomplished by binding the particles to the cells. The ratio ranges from 1:100 to 100:1, and any integer value therebetween, and In certain embodiments, the ratio ranges from 1:9 to 9:1, including any integer value therebetween. These may also be used to stimulate T cells. Anti-CD3 / anti-CD28 coupled particles to T cells that result in stimulation of T cells is as mentioned above. As mentioned above, the values ​​can vary, but certain values ​​are 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1: 2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10 :1, and 15:1, with one ratio being at least 1:1 of T particles to cells. In one embodiment, the ratio is 1:1 or less. In one particular embodiment, a particle to cell ratio of less than: In a further embodiment, the ratio of particles to cells varies depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 1:1 on day 1. 0:1 and then inject additional particles into the cells daily for up to 10 days. Add cells every other day to achieve a final ratio of 1:1 to 1:10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on the first stimulation day. and adjusted to 1:5 on the third and fifth stimulation days. particles at a final ratio of 1:1 on day 1, and on the third and fifth stimulation days. In another embodiment, the particles are added to the cells at a ratio of 1:5 on a daily or every other day basis. The ratio was 2:1 on the first stimulation day, and 1:1 on the third and fifth stimulation days. In another embodiment, the particles are adjusted to a final ratio of 1:1 on day 1. , and 1:10 on the third and fifth stimulation days, followed daily or every other day. Those skilled in the art will appreciate that various other ratios may also be suitable for use in the present invention. In particular, the ratio will vary depending on the size of the particle and the size and type of cell. There will be.

[0264] In further embodiments described herein, immune effector cells, such as T cells, Combining with drug-coated beads, then separating the beads and cells, and then In an alternative embodiment, the drug-coated beads and the cells are mixed prior to culturing. In a further embodiment, the beads and cells are first cultured together without separation. which results in enrichment of cell surface marker ligation by applying force This leads to an increase in the amount of ATP, which induces cell stimulation.

[0265] By way of example, anti-CD3 and anti-CD28 conjugated paramagnetic beads (3x 28 beads) to ligate cell surface proteins. In one embodiment, cells (for example, 10 to 10 T cells) and 1:1 ratio, DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads or MACS (registered trademark) from Miltenyi Biotec MicroBeads) in a buffer solution, e.g., PBS (calcium and magnesium stearate). (without divalent cations such as magnesium). It can be readily understood that any cell concentration can be used. For example, is extremely rare in samples, sometimes containing only 0.01% of the sample, and Therefore, any number of cells may contain the target cells. is within the context of the present invention. In certain embodiments, the particles and cells are mixed together. By significantly decreasing the volume (i.e., increasing the cell concentration), the maximum cell-particle ratio was It may be desirable to ensure contact. For example, in one embodiment, about 10 cells per ml A concentration of 2 billion is used. In another embodiment, greater than 100 million cells per ml. In further embodiments, 10 million, 15 million, 20 million, or 30 million cells per ml are used. 0 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 500 In yet another embodiment, a concentration of 75 million cells per ml is used. , 80 million, 85 million, 90 million, 95 million, or 100 million cell concentrations are used. In further embodiments, 125 million or 150 million cells per ml The use of higher concentrations may improve cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations may result in increased proliferation of CD28-negative T cells. This allows for more efficient capture of cells that may have low expression of the target antigen of interest, such as In certain embodiments, such cell populations may have therapeutic value and may be useful in For example, the use of high cell concentrations may be beneficial in cases where CD28 expression is normally low. , CD8 + This allows for more efficient selection of T cells.

[0266] In one embodiment described herein, the mixture is refrigerated for a period of from several hours (about 3 hours) to about 14 days. or any integer value between these. The mixture can be cultured for 21 days. The beads and T cells are cultured together for about 8 days. The cells are cultured together for 2-3 days. The T cell culture period is 60 days or To exceed this, stimulation over several cycles may also be desirable. The appropriate conditions include factors necessary for growth and survival, such as serum (e.g., fetal bovine serum or human serum), interleukin 2 (IL-2), insulin, IFN-gamma, IL-4 , IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and T NF-alpha, or any other additives for cell growth known to those skilled in the art. The appropriate medium (e.g., minimal essential medium or RPMI medium 1640) may contain factors including: Other additives for cell growth include: The surfactant, plasmanate, and N-acetylcysteine ​​and 2-mercapto The media may be RPMI 1640, ethanol, or other reducing agents. AIM-V, DMEM, MEM, Alpha-MEM, F-12, X-Vivo 15 and and X-Vivo 20, Optimizer, added amino acids, sodium pyruvate, and vitamins, serum-free or adequate serum (or plasma) supplementation, or prescribed a set of hormones and / or sites in sufficient quantities for T cell proliferation and expansion Antibiotics, such as penicillin and streptomycin, may be used in laboratory cultures. The target cells are only incorporated into the cell culture that is infused into the subject. The conditions necessary to support the growth of the cells, e.g., an appropriate temperature (e.g., 37°C) and atmosphere (e.g., For example, keep them under a constant air / 5% CO2 atmosphere.

[0267] T cells exposed to varying stimulation times can exhibit different characteristics. For example, typical Blood products or apheresis peripheral blood mononuclear cell products are used to generate cytotoxic T-cell populations. There are more helper T cell populations (T) than suppressor T cell populations (TC, CD8+). H, CD4+). By stimulating the CD3 receptor and CD28 receptor, In vivo T cell expansion begins approximately 8-9 days after the initial induction of T cells, which are primarily composed of TH cells. However, after about 8-9 days, the T cell population contains an increasingly large population of TC cells. Therefore, depending on the purpose of treatment, subjects may be infused with a T cell population that contains primarily TH cells. Similarly, when antigen-specific subsets of TC cells are isolated, , it may be beneficial to expand and propagate this subset to a greater extent.

[0268] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers were also significantly fluctuates, but for the most part, fluctuates reproducibly during the course of the cell expansion process. Therefore, such reproducibility is essential for tailoring activation of T cell products for specific purposes. This allows for the ability to:

[0269] Optionally, immune effector cells (e.g., T cells) of the embodiments are administered as AaPCs ( activating and propagating cells) AaPCs are also called artificial antigen-presenting cells (AaPCs). For example, antigen-presenting cells (APCs) can be used in conjunction with the therapeutic compositions and cells of the embodiments. Useful in the preparation of therapeutic products. In one embodiment, AaPC ​​is a genetically modified K For general guidance on the preparation and use of antigen-presenting systems, see See, for example, U.S. Pat. No. 6,225,042, each of which is incorporated by reference. The details, specifications of Nos. 6,355,479, 6,362,001, and No. 6,790,662, U.S. Patent Application Publication No. 2009 / 0017000, and and International Publication No. 2009 / 0004142, and International Publication No. 2007 / 10300 See Brochure No. 9. In still further aspects of the embodiment, the genetic modification Culture of modified CAR cells stimulates the expansion of CAR-expressing immune effector cells dendritic cells or AaPCs (activating and propagating The method includes culturing genetically modified CAR cells in the presence of a CAR-specific antibody. In a further embodiment, the AaPC ​​comprises a CAR-binding antibody or antibodies expressed on the surface of the AaPC. The AaPC ​​optionally activates or costimulates T cells. The additional molecule may optionally comprise a membrane-bound Cγ cytokine. In still yet a further aspect, the AaPCs are inactivated or irradiated. or have been tested for and found to be free of infectious agents In yet a further aspect, culturing genetically modified CAR cells in the presence of AaPC contains soluble cytokines such as IL-15, IL-21, and / or IL-2 The method comprises culturing the genetically modified CAR cells in a medium at a ratio of about 10:1 to about 10:1. 1:10, approximately 3:1 to approximately 1:5, approximately 1:1 to approximately 1:3 (immune effector cells to AaPCs) ), or any range of ratios derivable therein. For example, T cells and AaPCs co-culture can be at a ratio of about 1:1, about 1:2, or about 1:3.

[0270] In one embodiment, the AaPCs may express CD137L. The antigen targeted by the CAR cell, e.g., MUC16 (full length, truncated) In other embodiments, the AaPCs may further express: In certain embodiments, Aa PCs may be immunoreactive with at least one anti-CD3 antibody, such as OKT3 and / or UCHT1. In one embodiment, AaPCs can be inactivated (e.g., irradiated) to express somatic clones. In one embodiment, AaPCs can be used to eliminate the proliferation potential of infectious agents. In some cases, the product has been examined and found to be free of this substance. Methods for producing such AaPCs are known. In one embodiment, CARs associated with AaPCs are The modified T cell population may be cultured at a ratio of about 10:1 to about 1:10, about 3:1 to about 1:5, or about 1:1 Culture at a ratio of 1:1 to approximately 1:3 (T cells to AaPCs), or any range derivable therein. For example, co-culture of T cells and AaPCs can include co-culturing at a ratio of about 1:1, about 1:2, or In one aspect, the culturing step may be performed in a ratio of about 1:3 or about 1:3. The method may further include culturing the cells with a therapeutic agent (e.g., zoledronic acid).

[0271] In one embodiment, the population of genetically modified CAR cells is immediately infused into a subject or frozen. In another embodiment, the population of genetically modified CAR cells is subjected to sequestering prior to infusion into the subject. In a further embodiment, the population of genetically modified CAR cells is maintained in a cytokine bath. , 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 42 days, 49, 56, 63, In one embodiment, the cells are cultured and / or stimulated for up to 70 days. Co-culture of genetically modified CAR T cells with AaPCs to promote proliferation of R-positive T cells In another embodiment, the population of genetically modified CAR cells is treated with one stimulation, two stimulations, or two stimulations. Stimulation 3 times, stimulation 4 times, stimulation 5 times, stimulation 5 times, stimulation 6 times, stimulation 7 times, stimulation 8 times, stimulation 9 times, or stimulated for no more than 10 times. are not cultured ex vivo in the presence of AaPCs. The method of the embodiment may further comprise, after the transfection step and / or the culturing step, , enriching the cell population for immune effector cells (e.g., T cells) expressing the CAR. The enrichment step may further comprise the step of enriching the cells by fluorescence activated cell sorting (FACS). In a further embodiment, the method may include sorting for cells expressing the CAR. Sorting for expressing cells involves the use of CAR-binding antibodies. The process may also include depletion of CD56+ cells. In some embodiments, the method further includes a cryopreserved sample of the population of genetically modified CAR cells. nothing.

[0272] In some cases, AaPCs are prepared by cleaving peptides, MHC fragments, and the like without further processing. The antibody is incubated with a peptide of optimal length that allows direct binding to the molecule. Alternatively, the cells may express the antigen of interest (i.e., in the case of MHC-independent antigen recognition). Additionally, in some cases, the APC may express a specific CAR polypeptide or a general CAR. R polypeptide (e.g., uAaPC (universal activating a and propagating cells). The method is described in WO 2014 / 190273, which is incorporated herein by reference. In addition to peptide-MHC molecules or antigens of interest, Thus, the AaPC ​​system may also include at least one exogenous support molecule. Supporting molecules can be used in combination with other molecules, such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules can be selected from supportive molecules such as CD70 and B7 .1 (B7.1 was formerly known as B7 and also as CD80) These include, inter alia, CD28 molecules on the surface of T cells and / or CT It binds to the LA-4 molecule, which induces, for example, T cell expansion, Th1 differentiation, and short-term It affects the survival of T cells and the secretion of cytokines such as interleukin (IL) 2. Adhesion molecules include carbohydrate-binding glycoproteins such as selectins, and integrins. Transmembrane-bound glycoproteins, calcium-dependent proteins such as cadherins, and e.g. For example, intercellular adhesion molecules (ICAMs) promote cell-cell or cell-matrix contact. ) may include single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Selection, cloning, preparation, and expression of exemplary support molecules, including molecules and adhesion molecules For techniques, methods, and reagents useful in U.S. Patent Nos. 6,225,042, 6,355,479, and No. 6,362,001.

[0273] The cells selected to become AaPCs preferably have intracellular antigen processing capabilities, Intracellular peptide trafficking and / or MHC class I or class II molecules These may be due to defects in intracellular peptide loading in offspring or due to the poikilothermic (i.e., are less sensitive to temperature changes than mammalian cell lines) or are defective and mutated. Preferably, the cells selected to become AaPCs also have both inflammatory and metabolic properties. Exogenous MHC class I or class II molecules and supporting molecules introduced into the At least one endogenous counterpart (e.g., an endogenous MHC class I component) to the component. expressing Class II or Class II molecules, and / or endogenous support molecules as described above Furthermore, AaPCs lack the ability to differentiate into cells prior to their modification to generate AaPCs. Preferably, the AaPCs retain the defective and poikilothermic properties that the cells possessed. Insect cell lines, etc., TAP (transporter associated with Constitute or are derived from cell lines deficient in antigen processing Exemplary poikilothermic insect cell lines include the Drosophila Drosophila cell lines (see, e.g., Schneider 1972). Exemplary methods for the preparation, growth, and culture of eider 2 cells are described in U.S. Pat. 225,042, 6,355,479, and 6,362,0 This is presented in Specification No. 01.

[0274] In one embodiment, the AaPCs are also subjected to freeze-thaw cycles. In the freezing cycle, a suitable receptacle containing AaPCs is placed in a suitable container so that freezing occurs rapidly. , a suitable amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or similar cryogenic material The AaPCs can be frozen by contacting them with the frozen AP. C, by excision of AaPC ​​from cryogenic material and exposure to ambient room temperature conditions, or Accelerated melting process using a lukewarm water bath or the warmth of your hands to help reduce melting time Additionally, AaPCs can be frozen and stored for extended periods of time prior to thawing. Frozen AaPCs can also be thawed and then resuspended in frozen form before further use. It can also be dried. Preferably, the solvent is dimethyl sulfoxide (DMSO), polyethylene Preservatives that may have a detrimental effect on the freeze-thaw procedure, such as polyethylene glycol (PEG), Preservatives that are used in AaPCs that undergo freeze-thaw cycles should be absent or should be removed from the media containing the AaPCs. These preservatives can be essentially removed, such as by introducing AaPCs into a medium essentially lacking such preservatives. do.

[0275] In a further embodiment, after inactivation, cell growth, replication, or nucleic acid expression is essentially Cross-linking inactivates xenogenic nucleic acids and nucleic acids endogenous to the AaPC ​​so that they do not occur in the AaPC. In one embodiment, AaPCs can be derived from cells expressing exogenous MHC and supporting molecules, The presentation of such molecules on the surface of AaPCs and the binding of selected MHC molecules to the presented molecules. , and is inactivated at a time point after loading of one or more peptides. Such inactivated AaPCs loaded with selected peptides are capable of proliferation or replication. Although qualitatively impossible, the presentation function of the selected peptide is retained. Crosslinking also prevents bacterial and viral infection without substantially reducing the antigen-presenting cell function of AaPCs. Crosslinking also results in AaPC ​​that are essentially free of contaminating microorganisms such as bacteria. Help alleviate safety concerns about cell therapy products developed using aPCs While maintaining important AaPC ​​functions. Methods for crosslinking and AaPC ​​are described in detail in See, for example, U.S. Patent Application Publication No. 20090017000, which is incorporated herein by reference. Please refer to the specification.

[0276] In certain embodiments, engineered antigen-presenting cells (APCs) are further provided. To propagate immune effector cells in ivo, such cells can be added to, e.g., In a further embodiment, the engineered APCs themselves can be used as described above. This induces the expansion and proliferation of immune effector cells in vivo. The engineered APCs of the embodiment can themselves be used as therapeutic agents. In other embodiments, the engineered APCs are endogenous immune effector cells specific for the target antigen. stimulate activation of and / or adoptively transferred immune effectors specific for the target antigen They can be used as therapeutic agents that can increase the activity or prolong the survival of cells. do.

[0277] As used herein, the term "engineered APC" refers to an APC that contains at least a first transgene The first transgene encodes an HLA. C is for the expression of antigens so that they are presented on the surface of APCs in complex with HLA. In some embodiments, the engineered APCs may further comprise a second transgene. In a further embodiment, the engineered APCs may be T cells. or cell types that do not normally present antigen, such as T cell precursors (referred to as "T-APCs" ) Thus, in some aspects, the engineered APC of the embodiment can be made from: The target antigen is expressed on the surface of the engineered APC in complex with an epitope of the target antigen. The first transgene encodes an antigen and the second encodes a human leukocyte antigen (HLA). In certain specific embodiments, the HL transgene expressed in the engineered APCs A is HLA-A2.

[0278] In some aspects, the engineered APC of the embodiments comprises at least a third gene encoding a costimulatory molecule. The costimulatory molecule may be a membrane-bound Cγ cytokine. In certain aspects, the costimulatory cytokine may be a membrane-bound cytokine. In some further aspects, the engineered APCs are IL-15, such as recombinant IL-15. (or deleted) genes, e.g., PD-1, LIM-3, CTLA-4, or Inhibitory genes such as TCR are edited to reduce or eliminate gene expression. The engineered APCs of the embodiments can be engineered to carry a transcript encoding any target antigen of interest. The gene may further comprise a nucleotide sequence.

[0279] Point-of-care (POC) In one embodiment of the present disclosure, the immune effector cells described herein are administered as POC (p Modification in a point-of-care facility. In some cases, modified immune effector cells are also called engineered T cells. ) The facility is located at a hospital or institution (e.g., medical facility) near the subject requiring treatment. Subjects undergo aphaeresis to obtain peripheral blood mononuclear cells (PBMCs) or a subpopulation of PBMCs. can be concentrated, for example, by wet fractionation or Ficoll separation. The PBMCs or subpopulations of PBMCs may be stored in any suitable cryopreservation medium prior to further processing. In one case, the cells can be stored frozen in a preservative solution containing human serum albumin. The wet classification process is carried out using a buffer containing the following: Immune effector cells, such as T cells, The T cells can be isolated by the selection methods described herein. The selection method for these antibodies is to use beads specific for CD3 on T cells or beads specific for CD4 and CD8. In one case, the beads may be paramagnetic beads. Effector cells can be cryopreserved in any suitable cryopreservation solution prior to modification. Immune effector cells can be incubated for up to 24, 36, or 48 hours prior to infusion. The cells can be thawed for 1 hour, 72 hours, or 96 hours. Prior to incubation, supplement cell culture buffer, e.g., with fetal bovine serum (FBS) or human serum AB. Cell culture buffer (e.g., RPMI) containing steroids such as IL-2 and IL-21 In another embodiment, the collected immune effector - Cells can be rapidly modified without the need for cryopreservation.

[0280] Optionally, the chimeric receptor, one or more cell tags, and / or cytokines By manipulating / introducing the gene into immune effector cells, immune effector cells can be improved. The resulting antibody is then rapidly infused into the subject. Sources can include both allogeneic and autologous sources. In one case, immune effectors The chimeric receptor may be a T cell or a NK cell. In one instance, the chimeric receptor is a MUC1 receptor. 16 CAR. Alternatively, the cytokine may be mbIL-15. In one instance, the mbIL-15 is the mbIL-15 of SEQ ID NO: 69, or a variant thereof. or fragments thereof. In yet another alternative, expression of mbIL-15 is controlled by the use of the methods described herein. It is modulated by a ligand-inducible gene switch expression system. A ligand such as MEX is delivered to a subject to modulate the expression of mbIL-15. In other cases, the cytokine can be IL-12. Expression of IL-12 can be regulated by the ligand-inducible gene switch expression system described herein. For example, a ligand such as berezimex can be delivered to a subject to induce I The expression of L-12 can be modulated.

[0281] In another embodiment, berezimex is administered at 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, Administered at 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg In a further embodiment, low doses of berezimex are administered, for example, at 0.5 mg, 1 mg, 5 mg In one embodiment, berezimex is administered at a dose of 10 mg, 15 mg, or 20 mg. Infusing immune effector cells , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days prior to the date of the In a further embodiment, berezimex is administered in combination with an infusion of engineered immune effector cells. Thereafter, for the duration of the validity period, approximately once every 12 hours, approximately once every 24 hours, approximately once every 36 hours In one embodiment, berezimec is administered to a subject once every 48 hours or about once every 48 hours. The effective period for administration of the drug is approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, In other embodiments, after a rest period following a drug holiday or if the subject has not experienced a relapse, the treatment is continued for 30 days. If symptoms persist, berezimex can be readministered.

[0282] In certain cases where adverse effects on the subject are observed or no treatment is required, the truncated forms described herein may be used for conditional ablation of engineered immune effector cells in vivo, e.g., via cetuximab. epithelium Cell tags can be activated, including cell tags such as growth factor receptor tags.

[0283] In some embodiments, such immune effector cells are constructed as described in Figures 1-2B. The construct is modified via electroporation. In one case, electroporation was performed using a Lonza This is performed with an electroporator such as the Nucleofector™ electroporator. In embodiments, the vector containing the above-described construct is a non-viral vector or a viral vector. In one case, the non-viral vector is a Sleeping Beauty vector. In one case, specific sequences are used to identify the target gene. Electroporate immune effector cells. For example, A second transposon is introduced into the vector followed by DNA encoding the transposase. Alternatively, immune effector cells can be perforated with all transposons. Alternatively, the immune enzyme and the transposase can be electroporated simultaneously. Infector cells are transposase-transduced followed by transfection with both transposons or one transposon. Sposon can be electroporated simultaneously. The vector cells can be allowed to rest for a period of time before the next electroporation step. Cut.

[0284] In some cases, the engineered immune effector cells undergo a propagation step and an activation step. Optionally, the engineered immune effector cells are not subjected to an incubation step or No culture step (e.g., ex vivo propagation) is performed. Prior to infusion, the engineered immune effector cells are placed in a buffer containing IL-2 and IL-21. In other cases, prior to infusion, the engineered immune effector cells are incubated in a cell culture buffer, e.g. , in cell culture buffer (e.g., RPMI) supplemented with fetal bovine serum (FBS), or Prior to infusion, the engineered immune effector cells are harvested, washed, and , in a saline buffer, into a preparation for infusion into a subject.

[0285] In one case, the subject is lymphodepleted prior to the infusion. No depletion is required and the modified immune effector cells are rapidly infused into the subject. Exemplary lymphodepletion regimens are listed in Tables 4 and 5 below.

[0286] [Table 2]

[0287] [Table 3]

[0288] In further instances, the subject undergoes minimal lymphodepletion. Lymphocyte depletion regimens are those that can be infused into subjects within 1, 2, or 3 days after a lymphodepleting regimen. Refers to a reduced lymphodepletion protocol. In one case, a reduced lymphodepletion protocol may include low doses of fludarabine and / or cyclophosphamide. A reduced lymphodepletion protocol involves a short-term reduction in lymphocyte count, e.g., over one or two days. This may include pa depletion.

[0289] In one embodiment, the chimeric receptor and cytokine are engineered into immune effector cells. / transduction to modify immune effector cells, which are then rapidly infused into the subject In other cases, chimeric receptors and cytokines are engineered / transduced into cells. and then modifying the immune effector cells by at least 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, Inject the drug into the subject within 46, 47, 48, 49, or 50 hours. By engineering / transducing mela receptors and cytokines into immune effector cells , immune effector cells were modified, and then the following were performed: 0 days, <1 day, <2 days, <3 days, < Subjects are infused within 4 days, <5 days, <6 days, or <7 days.

[0290] In some embodiments, an amount of engineered effector cells is administered to a subject in need thereof. The dose is determined based on efficacy and potential to induce cytokine-related toxicity. In another embodiment, the engineered effector cell comprises a CAR + cells and CD3 + cell In some cases, the amount of engineered effector cells is about 10 cells 4 ~about 10 9 In some cases, the amount of modified effector cells is 1 kg Approximately 10 engineered effector cells per 4 ~about 10 5 In some cases, modified effects The amount of engineered effector cells was approximately 10 per kg. 5 ~about 10 6 Includes pieces. In some cases, the amount of engineered effector cells is about 1 engineered effector cell per kg. 0 6 ~about 10 7 Optionally, the amount of modified effector cells may be Engineered effector cells >10 4 Pieces and less than or equal to approximately 10 5 In some cases, modified F The amount of engineered effector cells is >10 per kg. 5 Pieces and less than or equal to approximately 10 6 In some cases, the amount of engineered effector cells is 100 mg / kg of engineered effector cells. Tar cells >10 6 Pieces and less than or equal to approximately 10 7 Includes pieces.

[0291] In one embodiment, the engineered immune effector cells are delivered via localized delivery directly to the tumor tissue. For example, in ovarian cancer, engineered immune effector cells can be delivered to the abdomen or The chemotherapy drug may be delivered intraperitoneally (IP) into the peritoneal cavity. Such IP delivery is a convenient method for delivering chemotherapy drugs. This can be done through a port installed for delivery or an existing port. Other methods for local delivery of cytotoxic cells include catheter infusion into the resection cavity and ultrasound-guided intratumoral administration. , coronary infusion, or intrapleural delivery.

[0292] In one embodiment, a subject in need thereof is administered a modified first dose delivered via IP. The immune effector cells are then combined with a second dose of modified immune effector cells delivered via IV. In a further embodiment, treatment can be initiated with a second dose of vector cells. The engineered immune effector cells may be administered in subsequent doses, which may be delivered via IV or IP. In one embodiment, the period between the first dose and the second or further subsequent dose is: Approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2 In one embodiment, the first dose and the second dose or further consecutive doses may be administered for 9, 30 days. The time period between doses is approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months In another embodiment, the period between the first dose and the second or further subsequent dose is The period can be about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0293] In another embodiment, the catheter contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 doses of The modified immune effector cells may be placed at the site of a tumor or metastasis for further administration. In some cases, the dose of engineered effector cells is approximately 1 engineered effector cell per kg. 0 2 ~about 10 9 If toxicity is observed, the dose of modified effector cells may be adjusted to Approximately 10 engineered effector cells per kg 2 ~about 10 5 In some cases, The dose of engineered effector cells is approximately 10 engineered effector cells per kg. 2 Start with pieces and subsequent dosing is approximately 10:10 modified effector cells per kg. 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 The number of entries can be increased up to 100.

[0294] In another embodiment, the method for stimulating the proliferation and / or survival of engineered cells comprises: obtaining from a subject, cells of the cell sample comprising one or more transposons; and transfecting one or more polynucleotides. In this study, the transposon may be a chimeric antigen receptor (CAR), a cytokine, or one or more and integrating the cell tag and the one or more polynucleotides into the genome of the cell. and encodes a transposase effective to produce a population of engineered cells. In some embodiments, the transposon may be a chimeric antigen receptor (CAR), a cytokine, or one or more cell tags, gene switch polypeptides for ligand-induced control of cytokines, and integrating said one or more polynucleotides into the genome of said cell to manipulate said one or more polynucleotides. The gene encodes a transposase effective to generate a population of cells. The gene switch polypeptide is i) fused to a first nuclear receptor ligand-binding domain. ii) a first gene switch polypeptide comprising a DNA-binding domain associated with said first gene switch polypeptide; and comprising a transactivation domain fused to two nuclear receptor ligand binding domains, In some embodiments, the first gene switch polypeptide comprises a second gene switch polypeptide. The polypeptide and the second gene switch polypeptide are connected by a linker. In one instance, lymphodepletion is not required prior to administration of the engineered cells to a subject.

[0295] In one case, the method of in vivo propagation of engineered cells involves transferring a cell sample to a subject and injecting cells of the cell sample with one or more transposons containing the one or more transposons. In one embodiment, the method comprises transfecting a plurality of polynucleotides. The transposon may contain a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, and integrating said one or more polynucleotides into the genome of said cell to manipulate said one or more polynucleotides. In one embodiment, the transposase encodes a transposase effective to generate a population of transforming cells. The transposon may be a chimeric antigen receptor (CAR), a cytokine, or one or more cell types. Patent application title: GENE SWITCH POLYPEPTIDES FOR LIGAND-INDUCED REGULATION OF CYTOKINES AND THEIR FORMATION integrating one or more polynucleotides into the genome of said cells to form a population of engineered cells; In one embodiment, the gene switch encodes a transposase effective to effect The polypeptide comprises: i) a DNA fragment fused to a first nuclear receptor ligand-binding domain; a first gene switch polypeptide comprising a nuclear receptor binding domain; and ii) a second nuclear receptor binding domain. a second gene comprising a transactivation domain fused to a receptor ligand binding domain; In some embodiments, the first gene switch polypeptide The gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In some cases, lymphodepletion is not required prior to administration of the engineered cells to a subject.

[0296] In another embodiment, the survival of the engineered cells in vivo in a subject in need thereof. The method for enhancing the activity of a cell includes the steps of obtaining a cell sample from a subject, and administering to cells of the cell sample one or more Transfecting one or more polynucleotides, including multiple transposons Optionally, the one or more transposons are (CAR), cytokine, one or more cell tags, and DNA are added to the genome of the cells. encodes a transposase effective for integration into the genome to result in a population of engineered cells Optionally, the one or more transposons are selected from the group consisting of chimeric antigen receptors (CARs), cytoplasmic receptors (CRRs), and cytoplasmic receptors (CRRs). cytokine, one or more cell tags, gene for ligand-induced regulation of cytokine The switch polypeptide and DNA are integrated into the genome of the cell, In some cases, gene switches encode transposases that are effective in generating a population. The polypeptide comprises: i) a DNA fragment fused to a first nuclear receptor ligand-binding domain; a first gene switch polypeptide comprising a nuclear receptor binding domain; and ii) a second nuclear receptor binding domain. a second gene comprising a transactivation domain fused to a receptor ligand binding domain; a gene switch polypeptide, In one case, the engineered cell is connected to the switch polypeptide by a linker. In one embodiment, lymphodepletion is not required prior to administration to a subject. Lymphatic depletion is not required prior to administration to elephants.

[0297] In another embodiment, the method of treating a subject with a solid tumor comprises obtaining a cell sample from the subject. and injecting the cells of the sample with one or more poisomers containing one or more transposons. and administering the population of engineered cells to a subject. In one instance, lymphodepletion is required prior to administration of the engineered cells to the subject. Optionally, the one or more transposons are not chimeric antigen receptors (CARs). , cytokines, one or more cell tags, and DNA integration into the genome of the cell. Optionally, one or more transposons The antibody may comprise a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, a cytokine gene switch polypeptides for ligand-induced control of ATP, and DNA in cells It encodes a transposase that is effective in integrating into the genome. The switch polypeptide comprises: i) a D fused to a first nuclear receptor ligand-binding domain; a first gene switch polypeptide comprising an NA-binding domain; and ii) a second nucleic acid sequence. a second domain comprising a transactivation domain fused to an endoreceptor ligand binding domain; a gene switch polypeptide, The child switch polypeptide is connected to the cell by a linker. Transfected via electroporation. Optionally, a gene switch polypeptide The polynucleotide encoding the gene is modulated by a promoter. The promoter may be a tissue-specific promoter or an EF1A promoter, or Optionally, the tissue-specific promoter is a T cell-specific promoter. Optionally, the cytokine comprises a NFAT response element or an NFAT response element. IL-1, IL-2, IL-15, IL-12, IL-21, IL-15, IL-15R IL-15 α, or a fusion of an IL-15 variant. In some cases, cytokines are in secreted form. In other cases, cytokines are in membrane-bound form. In some cases, the cells are NK cells, NKT cells, T cells, or T cell precursors. Alternatively, the cells can be administered to a subject (e.g., by transfusing the engineered cells into the subject). Optionally, the method includes administering an effective amount of a ligand (e.g., berezimex). In some cases, the CAR further comprises inducing expression of a cytokine. Optionally, the transposase is capable of binding to at least MUC-16. Salmonid-type Tc1-like transposase. In some cases, the transposase is SB1 1 or SB100x transposase. In other cases, the transposase is P Optionally, the cell tag is a truncated HER1 mutant or comprises at least one of the CD20 truncated variants.

[0298] therapeutic application In some embodiments described herein, Sleeping Beauty transformers Transduced by poson and Sleeping Beauty transposase, Immune effector cells (e.g., T cells) are presented. For example, Sleeping B A beauty transposon or transposon, as described herein, is a MU The antigen recognition domain of C16 was linked to the CD8 alpha hinge and stalk domain of these mutants. the intracellular domain of CD1, CD3 zeta, CD28, 4-1BB, or any combination thereof and a CD3 zeta intracellular domain, one or more cell tags, one or more subunits, CARs in combination with cytokines and, optionally, components of a gene switch system. Thus, in some cases, the transduced T cells may comprise CAR-mediated T cells. may elicit a response.

[0299] In some embodiments described herein, the specificity of primary T cells is determined by the MUC16 surface antigen. The present invention also provides a method for the treatment of T cell-mediated leukemia using CARs. for stimulating a mediated immune response and targeting cell populations or tissues in mammals 2. A method of administering a CAR to mammalian T cells expressing the CAR, comprising administering the CAR to mammalian T cells expressing the CAR. R is MUC16, the stalk domain, e.g., the intracellular domain of human CD3 zeta, and and a binding moiety that specifically interacts with a zeta chain portion that includes a costimulatory signaling domain. Well, I'll also show you how.

[0300] In one embodiment, the present disclosure provides a method for the production of T cells in which the T cells are genetically modified to express the MUC16-specific CA The cell therapy involves transfusing CAR T cells into recipients in need. The infused cells kill MUC16-overexpressing cells in the recipient. Unlike antibody therapy, the CAR T cells described herein can be used in vivo. ivo and have long-term persistence that may result in persistent effects on tumor cells. can result in:

[0301] The present invention relates to a method for detecting a disease involving overexpression of MUC16 in a subject. a) providing i) a sample from a subject, and ii) an antibody, or antibodies thereof, as described herein; b) providing any one or more of the antigen-binding fragments of the antibody; and c) contacting the sample with the antibody under conditions for specific binding of the antibody to the disease. detecting increased levels of antibody binding to the sample compared to a control sample that does not contain the antibody; In one embodiment, a method is provided comprising detecting a disease in a subject. In a preferred embodiment, the disease is cancer. In a preferred embodiment, the cancer is selected from the group consisting of ovarian cancer and breast cancer. While not intending to limit the method of detection, in one embodiment, the sample The step of detecting antibody binding to the target protein can be performed using immunohistochemistry, enzyme-linked immunosorbent assay (EL ISA), fluorescence-activated cell sorting (FACS), Western blot, immunoprecipitation, and and / or radiographic imaging.

[0302] Provided herein are methods for treating diseases involving overexpression of MUC16, comprising administering to a subject a therapeutically effective amount of the compound described herein. A therapeutically effective amount of any one or more of the antibodies, or antigen-binding fragments thereof, described Also provided is a method comprising administering to a subject having a disease. The disease is cancer exemplified by ovarian cancer and breast cancer.

[0303] In one embodiment, the MUC16 CAR T cells described herein demonstrate robust in Able to undergo T cell expansion in vivo and persist for extended periods of time In another embodiment, the CAR T cells described herein are capable of being reactivated. They can evolve into memory T cells.

[0304] The CAR-modified T cells described herein can also be expressed ex vivo in mammals. It may also serve as a form of vaccine for in vivo immunization and / or therapy. In some embodiments, the mammal is a human. and, prior to the step of administering the immune effector cells to the mammal, at least one of the following is performed: At least one of these occurs in vitro: i) cell expansion, ii) CAR co-transfection, and and / or iii) cryopreservation of the cells.

[0305] Ex vivo procedures are well known and are discussed more fully below. In one embodiment, the cells are isolated from a mammal (e.g., a human) and express a CAR as disclosed herein. Genetically modified (i.e., transduced or transduced in vitro) by a vector containing The CAR-modified cells are administered to a mammalian recipient to demonstrate therapeutic efficacy. The mammalian recipient may be a human, and the CAR-modified The cells can be autologous to the recipient. Alternatively, the cells can be autologous to the recipient. The cells may be homogeneous, homogeneous, or heterogeneous.

[0306] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in detail in the present specification. The cells of the present invention are described in U.S. Pat. No. 5,199,942, which is incorporated herein by reference. Other suitable methods are known in the art, and therefore the present invention is not limited to the above. The present invention is not limited to any particular method of ex vivo expansion of cells. In vivo culture and expansion of T cells can be achieved by (1) peripheral blood collection or bone marrow grafting. recovering mammalian-derived CD34+ hematopoietic stem and progenitor cells from the mammal; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in US Pat. No. 5,199,942, flt3-L, IL-1 Other factors such as IL-1, IL-3, and c-kit ligand contribute to the growth and expansion of cells. It can be used for:

[0307] In addition to using cell-based vaccines for ex vivo immunization The present invention also provides an in vivo method for eliciting an antigen-directed immune response in a patient. Also provided are compositions and methods for immunization against rabies.

[0308] Generally, cells activated and expanded as described herein are used to treat immunocompromised In particular, the CAR modifications of the present invention can be used to treat and prevent diseases occurring in individuals. T cells are used in the treatment of MUC16 malignancies, such as MUC16. In embodiments, the cells of the invention are used to treat patients at risk of developing MUC16. Thus, methods for treating or preventing MUC16 include administering a therapeutically effective amount of a compound of the present invention. Administering the CAR-modified T cells to a subject in need thereof. In some embodiments, cells activated and expanded as described herein may be treated with MUC16. It can be used for.

[0309] Briefly, the pharmaceutical compositions described herein are intended to be used to treat or inhibit the target cell populations described herein. with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain a combination of neutral buffered saline, phosphate buffered saline, Buffer solutions such as glucose, mannose, sucrose or dextran, mannitol carbohydrates such as ethanol; proteins; polypeptides or amino acids such as glycine; antioxidants chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide In some embodiments, the compositions of the present invention are administered intravenously. It is formulated for administration.

[0310] The pharmaceutical compositions described herein may be administered in a manner appropriate for the disease being treated (or prevented). The amount and frequency of administration will depend on the condition of the patient, as well as the type and severity of the patient's disease. The appropriate dosage will depend on factors such as severity, but can be determined through clinical trials.

[0311] "Immunologically effective amount," or "therapeutic amount," as used herein, refers to the amount administered. The exact amount of composition to be administered will depend on individual differences in age, weight, and condition of the patient (subject). Generally, the pharmaceutical compositions comprising the T cells described herein comprise: 10 per kg body weight, including all integer values ​​within these ranges 4 ~10 9 cells, kg body Weight per 10 5 ~10 6 The T cell compositions may also be administered in doses of 100 cells. The cells can be administered multiple times at a dose of 100 mg / kg or more. The cells can be administered using infusion techniques commonly known in immunotherapy. (e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, (1988). The optimal dosage and administration regimen for a particular patient will depend on the patient. monitoring symptoms of the disease and adjusting treatment accordingly, can be more easily determined.

[0312] In certain embodiments, activated T cells are administered to a subject, and then blood is drawn again (or or apheresis), activating the T cells derived therefrom, and It may be desirable to reinfuse the expanded T cells into the patient. This process may be repeated every few weeks. In certain embodiments, T cells are obtained from a blood draw of 10 cc to 400 cc. In certain embodiments, T cells can be activated from 20cc, 30cc, 40cc, c, from a 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc blood draw Without being bound by theory, this multiple blood draw / multiple reinfusion protocol The use of a cloning tool can contribute to the selection of specific populations of T cells. The activity of the subject compositions is determined after lymphodepletion of the patient, either through radiation or chemotherapy. It may be desirable to administer engineered T cells.

[0313] Administration of the compositions described herein can be by aerosol inhalation, injection, ingestion, transfusion, implantation, or the like. This may be done in any convenient way, including by implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intravenously. It may be administered by intravenous (iv) injection or intraperitoneally. The T cell composition is administered to the patient by intradermal or subcutaneous injection. The MUC16 CAR-T cell compositions of the present invention are administered by iv injection. The T cell composition may be injected directly into a lymph node or into the site of a primary tumor or metastasis. do.

[0314] The dosage of the above treatments administered to a patient will depend on the exact nature of the condition being treated and the severity of the treatment. Scaling of dosages for human administration will depend on the recipient. The dosage of the above treatments may be administered in accordance with accepted practices in the field. For example, the dosage of the above treatments may be administered in the range of 100 mg / kg. 1 x 10 CAR+ cells 4 5 x 10 CAR+ cells per kg 6 It is within the range of An exemplary dose is 1 x 10 CAR+ cells per kg. 2 CA per piece, 1 kg R+ cells 1×10 3 1 x 10 CAR+ cells per kg 4 CA per piece, 1 kg R+ cells 1×10 5 3 x 10 CAR+ cells per kg 5 CA per piece, 1 kg R+ cells 1×10 6 5 x 10 CAR+ cells per kg 6 CA per piece, 1 kg R+ cells 1×10 7 1 x 10 CAR+ cells per kg 8 per piece or per kg 1 x 10 CAR+ cells 9The appropriate dose may be adjusted for adult or pediatric patients. It can be done.

[0315] Alternatively, a typical example of immune effector cells administered to a mammal (e.g., a human) is The amount can range, for example, from 100, 1000, 10,000, 1 million to 100 billion cells. However, amounts below or above this exemplary range are also within the scope of the invention. The host cell dose is about 1 million to about 50 billion cells (e.g., about 5 million cells, Approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 0 billion cells, approximately 30 billion cells, approximately 40 billion cells, or any two of the preceding values (range defined by) about 10 million to about 100 billion cells (e.g., about 2000 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells Cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells cells, or a range defined by any two of the preceding values), from about 100 million cells to about 500 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells) 0 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million Cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells, or a range defined by any two of the preceding values).

[0316] Therapeutic or prophylactic efficacy may be monitored by periodic evaluation of the treated patient. For repeated administration over several days or longer, depending on the condition, treatment may be continued until the disease symptoms subside. Repeated doses are administered until the desired suppression of symptoms occurs. However, if other dosing regimens are available, The desired dose can be administered by a single bolus injection of the composition. The delivery may be by multiple bolus administration of the composition or by continuous infusion of the composition.

[0317] Compositions comprising immune effector cells expressing the nucleic acid sequences of the present disclosure, or these nucleic acids The vector containing the sequence can be administered with one or more additional therapeutic agents, By "co-administration," one or more additional therapeutic agents and and a composition comprising the host cell of the present invention or a vector of the present invention administered sufficiently close in time to or potentiating the effect of one or more additional therapeutic agents, or vice versa. In this regard, compositions comprising the immune effector cells described herein or The described vectors may also be administered simultaneously with one or more additional therapeutic agents, may be administered first and one or more additional therapeutic agents may be administered second, or Alternatively, compositions comprising the disclosed immune effector cells or The vector described herein and one or more additional therapeutic agents are administered simultaneously. It is possible.

[0318] may be included in a composition comprising a host cell of the invention and / or a vector of the invention, or Examples of therapeutic agents that may be co-administered with the composition (or included in the kit) include interleukins, These include steroids, cytokines, interferons, adjuvants, and chemotherapeutic agents. In embodiments, the additional therapeutic agent is IFN-alpha, IFN-beta, IFN-gamma , GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors , and hGH, human Toll-like receptors TLR1, TLR2, TLR3, TLR4, and TLR It is a ligand for TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

[0319] "Anti-foaming agent" is used to prevent foaming during processing, coagulation of aqueous dispersion, and air bubbles in the finished thin film. This reduces foaming, which can result in or generally impair processing. The foaming agent is a silicon emulsion or sesquioleate. Contains sorbitan sesquoleate.

[0320] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, Contains sodium thorium, ascorbic acid, sodium metabisulfite, and tocopherol. In certain embodiments, antioxidants enhance chemical stability when needed.

[0321] The formulations described herein contain antioxidants, metal chelators, thiol-containing compounds, and These may benefit from the addition of stabilizers such as benzophenone, benzophenone-3, benzophenone-4, benzophenone-5, benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-20, benzophenone-31, benzophenone-32, benzophenone-33, benzophenone-44, benzophenone-4 (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM (e) about 0.01% to about 2% w / v ascorbic acid; (f) about 10 mM EDTA; 0.003% to approximately 0.02% w / v polysorbate 80, (g) 0.001% to approximately 0. 0.5% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextromethorphan sulfate (k) cyclodextrin, (l) pentosan polysulfate, and other heparins (m) divalent cations such as magnesium and zinc, or (n) combinations thereof This includes, but is not limited to:

[0322] "Binders" impart cohesive properties and are, for example, alginic acid and its salts; carboxylates; Methylcellulose, methylcellulose (e.g., Methocel®), hydro Hydroxypropyl methylcellulose, Hydroxyethyl cellulose, Hydroxypropyl cellulose cellulose (e.g., Klucel®), ethyl cellulose (e.g., Ethyl cellulose cel®), and microcrystalline cellulose (e.g., Avicel®). Any cellulose derivative; microcrystalline dextrose; amylose; magnesium aluminum silicate Sodium; Polysaccharide acid; Bentonite; Gelatin; Polyvinylpyrrolidone / vinyl acetate copolymer Crospovidone; Povidone; Starch; Pregelatinized starch; Tragacanth, dextrin sugar, sucrose (e.g., Dipac®), glucose, dextrose, Honey, mannitol, sorbitol, xylitol (e.g., Xylitab® ), and sugars such as lactose; acacia, tragacanth, ghatti gum, isapor lotus Mucus of the cucumber, polyvinylpyrrolidone (e.g., Polyvidone® CL, K ollidon® CL, Polyplasdone® XL-10) , larch-derived arabinogalactan (Veegum brand), poly These include natural or synthetic gums such as ethylene glycol, waxes, and sodium alginate.

[0323] "Carrier" or "carrier material" includes any excipient commonly used in pharmaceuticals, Compatibility with compounds disclosed herein, such as lutinib and anti-cancer compounds, and The carrier materials should be selected based on the release profile characteristics of the desired dosage form. Examples of suitable additives include binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, "Pharmaceutically compatible carrier materials" include acacia, gelatin, etc. , colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodex Torin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol Cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurochol Acid, Phosphatidylcholine, Sodium Chloride, Tricalcium Phosphate, Dipotassium Phosphate , cellulose and cellulose conjugates, sugar, sodium stearoyl lactylate, color These may include geenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, but not limited to, Remington: The Science and Practice of Pharmacy, Nineteenth Edition. h Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberm. an, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, S See eventh Ed. (Lippincott Williams & Wilkins 1999).

[0324] "Dispersing agents" and / or "viscosity modulating agents" are used in the liquid medium or granulation process. It also includes materials that control the diffusion and homogeneity of the drug through blending. In some cases, these agents may also be incorporated into the coating or eroding matrix. Exemplary diffusion enhancers / dispersants include, for example, hydrophilic polymers, Solubilizers, Tween® 60 or Tween® 80, PEG, polyvinyl alcohol PVP (commercially known as Plasdone®) ), and for example, hydroxypropyl cellulose (e.g., HPC, HPC-SL, and and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100 , HPMC K4M, HPMC K15M, and HPMC K100M), carboxy Methylcellulose sodium (carboxymethylcellulose sodium), methylcellulose, Hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl phthalate Hydroxypropyl methylcellulose, hydroxypropyl methylcellulose stearate acetate (HPM CAS), amorphous cellulose, carbohydrate-based dispersants, magnesium aluminum silicate Sodium, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / Vinyl acetate copolymer (S630), with ethylene oxide and formaldehyde, 4- (1,1,3,3-tetramethylbutyl)-phenol polymer (as tyloxapol) are also known), poloxamers (e.g., bromines of ethylene oxide and propylene oxide), Pluronics F68®, a methacrylic copolymer, Pluronics F88®, and Pluronics F108®); and oxamine (e.g., the sequential synthesis of propylene oxide and ethylene oxide to ethylenediamine) Poloxamine 90, a tetrafunctional block copolymer derived from the catalytic addition of 8® (BASF Corporation, Parsippany, NJ Tetronic 908®, also known as PEG-100, polyvinylpyrrolidone K12, Polyvinylpyrrolidone K17, Polyvinylpyrrolidone K25, or Polyvinyl Pyrrolidone K30, Polyvinylpyrrolidone / Vinyl Acetate Copolymer (S-630), Poly Ethylene glycol (e.g., polyethylene glycol is about 300 to about 6000, may have a molecular weight of about 3350 to about 4000, or about 7000 to about 5400), Sodium carboxymethylcellulose, methylcellulose, polysorbate 80, algin sodium carbonate, e.g., gums such as tragacanth and acacia gum, guar gum, xanthan, including santhang gum; sugars, such as sodium carboxymethylcellulose; Cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, poly Resorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate , polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol Cellulose includes cellulose (PVA), alginate, chitosan, and combinations thereof. Alternatively, a plasticizer such as triethyl cellulose can also be used as a dispersant. Dispersing agents that are particularly useful in liposomal and self-emulsifying dispersions are dimyristoyl Phosphatidylcholine, natural phosphatidylcholine derived from chicken eggs, natural phosphatidylcholine derived from chicken eggs Phosphatidylglycerol, cholesterol, and isopropyl myristate .

[0325] Combinations of one or more erosion promoters with one or more diffusion promoters may also be used. can be used in the present composition.

[0326] The term "diluent" refers to a compound used to dilute a compound of interest prior to delivery. Diluents can also provide a more stable environment, which may help stabilize the compound. In the art, salts dissolved in buffers (which also act as p H) in a variety of solutions, including but not limited to phosphate buffered saline solutions. In certain embodiments, the diluent is used as an unspecified diluent to provide the bulk of the composition. The amount of hydroxybenzoate in the mixture may be increased to facilitate compression or to provide a homogeneous blend for capsule filling. Such compounds are, for example, lactose, starch, mannitol, microcrystalline cellulose such as cellulose, sorbitol, dextrose, Avicel®; Dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, phosphate Calcium; Anhydrous lactose, spray-dried lactose; Pregelatinized starch, Di-Pac( Compressible sugars such as Amstar®; mannitol, hydroxypropyl methylcellulose, cellulose, hydroxypropyl methylcellulose stearate acetate, sucrose-based Diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate Sodium trihydrate, dextrate; hydrolyzed cereal solids, amylose; flour Powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; Contains inositol, bentonite, etc.

[0327] "Fillers" are lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate Sodium, calcium sulfate, crystalline cellulose, cellulose powder, dextrose, dextrose Dextrate, dextran, starch, alpha starch, sucrose, xylose Lithitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene Contains compounds such as glycols.

[0328] "Lubricants" and "glidants" are additives that prevent, reduce, or inhibit adhesion or friction of materials. Exemplary lubricants are, for example, stearic acid, calcium hydroxide, lubricating Contains no hydrocarbons such as stone, sodium stearyl fumarate, mineral oil, or hydrogenated soybean oil (S hydrogenated vegetable oils such as terotex®, high fatty acids, and aluminum, Alkali metal salts and alkaline earth metals such as calcium, magnesium, and zinc Salt, Stearic Acid, Sodium Stearate, Glycerol, Talc, Wax, Stearow et®, boric acid, sodium benzoate, sodium acetate, sodium chloride, isopropyl alcohol, polyethylene glycol (e.g., PEG-4000), or Carbohydrate x (trademark) and other methoxypolyethylene glycols, sodium oleate, sodium benzoate Sodium, Glyceryl Behenate, Polyethylene Glycol, Magnesium Lauryl Sulfate or sodium lauryl sulfate, Syloid™, Cab-O-Sil® colloidal silica, starch such as corn starch, silicone oil, surfactants Includes sexually active ingredients.

[0329] "Plasticizer" means a compound used to soften the microencapsulated material; is a film coating that reduces the brittleness of the microencapsulated material. Examples of the copolymerizing agent include PEG 300, PEG 400, PEG 600, and PEG 1450. polyethylene glycols such as PEG 3350, and PEG 800, stearin Acid, propylene glycol, oleic acid, triethylcellulose, and triacetin In some embodiments, the plasticizer may also function as a dispersing agent or humectant.

[0330] "Solubilizers" include triacetin, triethyl citrate, ethyl oleate, and caprylic acid. Ethyl, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone Polyvinylpyrrolidone, Hydroxypropylmethylcellulose, Hydroxypropyl Cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol terol, bile salts, polyethylene glycol 200-600, glycofurol, trans These include compounds such as methacrylate, propylene glycol, and dimethyl isosorbide.

[0331] "Stabilizers" include compounds such as any antioxidants, buffers, acids, preservatives, and the like.

[0332] "Suspending agents" include polyvinylpyrrolidones, e.g., polyvinylpyrrolidone K12 ... Polyvinylpyrrolidone K17, Polyvinylpyrrolidone K25, or Polyvinylpyrrolidone K3 0, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol ( For example, polyethylene glycol may have a molecular weight of about 300 to about 6000, or about 3350 to about 40 00, or may have a molecular weight of about 7000 to about 5400), carboxymethyl cellulose Sodium phosphate, methylcellulose, hydroxypropyl methylcellulose, stearic acid Hydroxymethylcellulose acetate, Polysorbate 80, Hydroxyethylcellulose, Sodium alginates, such as gum tragacanth and gum acacia, guar gum, etc. Gums, xanthans including xanthan gum; sugars, e.g., carboxymethylcellulose sodium sodium, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose derivatives such as methyl cellulose and hydroxyethyl cellulose, polysorbate Glutamate 80, Sodium Alginate, Polyethoxylated Sorbitan Monolaurate, Monolaurate These include compounds such as polyethoxylated sorbitan urate and povidone.

[0333] "Surfactants" include sodium lauryl sulfate, sodium docusate, T Tween 60 or Tween 80, Triacetin, Vitamin E TPGS, Mono- Sorbitan oleate, polyoxyethylene sorbitan monooleate, polysorbate ( polysorbate), poloxamer, bile salts, glyceryl monostearate, oxidized Copolymers of ethylene and propylene oxide, such as Pluronic® (B Some other surfactants include compounds such as polyoxyethylene glycerin. Fatty acid esters and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, for example: Octoxynol 10, Octoxynol 40. In some embodiments, surfactants can be incorporated to enhance physical stability or for other purposes.

[0334] "Viscosity enhancers" include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, stearyl Hydroxypropylmethylcellulose acrylate acetate, Hydroxypropylmethyl phthalate Cellulose, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0335] "Moisturizing Agents" are oleic acid, glycerin monostearate, sorbitan monooleate , Sorbitan monolaurate, Triethanolamine oleate, Polyisobutene monooleate Polyoxyethylene sorbitan monolaurate, sodium hydroxide Docusate, Sodium Oleate, Sodium Lauryl Sulfate, Sodium Dodecyl Glucoside Docusate, Triacetin, Tween 80, Vitamin E TPGS, This includes compounds such as ammonium salts.

[0336] Kits and Compositions One aspect of the present disclosure is a method for administering a MUC16-specific CAR of the present invention and, optionally, for safety reasons, , such as HER1t or HER1t-1 and functional variants thereof, or CD2 0 or CD20t-1, and genes containing functional variants thereof The present invention relates to kits and compositions comprising a first vector containing a gene region. The product may further comprise a cytokine. In another embodiment, the kit and composition comprise a RHEO These kits and combinations may include SWITCH® gene switch components. The composition may comprise multiple proteins, each encoding a different protein or subset of proteins. These vectors may be viral, non-viral, episomal, or In some embodiments, the vector may be integrated with a transposon, e.g., Slee ping Beauty transposon.

[0337] In some embodiments, the kits and compositions include not only vectors but also cells and Interleukins, cytokines, interleukins and chemotherapeutic agents, adjuvants, infiltration In one embodiment, the cells are T cells. In one embodiment, the kits and compositions include IL-2. In one embodiment, the kit and composition comprise Bcl-2, STAT In some embodiments, the kit comprises an IL-15 or STAT5 inhibitor. Contains mbIL-15.

[0338] In certain embodiments herein, one or more of the methods described herein Disclosed are kits and articles of manufacture for use by the method. Such kits include vials, test One or more vessels, such as tubes, each of which may be used in the methods described herein. A container compartmentalized to receive a container containing one of the individual elements used in the Suitable containers include, for example, bottles, vials, and the like. In one embodiment, the container is made of glass or plastic. They are made from a variety of materials, including quartz.

[0339] The products presented herein contain packaging materials. Pharmaceutical Packaging Materials Examples of materials include blister packs, bottles, test tubes, bags, containers, bottles, as well as selected Any packaging materials appropriate for the formulation to be packaged and the intended mode of administration and treatment. Including but not limited to:

[0340] For example, the container may contain CAR-T cells (e.g., MUC16-specific CTLs described herein). AR-T cells), and optionally, in addition, cytokines and / or Such kits optionally comprise a method for administering a therapeutic agent to a subject, the subject, or a chemotherapeutic agent. This includes confirmation of the contents of any description, labeling or instructions regarding its use in the

[0341] Kits typically include a label listing the contents and / or instructions for use, etc. and a package insert with instructions for use. Typically, a set of instructions also It is expected to be accepted.

[0342] In some embodiments, the indicia is affixed to or associated with the container. In embodiments, letters, numbers, or other symbols forming the indicia are adhered to the container itself. If the marking is printed, cast or etched, the marking shall be affixed to the container. The display may also be displayed in a receptacle or carrying case that holds the container, e.g. , if present as a package insert, is associated with the container. Used to indicate that the contents are to be used for a specific therapeutic application. The label also points to directions for use of the contents, such as in the methods described herein. vinegar. [Example]

[0343] These examples are provided for illustrative purposes only and are not intended to be limiting of the scope of the patents presented herein. It is not intended to limit the scope of the claims.

[0344] [Example 1] Nucleofection of T cells with Sleeping Beauty To create genetically modified T cells, cryopreserved pan-T cells are thawed, washed, and then pre-injected. Prewarmed phenol red-free RPMI supplemented with FBS and Glutamax Resuspend in 1640 medium (R20 medium) and incubate in a humidified incubator at 37°C with 5% CO Cells were counted, centrifuged, and resuspended in nucleofection buffer. To generate CAR-T cells, a specific number of T cells (typically 5 per cuvette) are injected. ~40×10 6 For each nucleofection cuvette containing a reaction (range of 1000), use A total of 15 μg of transposon plasmid containing the R construct was added to the SB transposase. T cell electroporation was performed using Amexa 2 b Nucleofection device or 4D Nucleofector (Lonza, Walker After electroporation, the contents from each cuvette were The cells were transferred to pre-warmed R20 medium and placed in an incubator at 37°C. Samples of cell cultures were taken for flow cytometry analysis and at specific time points where applicable. The expression of AR, HER1t, and mbIL-15 was characterized. For certain experiments, CAR + T cells were numerically expanded ex vivo for further characterization. The CAR that was created + Due to the ex vivo numerical expansion of T cells, T The cells were further treated with AaPCs (activating and propagating Briefly, truncated MUC16 (MUC16t cells) were co-cultured with ) K56 engineered to express CD86, 41BBL, and mbIL15 along with antigens Irradiated AaPCs from the two cell lines were incubated with IL-21 and IL-21 for subsequent weekly AaPC ​​additions. CAR in complete medium with IL-2 + They were co-cultured with T cells.

[0345] Flow cytometry analysis of CAR, HER1t1, and mbIL15 expression was performed using electrophoresis. Day 1 after air perforation, and HiLyte™ Fluor 647 conjugate, respectively. Recombinant MUC16t-Fc fusion protein or AF647-labeled Protein nL, phycoerythrin (PE)-conjugated cetuximab and fluorescent isothiocyanate Use FITC-conjugated anti-IL-15 and anti-IL-15RA antibodies , performed before each AaPC ​​stimulation.

[0346] [Example 2] Generation of MUC16 CAR-T cells CAR-T cells expressing MUC16-2 CAR were generated using the SB-based promoter described in Example 1. MUC16-2 CAR-HER1t1 T cells were generated by electroporation of plasmids. SB transposon encoding MUC16-2 CAR and HER1t1 plus Electroporation of the SB11 transposase plasmid into human T cells MUC16-2 CAR-mbIL15-HER1t1 T cells were generated using the following methods: (1) SB transfectants encoding MUC16-2 CAR, mbIL15, and HER1t1 (2) SB11 transposase plasmid, and (3) SB11 transposase plasmid in human T cells The expression of CAR, mbIL15, and HER1t1 was One day after nucleofection, multiparameter flow cytometry was performed. was used to quantify.

[0347] Table 4 shows the results of MUC16-2 CAR and HE at day 1 after nucleofection. R1t1 gene (MUC16-2 CAR-HER1t1 T cells) or MUC16- 2 CAR, mbIL15 and HER1t1 (MUC16-2 CAR-mbIL15 -HER1t1 T cells) using a transposon expressing SB-based plasmid Transgene-expressing cells in different donor T cells after nucleofection Transfection efficiency, measured by %, is shown. Cells were transfected with 100% of the CD3+ live population. And then the gate was closed...

Claims

1. 1. A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a MUC16 antigen-binding domain comprising: (i) a VL domain having at least 90% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 90% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:6; (b) a transmembrane domain; and (c) intracellular signaling domain A nucleic acid comprising:

2. 2. The nucleic acid of claim 1, wherein the MUC16 antigen-binding domain comprises: (i) a VL domain having at least 95% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 95% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:

6.

3. 2. The nucleic acid of claim 1, wherein the MUC16 antigen-binding domain comprises: (i) a VL domain having at least 98% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 98% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:

6.

4. 2. The nucleic acid of claim 1, wherein the MUC16 antigen-binding domain comprises: (i) a VL domain having at least 99% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 99% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:

6.

5. The nucleic acid of claim 1, wherein the MUC16 antigen-binding domain comprises: (i) a VL domain having the sequence of SEQ ID NO: 5; and (ii) a VH domain having the sequence of SEQ ID NO:

6.

6. The nucleic acid of claim 1, wherein the CAR further comprises a stalk domain.

7. 7. The nucleic acid of claim 6, wherein the stalk domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

16.

8. The nucleic acid of claim 1, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

9. The nucleic acid of claim 8, wherein the CD3 zeta signaling domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

26.

10. The nucleic acid of claim 1, wherein the intracellular signaling domain comprises a 4-1BB costimulatory signaling domain or a CD28 costimulatory signaling domain.

11. The nucleic acid of claim 1, wherein the intracellular signaling domain comprises a 4-1BB costimulatory signaling domain.

12. 11. The nucleic acid of claim 10, wherein the 4-1BB costimulatory signaling domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

22.

13. The nucleic acid of claim 1, wherein the transmembrane domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

20.

14. (a) the MUC16 antigen-binding domain comprises: (i) a VL domain having at least 95% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 95% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:6; (b) the transmembrane domain comprises a polypeptide having at least 95% identity to SEQ ID NO: 20; and (c) the intracellular signaling domain comprises: (i) a first polypeptide having at least 95% identity to SEQ ID NO:22; and (ii) a second polypeptide having at least 95% identity to SEQ ID NO:26; 2. The nucleic acid of claim 1, wherein the CAR further comprises a stalk domain having at least 95% identity to SEQ ID NO:

16.

15. (a) the MUC16 antigen-binding domain comprises: (i) a VL domain having at least 98% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 98% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:6; (b) the transmembrane domain comprises a polypeptide having at least 98% identity to SEQ ID NO: 20; and (c) the intracellular signaling domain comprises: (i) a first polypeptide having at least 98% identity to SEQ ID NO:22; and (ii) a second polypeptide having at least 98% identity to SEQ ID NO:26; 2. The nucleic acid of claim 1, wherein the CAR further comprises a stalk domain having at least 98% identity to SEQ ID NO:

16.

16. (a) the MUC16 antigen-binding domain comprises: (i) a VL domain having the sequence of SEQ ID NO:5; and (ii) a VH domain having the sequence of SEQ ID NO:6; (b) the transmembrane domain comprises a polypeptide having the sequence of SEQ ID NO: 20; and (c) the intracellular signaling domain comprises: (i) a first polypeptide having the sequence of SEQ ID NO:22; and (ii) a second polypeptide having the sequence of SEQ ID NO:26; 2. The nucleic acid of claim 1, wherein the CAR further comprises a stalk domain having the sequence of SEQ ID NO:

16.

17. The nucleic acid of claim 1, wherein the CAR comprises a polypeptide having at least 90% identity to SEQ ID NO: 31, and the MUC16 antigen-binding domain comprises all three CDRs of SEQ ID NO: 5 and all three CDRs of SEQ ID NO:

6.

18. 2. The nucleic acid of claim 1, wherein the CAR comprises a polypeptide having at least 95% identity to SEQ ID NO: 31, and the MUC16 antigen-binding domain comprises all three CDRs of SEQ ID NO: 5 and all three CDRs of SEQ ID NO:

6.

19. The nucleic acid of claim 1, wherein the CAR comprises a polypeptide having at least 98% identity to SEQ ID NO: 31, and the MUC16 antigen-binding domain comprises all three CDRs of SEQ ID NO: 5 and all three CDRs of SEQ ID NO:

6.

20. The nucleic acid of claim 1, wherein the CAR comprises a polypeptide having the sequence of SEQ ID NO:

31.

21. The nucleic acid of claim 1 , further encoding a cell tag.

22. 22. The nucleic acid of claim 21, wherein the cell tag comprises a truncated epidermal growth factor receptor (EGFR).

23. 23. The nucleic acid of claim 22, wherein the truncated EGFR comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

66.

24. The nucleic acid of claim 1 further encoding a cytokine.

25. The nucleic acid of claim 24, wherein the cytokine is membrane-bound interleukin 15 (IL-15).

26. 26. The nucleic acid of claim 25, wherein the membrane-bound IL-15 comprises a polypeptide having at least 90% identity to SEQ ID NO:

69.

27. A vector comprising the nucleic acid of any one of claims 1 to 26.

28. 28. The vector of claim 27, wherein the vector is a lentiviral vector, a retroviral vector, or a non-viral vector.

29. 28. The vector of claim 27, wherein the vector comprises a backbone that is a Sleeping Beauty transposon DNA plasmid.

30. 28. The vector of claim 27, further comprising a promoter.

31. 31. The vector of claim 30, wherein the promoter is the hEF1a1 promoter.

32. An immune effector cell comprising the nucleic acid of any one of claims 1 to 26.

33. 33. The immune effector cell of claim 32, wherein the cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell.

34. An immune effector cell comprising a CAR, wherein the CAR is (a) a MUC16 antigen-binding domain comprising: (i) a VL domain having at least 90% identity to SEQ ID NO:5 and comprising all three CDRs of SEQ ID NO:5; and (ii) a VH domain having at least 90% identity to SEQ ID NO:6 and comprising all three CDRs of SEQ ID NO:6; (b) a transmembrane domain; and (c) intracellular signaling domain immune effector cells, including

35. The immune effector cell of claim 34, further comprising membrane-bound IL-15.

36. The immune effector cell of claim 34, further comprising a cell tag.

37. 28. A method of expressing a CAR in an immune effector cell, comprising contacting the immune effector cell with the vector of claim 27.

38. 38. The method of claim 37, further comprising contacting the immune effector cells with a Sleeping Beauty transposase.

39. 39. The method of claim 38, wherein the Sleeping Beauty transposase is SB11, SB100X, or SB110.

40. 33. A pharmaceutical composition for treating ovarian or breast cancer in a patient comprising the immune effector cells of claim 32, wherein one or more doses are administered to the patient.

41. 41. The pharmaceutical composition of claim 40 for treating ovarian cancer.

42. 41. The pharmaceutical composition of claim 40 for treating breast cancer.

43. A composition comprising the immune effector cells of claim 32.

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