Human applications of engineered chimeric antigen receptor (CAR) T cells

Abstract

The present invention relates to methods and compositions for the immunotherapy of modified T cells using a chimeric antigen receptor (CAR). In a specific aspect, electroporation combined with a transposon-based integration system is used to produce T cells expressing CAR, to produce a colony of cells expressing CAR, the cells expressing CAR require minimal ex vivo expansion or can be directly administered to a patient for disease (e.g., cancer) treatment.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 823,253, filed May 14, 2013, the entire contents of which are incorporated herein by reference.

[0002] This invention was made with Government support under Grant No. W81XWH-11-1-0002-01 awarded by the Department of Defense. The Government has certain rights in this invention.

[0003] Incorporation of Sequence Listing

[0004] The sequence listing contained in the file is called "UTFC.P1222WO_ST25.txt" (which is in the Microsoft , measuring 37 KB and created on May 14, 2014) is submitted electronically herewith and is incorporated herein by reference. Background of the Invention

[0005] 1. Field of the Invention

[0006] The present invention generally relates to the fields of medicine, immunology, cell biology and molecular biology. In some aspects, the field of the present invention relates to immunotherapy. More specifically, it relates to the production of clinical-grade chimeric antigen receptor (CAR) T cells and methods of treating such cells.

[0007] 2. Description of Related Fields

[0008] The efficacy of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer biological products with the potential to achieve superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential to migrate to the tumor site, and (iv) the ability to recirculate effector functions within the tumor microenvironment. Such gene therapy combined with immunotherapy can redirect T cells to be specific for B-lineage antigens, and patients with advanced B-cell malignancies benefit from infusion of such tumor-specific T cells (Jena et al., 2010; Till et al., 2008; Porter et al., 2011; Brentjens et al., 2011; Cooper and Bollard, 2012; Kalos et al., 2011; Kochenderfer et al., 2010; Kochenderfer et al., 2012; Brentjens et al., 2013). Most genetic manipulation methods for human application engineered T cells have used retroviruses and lentiviruses to stably express chimeric antigen receptors (CAR) (Jena et al., 2010; Ertl et al., 2011; Kohn et al., 2011). This method, although in compliance with current good manufacturing practices (cGMP), can be expensive because it relies on the manufacture and release of clinical-grade recombinant viruses from a limited number of production facilities. New methods are needed to produce genetically modified clinical-grade T cell products with specificity for hematological malignancies and solid tumors. SUMMARY OF THE INVENTION

[0009] In a first embodiment, a method of providing a T cell response in a human subject having a disease is provided, comprising obtaining a cell sample from the subject (comprising T cells or T cell progenitors); transfecting the cells with a nucleic acid encoding a chimeric T cell receptor (CAR) that is capable of integrating into the genome of the cells; and administering to the subject an effective amount of the transgenic cells to provide a T cell response.

[0010] Therefore, in some aspects, the method of the embodiment includes: (a) obtaining a cell sample from a subject, the sample comprising T cells or T cell progenitors; (b) transfecting cells with a chimeric antigen receptor (CAR) encoding a transposon and a transposase that effectively integrates the DNA encoding CAR into the genome of the cell to provide a population of cells expressing transgenic CAR; (c) optionally culturing a population of transgenic CAR cells in vitro in a culture medium that selectively enhances the proliferation of T cells expressing CAR, wherein the transgenic CAR cells are cultured, if any, for no more than 21 days; and (d) administering an effective amount of transgenic CAR cells to the subject to provide a T cell response. Therefore, in some aspects, the transgenic CAR cells are cultured in vitro for less than 21 days, such as less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. In certain aspects, CAR cells are cultured in vitro for no more than 3 to 5 days. In another aspect, steps (a)-(d) of the method are completed (i.e., obtaining a cell sample for administration of CAR T cells) in no more than 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days.

[0011] In another aspect, the method of providing a T cell response in a human subject with a disease according to an embodiment includes: (a) obtaining a cell sample from the subject, the sample comprising T cells or progenitor cells of T cells and having an initial volume of about 20 to 200 ml when obtained from the subject; (b) transfecting cells with a chimeric antigen receptor (CAR) encoding a transposon flanking the transposon and a transposase that effectively integrates the DNA encoding the CAR into the genome of the cell, to provide a T cell population expressing a transgenic CAR; (c) optionally culturing a population of transgenic CAR cells in vitro in a culture medium that selectively enhances the proliferation of T cells expressing CAR; and (d) administering an effective amount of transgenic CAR T cells to the subject to provide a T cell response. For example, the cell sample from the subject can be a peripheral blood or umbilical cord blood sample of less than about 200 ml. In some aspects, the sample can be collected by plasmapheresis. However, in certain preferred aspects, the sample is collected by a method that does not involve plasmapheresis (e.g., by venipuncture). In another aspect, the cell sample has an initial volume of less than about 175, 150, 125, 100, 75, 50, or 25 ml (e.g., when obtained from a subject, the cell sample has an initial volume of about 50 to 200 ml, about 50 to 100 ml, or 100 to 200 ml).

[0012] In another embodiment, there is provided a separated transgenic cell, which comprises the CAR for the expression of the envelope protein targeting HERV-K. In some aspects, the cell comprises the DNA encoding the CAR integrated into the genome of the cell (for example, CAR DNA flanking transposon repeats). For example, the CAR sequence may include the CDR sequence (for example, CDR 1-6) of monoclonal antibody 6H5 or the scFV sequence of monoclonal antibody 6H5. In some aspects, HERV-K- targeting CAR and SEQ ID NO:4 amino acid sequence has at least 85% homogeneity (for example, with SEQ ID NO:4 has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity sequence). In some aspects, the cell of the embodiment (for example, human HERV-K- targeting CAR cell) can be used to treat a subject with a cancer expressing HERV-K (or provide an immune response in a subject).

[0013] In another embodiment, there is provided an isolated transgenic cell comprising expressed CAR and expressed membrane-bound IL-15. For example, in some aspects, the membrane-bound IL-15 includes a fusion protein between IL-15 and IL-15Rα. In another aspect, the membrane-bound IL-15 comprises an amino acid sequence with at least about 85% to SEQ ID NO: 6 (referred to herein as mIL15), 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. As described in further detail herein, in some cases, the membrane-bound IL-15 is encoded by RNA or DNA (e.g., extrachromosomal or additional vectors). In certain aspects, the cell comprises DNA encoding membrane-bound IL-15 integrated into the genome of the cell (e.g., DNA encoding transposon repeats). In certain aspects, the cells of the embodiments (e.g., human CAR cells, expressing membrane-bound cytokines) can be used to treat (or provide an immune response in) subjects with low levels of target antigen, such as subjects with minimal residual disease (as further described herein).

[0014] In some aspects, the method of the embodiment relates to transfecting cells with DNA encoding chimeric T cell receptors (CAR) and in some cases transposases. Methods for transfecting cells are well known in the art, but in some aspects, efficient transfection methods such as electroporation are used. For example, a nuclear transfection device can be used to introduce nucleic acid into cells. Preferably, the transfection step does not involve infecting or transducing cells with viruses, which can cause genotoxicity and / or lead to an immune response against cells containing viral sequences in the treated subject.

[0015] Other aspects of the embodiment relate to transfecting cells with expression vectors encoding CAR. Many CAR constructs and expression vectors therefor are known in the art and are further described in detail herein. For example, in some aspects, the CAR expression vector is a DNA expression vector such as a plasmid, a linear expression vector or an episome. In some aspects, the vector comprises additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, polyadenylic acid signals and / or one or more introns. In preferred aspects, the CAR coding sequence is flanked by a transposon sequence so that the presence of a transposase allows the coding sequence to be integrated into the genome of the transfected cell.

[0016] As mentioned above, in some aspects, cells are further transfected with a transposase that promotes the integration of CAR encoding sequences into the genome of transfected cells. In some aspects, transposase is provided as a DNA expression vector. However, in preferred aspects, transposase is provided as expressible RNA or protein so that the long-term expression of transposase does not occur in transgenic cells. For example, in some aspects, transposase is provided as mRNA (for example, mRNA comprising a cap and a polyadenylic acid tail). Any transposition system can be used according to the embodiment. However, in some aspects, transposase is salmon type Tc1 sample transposase (SB). For example, transposase can be so-called "Sleeping Beauty" transposase, see, for example, United States Patent (USP) 6,489,458, which is incorporated herein by reference. In some aspects, transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposase include, but are not limited to, SB10, SB11 or SB100x transposase (see, for example, Mates et al., 2009, incorporated herein by reference). For example, the method may involve electroporating cells with mRNA encoding SB10, SB11, or SB100x transposase.

[0017] In another aspect, the transgenic CAR cells of the embodiment also include an expression vector for expressing a membrane-bound cytokine that stimulates T cell proliferation and / or survival. Specifically, the CAR cells comprising such cytokines can be due to the stimulation provided by cytokine expression and can be propagated and / or maintained in the case of in vitro culture with little or no activation and proliferation cells (AaPC) or artificial antigen presenting cells (aAPC). Similarly, such CAR cells can also be propagated in vivo even when a large number of antigens recognized by CAR are not present (for example, such as in the case of a cancer patient in remission or a patient with minimal residual disease). In some aspects, CAR cells include DNA or RNA expression vectors for C γ cytokines and elements (for example, transmembrane domains) expressed to provide surface expression of cytokines. For example, CAR cells can include membrane-bound forms of IL-7, IL-15 or IL-21. In some aspects, cytokines are tethered to membranes by the fusion of cytokine coding sequences and cytokine receptors. For example, the cell may include a vector for expressing an IL-15-IL-15Rα fusion protein (e.g., a protein comprising a sequence of SEQ ID NO: 6). In another aspect, the vector encoding the membrane-bound Cγ cytokine is a DNA expression vector, such as a vector or an extrachromosomal vector (e.g., and an additional vector) that is integrated into the genome of the CAR cell. In another aspect, the expression of the membrane-bound enzyme Cγ cytokine is under the control of an inducible promoter (e.g., a drug-inducible promoter) so that the expression of the cytokine in the CAR cell (and thus the proliferation of the CAR cell) can be controlled by inducing or inhibiting promoter activity.

[0018] Aspects of the embodiment relate to obtaining a sample comprising NK cells, NKT cells, T cells or the progenitor cells of T cells from a patient. For example, in some cases, sample is a cord blood sample, a peripheral blood sample (for example, a mononuclear cell fraction) or a sample comprising pluripotent cells from a subject. In some respects, the sample from the subject can be cultivated to produce induced pluripotent stem (iPS) cells, and these cells are used to produce NK cells, NKT cells or T cells. Cell sample can be directly cultivated from the subject, or it can be cryopreserved before use. In some respects, obtaining a cell sample includes collecting a cell sample. In other respects, sample is obtained by a third party. In yet another aspect, the sample from the subject can be processed to purify or enrich the T cells or the progenitor cells of T cells in the sample. For example, sample experience gradient purification, cell culture selection and / or cell sorting (for example, by fluorescence activated cell sorting (FACS)) can be made.

[0019] In some aspects, the method of the embodiment also includes, obtains, produces or uses antigen presenting cells.For example, the antigen presenting cells can be dendritic cells, activation and proliferating cells (AaPC), or inactivated (for example, irradiated) artificial antigen presenting cells (aAPC). The method for producing such aAPC is known in the art and is further described in detail herein. Therefore, in some aspects, transgenic CAR cells are co-cultured with inactivated aAPC for a limited time period in vitro to amplify the CAR cell colony. The step of co-culturing CAR cells and aAPC can be carried out in a culture medium comprising, for example, interleukin-21 (IL-21) and / or interleukin-2 (IL-2). In some aspects, the ratio of about 10:1 to about 1:10, about 3:1 to about 1:5 or about 1:1 to about 1:3 CAR cells and inactivated aAPC is co-cultured. For example, CAR cells and aAPC can be co-cultured with a ratio of about 1:1, about 1:2 or about 1:3.

[0020] In some aspects, cells such as AaPC ​​or aAPC used for the culture of CAR cells are engineered to express specific polypeptides that enhance the growth of CAR cells. For example, the cells may contain (i) an antigen targeted by the CAR expressed on the transgenic CAR cell; (ii) CD64; (ii) CD86; (iii) CD137L; and / or (v) membrane-bound IL-15 expressed on the surface of the aAPC. In some aspects, the AaPC ​​or aAPC comprises a CAR binding antibody or fragment thereof expressed on the surface of the AaPC ​​or aAPC. Preferably, the AaPC ​​or aAPC used in this method is tested for infectious material and confirmed to be absent and / or the AaPC ​​or aAPC is tested and confirmed to be inactivated and non-proliferative.

[0021] Although the amplification of AaPC ​​or aAPC can increase the number or concentration of CAR cells in culture, the method is too laborious and expensive. In addition, in some aspects, the subject in need of treatment should be re-infused with genetically modified CAR cells in the shortest possible time. Therefore, in some aspects, the ex vivo culture of genetically modified CAR cells (c) is no more than 14 days, no more than 7 days or no more than 3 days. For example, ex vivo culture (for example, in the presence of AaPC ​​or aAPC) can be performed to achieve less than one population doubling of genetically modified CAR cells. In another aspect, the genetically modified cells are not cultured in vitro in the presence of AaPC ​​or aAPC.

[0022] In another aspect, the method of the embodiment includes a step for enriching a cell population for CAR-expressing T cells after transfection of the cell (step (b)) or after ex vivo expansion of the cell (step (c)). For example, the enrichment step may include sorting of the cells (e.g., by FACS), for example, by using an antigen bound by a CAR or CAR-binding antibody. In another aspect, the enrichment step includes depleting the non-T cells or depleting cells lacking CAR expression. For example, CD56 T cells may be depleted from a cell population. + In another aspect, a sample of CAR cells is stored (or maintained in culture) when the cells are administered to a subject. For example, the sample can be frozen for later expansion or analysis.

[0023] In certain aspects, the expression of endogenous T cell receptors and / or endogenous HLA of the transgenic CAR cells of the embodiments is inactivated. For example, T cells can be engineered to eliminate the expression of endogenous α / β T cell receptors (TCR). In specific embodiments, the CAR + T cell genetic modification is to eliminate the expression of TCR. In some aspects, zinc finger nuclease (ZFN) is used to destroy the T cell receptor α / β in the T cell expressing CAR. In some aspects, for example, by using zinc finger nuclease to knock out the T cell receptor αβ chain in the T cell expressing CAR.

[0024] As further described herein, the CAR cells of the embodiment can be used to treat many diseases and conditions. It is possible to treat substantially any disease involving the specificity of a particular antigen or the expression of enhancement by targeting CAR cells to antigens. For example, autoimmune diseases, infections and cancers can be treated with the methods and / or compositions of the present invention. These diseases include cancers, such as primary cancers, metastatic cancers, recurrent cancers, cancers sensitive to treatment, refractory cancers (for example, chemotherapy-refractory cancers). Cancer can be blood cancer, lung cancer, brain cancer, colon cancer, prostate cancer, breast cancer, liver cancer, kidney cancer, gastric cancer, cervical cancer, ovarian cancer, testicular cancer, pituitary cancer, esophageal cancer, spleen cancer, skin cancer, bone cancer, etc. (for example, B cell lymphoma or melanoma). In the case of cancer treatment, CAR cells typically target cancer cell antigens (also referred to as tumor-associated antigens (TAA)).

[0025] In another aspect, the transgenic CAR cells of the embodiment can be used to treat subjects with minimal residual disease (e.g., subjects with very low amounts of CAR targeting antigens present), such as cancer patients in significant remission. By using highly sensitive diagnostic techniques, cancer-associated antigens (or cancer cells) can be detected in patients who do not show obvious cancer symptoms. Such patients can be treated by this method to eliminate residual disease by utilizing antigen-targeting CAR cells. In a preferred embodiment, the transgenic CAR cells for targeting residual disease also include the expression of membrane-bound proliferative cytokines, because these cells will retain the ability to amplify (although in small amounts) in vivo to target antigens.

[0026] The methods of the embodiments can be used to manufacture (e.g., for clinical trials) CARs for a variety of tumor antigens (e.g., CD19, ROR1, CD56, EGFR, CD123, c-met, GD2). + T cells. CARs produced using this technology + T cells can be used to treat patients with leukemia (AML, ALL, CML), infection and / or solid tumors. For example, the method of the embodiment can be used to treat cell proliferative diseases, fungal, viral, bacterial or parasitic infections. Pathogens that can be targeted include, but are not limited to, Plasmodium, Trypanosoma, Aspergillus, Candida, HSV, RSV, EBV, CMV, JC virus, BK virus or Ebola pathogens. Other examples of antigens that can be targeted by the CAR cells of the embodiment include, but are not limited to, CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin (meothelin), GD3, HERV-K, IL-11R α, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII or VEGFR2. In certain aspects, the method of the embodiment relates to the targeting of CD19 or HERV-K-expressing cells. For example, HERV-K targeting CAR cells may include a CAR comprising the scFv sequence of monoclonal antibody 6H5. In another aspect, the CAR of the embodiments can be conjugated or fused to a cytokine such as IL-2, IL-7, IL-15, IL-21, or a combination thereof.

[0027] In some embodiments, a method of treating an individual with a medical condition is provided, comprising providing an effective amount of cells from a cell population described herein, in some aspects comprising more than once, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days apart. In specific embodiments, the cancer is lymphoma, leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or a B cell-related autoimmune disease.

[0028] In another embodiment, an isolated or recombinant polypeptide comprising a CD19 targeting CAR is provided, the CAR comprising an amino acid sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. In a related embodiment, an isolated or recombinant polynucleotide sequence encoding a CD19 targeting CAR is provided (e.g., encoding an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1). For example, in some aspects, the polynucleotide sequence is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3 (which encodes CD-19 targeting CAR) or SEQ ID NO: 4 (which encodes CD-19 targeting CAR, expression control sequences and flanking transposon repeats). In another embodiment, a host cell comprising a polypeptide encoding CD19 targeting CAR and / or a polynucleotide encoding CD19 targeting CAR of the embodiment is provided. For example, the host cell can be a T cell or a T cell precursor. Preferably, the host cell is a human cell. Those skilled in the art will recognize that any of the aforementioned polypeptides, polynucleotides or host cells can be used according to the methods described herein.

[0029] In another embodiment, there is provided an isolated or recombinant polypeptide comprising a HERV-K targeting CAR, the CAR comprising an amino acid sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4. In a related embodiment, there is provided an isolated or recombinant polynucleotide sequence encoding a HERV-K targeting CAR (e.g., encoding an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4). For example, in some aspects, the polynucleotide sequence has 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5 (which encodes HERV-K targeting CAR, expression control sequences and flanking transposon repeats). In another embodiment, a host cell comprising a polypeptide encoding a HERV-K targeting CAR and / or a polynucleotide encoding a HERV-K targeting CAR of the embodiment is provided. For example, the host cell can be a T cell or a T cell precursor. Preferably, the host cell is a human cell. Those skilled in the art will recognize that any of the aforementioned polypeptides, polynucleotides or host cells can be used according to the methods described herein.

[0030] In another embodiment, an isolated or recombinant polypeptide is provided comprising membrane-bound IL-15 comprising an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In a related embodiment, an isolated or recombinant polynucleotide sequence encoding membrane-bound IL-15 (e.g., encoding an amino acid sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6) is provided. For example, in some aspects, the polynucleotide sequence is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 (membrane-bound IL-15, expression control sequence, and flanking transposon repeats). In another embodiment, a host cell comprising a polypeptide encoding membrane-bound IL-15 and / or a polynucleotide encoding membrane-bound IL-15 of the embodiments is provided. For example, the host cell can be a T cell, a T cell precursor, or an aAPC. Preferably, the host cell is a human cell. One skilled in the art will recognize that any of the aforementioned polypeptides, polynucleotides, or host cells can be used in accordance with the methods described herein.

[0031] As used in this specification, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more than one.

[0032] Although this disclosure supports definitions referring to only alternatives and "and / or," use of the term "or" in the claims is used to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive. As used herein, "another" can mean at least a second or more.

[0033] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value, or the variability that exists among the study subjects.

[0034] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments provided herein.

[0036] Figure 1 : Summarize the CAR from PB and UCB + Steps in the process of electroporation and propagation of T cells.

[0037] Figure 2: Characterization of genetically modified T cells from PB. (A) EGFP expression on day 0 of the first stimulation cycle to assess the efficiency of gene transfer. (B) Expression of EGFP on day 0 of the first stimulation cycle to assess the efficiency of gene transfer. (C) Expression of EGFP on day 0 of the first stimulation cycle to assess the efficiency of gene transfer. + 、CD8 + and CD4 + Expression of a CD19-specific CAR (CD19RCD28) on T cells assessed by flow cytometry. Similar CAR expression was observed for UCB-derived T cells. (D) Kinetics of CAR expression.

[0038] Figure 3 : PB-derived CAR +Proliferation of T cells. CD3 T cells derived from PB were cultured repeatedly on γ-irradiated aAPC in the presence of recombinant human soluble IL-2 and IL-21. + and CAR + Figure 3. Digital expansion rate of T cells. Upward arrows indicate the addition of gamma-irradiated aAPCs, marking the beginning of each stimulation cycle. UCB-derived CAR + T cells displayed similar numerical expansion rates.

[0039] Figure 4 Genetic modification and propagation of CARs derived from PB and UCB using the SB and aAPC systems + Schematic diagram of the T cell production process. CD19-specific CAR+ T cells were generated by electrotransfer of SB-derived supercoiled DNA plasmids and subsequent co-culture on K562-derived aAPCs (clone #4) in the presence of recombinant human soluble IL-2 and IL-21.

[0040] Figure 5: Collection and characterization of aAPC. (A, B) Sepax volume reduction. aAPC clone #4 grown in a VueLife bag was collected using the CS-490.1 kit in Sepax II. Sepax collection (S, n = 4) was compared to the manual (M, n = 1) procedure. Mean pre- / post-treatment cell counts (4.9 x 10 8 Compare 5x10 8 ) is similar to that using the Sepax system. (C) Phenotype of aAPC (clone #4). Flow cytometric analysis showing the expression of CD19, CD64, CD86, CD137L, and a membrane-bound form of IL-15 (peptide fused to a modified IgG4 Fc region) co-expressed with EGFP (mIL-15-EGFP) on K562 aAPC and K562 parental controls.

[0041] Figure 6 : Schematic diagram of the process of generating clinical grade CD19 specific T cells. MCB (PACT) and WCB (MDACC) were used to generate K562-derived aAPC (clone #4). To generate CAR +T cells, digitally amplified aAPCs in bags, collected using the Sepax II system, irradiated (100 Gy) and cryopreserved for later use. CD19-specific T cells were produced as follows; PBMCs were isolated from normal donor plasmapheresis products using the Sepax II system and cryopreserved. PBMCs were subsequently thawed, electroporated using the Nucleofector system using SB DNA plasmids (CD19RCD28 CAR transposon, SB11 transposase), and co-cultured with thawed irradiated aAPCs along with cytokines (IL-2 and IL-21) for a 28-day culture period, followed by cryopreservation.

[0042] Figure 7: CAR + T cell phenotypes. (A) T cell phenotypes on the day after electroporation (culture day 1) and in the absence of CD19 + Expression of CD19RCD28 CAR on T cells after 28 days of co-culture on aAPC clone #4 in the presence of aAPC. (B) CAR expression analyzed by Western blot using a CD3-ζ specific antibody. Whole cell lysates were electrophoresed on SDS-PAGE under reducing conditions. Molecular weight marker (M), parental Jurkat cells (lane 1), CD19RCD28 + JurkaT cells (lane 2), CAR neg Control primary T cells (lane 3) and CD19RCD28 + T cells (lane 4). (C) CD3 in lymphocyte gates in culture over time + 、CD4 + CAR + and CD8 + CAR + Percentage of T cells expressing CAR. Each symbol represents a separate experiment; the solid line is the average of three validation experiments. (D) Memory / naive CAR at the end of co-culture (d28) + T cell immunophenotype, adhesion, activation, cell lysis, and exhaustion markers.

[0043] Figure 8: CAR + Amplification kinetics and redirection specificity of T cells. Genetically modified T cells were co-cultured with aAPC clone #4 for 28 days. At the end of each stimulation cycle (7 days), cells were counted and stained for the expression of CAR and CD3 of the cells. 3 validation runs (V1, V2 and V3) were performed, and the figure represents the inferred (A) CAR + T cells, (B) CD3 +T cells, (C) total viable cells over time. Arrows indicate the addition of aAPC to the culture. (D) Chromium release assay using CAR for 4 hours + T cell assay, compared to CD19 neg Background cleavage of CD19 by EL-4 + Targets (Daudiβ2m, NALM-6, CD19 + Mean ± SD of each validation run is presented.

[0044] Figure 9: Safety profile associated with the SB system. (A) Telomere length of cells was measured using fluorescence in situ hybridization and flow cytometry (Flow-FISH) assays. The dominant T cell populations (V1 and V2, CD8 + T cells; V3, CD4 + (B) Amplification of the CAR from day 28 using primers and probes specific for the CD19RCD28 CAR. + T cell genomic DNA. Relative quantity (RQ) analysis of CD19RCD28 target copy number was determined using CD19RCD28 CAR-transduced Jurka cells (known to have one CD19RCD28 CAR per genome based on FISH analysis) as a reference and endogenous RNase P as a normalizer. (C) CAR on days 28 and 35 + TCR Vβ analysis of T cells. Data show 3 validation runs of CAR compared to unmanipulated controls at day 0. + Mean ± SD of T cells. (D) Genomic PCR showing the absence of SB11 transposase integration. Genomic DNA (20 ng) was amplified using SB11 or GAPDH primers. CAR amplified using SB11 primers neg Control T cells (lane 5) and CAR + T cells (lane 7); CAR amplified using GAPDH primers neg Control T cells (lane 6), CAR + T cells (lane 8) and Jurkat cells stably expressing SB11 (lane 4). Jurkat cells stably expressing SB11 (Jurkat / SB11-IRES2-EGFP) amplified using SB11 primers (lane 3) and linear plasmid pKan-CMV-SB11 (lane 2) were used as positive controls. (E) CARs from 3 validation runs +G-banded karyotype of T cells showed no structural or numerical changes.Representative chromosome spreads from validation 2 are shown.

[0045] Figure 10 : Generation of CD19RCD28 CAR transposon. CD19RCD28mz (CoOp) / pEK vector and SB DNA plasmid pT-MNDU3-EGFP (Singh et al., 2008; Hollis et al., 2006) containing a codon-optimized chimeric antigen receptor (CAR) were digested with SpeI and NheI and SpeI and NruI to release CAR and EGFP fragments, respectively. The pT-MNDU3 vector lacking EGFP was then ligated with the CAR fragment to produce the CD19RCD28mz (CoOp) / pT-MNDU3 vector. The kanamycin resistance gene and the ColE1 replication origin obtained by digestion of the pEK vector with AseI and PacI were further ligated into the CD19RCD28mz (CoOp) pT-MNDU3 vector digested with SalI and ZraI to produce Pre-CD19RCD28mz (CoOp) / pSBSO. In the final step, the MNDU3 promoter was released from Pre-CD19RCD28mz(CoOp) / pSBSO using digestion with NheI and NsiI and replaced with the hEF-1a promoter fragment obtained from the pVitro4 vector using XhoI and NheI to generate the final vector CD19RCD29mz(CoOp) / pSBSO.

[0046] Figure 11 : Generation of SB11 transposase. The SB transposase vector pCMV-SB11 was digested with PvuII to release a fragment containing the CMV promoter / enhancer and the SB transposase encoding gene, which was ligated to a fragment containing the kanamycin resistance gene and the ColE1 replication origin from the pEK vector to generate the pKan-CMV-SB11 vector.

[0047] Figure 12: Schematic diagram of the CD19 expression plasmid ΔCD19CoOp-F2A-Neo / pSBSO. The DNA fragment encoding the CD19RCD28 CAR from the plasmid CoOpCD19RCD28 / pSBSO was exchanged with a DNA fragment encoding the neomycin resistance gene (NeoR) fused to a codon-optimized (GENEART) truncated CD19 (ΔCD19, [Serrano et al., 2006; Mahmoud et al., 1999]) via an F2A linker (amino acids, VKQTLNFDLLKLAGDVESNPGP; [Szymczak et al., 2004; Yang et al., 2008; Kim et al., 2011]) [PCR cloned from pSelect-Neo (InvivoGen)] to generate ΔCD19CoOp-F2A-Neo / pSBSO. EF1α promoter, elongation factor-1α promoter; NeoR, neomycin resistance gene; bGHpAn, polyadenylation signal from bovine growth factor hormone; ColE1, ori; KanR, kanamycin resistance gene; IR, SB inverted / direct repeat.

[0048] Figure 13 :CD19-specific CAR + The rate of digital expansion of T cells. Genetically modified T cells were co-cultured with aAPCs during a 7-day stimulation cycle, and the total T cells, CD3 + and CAR + Weekly fold expansion rates from each validation run at the end of each stimulation cycle of T cells. Mean fold expansion (n=3) is shown.

[0049] Figure 14 :CD19-specific CAR + Specificity of T cell redirection. CAR+ T cells generated in three validation runs (V1, V2, V3) were specific for CD19 in a standard 4-hour chromium assay. + Tumor targets (Daudiβ2m, NALM-6, CD19 + EL-4) CD19 specific cleavage. neg The background autologous lysis of the control was 1.5%.

[0050] Figure 15 : Safety with respect to chromosomal aberrations. CARs generated from validation runs (V1 and V3) + The G-banded karyotype of T cells showed no structural or numerical changes.

[0051] Figure 16: (A) Representative images of tumor cells (200X) with varying intensities (scored 0-3) of HERV-K expression (top panel) when compared to isotype IgG2a control staining (bottom panel). (B) Images of tumor cells (400X) showing HERV-K staining that is dotted and adjacent to the cell membrane (solid arrows) or more diffuse cytoplasmic staining (dashed arrows). (C) Dot plots representing the H-index for each patient show significant differences between benign and tumor tissues (p < 0.0267). (D) No significant differences were seen between malignant and metastatic tumors. Significant differences were seen between benign tumors and malignant or metastatic tumors.

[0052] Figure 17: (A) HERV-K specific CAR encoding SB plasmid. (B) Representative CD3 + HERV-K-specific CAR + Flow plot of CAR (Fc) expression on T cells. The quadrant percentage of the flow plot is in the upper right corner. CAR, chimeric antigen receptor. (C) HERV-K-specific CAR + T cells and nonspecific CD-19 CAR + There were no significant differences in total cell growth between T cells. (D) By day 21, all HERV-K-specific CAR cells were CD3 + T cells. (E) CAR integration analysis shows HERV-K specific CAR + T cells had less than 2 integrations per cell. Data represent the mean of two independent experiments performed in triplicate using three different donors. (F) HERV-K specific CAR + The phenotype of T cells is CD3 producing high levels of granzyme B. + CD56 + CD45RO hi CD45RA lo CD27 + CD62L + T cells. All data represent the mean of 4 donors.

[0053] Figure 18A -C: (A) Histogram representing HERV-K antigen expression (in red) on tumor cell surface compared to isotype control (in blue). (B) Expression of HERV-K specific CAR using different dilutions compared to control T cells without DNA (in dotted line). +Figure 2. Standard 4-hour CRA of melanoma tumor targets performed by T cells (indicated by solid line). Data are mean ± SD (mean of triplicate measurements for each donor) from four healthy donors pooled from two independent experiments. + Two-way ANOVA with Bonferroni post hoc test was performed between T cells and control cells without DNA. CAR, chimeric antigen receptor; CRA, chromium release assay; E:T, effector to target ratio. (C) When incubated with target, CAR + IFN-γ production by T cells. PMA-ionomycin was used as a positive control.

[0054] Figure 19: HERV-K specific CAR + T cell specificity. (A) and HERV-K neg Histogram of EL4 cells artificially expressing HERV-K antigen (indicated in black) plotted alongside EL4 parental (blue) and isotype control staining (orange). (B) 4-hour CRA showing HERV-K-specific CAR at different E:T ratios. + T cells showed a significant increase in killing of antigen-expressing EL4 cells compared to the parental cells (p < 0.001). (C) Immunoblot assays were performed to show HERV-K env-specific shRNA-mediated knockdown in A888 cells when compared to A888 parental cells or A888 treated with scrambled shRNA. The lower panel shows actin expression as a control. (D) HERV-K-specific CAR + CRA of T cells with A888 HERV-K KD cells, A888 parent (A888P) and A888 scrambled control (A888 scra) showed significant antigen-specific killing by T cells. All data represent the mean of two independent experiments (performed in triplicate using 3 donors). Two-way ANOVA with Bonferroni post-hoc test was used for (B) comparison of EL4 parent with HERV-K + EL4 and one-way ANOVA with Newman-Keuls multiple comparison test were used for (C) comparison of A888 KD with A888 P and A888 scra.

[0055] FIG20 : To determine the HERV-K specific CAR over a 15 hour period + T cell activation, target cells and effector cells were mixed at a ratio of 1:5 (Invitrogen, dead cell stain) were plated in culture medium. 50 images of each target and effector cell were recorded every 7 minutes during this period. (A) Represents HERV-K+ Melanoma cells (A888 and A375) and HERV-Kneg control (HEK293 parental) cells were stained with CAR + T cells are imaged at different time points. Cells that turned green were recorded as dead cells and the intensity of fluorescence was measured. (B, C, D) represent the mean fluorescence intensity of target cells. The upper line represents dead cells, while the lower line represents the baseline intensity of live cells. (E) represents the curve showing the significant difference (*p < 0.05) in the mean fluorescence intensity compared to the HEK293 parental cells at the 15-hour time point. Data represent the average of two independent experiments, with 50 images per experiment. A one-way ANOVA with Tukey's post hoc test was performed.

[0056] Figure 21: HERV-K specific CAR + In vivo anti-tumor activity of T cells. (A) Schematic diagram of the experiment. (B) Representative images of mice from day 3 to day 25. (C) mKate-derived + rRLuc + HERV-K + BLI of A375-SM tumors and (D) post-hoc analysis of liver tissues, where red dots represent mKate + Tumor metastases. Data are mean ± SD (n = 5-6 mice / group). Comparisons were performed using a two-way ANOVA (with Bonferroni post hoc test) between treated and untreated mice (Figure 5E). **P < 0.01 and ***P < 0.001. ANOVA, analysis of differences; BLI, bioluminescence imaging; CAR, chimeric antigen receptor; IL, interleukin.

[0057] Figure 22: (A) shows representative images (200X) of HERV-K antigen expression on tissue sections from 29 normal organs. The H-index was calculated as 0 because no staining was observed in any of these tissues. (B) The H-index of malignant tissues from various organs and different patients is shown in a dot plot.

[0058] Figure 23: (A) shows CD4 + Comparison with CD8 + HERV-K-specific CAR + T cell growth. (B) represents HERV-K specific CAR + nCounter analysis of the expression of various genes in T cells compared to control cells without DNA. Red indicates high expression, while green indicates low mRNA expression levels. (C) In HERV-K specific CAR + Ingenuity pathway analysis of highly expressed genes in T cells.

[0059] Figure 24 : Using melanoma and CD19-specific tumor targets and CD19 CAR + 4-hour standard CRA performed on T cells. All data are representative of two independent experiments (performed with an average of 6 donors and analyzed using two-way ANOVA with Bonferroni post-test).

[0060] Figure 25 : A bidirectional SB plasmid encoding a HERV-K antigen under the hEF-1α promoter and a neomycin resistance gene under the CMV promoter.

[0061] Figure 26 : Images representing HERV-K-specific CAR (shown in green) engaged with HERV-K antigen (shown in red) on the surface of tumor cells.

[0062] Figure 27: (A) SB plasmid encoding myc-ffLuc with neomycin resistance gene. (B) HERV-K specific CAR + T cells and HERV-K-specific CAR-ffLuc + Total cell growth of T cells. (C) Using HERV-K specific CAR-ffLuc + 4-hour CRA of A375SM and EL4 parental cells performed with T cells. (D) Images of mice showing ffLuc activity. (E) Lentiviral plasmids encoding RLuc and mKate for tumor cell imaging.

[0063] Figure 28A -B: Schematic representation of mIL15. A) The mIL15 construct is flanked by inverted repeats that are components of the "Sleeping Beauty" expression plasmid. The mIL15 mutein is a fusion protein of IL-15 with full-length IL-15Rα via a flexible serine-glycine linker. B) Schematic representation of the expressed protein structure of mIL15.

[0064] Figure 29A -B: CAR and mIL15 expression in genetically modified T cells after ex vivo expansion on aAPCs after 5 stimulation cycles. A) Expression of representative samples from 5 donors. B) Expression of the indicated markers (CAR and / or mIL15) in genetically modified T cells.

[0065] Figure 30 : Verification of mIL15 functionality by phosflow of pSTAT5. Unless otherwise indicated, cells were incubated for 5 h in serum and without cytokines to obtain baseline and IL-15-mediated phosphorylation. Representative graphs (n=6).

[0066] Figure 31 : CAR with or without co-expression of mIL15 after 4 stimulation cycles on aAPC + Inferred counts of T cells. CARs were cultured with soluble IL-2 and IL-21 (standard culture conditions) or IL-15 and IL-21 (soluble cytokine control). + T cells and the use of IL-21 to culture mIL15 + CAR + T cells. Data are mean ± SD, n = 4.

[0067] Figure 32A -B: ex vivo expanded mIL15 + CAR + T cell phenotype and specific lytic capacity. A) Percentage of surface expression of certain T cell activation and differentiation markers after 4 stimulations on aAPC. Horizontal lines represent mean values. *P = 0.047, paired t-test, n > 4. B) CD19 expression on aAPC + or CD19 neg CAR from a 4-hour chromium release assay after 5 stimulations of the target + T cells (left) and mIL15 + CAR + T cell (right panel) specific lysis. Data are expressed as mean ± SD, n = 3.

[0068] Figure 33: mIL15 maintains functional activity and resistance to AICD + CAR + Long-term persistence of T cells in vitro. A) mIL15 cells expanded in vitro after 4 aAPC stimulations + / - CAR + T cells underwent withdrawal from antigen restimulation to assess long-term in vitro persistence and to observe expansion kinetics over 60 days. + CAR + T cells do not receive any exogenous cytokine support, and CAR + T cells did not receive cytokines, IL-2, or IL-15. Data are logarithms of the mean ± SD, ****P < 0.0001, RMANOVA, n = 3. B) By comparing with: no target, CD19 + / - EL4, CD19 +Nalm-6 or LAC were incubated together for 6 hours to test the antigen responsiveness of surviving T cells for more than 75 days after antigen exposure. Analysis was performed by flow cytometry using IFNγ intracellular staining. Representative flow graphs are shown, n=3. C) Surviving T cells 75 days after withdrawal were tested, stimulated with aAPC in a 1:1 ratio as previously described, and supplemented with the addition of IL-21 to supplement the culture medium using the cytokines (if any) used in the withdrawal culture maintenance process. After 8 days, T cells were stained with Annexin V to determine the ratio of viable cells to apoptotic / necrotic cells in the stimulated culture. Representative flow graphs are shown, n=3.

[0069] Figure 34A -C: Long-term maintenance of mIL15 + CAR + T cells display forked CD45RA + CCR7 + / - Phenotype. A) mIL15 from stimulation 4 + CAR + Representative flow cytometry of CD45RA and CCR7 populations of T cells and those maintained 75 days after the last antigen stimulation, n = 7. B) Frequency of population subsets from (A). ***P < 0.001 and ****P < 0.0001, RM ANOVA, n = 7. C) shows CCR7 from stimulation 4 neg and CCR7 + mIL15 + CAR + Representative histograms of Annexin V levels in T cells and those maintained 75 days after the last stimulation, n = 3. Histograms were gated on the lymphocyte population to avoid nonspecific CCR7 staining.

[0070] Figure 35A -E: Graph showing the expression of mIL15 by flow cytometry analysis + CAR + Additional characterization of T cells. Figure 35A WD-mIL15 from 3 normal donors that have been cultured for 1-2 years without antigen restimulation + CAR + Steady-state proliferation levels of T cells (PKH dilutions 10 days after staining) (top panel) and the proliferative capacity of these cells after antigen restimulation with aAPCs (bottom panel). Figure 35B , submit long-term removal of mIL15 + CAR +The phenotype of T cells (CD3 and mIL15 surface expression) was used for karyotyping. T cells were first withdrawn with restimulation of aAPCs and subsequently genotyped and submitted for karyotyping. Figure 35C, long-term (1.5-2.46 years) maintenance of mIL15 in vitro in the absence of antigen restimulation and exogenous cytokines. + CAR + Normal karyotype (G banding) of T cells. Representative metaphase chromosome spreads from four normal donors are shown. Figure 35D , memory dynamics after cell stimulation with K562 aAPC. Figure 35E , using 1 vs. 2 CAR and mIL15 + CAR + Stimulated memory dynamics of T cells.

[0071] Figure 36 :Molecular characterization shows that long-term maintenance of mIL15 + CAR + T cells displayed features associated with less differentiated T cell subsets. Based on gene ontology information, mIL15 from stimulation 4 was classified under broad categories. + CAR + T cells were consistently maintained in the withdrawal condition compared to those mIL15 + CAR + All genes significantly differentially expressed between T cells were functionally classified. + CAR + Genes in the up- or down-regulated distribution types in T cells.

[0072] Figure 37 : Validation of transcription factors associated with T cell differentiation states showed that long-term maintenance of mIL15 + CAR + T cells display a poorly differentiated state. Top panel: Selected differentially expressed genes (Tcf-7, Blimp-1, and T-bet) validated by intracellular staining and analyzed by flow cytometry. Representative flow plots are shown, n=5. Bottom panel: Normalized mRNA copy number output from the nCounter analysis system.

[0073] FIG38 : Validation of surface markers associated with T cell differentiation status indicates long-term persistence of mIL15 + CAR +T cells showed low differentiation. Top panel: Select differentially expressed genes, IL-7Ra and CCR7, were validated by staining and analyzed by flow cytometry. *P = 0.0156 and **P < 0.001, one-tailed Wilcoxon paired signed-rank test and paired one-tailed T-test, respectively. Representative flow plots are shown, n = 5-7. Bottom panel: Normalized mRNA copy number from the nCounter analysis system output, n = 3.

[0074] Figure 39: mIL15 + CAR + Acquisition of IL-2 production capacity of T cells. A) Maintained antigen withdrawal mIL15 cells stimulated for 4 h and left unstimulated or activated with lymphocyte activation cocktail (LAC) for 6 h, followed by intracellular IL-2 staining and flow cytometric analysis. + CAR + Representative histograms of IL-2 intracellular staining of T cells (WD-mIL15-CAR). B) Frequency of IL-2-producing LAC-stimulated T cells from (A). ****P<0.0001, n=6, paired t-test.

[0075] Figure 40A -D:mIL15 + CAR + In vivo persistence and antitumor activity of T cells in an environment rich in tumor antigens. A) Schematic diagram of the experiment. B) mIL-15 derived T cells adoptively transferred into mice after Nalm-6 tumor introduction. + / - CAR + ffluc + BLI of T cells (n=5). C) Analysis of the presence of human T cells in spleen (top panel) and bone marrow (bottom panel) on day 14 by staining with human CD3 and detection by flow cytometry. Representative flow plots (n=5). D) CD3 in peripheral blood by flow cytometry + and CD19 + Analysis of the presence of cells in the filtrate-eluted mouse CD45 + Frequency of cells obtained after 48 h. Data depict individual mice and mean ± SD. ***P < 0.001, one-way ANOVA, n = 3-5.

[0076] Figure 41A -F: Evaluation of mIL15 in a low-antigen setting + CAR + In vivo model of T cell persistence and anti-tumor efficacy. A) Schematic diagram of the experiment. B) CAR + T cells or mIL15 + CAR +Adoptive transfer of T cells followed by CD19 T cell transplantation 6 days after T cell transplantation + T cells from mice injected with Nalm-6 tumors (ffLuc + ) BLI. C) Longitudinal BLI monitoring Nalm-6 loading. Images represent photon flux from Nalm-6 cell-derived rLuc activity. D) Using CAR + T cells, mIL15 + CAR + Tumor flux (rLuc) over time in mice treated with T cells or without T cells. Data are mean ± SD. ****P < 0.0001, one-way ANOVA, n = 4-5. E) Human T cells (human CD3 + ) and Nalm-6 tumor cells (human CD19 + ) were analyzed for the collected tissues and blood. F) By using a low tumor model, the long-term survival of mice was assessed to day 98. As previously described for a low tumor model in which mice were transplanted with mIL15-CAR T cells (n=7), CAR T cells (n=8), or without T cells (n=8), followed by NALM-6 tumor stimulation, the experimental conditions were similarly performed. The scores within the arc represent the proportion of mice that survived to day 98. * P = 0.045 (mIL15-CAR vs. CAR T cell treatment), log-rank test (Mantel-Cox).

[0077] Figure 42A-E: The persistence and maintained function of mIL15+CAR+ T cells that are independent of antigen. A) Schematic diagram of the experiment. B) BLI of ffLuc+ T cells in mice treated with CAR+ and mIL15+CAR+ T cells in the presence of tumor antigens. C) Analysis of bone marrow, spleen, and peripheral blood of human CD3+ T cells and CD19+ tumor cells, as detected by flow cytometry. Representative flow graphs are shown (left panel), and the frequency of human CD3+ T cells is plotted (right panel). Data are expressed as mean ± SD, n = 5. **P = 0.0027 (bone marrow), **P = 0.0081 (spleen), ns = not significant, unpaired t-test. D) Longitudinal mapping of T cell flow (ffluc). Background luminescence (shaded in gray) was determined by obtaining flow from mice that did not receive ffluc+ T cells. Data are expressed as mean ± SD, n = 5. *P = 0.0128, **P + 0.00231, unpaired t-test. E) Cells isolated from spleen, liver or bone marrow were expanded ex vivo on aAPC to generate sufficient cell numbers to assess intracellular IFNγ production in response to CD19- and CD19+ targets (as previously described), n=7 from 3 tissue sources and 4 mice. Histograms gated for CD3.

[0078] Figure 43 : Schematic diagram showing an exemplary protocol for CAR T cell generation using SB transposase provided in mRNA form. Effective quantities of active CAR T cells can be generated in two weeks or less (e.g., 16 days).

[0079] Figure 44A -D: Figure 44A , graph (left) and flow cytometry histogram (right) show that the percentage of T cells stably expressing CAR increased significantly from day 9 (8.3%) to day 16 (66.2%). Figure 44B , the graph shows that T cells grew rapidly and expanded 20-fold by day 16 after electroporation. Figure 44C , the graph shows the results of a chromium release assay using T cells on day 16. The generated CAR T cells provide CD-19-specific cytotoxicity against target cells (left panel). Essentially no cytotoxic activity was seen for unmodified T cells (right panel). Figure 44D , Flow cytometry histograms showing the number of central memory T cells at day 9 (left) and day 17 (right) after electroporation. The cells used for these studies were from a donor designated #O and SB11 mRNA was used for electroporation.

[0080] Figure 45A -B: Figure 45A, graphical display is from the result of the chromium release assay using the 9th day (upper figure) and the 15th day (lower figure) T cells.The CAR T cells produced provide CD-19 specific cytotoxicity (left figure) for target cells.Cytotoxic activity (right figure) is not seen substantially for unmodified T cells.Modified T cells kill CD19 positive target cells at the 9th and 15th days with similar efficiency, although the number of CAR positive cells is different.Figure 45B, graphical display CAR copy number (left figure) and CAR expression (right figure) in electroporated cells.The result shows that CAR DNA copy number is reduced from 1.5 to 0.9 from the 15th day to the 22nd day, and remains stable after this time point.The cells used for these studies are from the donor named #1, and SB100x mRNA is used for electroporation.

[0081] Figure 46 Figure 3: Flow cytometry data showing cell viability after electroporation. Following electroporation with DNA / mRNA, the total cell count initially decreased (days 1 and 2), followed by cell growth. Based on cellometer counts, cell number decreased by 59%-76% (viability 24-41%) by day 2 post-electroporation.

[0082] Description of Illustrative Embodiments

[0083] Clinical trials have demonstrated anti-tumor effects in patients who have received T cells genetically modified to have the desired specificity. + A novel approach to T cells. The system combines a highly efficient transfection system with a transposon and transposase system for CAR gene integration. These non-viral approaches have significant advantages as an alternative to viral-mediated transduction, as clinical-grade CARs + T cells should preferably not include added viral sequences. High-quality cGMP CAR T cells are obtained by electrotransferring DNA plasmids derived from transposon systems, such as from Sleeping Beauty or piggyBac, and amplifying the genetically modified T cells on aAPCs. The method results in clinically attractive numbers of CARs that show specificity for their target antigens (e.g., CD19). + The generation of T cells. Importantly, the cells meet the release criteria established by the FDA for use in clinical trials. These methods avoid the genotoxicity caused by virus-mediated transduction and the immunogenicity caused by the use of viruses.

[0084] Chimeric antigen receptors (CARs) do not rely on human leukocyte antigens (HLA) to recognize cell surface tumor-associated antigens and use one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production (Jena et al., 2010). Adoptive transfer of CAR-expressing T cells has shown promising results in multiple clinical trials. It is now possible to use a modular approach to manufacture clinical-grade genetically modified T cells. In certain embodiments, the platform technology disclosed herein includes (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) transposition (e.g., SB transposon, see, U.S. Patent 6,489,458, incorporated herein by reference), (iii) CARs that signal through an internal domain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ or other combinations), (iv) CARs with variable lengths connecting antigen recognition domains to extracellular domains on the cell surface and, in some cases, (v) CARs that can be robustly and digitally amplified. + T cells were derived from K562 artificial antigen-presenting cells (aAPC) (Singh et al., 2008; Singh et al., 2011; Huang et al., 2012).

[0085] In some embodiments, the currently disclosed method can be used to genetically modify T cells derived from peripheral blood and / or umbilical cord blood to express CARs that can be digitally amplified in vitro using aAPCs (Singh et al., 2008; Singh et al., 2011). Due to the relative ease of DNA plasmid production, electroporation, and the use of thawed gamma-irradiated master-bank aAPCs, the method has been involved in cell and gene therapy and can be easily transferred to facilities that operate according to current good manufacturing practices (cGMP) for phase I / II trials. The disclosed method for producing T cells is unique, at least because it does not use (i) viral transduction or (ii) naked DNA electroporation, followed by rapid amplification on PBMC / LCL feeder layers. As an example, a method for targeting CD19 by forced expression of a CAR that does not rely on HLA recognition of CD19 is disclosed. These T cells meet the release criteria defined by sterility, phenotype, viability, and cell number. In-process testing revealed that electroporated / propagated T cells expressed the CAR in the memory / naive population, had a normal karyotype, preserved the TCR Vβ repertoire, and were able to recognize and lyse CD19 in a CAR-dependent manner. + Tumor target.

[0086] Electrotransfer of non-viral plasmids is an attractive alternative to transduction because clinical-grade DNA species can be produced at a cost of about 1 / 10 of recombinant GMP-grade viruses. In order to improve the efficiency of integration, the present inventors transformed the "Sleeping Beauty" (SB) transposon and transferase to adapt it to human applications (Aronovich et al., 2011; Hackett et al., 2010; Izsvak et al., 2010; Kebriaei et al., 2012; Williams, 2008). In addition, they have used piggyBac transposon / transposase to force CAR expression (Manuri et al., 2010). The inventor's SB system uses two DNA plasmids, and the plasmid includes a transposon encoding a target gene (e.g., 2nd generation CD19 Specific CAR transgene, such as specified CD19RCD28) and a transposase (e.g., SB11), which inserts the transgene into the TA dinucleotide repeat sequence in the target cell genome (Geurts et al., 2006; Ivics et al., 1997; Izsvak and Ivics, 1997). In order to increase therapeutic potential, the inventor's 2nd generation CAR (Kowolik et al., 2006) signals through CD28 and CD3-ζ, except that this will support T cell proliferation and recirculation effector function in vivo. In addition, the SB system can be used to express CARs with different extracellular lengths and different endodomain signaling motifs.

[0087] To recover T cell elements that stably express CARs, K562 aAPCs (clone #4) expressing the desired antigen (e.g., CD19) along with costimulatory molecules such as CD86, CD137L, membrane-bound forms of interleukin (IL)-15 (peptides fused to modified IgG4 Fc regions or cytokine peptides fused to IL-15 receptor α), and CD64 (Fc-γ receptor 1) were developed to select T cells capable of supporting CAR-mediated proliferation in vitro. This powerful technology has allowed the manufacture of clinically relevant numbers (up to 10) of T cells suitable for human applications. 10 ) of CAR + T cells. If necessary, additional stimulation can be performed to generate larger numbers of genetically modified T cells. In addition, if fewer CARs are needed +T cells, T cells with fewer cuvettes and digital amplification carrying only one subset can be used to scale down the electroporation and proliferation method by performing 0, 1 or more rounds of proliferation on aAPC (added at the beginning of each stimulation cycle). Typically, at least 90% of the propagated T cells express CAR and are cryopreserved for infusion. In addition, the method can be used to produce T cells against a variety of tumor types by pairing the specificity of the introduced CAR with the expression of tumor-associated antigens (TAA) recognized by the CAR on the aAPC. The ex vivo expansion platform has also been modified to be suitable for the manufacture of NK cells, NK T cells and γδT cells.

[0088] CD4 expressing second-generation CAR + and CD8 + The generation of T cells included cells with stem cell / memory / naive phenotypes and displayed three hallmarks of redirected specificity. First, the genetically modified T cells specifically lysed CD19 + targets. Second, they respond to CD19 + Stimulate cells to produce cytokines (e.g., IFN-γ). Third, they all respond to CD19 in a CAR-dependent manner. + aAPCs and tissue culture environments (e.g., addition of IL-21) have been modified to generate patient- and donor-derived CD19-specific T cells for infusion following hematopoietic stem cell transplantation (Singh et al., 2011; Singh et al., 2008). The present inventors can generate CARs from peripheral blood obtained simply by venipuncture. + This avoids the cost, discomfort, and inconvenience of obtaining mononuclear cells through plasmapheresis. + The ability of T cells is particularly attractive for infusion after allogeneic umbilical cord blood transplantation. The small size and anonymity of neonatal donors avoid re-evaluation of the individual at a later time point, and only a limited number of collected mononuclear cells can be used as starting material for T cell production to avoid interfering with hematopoiesis. Other advances in the production method include a high-throughput electroporation device coupled to a fully enclosed WAVE bioreactor to minimize handling.

[0089] Current production methods include implementing processing and culture systems to reduce workload and prevent aseptic violations. To this end, the inventors co-cultured T cells with gamma-irradiated aAPCs in bioreactors and / or bags rather than culture flasks. This transition typically occurred on day 14 after electroporation. In addition, aAPCs as source material were digitally amplified in bioreactors and / or bags, and the inventors modified the Sepax device to adapt the processing of aAPCs for cryopreservation. When using large volumes of culture medium (>900 mL), the Sepax collection method has an additional advantage over automation because it reduces the additional centrifugation steps required for manual processing.

[0090] I. Definition

[0091] As used herein, the term "chimeric antigen receptor (CAR)" may refer to, for example, artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors, and includes engineered receptors that are transplanted artificially to specific immune effector cells. CAR can be used to confer the specificity of monoclonal antibodies to T cells, thereby allowing a large number of specific T cells to be produced, for example, for adoptive immune cell therapy. In a specific embodiment, CAR, for example, directs the specificity of cells to tumor-associated antigens. In some embodiments, CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In a specific aspect, CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused with CD3-ζ (a transmembrane domain and an inner domain). The specificity of other CAR designs can be derived from a ligand (e.g., a peptide) of a receptor or from a pattern recognition receptor such as Dectin. In a specific embodiment, malignant B cells can be targeted by using a CAR specific for B lineage molecule CD19 to redirect the specificity of T cells. In some cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In some cases, CAR comprises a domain for additional costimulatory signaling such as CD3-ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40. In some cases, molecules can be co-expressed with CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally eliminate T cells after the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0092] As used herein, the term "T cell receptor (TCR)" refers to a protein receptor on T cells that is composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, the TCR is composed of gamma and delta (γ / δ) chains. In embodiments of the present invention, for example, the TCR can be modified, for example, on any cell comprising a TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0093] The terms "tumor-associated antigen" and "cancer cell antigen" are used interchangeably herein. In each case, the terms refer to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0094] II. Chimeric Antigen Receptors

[0095] As used herein, the term "antigen" is a molecule that can be bound by an antibody or a T cell receptor. Antigens are additionally capable of inducing a humoral immune response and / or a cellular immune response, thereby leading to the production of B and / or T lymphocytes.

[0096] Embodiments of the present invention relate to nucleic acids, including nucleic acids encoding antigen-specific chimeric antigen receptor (CAR) polypeptides (including CARs (hCARs) that have been humanized to reduce immunogenicity, which include intracellular signaling domains, transmembrane domains, and extracellular domains comprising one or more signaling motifs). In certain embodiments, the CAR can recognize an epitope consisting of a shared space between one or more antigens. Pattern recognition receptors, such as Dectin-1, can be used to infer specificity for carbohydrate antigens. In certain embodiments, the binding region may include a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to the receptor. A complementarity determining region (CDR) is a short amino acid sequence found in the variable domain of an antigen receptor (e.g., immunoglobulin and T cell receptor) protein that is complementary to the antigen and thereby provides the receptor with its specific needle for that particular antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3). Since antigen receptors are typically composed of two polypeptide chains, there are six CDRs for each antigen receptor that can contact the antigen—three for each heavy and light chain. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable domains. Of these, CDR3 displays the greatest variability because it is encoded by recombination of the VJ (VDJ in the case of heavy and TCR αβ chains) regions.

[0097] It is expected that human CAR nucleic acids are human genes that enhance cellular immunotherapy in human patients. In specific embodiments, the present invention includes full-length CAR cDNA or coding regions. The antigen binding region or domain may comprise a V domain of a single chain variable fragment (scFv) derived from a specific human monoclonal antibody. H and V L The fragments of the present invention can be fragments of the human antigen-binding domains of the human antigen-specific antibody, such as those described in U.S. Patent No. 7,109,304 (incorporated herein by reference). The fragments can also be many different antigen-binding domains of human antigen-specific antibodies. In more specific embodiments, the fragments are antigen-specific scFvs encoded by sequences optimized for human codon usage for expression in human cells.

[0098] The arrangement can be multimeric, such as a diabody or a multimer. The multimer is most likely formed by cross-pairing the variable portions of the light and heavy chains into a substance that has been called a diabody by Winters. The hinge portion of the construct can have multiple alternatives ranging from being completely deleted to having a maintained first cysteine, to being substituted with proline instead of serine, to being truncated until the first cysteine. The Fc portion can be deleted. Any protein that is stable and / or dimerizing can be used for this purpose. Only one of the Fc domains can be used, for example, the CH2 or CH3 domains from a human immunoglobulin. The hinge, CH2 and CH3 regions of a human immunoglobulin that has been modified to increase dimerization can also be used. Only the hinge region of an immunoglobulin can also be used. Parts of CD8α can also be used.

[0099] The intracellular signaling domain of the chimeric receptor of the present invention is responsible for the activation of at least one normal effector function of the immune cell in which the chimeric receptor has been placed. The term "effector function" refers to a specialized function of a differentiated cell. The effector function of a T cell, for example, can be cytolytic activity or helper activity, including the secretion of cytokines. Effector function in naive, memory, or anamnestic T cells includes antigen-dependent proliferation. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain will generally be used, in many cases, it is not necessary to use the entire intracellular polypeptide. To the extent that truncated portions of the intracellular signaling domain can be used, such truncated portions can be used in place of the intact chain as long as they still transduce the effector function signal. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal. Examples include the ζ chain of the T cell receptor or any of its homologs (e.g., η, δ, γ or ε), MB1 chain, B29, Fc RIII, Fc RI and combinations of signaling molecules, such as CD3ζ and CD27, 4-1BB, DAP-10, OX40 and combinations thereof, as well as other similar molecules and fragments. The intracellular signaling portion of other members of the family of activating proteins can be used, such as FcγRIII and FcεRI. For disclosures on cTCRs using these alternative transmembrane and intracellular domains, see Gross et al. (1992), Stancovski et al. (1993), Moritz et al. (1994), Hwu et al. (1995), Weijtens et al. (1996) and Hekele et al. (1996). In a preferred embodiment, the human CD3ζ intracellular domain is used for activation.

[0100] The antigen-specific extracellular domain and the intracellular signaling domain can be connected by a transmembrane domain such as the human IgG4 Fc hinge and Fc region. Alternatives include the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3 ζ domain or the cysteine-mutated human CD3 ζ domain, or other transmembrane domains from other human transmembrane signaling proteins such as CD16 and CD8 and the erythropoietin receptor.

[0101] In some embodiments, CAR nucleic acid includes sequences encoding other co-stimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains. Other co-stimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to the initial signal triggered by CD3ζ, the additional signal provided by the human co-stimulatory receptors inserted in human CAR is important for the complete activation of T cells and can help improve the success of persistence and adoptive immunotherapy in vivo.

[0102] In a specific embodiment, the present invention relates to an isolated nucleic acid fragment and an expression cassette incorporating a DNA sequence encoding a CAR. The vector of the present invention is designed to deliver the desired gene primarily to immune cells, preferably T cells, under the control of a regulated eukaryotic promoter such as the MNDU3 promoter, the CMV promoter, the EF1α promoter, or the ubiquitin promoter. Similarly, the vector may also contain a selection marker, if there is no other reason, to facilitate its in vitro manipulation. In other embodiments, the CAR can be expressed from mRNA transcribed in vitro from a DNA template.

[0103] Chimeric antigen receptor molecules are recombinants and are characterized in that they bind antigens and conduct activation signals by the immune receptor activation motif (ITAM) present in their cytoplasmic tails. The receptor construct utilizing antigen binding moiety (for example, produced from single-chain antibodies (scFv)) is provided as the additional advantage of "universal", because they bind to the natural antigens on the target cell surface in a manner independent of HLA. For example, several laboratories have reported scFv constructs (Eshhar et al., 1993) fused with sequences of the intracellular portion of the ζ chain (ζ) of the coding CD3 complex, Fc receptor γ chain and sky tyrosine kinase; Fitzer-Attas et al., 1998). Redirected T cell effector mechanisms (including tumor recognition and cracking by CTL) have been recorded in several mouse and human antigen-scFv:ζ systems (Eshhar, 1997; Altenschmidt et al., 1997; Brocker et al., 1998).

[0104] So far, non-human antigen binding regions are generally used to construct chimeric antigen receptors. About using non-human antigen binding regions, the potential problem such as mouse monoclonal antibodies is the lack of human effector function and the inability to penetrate into tumor mass. In other words, such antibodies may not mediate complement dependent lysis or lyse human target cells to destroy cells expressing CAR by antibody dependent cellular toxicity or Fc receptor mediated phagocytosis. In addition, non-human monoclonal antibodies can be recognized as foreign proteins by the human host, and therefore, repeated injections of such foreign antibodies can cause the induction of an immune response leading to harmful hypersensitivity reactions. For mouse-based monoclonal antibodies, this is often referred to as human anti-mouse antibodies (HAMA) responses. Therefore, the use of human antibodies is more preferred because they do not cause the same strong HAMA response as mouse antibodies. Similarly, using human sequences in CAR can avoid immune-mediated recognition and the resulting elimination produced by endogenous T cells present in the recipient and recognizing the processed antigen in the case of HLA.

[0105] In some embodiments, the chimeric antigen receptor comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.

[0106] In a specific embodiment, the intracellular receptor signaling domain in the CAR includes, for example, those intracellular receptor signaling domains in the T cell antigen receptor complex, such as the ζ chain of CD3, as well as FcγRIII costimulatory signaling domains, CD28, CD27, DAP10, CD137, OX40, CD2 (alone or in series with CD3ζ). In a specific embodiment, the intracellular domain (which may be referred to as a cytoplasmic domain) includes one or more of TCRζ chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12 and CD40. In some embodiments, any portion of the endogenous T cell receptor complex is used in the intracellular domain. One or more cytoplasmic domains can be used because the so-called third generation CAR has, for example, at least two or three signaling domains fused together to obtain an additive or synergistic effect.

[0107] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as an extracellular domain comprising an antigen binding region) comprises a tumor-associated antigen or a pathogen-specific antigen structural domain, including carbohydrate antigens recognized by pattern recognition receptors such as Dectin-1. Tumor-associated antigens may be of any type as long as they are expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigens, melanoma-associated antigens, mutated p53, mutated ras, etc. In certain embodiments, when there is a low amount of tumor-associated antigen, CAR can be co-expressed with membrane-bound cytokines to improve persistence. For example, CAR can be co-expressed with membrane-bound IL-15.

[0108] In certain embodiments, intracellular tumor-associated antigens can be targeted, such as HA-1, survivin, WT1, and p53. This can be achieved by expressing a CAR on universal T cells that recognizes processed peptides derived from intracellular tumor-associated antigens in the context of HLA. Additionally, universal T cells can be genetically modified to express a pair of T cell receptors that recognize intracellular processed tumor-associated antigens in the context of HLA.

[0109] Pathogen can be any kind, but in specific embodiments, pathogen is for example fungus, bacterium or virus.Exemplary viral pathogens include those viral pathogens of Adenoviridae, Epstein-Barr virus (EBV) Section, Cytomegalovirus (CMV) Section, Respiratory Syncytial Virus (RSV) Section, JC Viridae, BK Viridae, HSV, HHV Viridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae and Togaviridae.Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, varicella, Ebola and rubella.Exemplary pathogenic fungi include Candida (Candida), Aspergillus (Aspergillus), Cryptococcus (Cryptococcus), Histoplasma (Histoplasma), Pneumocystis (Pneumocystis) and Stachybotrys (Stachybotrys). Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, Escherichia coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes and Salmonella. In one embodiment, the pathogen receptor dectin-1 can be used to produce a CAR that recognizes the carbohydrate structure on the fungal cell wall. T cells genetically modified to express a specific CAR based on Dectin-1 can recognize Aspergillus and target hyphae growth. In another embodiment, antibodies can be produced based on the identification of viral determinants (e.g., glycoproteins from CMV and Ebola viruses) that interrupt viral infection and pathology.

[0110] In some embodiments, the pathogenic antigen is an Aspergillus carbohydrate antigen for which the extracellular domain in the CAR recognizes the carbohydrate pattern of the fungal cell wall, such as by Dectin-1.

[0111] Chimeric immunoreceptors according to the present invention can be produced by any method known in the art, although preferably they are produced using recombinant DNA technology. Nucleic acid sequences encoding several regions of the chimeric receptor can be prepared and assembled into the complete coding sequence using standard molecular cloning techniques (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.). The resulting coding regions can be inserted into expression vectors and used to transform appropriate expression host allogeneic T cell lines.

[0112] As used herein, nucleic acid construct or nucleic acid sequence or polynucleotide is intended to refer to a DNA molecule that can be transformed or transduced into a T cell and transcribed and translated to produce a product (eg, a chimeric antigen receptor).

[0113] In exemplary nucleic acid constructs (polynucleotides) for use in the present invention, promoters are operably linked to nucleic acid sequences encoding the chimeric receptors of the present invention, i.e., they are positioned so as to promote transcription of messenger RNA from the DNA encoding the chimeric receptor. The promoters may be of genomic origin or synthetically produced. Various promoters for T cells are well known in the art (e.g., the CD4 promoter disclosed by Marodonet et al. (2003)). The promoters may be, for example, constitutive or inducible (where induction is associated with a particular cell type or a particular level of maturation). Alternatively, many well-known viral promoters are also suitable. Target promoters include the β-actin promoter, the SV40 early and late promoters, immunoglobulin promoters, the human cytomegalovirus promoter, retroviral promoters, and the Friend spleen lesion formation virus promoter. The promoters may or may not be associated with enhancers, wherein the enhancer may naturally associate with a particular promoter or with a different promoter.

[0114] The sequence encoding the open reading frame of the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA or provide T cell-specific expression (Barthel and Goldfeld, 2003). Similarly, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0115] To express the chimeric antigen receptors of the present invention, the naturally occurring or endogenous transcriptional initiation region of the nucleic acid sequence encoding the N-terminal component of the chimeric receptor can be used to produce the chimeric receptor in the target host. Alternatively, an exogenous transcriptional initiation region can be used to allow constitutive or inducible expression, wherein expression can be controlled depending on the target host, the desired expression level, the nature of the target host, etc.

[0116] Similarly, the signal sequence that chimeric receptor is directed to surface membrane can be the endogenous signal sequence of the N-terminal assembly of chimeric receptor.Optionally, in some cases, it may be desirable to exchange this sequence with different signal sequences.However, selected signal sequence should be compatible with the secretory pathway of T cell, so that chimeric receptor is presented on the surface of T cell.

[0117] Similarly, the terminator region can be provided by a naturally occurring or endogenous transcriptional terminator region of the nucleic acid sequence encoding the C-terminal component of the chimeric receptor. Alternatively, the terminator region can be derived from a variety of sources. In most cases, the source of the terminator region is generally considered unimportant for the expression of the recombinant protein, and a variety of terminators can be used without adversely affecting expression.

[0118] As will be appreciated by those skilled in the art, in some cases, several amino acids (e.g., typically no more than 10, more typically no more than 5 residues) at the end of the antigen binding domain in the CAR may be deleted. Similarly, it may also be desirable to introduce a small number of amino acids at the border, typically no more than 10, more typically no more than 5 residues. The deletion or insertion of amino acids may be for structural needs, providing convenient restriction sites, ease of operation, increased expression levels, etc. Additionally, substitution of one or more amino acids with different amino acids may occur for similar reasons, typically replacing no more than about 5 amino acids in any one domain.

[0119] Chimeric constructs encoding the chimeric receptors according to the present invention can be prepared in a convenient manner. Since, in most cases, native sequences can be used, the native genes can be isolated and manipulated where appropriate to allow for the proper connection of the various components. Thus, the nucleic acid sequences encoding the N-terminal and C-terminal proteins of the chimeric receptor can be isolated using polymerase chain reaction (PCR) using appropriate primers that result in deletion of unwanted portions of the gene. Alternatively, restriction digestion of the cloned gene can be used to generate the chimeric construct. In both cases, the sequence can be selected to provide blunt-ended or complementary overlapping restriction sites.

[0120] The various manipulations used to prepare chimeric constructs can be performed in vitro, and in specific embodiments, standard transformation or transfection methods are used to introduce the chimeric constructs into vectors for cloning and expression in appropriate hosts. Thus, after each manipulation, the construct resulting from the ligation of DNA sequences is cloned, the vector is isolated, and the sequence is screened to ensure that it encodes the desired chimeric receptor. The sequence can be screened by restriction analysis, sequencing, etc.

[0121] The chimeric construct of the present invention is used for subjects suffering from cancer or suspected of having cancer by reducing the size of the tumor or preventing the tumor from growing or regrowing in these subjects. Therefore, the present invention also relates to a method for reducing tumor growth or preventing its formation in a subject, in which the chimeric construct of the present invention is introduced into the isolated T cells of the subject and the transformed T cells are reintroduced into the subject, thereby achieving an anti-tumor response to reduce or eliminate the tumor in the subject. Suitable T cells that can be used include cytotoxic lymphocytes (CTLs) or any cells with T cell receptors that need to be destroyed. As is well known to those of ordinary skill in the art, various methods can be easily obtained for separating these cells from a subject. For example, using cell surface marker expression or using commercially available kits (e.g., ISOCELL from Pierce, Rockford, 111) TM ).

[0122] It is contemplated that the chimeric construct can be introduced into the subject's own T cells as naked DNA or in a suitable vector. Methods for stably transfecting T cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding the chimeric receptor of the present invention contained in a plasmid expression vector for expression in an appropriate orientation. Advantageously, the use of naked DNA reduces the time required to generate T cells expressing the chimeric receptor of the present invention.

[0123] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce the chimeric construct into T cells. Suitable vectors for use in accordance with the methods of the present invention are non-replicating in the T cells of the subject. Many viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain cell viability. Illustrative vectors include the pFB-neo vector disclosed herein. and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0124] Once it is determined that the transfected or transduced T cells are capable of expressing the chimeric receptor as a surface membrane protein by the required regulation and at the required level, it can be determined whether the chimeric receptor has the function of providing the required signal induction in the host cell. Subsequently, the transduced T cells are reintroduced or administered to the subject to activate the anti-tumor response in the subject. For ease of administration, the transduced T cells according to the present invention can be made into a pharmaceutical composition or an implant suitable for in vivo administration with an appropriate carrier or diluent (which can also be pharmaceutically acceptable). Methods for preparing such compositions or implants have been described in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack edited (1980)). When appropriate, the transduced T cells can be formulated into a preparation in a semisolid or liquid form, such as a capsule, solution, injection, inhalant or aerosol, in the usual manner for their respective routes of administration. Methods known in the art can be used to prevent or reduce the release and absorption of the composition before it reaches the target tissue or organ, or to ensure the timed release of the composition. Ideally, however, a pharmaceutically acceptable form is used that does not neutralize the cells expressing the chimeric receptor.Thus, ideally, the transduced T cells are formulated into a pharmaceutical composition containing a balanced salt solution, preferably Hanks balanced salt solution or normal saline.

[0125] III. Methods and Compositions Related to the Embodiments

[0126] In certain aspects, the present invention includes a method of preparing and / or expanding antigen-specific redirected T cells, comprising transfecting T cells with an expression vector containing a DNA construct encoding an hCAR, and then optionally stimulating the cells with antigen-positive cells, recombinant antigens, or antibodies to receptors that cause cell proliferation.

[0127] In another aspect, a method for stably transfecting and redirecting T cells using naked DNA by electroporation or other non-viral gene transfer (such as, but not limited to sonoporation) is provided. Most researchers have used viral vectors to carry foreign genes into T cells. By using naked DNA, the time required to produce redirected T cells can be reduced. "Naked DNA" refers to DNA encoding a chimeric T cell receptor (cTCR) contained in an expression cassette or vector for expression in an appropriate orientation. The electroporation method of the present invention produces stable transfectants expressing and carrying a chimeric TCR (cTCR) on their surface.

[0128] "Chimeric TCR" means a receptor expressed by a T cell and comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain can specifically bind to an antigen that is not normally bound by a T cell receptor in this manner in a manner not constrained by MHC. Under appropriate conditions, the stimulation of T cells by antigens results in the proliferation (amplification) of cells and / or the production of IL-2. The exemplary CD19- and HERV-K specific chimeric receptors of the present application are examples of chimeric TCRs. However, the method is also applicable to the transfection of chimeric TCRs specific for other target antigens, such as for HER2 / Neu (Stancovski et al., 1993), ERBB2 (Moritz et al., 1994), folate binding protein (Hwu et al., 1995), renal cell carcinoma (Weitjens et al., 1996), and HIV-1 envelope glycoproteins gp120 and gp41 (Roberts et al., 1994). Other cell surface target antigens include, but are not limited to, CD20, carcinoembryonic antigen, mesothelin, ROR1, c-Met, CD56, GD2, GD3, alpha-fetoprotein, CD23, CD30, CD123, IL-11Rα, kappa chain, lambda chain, CD70, CA-125, MUC-1, EGFR and variants, epithelial tumor antigens, etc.

[0129] In some aspects, T cells are primary human T cells, such as PBMCs (PBMCs) derived from human peripheral blood, T cells of PBMCs, bone marrow or umbilical cord blood collected after stimulation with G-CSF. Conditions include the use of mRNA and DNA and electroporation. After transfection, cells can be immediately infused or stored. In some aspects, after transfection, cells can be propagated in vitro as a large group for several days, weeks or months within about 1, 2, 3, 4, 5 days or more after gene transfer into cells. In another aspect, after transfection, transfectants are cloned and clones of expression cassettes or plasmids that show the presence of a single integrated or episomal expression and expression of chimeric receptors are amplified in vitro. The clones selected for amplification show the ability to specifically recognize and lyse target cells expressing CD19. Recombinant T cells can be amplified by the stimulation of IL-2 or other cytokines (e.g., IL-7, IL-12, IL-15, IL-21, etc.) that bind to common γ chains. Recombinant T cells can be amplified by the stimulation of artificial antigen presenting cells. Recombinant T cells can be expanded on artificial antigen presenting cells or using antibodies such as OKT3 that crosslink CD3 on the surface of T cells. Subsets of recombinant T cells can be deleted on artificial antigen presenting cells or using antibodies such as Campath that binds CD52 on the surface of T cells. In another aspect, the genetically modified cells can be cryopreserved.

[0130] Post-infusion T cell proliferation (survival) can be assessed by: (i) q-PCR using primers specific for the CAR; (ii) flow cytometry using antibodies specific for the CAR; and / or (iii) soluble TAA.

[0131] Embodiments of the present invention also relate to targeting B cell malignancies or disorders involving B cells that have cell surface epitopes specific for CD19 using redirected immune T cells. Malignant B cells are excellent targets for redirected T cells because B cells can serve as immunostimulatory antigen presenting cells for T cells. Preclinical studies supporting the anti-tumor activity of adoptive therapy with donor-derived CD19-specific T cells with human or humanized CARs include (i) CD19 + Redirected killing of targets, (ii) in combination with CD19 + Redirected secretion / expression of cytokines after incubation with target / stimulator cells, and (iii) in vitro with CD19 + Target / stimulator cells continue to proliferate after incubation.

[0132] In certain embodiments of the present invention, CAR cells are delivered to individuals in need thereof, such as individuals suffering from cancer or infection. The cells then improve the individual's immune system to attack respective cancer cells or pathogenic cells. In some cases, one or more doses of antigen-specific CART- cells are provided to the individual. In the case where two or more doses of antigen-specific CAR T cells are provided to the individual, the duration between administrations should be sufficient to allow for time to be reserved for proliferation in the individual, and in a specific embodiment, the duration between doses is 1, 2, 3, 4, 5, 6, 7 days or more.

[0133] The source of the modified allogeneic T cells comprising two chimeric antigen receptors and lacking a functional TCR can be of any type, but in specific embodiments, the cells are obtained from, for example, a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells. A suitable dose for achieving a therapeutic effect, for example, preferably over a series of administration cycles, can be at least 10 5 or about 10 5 to about 10 10 An exemplary dosing regimen consists of four weekly cycles of increasing doses, starting on Day 0 with at least about 10 5 cells, for example gradually increasing up to about 10 cells within a few weeks of starting an intrapatient dose escalation regimen. 10 Suitable modes of administration include intravenous, subcutaneous, intracavitary (eg, via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0134] The pharmaceutical composition of the present invention can be used alone or in combination with other confirmed medicaments for the treatment of cancer.Whether it is delivered alone or in combination with other medicaments, the pharmaceutical composition of the present invention can be delivered to various parts in mammal (particularly human) body to achieve specific effects by various approaches. Those skilled in the art will recognize that, although more than one approach can be used, a specific approach can provide a more direct and more effective reaction than another approach. For example, for the treatment of melanoma, intradermal delivery (which is better than inhalation) can be advantageously used. Local or systemic delivery can be achieved by applying (including applying the preparation in the body cavity or instilling it into the body cavity, inhaling or blowing into an aerosol), or by introducing parenterally (including intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous or intradermal administration).

[0135] The compositions of the present invention can be provided in unit dosage form, wherein each dosage unit, for example, an injection, contains a predetermined amount of the composition alone or in appropriate combination with other active agents. As used herein, the term unit dosage form refers to physically separate units suitable for use as unit dosages in human and animal subjects, each unit containing a predetermined amount of the composition of the present invention alone or in combination with other active agents, the amount being calculated to be sufficient to produce the desired effect when combined with a pharmaceutically acceptable diluent, carrier, or vehicle, as appropriate. The technical specifications of the novel unit dosage form of the present invention depend on the specific pharmacodynamics associated with the pharmaceutical composition in a particular subject.

[0136] Ideally, an effective amount or sufficient number of isolated transduced T cells are present in the composition and introduced into a subject to establish a long-term specific anti-tumor response to reduce the size of the tumor or eliminate tumor growth or regrowth that would otherwise result in the absence of such treatment. Ideally, the amount of transduced T cells reintroduced into the subject results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% reduction in tumor size when compared to the same conditions in which the transduced T cells are not present.

[0137] Therefore, the amount of transduced T cells administered should take into account the route of administration and should be such that a sufficient number of the transduced T cells will be introduced to achieve the desired therapeutic response. In addition, the amount of each active agent included in the compositions described herein (e.g., the amount per cell to be contacted or the amount per a certain body weight) may vary in different applications. In general, the concentration of transduced T cells should ideally be sufficient to provide at least about 1×10 6 to about 1×10 9 transduced T cells, or even more ideally, about 1 × 10 7 to about 5×10 8transduced T cells, although any suitable amount may be used herein, for example greater than 5×10 8 cells, or less than, for example, less than 1×10 7 Dosing regimens can be based on established cell-based therapies (see, eg, Topalian and Rosenberg, 1987; US Patent No. 4,690,915), or alternative continuous infusion strategies can be used.

[0138] These values ​​provide a general guide to the range of transduced T cells to be used by a doctor after optimizing the method of the present invention for the practice of the present invention. The citation of such ranges herein in no way excludes the use of higher or lower amounts of components, which can be guaranteed in specific applications. For example, actual dosages and regimens may depend on whether the pharmaceutical composition is administered in combination with other pharmaceutical compositions, or vary depending on individual differences in pharmacokinetics, drug disposition, and metabolism. Those skilled in the art can easily make any necessary adjustments according to the urgency of a particular situation.

[0139] IV. Exemplary Human CD19-Specific Chimeric Antigen Receptor T Cells

[0140] CD19 (a cell surface glycoprotein of the immunoglobulin superfamily) is a potential attractive target for the antibody therapy of B cell-related malignancies. This antigen is not present in hematopoietic stem cells, and in healthy individuals, its presence is only limited to B system and possibly some follicular dendritic cells (Scheuermann et al., 1995). In fact, it is present in the B cells from the earliest recognizable B lineage cells during the developmental process to B cell blasts, but loses in the maturation to plasma cells. In addition, CD19 does not fall off from the cell surface and rarely loses (Scheuermann et al., 1995) during tumor transformation. The protein is expressed on most malignant B lineage cells, including cells from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL) (Uckun et al., 1988). CD19 is mainly used as the B cell co-receptor combined with CD21 and CD81. Upon activation, the cytoplasmic tail of CD19 is phosphorylated, which leads to binding by Src family kinases and recruitment of PI-3 kinase.

[0141] In one aspect, the compositions and methods of the embodiments relate to human CD19-specific chimeric T cell receptor (or chimeric antigen receptor, CAR) polypeptides (designated as hCD19CAR), the receptor polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a human CD19 binding region. In another aspect, the CD19 binding region is F(ab')2, Fab', Fab, Fv, or scFv. The binding region may comprise an amino acid sequence having at least, at most, or about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the wild-type amino acid sequence. The intracellular domain may comprise the intracellular signaling domain of human CD3ζ, and may further comprise a human CD28 intracellular segment. In certain aspects, the transmembrane domain is a CD28 transmembrane domain.

[0142] In another aspect, the compositions of the present invention comprise nucleic acids encoding the above-mentioned polypeptides.In certain aspects, the nucleic acid sequences are optimized for human codon usage.

[0143] In another aspect, the composition of the present invention includes cells expressing the polypeptides described herein. T cells may contain an expression cassette encoding an hCD19CAR polypeptide. The expression cassette may be contained in a non-viral vector, such as a transposon or a human transposon or a recombinant variant thereof. The expression cassette may be packaged and contained in a viral vector or a recombinant variant thereof. The expression cassette may be integrated into the genome or maintained in an episomal form or expressed from mRNA.

[0144] In another aspect, the present invention includes a method for producing a T cell expressing a human CD19-specific CAR, the method comprising introducing an expression cassette into a cell, wherein the expression cassette encodes a polypeptide comprising a human extracellular CD19 binding domain, a transmembrane domain, and one or more intracellular signaling domains. The method may further comprise using a CD19 + Cells, recombinant CD19 or antibodies against the receptor stimulate the cells to cause cell proliferation, killing and / or cytokine production; for example, CD19 + The artificial antigen-presenting cells stimulate the cells to proliferate or expand.

[0145] In certain aspects, the present invention includes a method for treating a human disease state associated with cells expressing endogenous CD19, comprising infusing a patient with a recombinant cell expressing a human CD19-specific CAR in an amount sufficient to treat the condition, wherein the human CD19-specific CAR comprises a human CD19 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The condition can be, for example, lymphoma, leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or a B cell-related autoimmune disease.

[0146] The present invention relates to the generation of human CD19 specific chimeric antigen receptor (hCD19RCD28 or hCAR).In some aspects, the recombinant cells expressing hCAR have improved in vivo maintenance and anti-tumor efficacy.Human hCAR has an immunogenicity reduced compared to mouse hCAR, and the mouse hCAR includes scFv segments derived from mouse CD19 specific monoclonal antibodies (mAbs). Anti-tumor effects can be enhanced by genetically modified cells (making them specific for CD19). Typically, T cell specificity is achieved by electrotransfer of the expression cassette encoding hCAR.

[0147] The hCAR can be a chimeric receptor comprising one or more activation endodomains such as an activation domain derived from CD3-ζ. Additional T cell activation motifs include, but are not limited to, CD28, CD27, OX-40, DAP10 and 4-1BB. In some aspects, the activation domain can also include CD28 transmembrane and / or activation domains. In another aspect, the hCAR coding region and / or expression cassette codons optimized for expression in human cells and subjects (for example, in one embodiment, the scFv regions obtained from the VH and VL sequences of CD19 specific human antibodies) are incorporated into the CD19 binding segments of hCAR (for example, referring to U.S. Patent No. 7,109,304, which are incorporated herein by reference as a whole). In another embodiment, the hCAR expression cassette is maintained or integrated into the genome of a recombinant cell in an episomal form. In some aspects, the expression cassette is included in nucleic acids, viral vectors (such as retroviral vectors) or non-viral vectors (such as transposon mechanisms) that can be integrated using an integrase mechanism. In another embodiment, the expression cassette is contained within a transposon-based nucleic acid.In a specific embodiment, the expression cassette is part of the two-component "Sleeping Beauty" (SB) or piggyBac system, which utilizes a transposon and a transposase for enhanced non-viral gene transfer.

[0148] The cell number expressing recombinant hCAR can be expanded to a clinically significant quantity. An example of such amplification uses artificial antigen presenting cells (aAPC). The cells expressing recombinant hCAR can be verified and identified by flow cytometry and western blot analysis. The T cells expressing recombinant hCAR of CD19 specific CAR can recognize and kill target cells expressing CD19. In another aspect, hCAR can be expressed into universal cells, which can then be infused through the transplant barrier to help prevent immunogenicity. The hCAR can be used together with human genes to perform imaging (such as by positron emission tomography, PET) and conditional elimination (if there is cytotoxicity) of T cells. The recombinant T cells of the present invention can be used for CD19 specific cell therapy.

[0149] V. Exemplary HERV-Targeted Chimeric Antigen Receptor T Cells

[0150] In the Human Genome Project, it was discovered that a group of ancient retroviruses called human endogenous retroviruses (HERVs) were stably integrated into the human genome, constituting 8.5% of the total human genome. Among HERVs, HERV-K was found to be an oncogenic allele variant involved in melanoma, breast cancer, ovarian cancer, teratocarcinoma and prostate cancer, as well as various autoimmune diseases (such as multiple sclerosis and rheumatoid arthritis) (Buscher et al., 2005; Dreyfus, 2011; Seifarth et al., 1995). The oncogenic potential of HERV-K is attributed to the envelope (env) and GAG protein (Rec). Recent studies have shown that HERV-K env protein is only expressed on the surface of tumor cells and not on normal skin cells (Wang-Johanning et al., 2008; Li et al., 2010). It was found that the expression of HERV-K env protein increased with more aggressive and metastatic type III and IV melanomas rather than with less aggressive and limited type I melanomas (Buscher et al., 2005; Hahn et al., 2008; Serafino et al., 2009). This selective expression of HERV-K env protein on melanoma cells can be used as a treatment strategy for patients with refractory or metastatic melanomas, who have a poor prognosis due to resistance to conventional therapies such as chemotherapy, irradiation and surgery (Bhatia et al., 2009). Therefore, new targeted therapy strategies are needed to improve treatment outcomes.

[0151] In order to produce T cell therapy for melanoma, the present inventors target TAAs derived from HERV, whose genome was stably integrated into humans millions of years ago. In order to target HERK-K, T cells are engineered to express CAR specific for env protein by replacing the antigen binding ectodomain of CD19 specific CAR with a single chain antibody (scFv) sequence of an anti-HERV-K env specific monoclonal antibody. The new CAR is cloned into the SB system as a transposon. DNA plasmids encoding HERV-K env specific CAR and SB transposase are electrotransferred into primary human T cells, and genetically modified CARs are selectively propagated on irradiated artificial antigen presenting cells (aAPCs) expressing HERV-K env and desired T cell costimulatory molecules. + After co-culture on γ-irradiated aAPC, 95% of CD3 + T cells express CAR and + In contrast to T cells, these CAR + T cells can specifically kill HERV-K env in vitro + Tumor targets, but does not kill HERV-K env - Tumor targets. By comparing HERV-K env - These CARs were expressed in antigen-negative EL4 mouse cells that preferentially killed parental cells. + T cell specificity, and HERV-K knockdown in A888-mel cells by shRNA (specifically targeting HERV-K env RNA) resulted in reduced killing compared to the parental CAR. + T cells also successfully slowed tumor growth and reduced metastasis of A375-supermetastatic (SM) tumor cells from the lung to the liver in vivo. + The mice bearing tumors with T cells survived longer and appeared healthier than the group of mice bearing tumors alone. Thus, using an approach that has translational appeal for clinical trials, T cells targeting active retroviruses could be used as an immunotherapy for melanoma.

[0152] The clonal evolution of melanoma cells can render TCR therapy ineffective due to their dependence on MHC complexes for tumor recognition. Unlike TCR-based T cell therapy, CAR can overcome this clonal evolution of melanoma cells by mediating cell death in a manner independent of MHC. HERV-K antigens are only expressed on the surface of tumor cells and not on normal cells. Therefore, HERV-K-specific CARs are not suitable for melanoma cell therapy. +T cells can specifically target and eliminate tumor cells without any adverse side effects. They can be infused to treat HERV-K cells at many stages along the cancer spectrum, from minimal residual disease to bulky tumors to disease that is refractory to conventional therapy. + Malignant tumor.

[0153] HERV-K is expressed on many tumor types, including but not limited to melanoma (Muster et al., 2003; Buscher et al., 2005; Li et al., 2010; Reiche et al., 2010; Serafino et al., 2009), breast cancer (Patience et al., 1996; Wang-Johanning et al., 2003; Seifarth et al., 1995), ovarian cancer (Wang-Johanning et al., 2007), lymphoma (Contreras-Galindo et al., 2008) and teratocarcinoma (Bieda et al., 2001; Lower et al., 1993). In addition, infected cells, including those infected by HIV (Jones et al., 2012), also express HERV-K. This provides an attractive opportunity: a CAR targeting HERV-K can be designed to treat a variety of cancers and infections.

[0154] VI. Exemplary Membrane-Bound IL-15 Co-Expressing Chimeric Antigen Receptor T Cells for Targeting Minimal Residual Disease

[0155] Chemotherapy treatment of adult and pediatric B-lineage acute lymphoblastic leukemia (B-ALL) has a disease relapse rate of 65% and 20%, respectively, due to drug-resistant residual disease. The high incidence of B-ALL relapse (especially in a poor prognosis population) has promoted the use of immune-based therapies using allogeneic hematopoietic stem cell transplantation (HSCT). This therapy relies on alloreactive cells present in the donor graft, which are used to eradicate the remaining leukemic cells, or minimize residual disease, to improve disease-free survival. Donor lymphocyte infusion has been used to improve the ability of transplanted T cells to target residual B-ALL after allogeneic HSCT, but this treatment method for such patients achieves a remission rate of less than 10% and is associated with a high degree of morbidity and mortality from the frequency and severity of graft-versus-host disease (GVHD). Because relapse is a common and lethal problem in these refractory malignancies, adoptive therapy with peripheral blood mononuclear cell (PBMC)-derived T cells after HSCT is being used to enhance the antitumor or graft-versus-leukemia (GVL) effect by retargeting the specificity of the donor T cells to tumor-associated antigens (TAAs).

[0156] Many formulations of CARs specific for target antigens have been developed, among which the CD19-specific CAR targets the CD19 antigen on the cell surface of B-ALL. + Long-term persistence of cells and achieving durable responses in patients across different clinical regimens remain key issues hindering the therapeutic efficacy of CAR-based therapies.

[0157] At present, the T cells modified by CAR rely on the survival signal conduction by CAR that only occurs when encountering tumor antigens. In the clinical scenario where these T cells modified by CAR are infused into patients with huge mass diseases, there are sufficient tumor antigens to provide enough activation and survival signal conduction via CAR. However, patients with relapsed B-ALL generally utilize myeloablative chemotherapy, followed by HSCT to condition, and present minimal residual disease (MRD). In this case, the patient has low tumor load and minute-level TAA strictly limits the signal conduction of the CAR mediation necessary for the T cells of support infusion, thereby damaging therapeutic potential. It is expected that the alternative method for enhancing T cell persistence does not rely on CAR to improve the migration of CAR-modified T cells.

[0158] Cytokines in the common gamma chain receptor family (γC) are important co-stimulatory molecules for T cells, which are crucial for lymphoid function, survival, and proliferation. IL-15 has several properties that are ideal for adoptive therapy. IL-15 is a homeostatic cytokine that supports the survival of long-lived memory cytotoxic T cells, promotes the eradication of established tumors by alleviating functional suppression of tumor-resident cells, and inhibits AICD.

[0159] IL-15 is tissue-restricted and is only observed in serum or systemically at any level under pathological conditions. Unlike other γC cytokines that are secreted into the surrounding environment, IL-15 is trans-donated by producing cells to T cells in the presence of IL-15 receptor α (IL-15Rα). This cytokine's unique delivery mechanism to T cells and other responder cells: (i) is highly targeted and localized, (ii) increases the stability and half-life of IL-15, and (iii) produces qualitatively different signaling achieved by soluble IL-15.

[0160] In one embodiment, the present invention provides a method for producing chimeric antigen receptor (CAR) modified T cells with long-lived in vivo potential for the purpose of treating, for example, leukemia patients who exhibit minimal residual disease (MRD). In general, the method describes how soluble molecules such as cytokines can be fused to the cell surface to enhance therapeutic potential. The core of the method relies on the use of a human cytokine mutant protein of interleukin-15 (IL-15) (hereinafter referred to as mIL15) to co-modify CAR T cells. The mIL15 fusion protein consists of a codon-optimized cDNA sequence of IL-15 fused to the full-length IL15 receptor alpha via a flexible serine-glycine linker. The IL-15 mutant protein is designed in such a way that: (i) mIL15 expression is restricted to the CAR + mIL15 expressing T cells is able to continue to support cytokine signaling, which is crucial for their survival after infusion. mIL15 produced by genetic modification of the non-viral "Sleeping Beauty" system and subsequent ex vivo expansion on a clinically applicable platform + CAR + T cells produced T cell infusion products with enhanced post-infusion persistence in murine models with high, low, or no tumor burden. + CAR + T cells also showed enhanced anti-tumor efficacy in high or low tumor burden models.

[0161] mIL15 in high tumor burden models + CAR + T cells versus CAR + The enhanced persistence and antitumor activity of T cells suggest that mIL15 + CAR + T cells are more effective than CARs in treating leukemia patients with active disease in which tumor burden is prevalent. + T cells are more effective. Therefore, mIL15 + CAR + T cells can replace CAR in adoptive therapy in the broadest application + T cells. mIL15 + CAR +The ability of T cells to survive independently of survival signaling via CARs enables these modified T cells to persist after infusion despite the absence of tumor antigens. Therefore, this is expected to have the greatest impact on treatment efficacy in the MRD treatment setting, especially in patients who have undergone myeloablative chemotherapy and hematopoietic stem cell transplantation. These patients are amenable to therapy with mIL15 + CAR + Adoptive T cell transfer of T cells to treat their MRD and prevent relapse.

[0162] Membrane-bound cytokines such as mIL-15 have a wide range of effects. In addition to membrane-bound IL-15, other membrane-bound cytokines are also contemplated. The membrane-bound cytokines can also be extended to cell surface expression of other molecules associated with activation and proliferation of cells for human applications. These molecules include, but are not limited to, cytokines, chemokines, and other molecules that contribute to cell activation and proliferation for human applications.

[0163] Membrane-bound cytokines such as mIL15 can be used ex vivo to prepare cells for human applications and can be expressed on cells (e.g., T cells) for infusion in human applications. For example, membrane-bound IL-15 can be expressed on artificial antigen-presenting cells (aAPCs) (such as those derived from K562) to stimulate T cells and NK cells (and other cells) to activate and / or propagate. T cell populations activated / proliferated by mIL15 on aAPCs include genetically modified lymphocytes, but also tumor-infiltrating lymphocytes and other immune cells. These aAPCs are not infused. Instead, mIL15 (and other membrane-bound molecules) can be expressed on infused T cells and other cells.

[0164] The therapeutic efficacy of MRD therapy using CAR-modified T cells is hampered by the lack of persistence of T cells after adoptive transfer. + CAR + The ability of T cells to survive long-term in vivo independent of tumor antigens suggests great potential for treating patients with MRD. In this setting, mIL15 and the persistent T cells it supports may address a need where current approaches for patients with MRD are insufficient. The persistence of infused T cells and other lymphocytes in patients with MRD exceeds that of CARs. +T cells. If a sustained therapeutic effect is to be achieved, any immune cell used to treat and prevent malignancies, infections, or autoimmune diseases must be able to persist over the long term. Therefore, the persistence of activated T cells, in addition to signals derived from endogenous T cell receptors or introduced immune receptors, is important for many aspects of adoptive immunotherapy. The expression of membrane-bound cytokines can thus be used to enhance the therapeutic potential and persistence of T cells and other immune cells infused for a variety of pathological conditions.

[0165] The present inventors have generated + A mutant protein of IL-15 expressed on T cells as a membrane-bound fusion protein of IL-15 and IL-15Rα (mIL15). The mIL15 construct and the CD19-specific CAR (on day 0) were co-electrotransferred into primary human T cells as two "Sleeping Beauty" DNA transposon plasmids. + Artificial antigen-presenting cells co-cultured with supplemented IL-21 produce clinically relevant amounts of mIL15 + CAR + T cells. Signaling through the IL-15 receptor complex in genetically modified T cells was verified by phosphorylation of STAT5 (pSTAT5), and these T cells were shown to be equivalent to CAR + CD19 on T cells + Redirected specific lysis of tumor targets. In addition, after antigen withdrawal, signaling through mIL15 increases the occupancy of T cells with a less differentiated / younger phenotype that possess memory-associated properties, including specific cell surface markers, transcription factors, and the ability to secrete IL-2. These characteristics are desirable traits in T cells for adoptive transfer because they are associated with T cell subsets that display the ability to persist long-term in vivo. + In immunocompromised NSG mice with leukemia, mIL15 + CAR + T cells showed persistence and anti-tumor effects, but their CAR + T cell counterparts were unable to maintain significant persistence despite the presence of TAAs. + / - CAR + T cells for 6 days, followed by the introduction of diffuse CD19 + In the preventive mouse (NSG) model of leukemia, only mIL15 was found + CAR + T cells persist and prevent tumor invasion. To test mIL15 + CAR +Can T cells persist independently of TAA stimulation? + / - CAR + T cells were adoptively transferred into tumor-free NSG mice. Only mIL15 + CAR + T cells can persist in this in vivo environment without exogenous cytokine support or the presence of CD19 TAA. These data suggest that mIL15 can be used in CAR + The fusion molecules were co-expressed in T cells, resulting in enhanced persistence in vivo without the need for TAAs or exogenous cytokine support. In summary, this cytokine fusion molecule: (i) provides a stimulatory signal via pSTAT5, resulting in enhanced T cell persistence in vivo while maintaining tumor-specific functionality, (ii) maintains T cell subsets that promote a memory-like phenotype, (iii) eliminates the need and expense of clinical-grade IL-2 for in vitro and in vivo T cell expansion and persistence, and (iv) reduces the need for clinical-grade soluble IL-15.

[0166] VII. Immune System and Immunotherapy

[0167] In some embodiments, medical conditions are treated by transferring redirected T cells that elicit a specific immune response. In one embodiment of the invention, B-cell lineage malignancies or conditions are treated by transferring redirected T cells that elicit a specific immune response. Therefore, a basic understanding of immune responses is essential.

[0168] The cells of the adoptive immune system are a type of white blood cell, called lymphocytes. B cells and T cells are the main types of lymphocytes. B cells and T cells are derived from the same pluripotent hematopoietic stem cells and can not be distinguished from each other until they are activated. B cells play a major role in humoral immune response, while T cells are closely involved in cell-mediated immune response. They can be distinguished from other lymphocyte types, such as B cells and NK cells, in that they differ in the presence of a specific receptor called T cell receptor (TCR) on their cell surface. In almost all other vertebrates, B cells and T cells are produced by stem cells in the bone marrow. T cells travel to the thymus from which their name is derived and develop therein. In humans, about 1%-2% of the lymphocyte pool recirculates every hour to optimize the chance of antigen-specific lymphocytes finding their specific antigens in secondary lymphoid tissues.

[0169] T lymphocytes are produced from hematopoietic stem cells in the bone marrow and typically migrate to the thymus to mature. T cells express unique antigen-binding receptors (T cell receptors) on their membranes that can only recognize antigens associated with major histocompatibility complex (MHC) molecules on the surface of other cells. There are at least two T cell populations called T helper cells and T cytotoxic cells. The main difference between T helper cells and T cytotoxic cells is that they display membrane-bound glycoproteins CD4 and CD8, respectively. T helper cells secrete various lymphokines that are crucial for the activation of B cells, T cytotoxic cells, macrophages, and other cells of the immune system. On the contrary, the T cytotoxic cells of the antigen-MHC complex identified proliferate and differentiate into effector cells called cytotoxic T lymphocytes (CTLs). CTLs eliminate cells of the body that display antigens, such as virus-infected cells and tumor cells, by producing substances that cause cell lysis. Natural killer cells (or NK cells) are a type of cytotoxic lymphocytes that constitute the main component of the innate immune system. NK cells play a major role in the rejection of tumors and virus-infected cells. The cells kill by releasing small cytoplasmic granules of proteins called perforins and granzymes, which cause the target cell to die by apoptosis.

[0170] Antigen presenting cells (which include macrophages, B lymphocytes and dendritic cells) differ in the expression of their specific MHC molecules. APCs internalize antigens and re-express parts of the antigen together with MHC molecules on their outer cell membranes. The major histocompatibility complex (MHC) is a large gene complex with multiple loci. The MHC locus encodes two major classes of MHC membrane molecules called class I and class II MHC. T helper lymphocytes typically recognize antigens associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, MHC is referred to as the HLA complex and in mice as the H-2 complex.

[0171] T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells), which is generally responsible for identifying antigens that are bound to major histocompatibility complex (MHC) molecules. It is a heterodimer composed of α and β chains in 95% of T cells, while 5% of T cells have a TCR composed of γ and δ chains. The engagement of TCR with antigen and MHC leads to the activation of its T lymphocytes through a series of biochemical events mediated by related enzymes, co-receptors and specialized auxiliary molecules. In immunology, the CD3 antigen (CD stands for cluster of differentiation) is a protein complex composed of four different chains (CD3γ, CD3δ, and 2 times of CD3ε) in mammals, which associates with a molecule called T cell receptor (TCR) and ζ chain to produce activation signals in T lymphocytes. TCR, ζ chain and CD3 molecule together constitute the TCR complex. CD3γ, CD3δ and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily, which contain a single extracellular immunoglobulin domain. The transmembrane regions of the CD3 chains are negatively charged, a property that allows these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular tail of the CD3 molecule contains a single conserved motif called the immunoreceptor tyrosine-based activation motif, or ITAM, which is essential for the signaling ability of the TCR.

[0172] CD28 is one of the molecules expressed on T cells that provides a co-stimulatory signal required for T cell activation. CD28 is a receptor for B7.1 (CD80) and B7.2 (CD86). When activated by Toll-like receptor ligands, B7.1 expression is upregulated in antigen presenting cells (APCs). B7.2 expression on antigen presenting cells is constitutive. CD28 is the only B7 receptor constitutively expressed on naive T cells. Stimulation by CD28 in addition to TCR can provide efficient co-stimulatory signals for T cells that produce various interleukins (particularly IL-2 and IL-6).

[0173] Strategies for isolating and expanding antigen-specific T cells as therapeutic interventions for human disease have been validated in clinical trials (Riddell et al., 1992; Walter et al., 1995; Heslop et al., 1996).

[0174] Malignant B cells appear to be excellent targets for redirecting T cells because B cells can serve as immunostimulatory antigen-presenting cells for T cells (Glimcher et al., 1982). Lymphomas, by virtue of their lymph node tropism, are anatomically ideal for T cell-mediated recognition and elimination. +Extensive localization of infused T cells to lymph nodes has been documented in HIV patients who received infusions of CTL clones. In these patients, evaluation of lymph node biopsy material revealed that the infused clones constituted approximately 2%-8% of the lymph node CD8 + Cells. Lymph node homing can be further improved by co-transfecting T cells with a cDNA construct encoding L-selection molecules under the control of a constitutive promoter, because the adhesion molecule guides circulating T cells back to the lymph nodes and is downregulated due to in vitro expansion (Chao et al., 1997). The present invention can provide a method for treating a human disease state associated with cells expressing endogenous CD19, the method comprising infusing a therapeutically effective dose of a cell expressing recombinant human CD19-specific CAR as described above to the patient. The human disease state associated with cells expressing endogenous CD19 can be selected from the group consisting of: lymphoma, leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia and B cell-related autoimmune diseases.

[0175] Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (multiplication) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases known as hematological neoplasms. Leukemia is a broad term encompassing a range of diseases. Clinically and pathologically, leukemia is divided into its acute and chronic forms.

[0176] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding makes it impossible for the bone marrow to produce healthy blood cells. Acute forms of leukemia can occur in children and adolescents. In fact, it is the most common cause of death in American children than any other type of malignant disease. Acute leukemia requires immediate treatment due to the rapid progression and accumulation of malignant cells (which then overflow into the bloodstream and spread to other organs of the body). Central nervous system (CNS) lesions are uncommon, although the disease can occasionally cause cranial nerve palsy. Chronic leukemia is distinguished by an excessive accumulation of relatively mature, but still abnormal, blood cells. It usually takes several months to several years to progress, and the cells are produced at a much higher rate than normal, resulting in many abnormal white blood cells in the blood. Chronic leukemia occurs mainly in the elderly, but in theory it can occur in any age group. However, acute leukemia must be treated immediately, and chronic forms are sometimes monitored for a period of time before treatment to ensure maximum efficiency of treatment.

[0177] Furthermore, the diseases are classified as lymphocytic or lymphoblastic, which means the cancer develops in a type of bone marrow cell that normally goes on to form lymphocytes; and myeloid or bone marrow, which means the cancer develops in a type of bone marrow cell that normally goes on to form red blood cells, some types of white blood cells, and platelets (see Lymphoid Cells vs. Myeloid Cells).

[0178] Acute lymphocytic leukemia (also called acute lymphoblastic leukemia, or ALL) is the most common type of leukemia in young children. The disease also affects adults, especially those 65 years of age and older. Chronic lymphocytic leukemia (CLL) most commonly affects adults over the age of 55. It sometimes occurs in younger adults, but it almost never affects children. Acute myeloid leukemia (also called acute myeloid leukemia, or AML) occurs more often in adults than in children. This type of leukemia was previously called "acute non-lymphocytic leukemia." Chronic myeloid leukemia (CML) occurs primarily in adults. Very rarely, it also occurs in children.

[0179] Lymphoma is a type of cancer that originates in lymphocytes (a type of white blood cell in the immune system of vertebrates). There are many types of lymphoma. According to the National Institutes of Health, lymphoma accounts for approximately 5% of all cancer cases in the United States, and Hodgkin lymphoma, specifically, accounts for less than 1% of all cancer cases in the United States. Because the lymphatic system is part of the human immune system, patients with weakened immune systems (such as those caused by HIV infection or certain medications or drugs) also have a higher incidence of lymphoma.

[0180] In the 19th and 20th centuries, the disease was called Hodgkin's disease because it was discovered by Thomas Hodgkin in 1832. In layman's terms, lymphomas are broadly divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma (all other types of lymphoma). Scientific classifications of the types of lymphoma are more specific. Although the older classifications were called histiocytic lymphomas, these lymphomas are now recognized as being of B, T, or NK cell lineage in newer classifications.

[0181] An autoimmune disease or autoimmunity is the inability of an organism to recognize its own constituent parts (down to the submolecular level) as "self," which results in an immune response directed against its own cells and tissues. Any disease caused by such an abnormal immune response is known as an autoimmune disease. Prominent examples include celiac disease, type 1 diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), Sjögren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA).

[0182] Inflammatory diseases, including autoimmune diseases, are also a class of diseases associated with B cell disorders. Examples of autoimmune diseases include, but are not limited to, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, and inflammatory bowel disease. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention. The present invention relates to the treatment of autoimmune diseases of the present invention.

[0183] VIII. Artificial Antigen Presenting Cells

[0184] In some cases, aAPCs are used to prepare therapeutic compositions and cell therapy products of the embodiments. For general guidance on the preparation and use of antigen presentation systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO 2007 / 103009).

[0185] Typically, aAPCs are incubated with peptides of optimal length that allow the peptides to bind directly to MHC molecules without further treatment. Alternatively, the cells may express the target antigen (i.e., in the absence of MHC-independent antigen recognition). In addition to peptide-MHC molecules or target antigens, the aAPC system may further comprise at least one exogenous auxiliary molecule. Any suitable number and combination of auxiliary molecules may be used. The auxiliary molecules may be selected from auxiliary molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD70 and B7.1 (B7.1 was formerly known as B7 and also referred to as CD80), which, among other things, bind to CD28 and / or CTLA-4 molecules on the T cell surface, thereby affecting, for example, T cell expansion, Th1 differentiation, short-term T cell survival, and cytokine secretion such as interleukin (IL) -2 (see Kim et al., 2004, Nature, Vol. 22 (4), pp. 403-410). Adhesion molecules can include sugar-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and proteins of the single-pass transmembrane immunoglobulin (Ig) superfamily, such as intercellular adhesion molecules (ICAMs), which promote, for example, contact between cells or between cells and the matrix. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents for selecting, cloning, preparing, and expressing exemplary accessory molecules (including costimulatory molecules and adhesion molecules) are listed in, for example, U.S. Patents 6,225,042, 6,355,479, and 6,362,001.

[0186] Cells selected to become aAPCs preferably have defects in intracellular antigen processing, intracellular peptide transport, and / or intracellular MHC class I or II molecule-peptide loading, or are poikilothermic (i.e., less sensitive to temperature than mammalian cell lines), or have both defects and poikilothermic properties. Preferably, cells selected to become aAPCs also lack the ability to express at least one endogenous counterpart (e.g., endogenous MHC class I or class II molecules and / or endogenous accessory molecules as described above) of the exogenous MHC class I or class II molecules and accessory molecule components introduced into the cell. In addition, aAPCs preferably retain the defects and poikilothermic properties possessed by the cells before they were modified to generate the aAPCs. Exemplary aAPCs constitute or are derived from transporters associated with antigen processing (TAP)-deficient cell lines, such as insect cell lines. An exemplary poikilothermic insect cell line is a Drosophila cell line, such as the Schneider 2 cell line (see, e.g., Schneider, J. Embryol. Exp. Morph. 1972, Vol. 27, pp. 353-365). Illustrative methods for the preparation, growth, and culture of Schneider 2 cells are provided in U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.

[0187] In one embodiment, the aAPCs are also subjected to freeze-thaw cycles. In an exemplary freeze-thaw cycle, the aAPCs can be frozen by contacting a suitable container containing the aAPCs with an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or a similar cryogenic material to allow freezing to occur rapidly. The frozen aAPCs are then thawed by removing the aAPCs from the cryogenic material and exposing them to ambient room temperature conditions, or by a convenient thawing process in which a warm water bath or warm hands are used to promote a shorter thawing time. In addition, the aAPCs can be frozen and stored for an extended period of time before thawing. The frozen aAPCs can also be thawed and then lyophilized before further use. Preferably, preservatives such as dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and other preservatives that may adversely affect the freeze-thaw process are not present in the medium containing the aAPCs that undergo freeze-thaw cycles, or the preservatives are substantially removed, such as by transferring the aAPCs to a medium that is substantially free of such preservatives.

[0188] In other preferred embodiments, heterologous nucleic acids and nucleic acids endogenous to the aAPCs can be inactivated by crosslinking such that substantially no cell growth, nucleic acid replication, or expression occurs following inactivation. In one embodiment, aAPCs are inactivated at a point after expression of exogenous MHC and accessory molecules, presentation of such molecules on the aAPC surface, and loading of the presented MHC molecules with the selected peptide. Thus, aAPCs loaded with such inactivated and selected peptides, while rendered substantially incapable of proliferation or replication, retain the selected peptide-presenting function. Preferably, the crosslinking also produces aAPCs that are substantially free of contaminating microorganisms, such as bacteria and viruses, without substantially reducing the antigen-presenting cell function of the aAPCs. Thus, crosslinking maintains the important APC functions of aAPCs while helping to alleviate safety concerns associated with cell therapy products developed using aAPCs. For methods related to crosslinking and aAPCs, see, for example, U.S. Patent Application Publication No. 20090017000, which is incorporated herein by reference.

[0189] IX. Kits of the Present Invention

[0190] Any composition described herein can be included in a kit. In some embodiments, allogeneic CAR T cells are provided in a kit, which may also include reagents suitable for expanding cells such as media, aAPC, growth factors, antibodies (e.g., for sorting or characterizing CAR T cells) and / or plasmids encoding CAR or transposase.

[0191] In non-limiting examples, a chimeric receptor expression construct, one or more reagents for producing a chimeric receptor expression construct, cells for transfection of the expression construct, and / or one or more instruments for obtaining allogeneic cells for transfection of the expression construct (e.g., the instrument can be a syringe, a pipette, forceps, and / or any such medically approved device).

[0192] In some embodiments, an expression construct for eliminating endogenous TCR α / β expression, one or more reagents for generating the construct, and / or a CAR are provided in a kit. + In some embodiments, an expression construct encoding a zinc finger nuclease is included.

[0193] In some aspects, the kit comprises reagents or devices for electroporation of cells.

[0194] The test kit may comprise one or more suitable equal portions of the composition of the present invention or produce a composition reagent of the present invention. The components of the test kit may be packaged in an aqueous medium or in a lyophilized form. The container means of the test kit may comprise at least one vial, test tube, culture bottle, bottle, syringe or other container means in which the components may be placed and preferably aliquoted. When there is more than one component in the test kit, the test kit will generally also contain a second, third or other additional container in which additional components may be placed individually. However, various combinations of ingredients may be included in the vial. The test kit of the present invention generally also includes a device for enclosing the chimeric receptor construct and any other reagent container in a sealed configuration for commercial sale. Such containers may comprise, for example, injection-molded or blow-molded plastic containers in which the required vials are retained.

[0195] X. Examples

[0196] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to be well suited for the practice of the present invention and are therefore considered to constitute preferred modes for its practice. However, it will be appreciated by those skilled in the art that, based on the disclosure of the present invention, many changes may be made in the specific disclosed embodiments while still achieving the same or similar results without departing from the spirit and scope of the present invention.

[0197] Example 1 - Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modified T Cells from Peripheral and Umbilical Cord Blood - Materials and Methods

[0198] Isolation of mononuclear cells (MNCs) from PB and UCB. On or before day 0, dilute PB with an equal volume of PBS-EDTA and UCB with 4 times the volume of PBS-EDTA. Slowly layer the diluted blood (25 mL) on Ficoll (12 mL) in a 50 mL centrifuge tube and centrifuge at 400 x g for 30-40 minutes (without interruption). Use a pipette to collect the mononuclear cell fraction (interface) and transfer it to a fresh 50 mL centrifuge tube. Add the volume to 50 mL with PBS-EDTA and centrifuge at 450 x g for 10 minutes. Aspirate the supernatant and then gently resuspend the cell pellet in 50 mL of complete culture medium (CCM). Centrifuge at 400 x g for 10 minutes. Gently resuspend the cell pellet and pool in CCM, and use the trypan blue exclusion method (Cellometer, PBMC program) to count the cells. MNCs can be used for electroporation (nucleofection) or cryopreserved for future use.

[0199] Preparation of T cells for electroporation on day 0. If using cryopreserved MNCs, quickly thaw enough cells for a full electroporation (2 × 10 8 , add approximately 20% to account for cell loss during centrifugation, and incubate for 2 hours). Gently resuspend the cells and transfer them to an appropriately sized centrifuge tube containing pre-warmed complete phenol-free RPMI medium (PF-RPMI), centrifuge at 200 × g for 10 minutes (without stopping), and aspirate the supernatant. Subsequently, if freshly isolated MNCs are used, resuspend the MNCs in PF-RPMI, perform a cell count (Cellometer), and centrifuge the cells at 10 6 Transfer the MNCs to a concentration of 10 cells / ml into a cell culture vessel of appropriate size. Incubate the cells in a humidified 37°C / 5% CO2 incubator for 2 hours ± 30 minutes. Transfer the MNCs to a sterile centrifuge tube and spin at 200 × g for 5 minutes (without stopping). Aspirate the supernatant and gently resuspend the cell pellet in PF-RPMI and combine them. Perform a cell count (Cellometer) and calculate the required cell suspension (2 × 10 8 The calculated volume was transferred to a sterile 50 mL centrifuge tube and spun at 200×g for 10 minutes (without a break). The supernatant was aspirated so that no residual medium remained and gently resuspended by tapping the side of the tube.

[0200] Electroporation (nucleofection) of MNCs was performed on day 0 (full scale method using 10 cuvettes). A sterile 12-well plate was pre-incubated in a humidified 37°C / 5% CO2 incubator with 10 wells containing 4 mL of warm PF-RPMI. A Lonza Nucleofector Solution Human T cell kit (reconstituted according to the manufacturer's instructions) was prepared and pre-warmed in a Biosafety Cabinet (BSC). www.lonza.com ) to ambient temperature. Prepare the Nucleofector solution / DNA premix by adding 100 μL of Nucleofector solution, 15 μg of transposon (supercoiled DNA plasmid designated CD19RCD28 / pSBSO), and 5 μg of transposase (supercoiled DNA plasmid designated pCMV-SB11) / reaction / cuvette. Disperse the MCN cell pellet by gently tapping the side of the centrifuge tube and plate it at 2 × 10 7Resuspend in the nucleofection solution / DNA premix at a final cell concentration of 10 cells / 100 μL. Carefully transfer 100 μL of the cell suspension to each of 10 Lonza nucleofection cuvettes, taking care to avoid air bubbles. Tap the cuvette once and perform electroporation using program U-014 (for unstimulated T cells). Transfer the cuvettes and 12-well plate to the BSC. Use an Amaxa fine-tip pipette to collect the electroporated cells from each cuvette by adding approximately 500 μL of pre-warmed culture medium from the corresponding well, then return the plate to a humidified 37°C / 5% CO2 incubator for 2 hours ± 30 minutes. After a 2-hour incubation, collect cells from all wells and transfer them to a sterile centrifuge tube. Wash the cells by centrifugation at 140 × g for 8 minutes at ambient temperature without interruption, discarding the supernatant so that no residual culture medium covers the cell pellet. Disperse the cell pellet by gently tapping the side of the centrifuge tube and then gently resuspend in CCM to obtain a single-cell suspension. Count the cells and adjust the cell concentration to 10 in CCM. 6 The cell suspension was transferred to a cell culture flask and placed in an incubator overnight. The same method was used for the control EGFP-transfected cells (5×10 6 cells / cuvette using 5 μg Amaxa control EGFP supercoiled plasmid, pmaxGFP).

[0201] Analysis of CAR expression by flow cytometry was performed on day 1 of the first and subsequent stimulation cycles. Electroporated cells were collected and counted using trypan blue exclusion (hemocytometer). Cells (1-2×10 6 ) were stained as a measure of CAR expression. Cells were acquired on a FACS Calibur and data were analyzed using FCS Express software to calculate CAR expression. CAR in the culture was calculated using the following formula + Cells: (total number of viable cells) x (% CAR + cells) = CAR + The number of cells.

[0202] Preparation of aAPC (clone #4) performed on day 1 of the first and subsequent stimulation cycles. aAPC (clone #4) were derived from K562 cells (parental line obtained from the American Type Culture Collection) to co-express the desired T cell co-stimulatory molecules. An aliquot of frozen 100 Gy irradiated aAPC was thawed in a 37°C water bath. Cells were washed twice by centrifugation at 400 × g for 10 minutes in CCM and then counted using a Cellometer (trypan blue exclusion method). The number of viable aAPC required for stimulation was calculated: (CAR+ Number of cells) x 2 = the number of irradiated aAPCs required

[0203] CAR on day 1 of the first and subsequent stimulation cycles + aAPC-mediated stimulation of T cells. Electroporated cells (expressing CAR) were mixed with γ-irradiated aAPC (clone #4) at a ratio of 1:2 (CAR + Cells: live aAPCs were mixed in CCM. Note that the aAPC ratio was adjusted based on CAR expression based on flow cytometry on the day after electroporation. IL-21 (30 ng / mL) was added to the cell suspension. 6 The concentration of cells / mL at T-75cm 2 Aliquot the culture flasks and / or VueLife culture bags and return them to the incubator.

[0204] CAR on days 3 and 5 + Continuous culture of T cells. Perform half medium change, supplement IL-21, and 6 T cells were maintained at a concentration of 10 cells / mL.

[0205] The first aAPC-mediated stimulation cycle ended on day 7. T cells were collected, counted, and stained for CD3, CD4, CD8, and Fcγ (CAR).

[0206] CD56 7 to 14 days after electroporation + If CD56 + CD3 neg Lymphocytes ≥10% were depleted for CD56 using paramagnetic beads.

[0207] During stimulation cycles #2, #3, and #4, corresponding to days 8→14, 15→21, and 22→28, aAPCs were recursively added to propagate T cells to clinically sufficient numbers. The stimulation process was repeated up to 4 times. IL-2 (50 U / mL) was added to the cultures starting on days 7, 14, and 21 and at each subsequent media change (3 times per week on a Monday-Wednesday-Friday schedule). Excess T cells were cryopreserved (archived) using a programmed freezer for release testing and infusion as needed.

[0208] Example 2 - Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Cord Blood - Results

[0209] Electrotransfer of DNA plasmids and expansion of T cells on γ-irradiated aAPCs can be used to generate clinically attractive numbers of T cells derived from PB and UCB for human applications. These genetically modified T cells express introduced CARs that recognize TAA CD19 independently of the major histocompatibility complex. Expression of (i) transposons, through CD28 and CD3-ε, has been previously described. 14 SB-derived DNA plasmids (Singh et al., 2011; Davies et al., 2010; Kebriaei et al., 2012) encoding (i) a second generation CAR (CD19RCD28) signaling and (ii) the transposase SB11 (Jin et al., 2011). Plasmids used in this study were commercially produced by the Waisman Clinical Biomanufacturing Facility (Madison, WI). aAPCs (clone #4) derived from K562 cells (the parental line obtained from the American Type Culture Collection) co-expressed the desired T cell co-stimulatory molecules (90% of each introduced molecule on the aAPC cell surface) as previously described (Manuri et al., 2010). Here, SB transposition can be used to introduce CARs, followed by the addition of aAPCs to digitally expand T cells in a CAR-dependent manner to generate CD19-specific T cells from mononuclear cells (MNCs) derived from PB and UCB ( Figure 1 and 4 ) (Singh et al., 2011; Singh et al., 2008). For each recipient, 15 μg of DNA plasmid encoding transposon (CAR) (CD19RCD28 / pSBSO) and 5 μg of DNA plasmid encoding transposase (SB11) (pCMV-SB11) were used to treat 10 cuvettes (2×10 7 The number of cuvettes can be reduced if MNCs are limited or scaled down for laboratory work. The day of electroporation is defined as "day 0" of stimulation cycle #1. As a control for flow cytometry and culture conditions, autologous T cells are mock electroporated (without DNA plasmid) and digitally expanded on gamma-irradiated aAPC (clone #4) that have been preloaded with OKT3 to crosslink CD3 to support T cell proliferation. T cells are routinely assessed for electrotransfer efficiency and viability on the day after electroporation ( Figure 2B ). The expression of EGFP from the control DNA plasmid (referred to as pmaxGFP) and the expression of CAR at this starting time point reflect protein expression from the integrated plasmid and the episomal plasmid. Typically, EGFP expression is measured to be 60% and CAR expression is measured to be approximately 40% on the day after electroporation ( Figure 2A), T cell viability was 40%-50%. Recursive addition of γ-irradiated aAPCs in the presence of soluble recombinant human IL-2 and IL-21 restored T cells stably expressing CAR (CD19RCD28). If the percentage of these NK cells was ≥10%, especially if the percentage of CAR expressed on T cells was low, CD3 was depleted from the culture using CD56-specific paramagnetic beads. neg CD56 + This depletion prevents interference with aAPC support of CAR + The ability of T cells to proliferate rapidly increases the number of NK cells. Occasionally, during the last two stimulation cycles, + T cells deplete NK cells, but this is because CD56 in some CAR + The T cells were grown in a functionally closed system using VueLife culture bags through day 14. A subset of genetically modified and propagated T cells was typically cryopreserved on day 14 or day 21 of co-culture on aAPC (end of stimulation cycle #2 or #3) to serve as a source of archival material for future analysis and to be thawed if unforeseen problems subsequently occurred during the manufacturing process. Typically, on or around day 28 of culture ( Figure 3 ) T cells routinely expressed >90% CAR and >80% were viable (Fig. 2C, D). After 4 weeks of co-culture on aAPC, CD3 + The average fold expansion of T cells was 19,800 ± 11,313, of which CAR + The expression was 90% ± 7.5% (Singh et al., 2011). These T cells were cryopreserved and subjected to in-process and release testing, which provides information on the safety and therapeutic potential of the manufactured product. Relevant testing was performed in accordance with the Clinical Laboratory Improvement Amendments (CLIA) to generate a certificate of analysis before infusion into recipients undergoing clinical trials.

[0210] Example 3 - Production of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptors using Sleeping Beauty and artificial antigen-presenting cells - Materials and methods

[0211] Generation of clinical-grade DNA plasmids. SB transposon CoOp CD19RCD28 / pSBSO expresses a human codon-optimized (CoOp) second generation WT human T-cell leukemia virus (HTLV) under the EF-1 / HTLV hybrid composite promoter (InvivoGen), which is composed of the elongation factor-1α (EF-1α) (Kim et al., 1990) and the 5' untranslated region of human T-cell leukemia virus (HTLV) (Singh et al., 2011; Davies et al., 2010).CoOp CD19RCD28 CAR (SEQ ID NO: 1). The derivation of this DNA plasmid is described in Figure 10. The SB transposase SB11 under the cytomegalovirus (CMV) promoter was expressed in cis from the DNA plasmid pCMV-SB11 (Singh et al., 2011). The derivation of this DNA plasmid is described in Figure 11 Both plasmids were sequenced in their entirety and manufactured by Waisman Clinical Biomanufacturing Facility (Madison, WI) using kanamycin for selection of the bacterial strain Escherichia coli (E. coli) DH5α. The release criteria for the DNA plasmids are shown in Table 1. DNA plasmid ΔCD19 was used. CoOp -F2A-Neo / pSBSO expressed CD19 ( FIG. 12 ).

[0212] Table 1: Release criteria for DNA plasmids encoding SB transposons and transposases

[0213]

[0214] Cell counting. Trypan blue exclusion was used to distinguish live from dead cells and counted using a Cellometer (Nexecelom Bioscience) (Singh et al., 2011).

[0215] The separation of PBMC. Leukocyte removal products from two male volunteer healthy donors were purchased from KeyBiologics LLC (Memphis, TN). Peripheral blood mononuclear cells (PBMC) were separated by transformation of the Biosafe Sepax system (Eysins, Switzerland) to be suitable for use in accordance with the work of cGMP. In brief, after closing all magnetic clips on the CS-900 test kit, 100mL Ficoll (GE Healthcare) was aseptically transferred to a density gradient culture medium bag (" sucrose bag (ficoll bag)") via a Luer lock connector by a 60mL syringe, and the infusion tubing was heat-sealed using a handheld sealer (Sebra, model 2380). The reagent cartridge was spike-connected to a 1,000 mL bag for washing (containing CliniMACS buffer (PBS / EDTA, Miltenyi, Cat#70026) and 20 mL of 25% human serum albumin (HSA) (Baxter) (2% v / v, wash buffer), a final product bag [a 300 mL transfer pack with a coupler (Baxter / Fenwal 4R2014)], and a reagent / blood bag. Using a density gradient-based separation protocol (v126), syringe plungers were loaded into the centrifuge chamber and the Sepax unit lid was closed. The reagent / blood bag and product bag were suspended and all pistons were placed on the rotating pins in the 'T' position. After connecting the pressure sensor line, the cartridge was validated by an automated single-use test and then manually primed using gravity flow. After the cycle was completed, the final product was aseptically transferred to a centrifuge tube and washed once with wash buffer and once with phosphate-buffered saline (PBS) (centrifugation at 400 g for 10 minutes). After counting, the final product was centrifuged in a programmable cooling apparatus (Planer Kryo Cells were cryopreserved using cryopreservation medium (50% HSA, 40% Plasmalyte, 10% DMSO) in CryoMACS freezing bags (Miltenyi) and vials (Nunc) using the BM5 program (4°C to -4°C at -2°C / min, -4°C to -60°C at -35°C / min, -60°C to -20°C at 8°C / min, -20°C to -45°C at -2.5°C / min, and -45°C to -80°C at -10°C / min).

[0216] Generation of aAPC (clone #4) master and working cell banks. aAPC (clone #4, designated CJK64.86.41BBL. GFP. IL-15. CD19) co-expressing (i) CD19, (ii) CD64, (iii) CD86, (iv) CD137L, and (v) membrane-bound IL-15 (mIL15) (as a bicistronic vector with EGFP) were generated at the University of Pennsylvania using lentiviral transduction of K562 cells. The cells were cultured in a 10% heat-inactivated, defined FBS (Hyclone) medium at 5 × 10 5 aAPCs were digitally expanded in HyQ RPMI 1640 (Hyclone) culture medium (CM) containing 2 mM Glutamax-1 (Life Technologies-Invitrogen) at 10 cells / mL for maintenance of cells. A master cell bank (MCB) of 320 vials was generated by Production Assistance of Cellular Therapies (PACT) (Table 2). Subsequently, a working cell bank (WCB) of 200 vials of clone 4 aAPC derived from the MCB was generated at MDACC and tested (Table 3).

[0217] Table 2: Release criteria for the K562-derived aAPC (clone #4) primary cell bank

[0218]

[0219]

[0220] Table 3: Release criteria for the K562-derived aAPC (clone #4) working cell bank

[0221]

[0222] Selective breeding of CAR +aAPC of T cells (clone #4). γ-irradiated aAPC is used to digitally amplify genetically modified T cells. Thawed aAPC from WCB are propagated in CM for up to 60 days in VueLife cell culture bags and the cells are collected using the Biosafe Sepax II collection method. In brief, the CS-490.1 test kit is connected to a 300mL output bag (transfer pack) via a Luer lock connection. The separation tank is installed in the small recess, and the infusion tube is inserted into the optical sensor and the piston aligned in the T position. After connecting the pressure sensor line, the product bag and the supernatant / plasma bag are hung on the support frame. Select the modified scheme PBSCv302 from the Sepax menu and set the volume (initial volume) of the output product to be processed to ≤840mL. After verification and test kit testing, the program is started. After completion, the bag is removed, the magnetic clamp is closed, and the test kit is removed. Cells were removed from the final product bag aseptically, washed twice with culture medium (10% HSA in Plasmalyte), and then counted. aAPCs were irradiated (100 Gy) using a CIS BIO International irradiator (IBL-437C#09433) and then cryopreserved in cryopreservation medium using a programmed cooling device (Planer Kryo 750) for later use.

[0223] OKT3-loading of aAPCs. aAPCs loaded with OKT3 (via CD64) (clone #4) were used to propagate controls (CARs) that had not undergone genetic modification. neg ) Autologous control T cells. aAPCs obtained from culture were incubated overnight in serum-free X-Vivo 15 (Cat. No. 04-744Q, Lonza) containing 0.2% acetylcysteine ​​(Acetadote, Cumberland Pharmaceuticals), referred to as loading medium (LM). The next day, the cells were washed and irradiated (100 Gy) using a Gamma Cell 1000 Elite Cs-137 irradiator (MDS Nordion). 6 The concentration of cells / mL was 1 μg / 10 6 Functional grade purified anti-human CD3 (clone-OKT3, 16-0037-85, eBioscience) was resuspended in LM and incubated with gentle agitation on a 3-D rotator (Lab-Line) at 4° C. for 30 minutes. After washing three times with LM, the cells were used for experiments or frozen in aliquots in liquid nitrogen in the vapor layer for later use.

[0224] CAR +T cell production. Thawed PBMCs were mixed with (ii) DNA plasmid encoding CD19RCD28 CAR transposon ( CoOp CD19RCD28 / pSBSO) (15 μg supercoiled DNA / 2×10 7 PBMC / cuvette) and (iii) DNA plasmid encoding SB11 transposase (pCMV-SB11) (5 μg supercoiled DNA / 2×10 7 PBMC / cuvette) were resuspended in (i) Human T Cell Kit (Cat. No. VPA-1002, Lonza; in 1 cuvette, 100 μL for 2 × 10 7 The mixture was immediately transferred to a cuvette (Lonza) and electroporated using a Nucleofector II (Amaxa / Lonza) (defined as culture day 0), allowed to stand for 2 to 3 hours in 10% RPMI complete medium, and incubated overnight at 37°C, 5% CO2 after a half-medium change. On the second day, cells were collected, counted, phenotyped by flow cytometry, and compared with 1:2 (CAR + T cell:aAPC ratios were co-cultured with γ-irradiated aAPCs, marking the start of the stimulation cycle on culture days 1 and 7. IL-21 (Cat. No. AF-200-21, PeproTech) and IL-2 (Cat. No. NDC 65483-116-07, Novartis) were added from day 1 to day 7 on a Monday-Wednesday-Friday schedule, respectively. NK cells can block CAR + T cells can be expanded numerically, especially if their overgrowth occurs early in the tissue culture process. neg CD56 + If cells are ≥10%, the cells were cultured in a solution containing 25% HSA (80 μL / 10 7 The cells were isolated and purified using CD56 beads (Cat. No. 70206, Miltenyi Biotech, 20 μL beads / 10 μL) on an LS column (Cat. No. 130-042-401, Miltenyi Biotech) in CliniMACS buffer (10 μL). 7 T cells were cryopreserved as a backup on day 21 of culture after electroporation and at the end of the third stimulation cycle using a program cooling device (Planer Kryo 750) and stored in liquid nitrogen (vapor layer). Total T cells, CD3 + and CAR +T cell counts were plotted over time, and the slope was determined using linear regression. Fold expansion results were compared using the Student's t-test. The CD4 / CD8 ratio was calculated for each time point and validation run, and the mean was calculated.

[0225] CAR neg Generation of control T cells. As a control, 5x10 6 Mock-transfected PBMCs were co-cultured with irradiated K562-derived aAPC clone #4 loaded with anti-CD3 (OKT3). All cultures were supplemented with IL-21 (30 ng / mL) starting on day 1 of culture and IL-2 (50 U / mL) starting 7 days after the start of culture. All cytokines were subsequently added on a Monday-Wednesday-Friday schedule.

[0226] Cell lines. CD19 cells were cultured as previously described (Singh et al., 2011). + Daudiβ2m [Burkitt lymphoma, co-expressing β2-microglobulin, (Rabinovich et al., 2008)] and CD19 + NALM-6 (pre-B cells). Using the construct ΔCD19 CoOp -F2A-Neo / pSBSO modified EL-4 cells (mouse T cell lymphoma cell line) from ATCC to express CD19. Briefly, 5×10 6 EL-4 cells were resuspended in 100 μL of SB transposon (ΔCD19 CoOp -F2A-Neo / pSBSO, 3 μg) and SB transposase (pCMV-SB11, 1 μg) were added to the Amaxa Mouse T cell Nucleofector Kit (Cat. No. VPA-1006) and electroporated using Nucleofector II (Lonza) (Program X-001). Transformants were cultured in a cytocidal concentration of G418 (0.8 mg / mL) and subjected to fluorescence-activated cell sorting (FACS) for uniform expression of CD19 to obtain a clone (clone #17). Jurkat cells were obtained from ATCC and electroporated using Amaxa / Lonza Nucleofector solution (Kit V). CoOpCD19RCD28mz (CoOp) / pSBSO was electroporated (Program T-14, Nucleofector II, Lonza). Two weeks after electroporation, Jurkat cells stably expressing CAR underwent FACS for uniform expression of CAR to obtain clones (clone # 12) (Maiti et al., 2013). The cell line was maintained in HyQ RPMI 1640 (Hyclone) supplemented with 2mM Glutamax-1 (Invitrogen) and 10% heat-inactivated fetal calf serum (FCS) (Hyclone; 10% RPMI). All cell lines were verified using STR analysis or karyotyping according to the institutional cell line authentication policy.

[0227] Immunophenotyping of cells. Cells were stained with antibodies (Table 4) in 100 μL FACS buffer (2% FBS, 0.1% sodium azide) for 30 minutes at 4°C. For intracellular staining, after fixation / permeabilization for 20 minutes, cells were stained with appropriate antibodies in hot wash buffer at 4°C. FACSCalibur (BD Bioscience) was used for acquisition and Cell Quest (BD Bioscience) or FCS Express 3.00.0612 (De Novo software, Thornhill, Ontario, Canada) was used for analysis.

[0228] Table 4: Antibodies used for flow cytometry.

[0229]

[0230]

[0231] Western blot. As previously described (Singh et al., 2008), the protein expression of chimeric CD3-ζ (73-kDa) derived from CD19RCD28 was assessed. Briefly, protein lysates were transferred to nitrocellulose membranes using the iBlot Dry blotting system (Invitrogen), incubated with anti-human CD3-ζ monoclonal antibodies (catalog number 551033, 0.5 μg / mL, BD Biosciences, CA), and subsequently incubated with goat anti-mouse IgG (catalog number 1858413, 1:10,000; Pierce, IL) conjugated to horseradish peroxidase (HRP), developed using SuperSignal West Femto Maximum Sensitivity substrate (Pierce, IL), and subsequently captured using a VersaDoc™ 4000 gel imaging system (BioRad, CA).

[0232] Telomere length analysis by fluorescence in situ hybridization and flow cytometry (Flow-FISH). According to the manufacturer's instructions, the telomere length of T cells was measured by using a telomere PNA kit (Telomere PNA kit) / FITC (DAKO) for flow cytometry. Briefly, isolated cells (CD4 or CD8) and control cells (catalog number 85112105, CEM-1301 cell line; ECACC) were mixed with an equivalent measure in a hybridization solution with or without a FITC-labeled telomere PNA probe for 10 minutes at 82 ° C; hybridized overnight in the dark at room temperature; washed twice with a washing solution at 40 ° C; resuspended in PBS containing 2% FCS and propidium iodide (1 μg / mL); and analyzed on a FACSCalibur (BD Biosciences). FITC-labeled fluorescence calibration beads (catalog number 824A, Quantum TM FITC MESF (Bangs Laboratories) was used for flow cytometer calibration. Relative telomere length (RTL) was determined by comparing T cells with the CEM-1301 cell line according to the following formula:

[0233]

[0234] Chromium release assay. 51 T cell cytotoxicity was assessed in a standard 4-hour chromium release assay using Cr-labeled target cells. T cells were plated at 1×10 5 , 0.5x10 5 , 0.25x10 5 , 0.125x10 5 and 0.0625x10 5cells / well and 5x10 3 Target cells were plated in triplicate in 96-well V-bottom plates (Costar). After incubation, 50 μL of supernatant was collected onto LumaPlate (Perkin Elmer), read in TopCount NXT (Perkin Elmer), and the percentage specific lysis was calculated as follows:

[0235]

[0236] Spontaneous and maximal release were determined by measuring chromium in the conditioned supernatant of target cells incubated with CM or 0.1% Triton X-100 (Sigma), respectively.

[0237] Endotoxin tested. Use according to manufacturer's guidelines - The PTS Portable Test System (Charles River Laboratories) was used to determine the endotoxin level in the final product. The test has a detection limit of 0.01-10 EU / mL, which can be converted to EU / patient weight.

[0238] Mycoplasma testing Mycoplasma testing by PCR was performed using the TaKaRa Mycoplasma Detection Kit (Clontech) according to the manufacturer's instructions.

[0239] T cell receptor Vβ library. A panel of 24 TCR Vβ-specific mAbs (Cat. No. IM3497, IO TEST Beta Mark TCR-Vβ Library Kit, Beckman Coulter) was used in combination with CD3-specific mAb (Cat. No. 340949, BD Biosciences, 10 μL) and isotype-matched control mAb (Cat. No. 552834, BD Biosciences) to determine CAR + T cell receptor (TCR)-Vβ was used on days 28 and 35 of T cell culture.

[0240] Real-time PCR for determining the copy number of integrated CAR. To determine the copy number of CD19RCD28 CAR integrated in genetically modified T cells, a Steponeplus real-time PCR system (Applied Biosystems) was used with the following primers: forward (5'-CAGCGACGGCAGCTTCTT-3'; SEQ ID NO: 8), reverse (5'-TGCATCACGGA GCTAAA-3'; SEQ ID NO: 9) and probe (5'-AGAGCCGGTGGCAGG-3'; SEQ ID NO: 10) in a PCR reaction (50°C, 2 minutes; 95°C, 10 minutes; followed by 40 cycles (95°C, 15 seconds and 60°C, 1 minute) to amplify 50-100 ng of genomic DNA (catalog number 80204, AllPrep DNA / RNA Mini kit, Qiagen). Primers for the PNA enzyme P gene (catalog number 4316844, Applied Biosystems) was used as an internal control. Serial dilutions of genomic DNA from genetically modified Jurkat cells (clone #12) containing 1 copy of a CAR from CD19RCD28mz (CoOp) / pSBS O DNA plasmid were used to generate a standard curve (Maiti et al., 2013). All primers, probes, and TaqMan gene expression master mixes were purchased from Applied Biosystems.

[0241] PCR for SB11 transposase. A thermal cycler (PTC-200, DNA Engineering, BioRad) was used to amplify the CAR-11 transposase using forward (5'-ATGGGAAAATCAAAAGAAATC-3'; SEQ ID NO: 11) and reverse (5'-CTAGTATTTGGTAGCATTGC-3'; SEQ ID NO: 12) primers in a PCR reaction (95°C, 5 min; 25 cycles of 95°C, 15 s; 58°C, 40 s; 72°C, 60 s; followed by a final extension at 72°C for 7 min). +DNA (20 ng) isolated from T cells (AllPrep DNA / RNA Mini Kit, Qiagen). GAPDH was used as a housekeeping gene and amplified using forward (5'-TCTCCAGAACATCATCCCTGCCAC-3'; SEQ ID NO: 13) and reverse (5'-TGGGCCATGAGGTCCACCACCCTG-3'; SEQ ID NO: 14) primers in the same PCR reaction. Linearized pCMV-SB11 plasmid DNA (1 ng) and genomic DNA (20 ng) from genetically modified Jurka T cells stably expressing SB11 and EGFP (expressed from the DNA plasmid SB11-IRES2-EGFP) (Ma iti et al., 2013) were used as positive controls. T cells mock-electroporated (no DNA) and propagated by OKT3-aAPC were used as negative controls.

[0242] Assay to assess unwanted autonomous T cell growth. To monitor aberrant T cell growth, 2x10 5 CARs collected after 4 cycles of aAPC-mediated stimulation (28 days after electroporation) + T cells were cultured in triplicate in 24-well tissue culture plates for an additional 18 days. (i) Positive control: presence of aAPC and cytokines (50 U / mL IL-2 and 30 ng / mL IL-21). (ii) Test: absence of aAPC and cytokines. When the total viable cells on day 18 (i) were > 2 x 10 5 cells (for CAR cultured in the presence of aAPC and cytokines + T cells) and (ii) <2x10 4 cells (for CAR cultured in the absence of aAPC and cytokines + T cells), the genetically modified T cells passed the assay.

[0243] G-banding karyotyping. CAR was collected at the end of co-culture. + T cells were isolated and the slides were stained with Giemsa stain using standard methods. A total of 20 G-banded metaphases were analyzed.

[0244] Example 4 - Generation of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptors using Sleeping Beauty and artificial antigen-presenting cells - Results

[0245] aAPC (clone #4). K562 cells acting as aAPC (clone #4) were used for selective propagation of CAR +T cells. Cultured aAPCs were collected from VueLife bags using a Sepax II device using a volume reduction method (which took approximately 40 minutes). The average pre-treatment volume and aAPC count were 575 mL (range 500-700 mL) and 4.9 x 10 8 (Range 3.2x10 8 to 7.7x10 8 After treatment with Sepax II, the average recovery of cells was 108% (range 75% to 136%), with an output volume of 125 mL (range 50-200 mL) resulting in 78.3% (range 60% to 91.6%). Figure 5A , B) Average volume reduction. Automated cell recovery was similar to the manual volume reduction method (82%), which took 45 minutes of continuous operation time and resulted in a 91% volume reduction. aAPCs were regularly monitored by flow cytometry for expression of >80% of the introduced transgenes encoding CD19, CD64, CD86, CD137L, and EGFP (as a marker for expression of mIL15). After the generation of MCBs and WCBs and at each addition of gamma-irradiated aAPCs to T cell cultures (marking the beginning of each stimulation cycle), the T cell cultures were resuspended in 1% of the transgenes encoding CD19, CD64, CD86, CD137L, and EGFP (as a marker for expression of mIL15). Figure 5C Immunophenotyping was performed after PCR. MCB and WCB of clone 4 aAPC tested negative for sterility and mycoplasma on cells and cell supernatants. No viruses were detected in biosafety testing, including adventitious virus testing, replication-competent retrovirus testing, and screening for a range of human pathogenic viruses. The aAPC (clone #4) was confirmed to be derived from K562 based on fingerprint analysis (Table 5).

[0246] Table 5: STR fingerprint analysis of K562 aAPC (clone #4)

[0247] STR K562 aAPC (clone 4) AMEL X CSF1PO 9,10 D13S317 8 D16S539 11,12 D18S51 15,16 D19S433 14,14.2 D21S11 29,30,31 D2S1338 17 D3S1358 16 D5S818 11 D7S820 9,11 D8S1179 12 FGA 21 TH01 9.3 TPOX 8,9 vWA 16

[0248] CAR + T cell production. CD19-specific CAR + Large-scale production of T cells was performed to confirm that PBMCs could be electroporated and propagated to clinically meaningful numbers (Huls et al., 2013) ( Figure 6 ) and met the pre-determined release criteria (Table 6). Two normal donor plasmapheresis products were processed using the Sepax cell processing system to isolate mononuclear cells (MNCs). The plasmapheresis product 201 mL (donor 1) and 202 mL (donor 2) were processed in two batches (approximately 100 mL / batch) to produce 50 mL of output product. The pre-processing counts of the plasmapheresis products were similar to the post-processing counts. 40.5% and 51% CD3+ isolated from donors 1 and 2, respectively.+ A total of 5.3x10 T cells 9 and 7.1x10 9 The sample was then divided into aliquots (4x10 7 Cells / mL) were cryopreserved in CryoMACS freezing bags (10 mL) and reference freezing vials (1 mL) for later use. Three separate validation experiments were performed and are summarized in Table 6. Cells from donor 1 were used for validation runs 1 and 2 (V1, V2), and cells from donor 2 were used for validation run 3 (V3). For each run, freshly thawed PBMCs were electroporated and digitally expanded in vitro in a separate culture process (Table 7). On day 0 of culture, 3x10 8 (V1) and 8x10 8 (V2, V3) cells were thawed (viability, 88.9% to 97.6%) and allowed to rest for 2 hours before electroporation. CD19RCD28mz (CoOp) / pSBSO transposon and pCMV-SB11 transposase DNA plasmids were used at 2×10 7 2 to 3 x 10 cells / cuvette 8 cells (V1=2x10 8 ; V2 and V3 = 3x10 8 ) were electroporated and co-expressed with aAPC clone #4 based on CAR expression on day 2 (day 1 of culture). The electroporation efficiency of the three validation runs was assessed on day 1 of culture, as measured by CAR expression (33.7%, 25.5%, and 47.1%). CAR expression at the end of co-culture (day 28 of culture) was 92%, 99.2%, and 96.7%, and the cultures contained an average of 95% ± 5.3% CD3 + T cells, contaminating CD19 + cells (mean = 0.7% ± 0.15%) and CD32 + aAPC (mean = 0.6% ± 0.6%, Figure 7A , Table 6) was negligible. The present inventors further confirmed CAR expression by Western blotting of whole cell lysates of electroporated / propagated T cells using CD3-ζ chain-specific mAb, showing the expected 73-kDa chimeric ζ chain (Singh et al., 2008) ( Figure 7B After observing the kinetics of T cells grown on aAPC, the inventors observed an accelerated rate of T cell proliferation at the end of the second week of culture (end of stimulation cycle 2), which was consistent with an increase in total T cells (p=0.01) and CD3 fold expansion compared to the first week of stimulation. +(p = 0.01) T cell fold expansion was consistent. Total T cells (p = 0.01, p < 0.001), CD3 + (p=0.03, p<0.001), CAR + (p = 0.02, p < 0.001) The weekly fold expansion of T cells at the end of the third and fourth weeks was consistently higher than that of the first week ( Figure 13 ).

[0249] Table 6: Acceptance criteria for release of electroporated and propagated T cells

[0250]

[0251]

[0252]

[0253] The present inventors observed that after the first week of stimulation, CD3 + With CAR + After 4 weeks of co-culture on γ-irradiated aAPC, there was an average of 545-fold expansion of CD3 T cells. + Digital expansion of T cells, CAR + T cells had a 1,111-fold expansion. Ex vivo expansion (day 28 of culture) resulted in an average of 2.86 x 10 10 CD3 + T cells, almost all of which are CAR + (2.65x10 10 Total T cells (p=0.18), CD3 + (p=0.17) and CAR + (p=0.2) T cell proliferation kinetics were similar ( Figure 8A , B, C). These data support the recursive addition of aAPCs for the selective generation of CD19-specific T cells.

[0254] Electroporation and propagation of CAR + T cell immunophenotyping. 2 of 3 validation runs resulted in CD8 + CAR + T cells (mean 76% ± 22%) compared to CD4 + CAR + Preferential growth of T cells (mean 16% ± 24%) Figure 7C ), which can be predicted by the inclusion of IL-21 (Singh et al., 2011). +The average CD4 / CD8 ratio of T cells was 3.3 at the beginning of co-culture on aAPC (day 1 of culture). + CAR + T cells predominated, resulting in equal numbers of CD4 + CAR + and CD8 + CAR + T cells (ratio = 0.9), after which the ratio decreased (day 21 of culture, ratio = 0.4; day 28 of culture, ratio = 0.3). The total CD4 / CD8 ratio followed a similar trend and decreased over time (Table 7). CAR at the end of the propagation period (day 28 of culture) + T cells were activated (CD69 expression, average 43.6%; HLA-DR expression, average 61.2%), capable of cytolysis (granzyme B expression, average 92.3%), and expressed markers of memory / naive T cells (CD62L expression, average 45.6%; CD28 expression, average 66.4%; CD27 expression, average 77.5%, CD45RA expression, average 99.7%). The present inventors were unable to detect cell surface markers of exhaustion / senescence (PD-1 expression, average 2.7%; CD57 expression, average 3.3%). Figure 7D These data support CAR + The generation of aAPCs from a heterogeneous population of T cells is consistent.

[0255] CAR + T cell redirection specificity. T cells generated from all three validation runs were able to specifically lyse CD19 + Tumor targets. At an effector to target ratio of 20:1, an average of 57% ± 4% (mean ± SD, range 61.2% to 53.8%) of Daudiβ2m and 49% ± 7% (mean ± SD, range, 41% to 54%) of NALM-6 were cleaved. At an effector / target ratio of 20:1, CD19-specific killing was shown to be effective for CD19. + Killing by EL-4 (range, 4.2 to 9.2-fold) was significantly higher for CD19 neg The average of 6.2±2.6 (mean±SD) times of parental EL-4 cells, among which CAR neg The (mock electroporated) control produced 1.4 ± 1 (mean ± SD) times background CD19-specific lysis ( Figure 8D ,14). This suggests that the CAR in electroporated and propagated T cells redirects killing towards CD19.

[0256] Lack of unwanted autonomous proliferation by genetically modified and propagated T cells. The inventors evaluated CAR in the presence / absence of K562 aAPC and cytokines (IL-2 and IL-21). + At the end of 18 days of culture, the inventors observed <2 × 10 T cells in the genetically modified T cells that did not receive cytokines and aAPCs. 4 cells (mean 2,800, range 0.0-5.6×10 3 ), while the control group receiving cytokines and aAPCs expanded to an average of 7.6×10 7 cells, ranging from 4.12×10 7 to 12.8×10 7 (Table 8). These data suggest that CAR + T cells cannot maintain expansion after withdrawal of growth factor and antigen stimulation.

[0257] Table 8: Lack of autonomous cell growth by genetically modified T cells

[0258]

[0259] a Culture days when T cells were inoculated

[0260] b Total number of T cells seeded in culture at the beginning of the experiment

[0261] c Total number of T cells counted in the absence of cytokines and aAPCs

[0262] d Total (inferred) number of T cells counted in the presence of cytokines and aAPC (positive control)

[0263] ePerfect fold change = [(c / b)÷(d / b)]*100

[0264] Table 9: In-process testing for electroporation and propagation of T cells

[0265]

[0266] CAR + Telomere length in T cells. Telomere length is an important measure of cell differentiation and progression to senescence. Therefore, in order to evaluate the effect of SB transposition and ex vivo digital expansion of CAR+ T cells on telomere length, the present inventors used Flow-FISH to assay the telomere length of cells from CAR+ T cells. + Telomere length of T cells (culture day 28) was compared with their respective matched unmanipulated controls (before electroporation).+ CAR + CD4 T cells and V3 predominant + CAR + The inventors used CD8 T cells to generate + T cells (for V1 and V2) and CD4 + Telomere length of T cells (for V3) was compared with that of CD8 + and CD4 + T cell comparison ( Figure 9A The average telomere length of T cells after ex vivo digital expansion (8.93% ± 1.33% on day 28 of culture) was similar to that of unmanipulated control T cells (7.77% ± 1.21% on day 0). These results indicate that CAR + Electroporation and propagation of T cells did not result in erosion of telomere length.

[0267] CAR transgene copy number. Using CD19RCD28 + The copy number of the integrated CD19RCD28 transgene was determined using Jurkat cell clone #12 as a reference and endogenous RNase P as a normalizer (Maiti et al., 2013). The average transgene copies per T cell generated in the validation run were 0.96 ± 0.09 (range, 0.75 to 1.07, Figure 9B These data indicate that SB transposition results in approximately one integrated copy of the CAR / T cell genome.

[0268] TCR Vβ usage. Electroporation and propagation of T cells can lead to the emergence of oligoclonal or clonal populations of T cells that can indicate preferential growth and thus be indicators of genotoxic events. Therefore, the inventors evaluated TCR Vβ usage by flow cytometry as a tool to assess library diversity. After 28 days of co-culture on aAPC, all 24 TCR Vβ families tested were maintained in T cells, which was similar to the library before electroporation. In addition, TCR Vβ families were maintained after extended culture time (day 35 of culture, Figure 9C These data indicate that cultured CAR + Broad TCR diversity in T cells was maintained and did not reveal an imbalance in TCR sequence usage.

[0269] CAR + Deficiency of SB11 transposase in T cells. Continued expression of SB11 transposase may lead to reactivation of the integrated CAR transgene. Therefore, the present inventors performed genomic PCR to exclude CAR +Inappropriate homologous recombination of DNA plasmid encoding SB11 in T cells. Within the limitations of the assay, the present invention could not detect in PCR reactions containing CAR from 28 days of culture. + The DNA band (about 1 kb) of T cells amplified using SB11-specific primers ( Figure 9D These data indicate that integration of the SB11 transposase is absent in electroporated and propagated T cells.

[0270] CAR + T cell karyotype. Chromosome structural integrity was assessed to exclude global genotoxicity associated with SB transposition. CARs from all three validation runs + G-banding of T cells (collected 28 days after electroporation) showed normal (male) karyotype in all metaphase chromosome spreads analyzed ( Figure 9E , 15).

[0271] Example 5 - Targeting Archaic Viruses Expressed in Cancer and Infection Using Adoptive T Cells Engineered to Express Chimeric Antigen Receptors - Methods

[0272] Immunohistochemistry. Tissue microarrays (#ME1004a, ME2082b, FDA998t) obtained from USBiomax (Rockville, MD) were hydrolyzed with DiH2O. Antigen retrieval using EDTA-free citrate buffer (pH 6) was performed. Slides were blocked using 3% hydrogen peroxide (Biocare Medical, Concord, CA), avidin (Biocare Medical), biotin (Biocare Medical), and polyvalent whole serum (Biocare Medical). Slides were incubated with HERV-K mAb (0.6 mg / ml) at a dilution of 1:20, followed by incubation with biotinylated anti-mouse IgG (Biocare Medical) and streptavidin-HRP (Sigma-Aldrich, St Louis, MO) for 30 minutes each and displayed using Mayer's hematoxylin counterstain (Sigma-Aldrich). Slides were similarly stained using an isotype control mouse IgG2a (0.25 mg / ml) antibody (BD Pharmingen, Franklin Lakes, NJ).

[0273] The scFv sequence against the HERV-K envelope protein (from mAb clone 6H5) was codon-optimized (CoOp) (Invitrogen, Carlsbad, CA) and then cloned into the SB transposon under the control of the human elongation factor-1α (hEF-1α) promoter, flanked by CoOp SB inverted repeat sequence of 6H5CD28 / pSBSO.

[0274] HERV-K antigens were expressed from an SB plasmid containing the bidirectional hEF-1α and cytomegalovirus (CMV) promoters. A codon-optimized full-length antigen sequence with the viral transmembrane domain was cloned under the control of the hEF-1α promoter, and the neomycin gene was transcribed in a bidirectional vector under the control of the CMV promoter. Transposase (SB11) was expressed in cis from the plasmid pCMV-SB11 (Davies et al., 2010).

[0275] To generate the SB plasmid for in vivo imaging of T cells, codon-optimized firefly luciferase was fused to a myc tag and expressed under the control of the CMV promoter. A lentiviral vector encoding mKate-Renilla luciferase under the control of the eEF1α promoter was used as an imaging vector for in vivo melanoma tumor cells.

[0276] Cell line and their breeding.A375-mel and A888-mel are the friendly gifts of Dr.Lazlo Radvanyi from The University of Texas MDAnderson Cancer Center (Houston, TX).A624-mel and EL4 parent generation are available from American Type Culture Collection (Rockville, MD).A375-SM (super metastatic melanoma cell line) is received from the CCGS core facility of University of Texas MDAnderson Cancer Center (Houston, TX).All cell lines are cultured in RPMI (Thermo Scientific, Rockford, IL) containing 10% FBS (Thermo Scientific) and 5% glutamax (GibcoLife technologies, Grand Island, NY).All cell lines are verified by morphology, cell fingerprint analysis and / or flow cytometry.Test their Mycoplasma and they are stored in the research cell bank.

[0277] Generation and expansion of T cells expressing HERV-K specific CAR. PBMCs were isolated from healthy donors by Ficoll-Paque density gradient centrifugation (GE Healthcare Bio-Sciences, Piscataway, NJ) and then electroporated with the HERV-K SB transposon and SB11 transposase using the Amaxa electroporation system. Briefly, 2 × 10 7 PBMC cells were then incubated for 2 hours in complete RPMI supplemented with 10% fetal bovine serum (Thermo Scientific) and 1% glutamax (Gibco Lifetechnologies). These cells were then resuspended in 100 μl of Amaxa Nucleofector solution (human T cell kit) along with HERV-K CAR transposons (6H5CD28 / pSBSO, 15 μg) and SB transposase (pCMV-SB11, 5 μg), transferred to a single cuvette, and subsequently electroporated using the U-14 program in Amaxa electroporator (Lonza, USA). Electroporated T cells were incubated at 37°C for 4 hours in RPMI (Thermo Scientific) completely free of phenol, followed by half-medium changes.

[0278] K562 expresses endogenous HERV-K antigen and is therefore used for the propagation of HERV-K-specific CARs. + Electroporated T cells cultured in RPMI with 10% FBS were supplemented with gamma-irradiated (100 Gy) K562-aAPCs at a T cell:aAPC ratio of 1:2. Irradiated aAPCs were added at the end of each week of T cell stimulation at the same ratio. Soluble IL-21 (eBioscience) and IL-2 (Chiron) cytokines were supplemented at concentrations of 30 ng / ml and 50 U / ml, respectively, to complete the RPMI medium every other day in post-electroporation culture. Mock-transfected DNA-free control T cells grown in the presence of OKT3-loaded K562 cells were used as negative controls. + T cells were cultured under the same conditions as those used in CoOp CD19CARCD28 / pSBSO and SB11 transposase electroporated CD19CAR + T cells were grown and the CD19CAR + T cells used as nonspecific CAR + T cell control.

[0279] Monitor CD3 T cell cultures weekly neg CD56+ The presence of aAPC cells was detected and the culture was depleted if the population exceeded 10% of the total population. This depletion usually occurred between days 10 and 14 of the initial co-culture with aAPCs. The depletion was performed using positive selection "possel" on an Automax (Miltenyi Biotech) using CD56 beads (Miltenyi Biotech Inc, Auburn, CA) according to the manufacturer's instructions.

[0280] T cell viability was assessed using a Cellometer automated cell counter (Automatic T4 Cell Counter, Nexcelom Bioscience, Lawrence, MA) based on the trypan blue exclusion method. Viability was analyzed during the electroporation and co-culture period using the programs "PBMC_human_frozen" and "activated T cells." Total cells, CD3 T cells, and T cells were counted at the end of the 7-day, 14-day, 28-day, and 32-day co-cultures for individual donors. + 、CD4 + 、CD8 + and CAR + The fold expansion of cells was compared with that of day 1 and the CD19CAR expression was compared using Student's t-test. + Average of 3 donors between T cells and no DNA control cells.

[0281] Flow cytometry. Unless otherwise mentioned, all reagents were obtained from BD Biosciences (Franklin Lakes, NJ). One million cells were stained with antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein conjugated to a cyanine dye (PerCPCy5.5), or allophycocyanin (APC). The antibodies used included anti-CD3 FITC (5 μl), anti-CD3 PerCPCy5.5 (2.5 μl), anti-CD3 PE (2 μl), anti-CD4 APC (2.5 μl), anti-CD8 PerCPCy5.5 (4 μl), anti-CD56 APC (2.5 μl), Annexin V (5 μl), anti-CD32 FITC (5 μl), anti-CD45RA FITC (5 μl), anti-CD45RO APC (2.5 μl), anti-granzyme B FITC (5 μl), anti-CD62L APC (2.5 μl), anti-IFN-γ APC (2 μl), anti-CD27 PE (2 μl), anti-αβTCR FITC (5 μl), anti-γδTCR PE (2 μl) and anti-CCR7PerCPCy5.5 (2.5 μl, Biolegend). F(ab')2 fragments of goat anti-human Fcγ conjugated with FITC (3 μl, Invitrogen) and PE (2.5 μl, Invitrogen) were used to detect the cell surface expression of HERV-K specific CAR. Non-specific antibody binding was blocked using FACS wash buffer (2% FBS and 0.1% sodium azide in PBS). Data were acquired using CellQuest version 3.3 (BD ​​Biosciences) in FACSCalibur (BD Biosciences). Median fluorescence intensity (MFI) was analyzed and calculated using FlowJo version 7.5.5.

[0282] nCounter Digital Gene Expression System. HERV-K specific CARs were evaluated using the nCounter Analysis System (model NCT-SYST-120, NanoString Technologies; Geiss et al., 2008). + Differences in gene expression between T cells and no-DNA control T cells. Briefly, 10 4 HERV-K specific CAR +T cells or DNA-free T cells were used and mRNA was hybridized with the reported code set and a custom designed capture code set using the nCounterGene expression assay kit at 65°C for 12 hours. The nCounter prep station was used for post-hybridization processing. Nanomir software was used to normalize mRNA levels using internal controls. The R-program, tree view, and cluster view were used to output data for statistical analysis. Normalized results were expressed as relative mRNA levels. Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Inc., New York, NY) was performed on statistically significant genes. www.ingenuity.com ) to understand their biological interactions based on databases derived from literature sources.

[0283] Integration analysis. HERV-K specific CAR was isolated using the QIAamp DNA mini kit (Qiagen). +Genomic DNA of T cells was collected and real-time PCR was performed as previously described (Maiti et al., 2013). Genomic DNA from Jurkat cells (clone #14) with a single integrated copy of the previously described CAR was used as a positive control (Maiti et al., 2013). The expression vectors were PCR amplified in triplicate using 10 μl of TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA), 1 μl (250 nM of 1× probe and 900 nM of 1× primer) of 20× FAM-labeled CAR-specific TaqMan probe primer set for IgG4 Fc [forward (5′-GAGGGCAACGTCTTTAGCTG-3′; SEQ ID NO: 15) and reverse (5′-GATGATGAAGGCCACTGTCA-3′; SEQ ID NO: 16) primers and carboxyfluorescein (FAM)-labeled probe (5′-AGATGTTCTGGGTGCTGGTC-3′; SEQ ID NO: 17)] and 1 μl (900 nM of 1× primer and 250 nM of 1× probe) of 20× VIC-labeled TaqMan RNase P probe primer set (Applied Biosystems). ng of genomic DNA were used in a total reaction volume of 20 μl. Primer hybridization occurred in the IgGFc4 portion of the CAR. Amplification cycles included 50°C for 2 minutes; 95°C for 10 minutes; and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Detection was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The autosomal RNase P gene, which exists at 2 copies per diploid cell, was used as an endogenous reference for normalization (Jin et al., 2011). ΔΔC T The RIPA method (Applied Biosystems, CA) was used to calculate the number of integrations for RNase P, and Jurkat clones were used as normalization controls.

[0284] Chromium release assay (CRA). CRA was performed as previously described (Maiti et al., 2013; Jin et al., 2011). Briefly, 51 Cr treatment of HERV-K +ve Target for 2 hours, followed by HERV-K specific CAR on day 35 + T cells or DNA-free control T cells were incubated together and the following formula was used to calculate the 51 Cr release percentage:

[0285]

[0286] Time-lapse bioimaging. (A) To visualize the engagement of CAR with antigens on tumor cells, time-lapse imaging was performed using the BioStation IMCell-S1 / Cell-S1-P system (Nikon, Melville, NY). K562 parental cells were stained with anti-HERV-K APC antibody (1 μg), and HERV-K-specific CAR was stained with FITC-labeled goat anti-human Fcγ antibody F(ab′)2 fragment (5 μl, BD Biosciences). + T cells were stained for CAR surface expression. T cells were mixed with target cells at a ratio of 5:1 and then plated on T-35mm glass bottom plates (Fisher Scientific, Hampton, NH) in complete RPMI medium. Cells were immediately imaged every 2 minutes at 37°C for up to 8 hours. Separately, phase contrast fluorescence channel 2 (to observe green HERV-K specific CAR) was used. + Each image was recorded with a 20X objective lens at 1600 × 1200 pixels at an exposure time of 1 / 125 and 1 / 5 seconds for 1 / 125 and 1 / 5 seconds for 1 / 125 and 1 / 5 seconds for 1 / 125 and 1 / 5 seconds for 1 / 125 and 1 / 5 seconds for 1 / 120. When the APC-labeled antigen overlaps with the green-labeled CAR, the engagement of the CAR with the antigen is seen.

[0287] (B) To visualize and quantify HERV-K-specific CAR + The time it takes for T cells to kill tumor cells was measured using a 1ng / mg Tumor cells were incubated with HERV-K specific CAR in complete RPMI (Invitrogen) + T cells were plated together in T-35 mm glass bottom plates. Tumor cell death was recorded as the time when the tumor cell wall was perforated and the cells turned green (by using the BiostationIM cell-S1-P system (Nikon)). The intensity of green fluorescence in each tumor sample was recorded using live cell imaging software (Nikon) over a 15-hour period.

[0288] Intracellular IFN-γ release assay. HERV-K specific CAR was injected into the cell wall of 200 μl complete RPMI medium. +T cells were co-cultured with tumor cells in a round-bottom 96-well plate at a ratio of 1: 10. Protein transport inhibitors (BD Golgi Plug containing Brefeldin A) were added to all wells to capture IFN-γ inside the cells. The co-cultures were incubated at 37°C for 4 hours, followed by staining for HERV-K specific CAR expression at 4°C for 20 minutes. The cells were subsequently washed, fixed, and permeabilized with 100 μl of Cytofix / Cytoperm buffer (catalog number 555028) at 4°C for 20 minutes. The permeabilized cells were subsequently stained for cytokines using an anti-IFN-γAPC antibody. The cells were washed and subsequently analyzed using FACSCalibur. T cells treated with PMA (phorbol 12 myristate 13 esters) and ionomycin (BD Biosciences) were used as positive controls for the assay. Similar assays were performed using DNA-free control T cells.

[0289] HERV-K +ve Development of the EL4 cell line. Two million EL4 cells were suspended in an AMAXA mouse T cell nucleofection solution (Amaxa, USA) containing an SB transfectant expressing a HERV-K antigen and SB11 transposase (2 μg of total DNA) to a final volume of 100 ml. The suspension was transferred to a single cuvette and electroporated using the C-09 program in an Amexa electroporator. The cells were incubated at 37°C in the electroporation medium provided with the kit supplemented with 10% FBS (Thermo Scientific Pierce) for 4 hours. The cells were then transferred to DMEM, 10% FBS (Thermo Scientific Pierce) and 5% glutamax (Gibco Life Technologies). The cells were then grown in the presence of 0.8 mg / ml of G418 and HERV-K antibodies were used to sort neomycin-resistant HERVK. +ve EL4 cells were grown to obtain pure cell populations.

[0290] shRNA-mediated HERV-K knockdown in A888-mel cells. A888-mel cells were grown to 90% confluence in 6-well plates. 100 μl of HERV-K specific shRNA or scrambled shRNA lentivirus and polybrene (5 μg / ml) were subsequently used to replace the culture medium, transduced at 37°C for 4 hours, and then the culture medium was replaced with conventional RPMI culture medium. Cells were subsequently sorted based on GFP expression and grown. Scrambled shRNA-transduced A888-mel cells were used as controls. Immunoblotting of cell line lysates was performed to determine the extent of HERV-K knockdown. 6H5 HERV-K antibody was used to detect HERV-K antigen expression on A888-mel cells or parental cells with HERV-K shRNA or scrambled shRNA. 10 million cells were lysed with RIPA buffer containing protease inhibitors (Roche Applied Science, San Francisco, CA). BCA assay was performed to detect protein concentration (Thermo Scientific Pierce). A 4%-20% gradient gel (Biorad, Hercules, CA) was used to run 10 μg of protein boiled in SDS loading buffer. The protein was then transferred to a nitrocellulose membrane and blocked with 5% milk in PBST and then incubated with 6H5 HERV-K antibody (mg / ml). Binding was detected using goat anti-mouse Fc-HRP (Sigma-Aldrich) and the ECL Westfemto TM The substrate was developed using the Versa doc Quantityone TM Blots were imaged using ImageJ software (Biorad) and quantified using ImageJ software.

[0291] In vivo analysis. Metastatic melanoma model: On day 0, 10 6 A375-SM cells were injected intravenously into 5-week-old female NOD.Cg-Prkdc scid Il2rg tm1wjl / SzJ (NSG) mice (Jackson Laboratories, Bar Harbor, ME). A375-SM cells were previously stably transduced with mKate-rRLuc and sorted for a homogeneous population of cells. Starting on days 7, 14, and 21, mice in the treatment group (n=7) received 2×10 6 HERV-K-specific CAR-FfLuc +T cells. IL-2 (600 U; eBioscience) was injected intraperitoneally (ip) three times a week during the treatment period. One group of tumor-bearing mice (n=6) received no treatment, while a group of tumor-free mice (n=3) received a similar number of CARs as in the control group. + T cells.

[0292] Flow Quantification: Bioluminescence imaging (BLI) was performed weekly to image tumor and T cell activity in vivo. Mice were anesthetized and placed in the anterior-posterior position for BLI using a Xeno IVIS 100 series system (Caliper Life Sciences) as previously described (Singh et al., 2007). + For imaging of T cell activity, 150 μl (200 μg / mouse) of D-luciferin potassium salt (Caliper Life Sciences) was injected intraperitoneally (ip). Emitted photons were quantified 10 minutes after injection using the Living Image 2.50.1 (Caliper Life Sciences) program. To image tumor cell activity, 100 μl of Endurin (Promega, Fitchburg, WA) was injected intraperitoneally. 20 minutes after injection, tumor activity was quantified similarly to ffLuc. An unpaired Student's t-test was performed to determine statistical significance of the flux.

[0293] Statistical analysis. For the analysis of statistical differences in antigen expression between melanoma of different grades and stages and normal tissues, Student's t-test and ANOVA were used. For the analysis of control versus HERV-K specific CAR + Differences between T cell expansion, phenotypic analysis, and functional assays were performed using Student's t-tests, and mean values, standard deviations, and 95% confidence intervals (CIs) were calculated. For the in vivo metastatic melanoma model, Student's t-tests were used to analyze the significance of flux data between tumor and treatment groups. All statistical tests were two-sided and performed using Graph Pad Prism software (GraphPad Software Inc, San Diego, CA). All P values ​​less than 0.05 were considered statistically significant.

[0294] Example 6 - Targeting Archaeretroviruses Expressed in Cancer and Infection Using Adoptive T Cells Engineered to Express Chimeric Antigen Receptors - Results

[0295] Expression of HERV-K in melanoma patient samples. In order to clarify the physiological relevance of HERV-K in melanoma invasion and metastasis in vivo, researchers sought to evaluate the expression of tumor antigen HERV-K envelope protein in biopsy tissues obtained from patients with melanoma of different stages. Malignant tumor tissues from 268 patients and benign skin and breast tissues from 40 patients were analyzed using IHC. Tumor tissue staining was graded based on the percentage and staining intensity of tumor cells positive for HERV-K, and their products were calculated to obtain the H-index. Tumor tissues showed different levels of staining intensity, and when compared with isotype control staining on the same tissue, the tumor tissues were scored as 0, 1, 2 or 3 points ( Figure 16A ). The antigen is expressed on the cells in an emphasized form along the cell surface, as shown by the solid arrow, or as a diffuse cytoplasmic staining, as shown by the dotted arrow (Figure 16B). This may suggest the circulation and accumulation of the antigen to the cell surface for the purpose of shedding viral proteins (REF). Tumor cells express a significantly higher H-index of HERV-K antigen than benign tumors ( Figure 16C Although there was no difference in the H-index between malignant and metastatic tumors, a significant difference was seen between tumors in stages I and II and those in stages III and IV ( Figure 16D , E). To further demonstrate the specificity of HERV-K expression in tumor cells rather than normal cells, tissues from 33 types of normal organs (each type obtained from 3 normal donors) were analyzed. No significant expression of HERV-K was observed in any normal organ tissue (Figure 22). These findings indicate that HERV-K upregulation specifically occurring in tumor cells can be used as a unique target marker for immunotherapy.

[0296] HERV-K-specific CAR + Propagation and characterization of T cells. HERV-K env-specific monoclonal antibodies (mAbs) were developed in mice, and the 6H5 mAb clone was found to be the most sensitive in detecting antigens in vivo (Wang-Johanning et al., 2003). The scFv sequence of the 6H5 mAb clone was used to construct CAR. The scFv box was fused to the IgG4 Fc region via a flexible linker, followed by the CD28 transmembrane domain and the CD28 and CD3z intracellular domains. The fusion construct was then cloned into the SB transposon vector ( Figure 17A ).

[0297] To generate CAR specific for HERV-K env antigen + T cells, the present inventors used SB transposon together with SB11 transposase to electroporate peripheral blood mononuclear cells (PBMCs), and then expressed endogenously derived HERV-K+ve The cells are propagated on K562aAPC. In order to selectively propagate the T cells with stably expressed CAR, these aAPCs endogenously expressing HERV-K antigens are genetically modified to co-express the required T cell co-stimulatory molecules CD86, 4-1BBL and membrane-bound IL-15 (co-expressed with enhanced green fluorescent protein EGFP) (Singh et al., 2008). PBMCs grown under the same culture conditions on aAPCPs loaded with OKT3 without any transposon electroporation were used as negative no DNA controls.

[0298] CAR expression was detected using a polyclonal Fc antibody specific for the IgG4 Fc region. CAR expression was detected using Fc antibody every 7 days before supplementing the culture with irradiated aAPC. + T cells were stained. Flow data showed that 95% of T cells expressed CAR on their surface, which was detectable for up to 35 days in culture ( Figure 17B ). When compared to no DNA control cells, HERV-K specific CAR + No significant differences were seen in the growth kinetics of T cells, and all HERV-K-specific CARs + On the 14th day of culture, T cells were CD3 + T cells ( Figure 17C ,D). CAR + The mean percentage of CD4 increased, however, CA R + CD8 cells decreased as the culture period progressed ( Figure 23A ). HERV-K specific C AR + Real-time PCR analysis of genomic DNA of T cells compared to cells without DNA showed that + There are fewer than two CARs integrated into the T cell genome ( Figure 17D ). Cultured CAR + T cells have an effector memory phenotype that includes CD3 with significant lytic potential as observed by granzyme B levels. + CD56 + CD45RO + TCRαβ + CD27 neg CCR7 neg cell( Figure 17F ).

[0299] Measurement of HERV-K-specific CARs from 3 normal donors +mRNA levels in T cells were compared to those in DNA-free control T cells from day 28 of culture using nCounter analysis. + T cells have significantly higher levels of chemoattractants, transcriptional regulators, and activators. + Elevated levels of perforin 1 and granzyme H in T cells indicate a higher lytic potential of these cells ( Figure 23B Ingenuity pathway analysis (IPA) (p<0.05) showed that these + Several of the genes upregulated in T cells are involved in NF-κB activation. Chemoattractants and cytokines are involved in the regulation of IL-10, IFN-γ, and IL-12 ( Figure 23C These data reinforce previous observations that HERV-K-specific CARs + T cells have a central effector phenotype.

[0300] HERV-K CAR + Characterization of T cell functionality. Antigen expression on melanoma cell lines such as A888, A375, A375-SM, and A624 was analyzed using monoclonal 6H5 antibody against HERV-K env protein. Figure 18A ). HERV-K antigen expression on melanoma cells was compared with isotype control (mouse IgG2a). In order to analyze HERV-K specific CAR + Functionality of T cells pulsed with radioactive chromium for HERV-K + Tumor cells and treated with different ratios of HERV-K specific CAR + T cells were cultured. A no-DNA control was used as a negative control for CRA. When compared with no-DNA control T cells, the HERV-K specific CAR + Significantly higher levels of HERV-K were observed in T cells + Tumor cell lysis ( Figure 18B ). Also irrelevant CARs such as CD19 specific CARs + T cells were used to perform CRA, and a baseline level of nonspecific killing was observed for HERV-K antigen-positive tumor cells when compared to CD19 antigen-positive tumor cells such as EL-4 cells with CD19 antigen ( Figure 24 ).

[0301] To further demonstrate that these HERV-K-specific CARs + T cell functionality was assessed by 4-hour IFN-γ release. Melanoma tumor targets were co-administered with HERV-K-specific CAR at a 1:10 ratio. +T cells or DNA-free control T cells were co-cultured, and intracellular cytokine levels were subsequently analyzed by flow cytometry. T cells cultured with PMA-ionomycin were used as a positive control. HERV-K specific CAR + T cells showed higher levels of IFN-γ release compared to DNA-free control T cells ( Figure 18C ) and the results are associated with CRA.

[0302] HERV-K-specific CAR + To demonstrate the specificity of HER-K-specific CAR, a bidirectional SB vector encoding HERV-K antigen under the hEF-1α promoter and neomycin resistance under the CMV promoter was used to target HERV-K. neg EL-4 cells were electroporated ( Figure 25 ). Then EL-4HERV-K +ve The cells were single-cell sorted and grown in the presence of the mammalian selection marker neomycin to obtain a pure population expressing HERV-K env. Interestingly, these cells lost HERV-K antigen expression within 10 days of culture due to post-translational modification and proteolytic cleavage. +ve Cells were subjected to 4 hours of CRA. EL-4 HERV-K cells pulsed with chromium were +ve Cells and HERV-K neg EL4 parental cells and different concentrations of HERV-K specific CAR + T cells were co-cultured and graded tumor-specific lysis was observed in an antigen-specific manner ( Figure 19B ).

[0303] To further demonstrate specificity, HERV-K env antigen was knocked down in A888 melanoma cells using shRNA lentivirus. Immunoblot analysis showed approximately 50% knockdown of HERV-K protein levels compared to A888 parental and control scrambled shRNAs ( Figure 19C HERV-K-specific CAR + T cells with HERV-K knockdown cells, A888 parental, and A888 with scrambled shRNA showed a significant reduction in killing of HERV-K knockdown tumor cells compared to parental and control tumor cells ( Figure 19D ).

[0304] Time-lapse imaging to visualize and quantify CAR + The time it takes for T cells to kill tumor targets and the number of tumor cells killed during a 15-hour period. Tumor cells and T cells were cultured at a ratio of 1:5 in the presence of Make the cells green. As melanoma target A888 and A375 cells are transfected with HERV-K specific CAR + T cell killing was reported with a sudden rise in green fluorescence around 5 hours, which gradually increased over a period of 15 hours. neg No killing was observed for the HEK293 parental target. Approximately 30% of A888 cells and 35% of A375 cells were killed by CAR over a 15-hour period. + T cell killing. To visualize antigen-specific CAR engagement, K562 cells were positive for HERV-K antigen staining (shown in red) and HERV-K specific CAR was stained green for Fc staining. Overlap of CAR and antigen was seen within 5 hours after antibody-bound antigen and CAR were internalized by cells ( Figure 26 ).

[0305] HERV-K-specific CAR + The tumor killing ability of T cells in vivo. PBMCs were double electroporated using SB vectors encoding HERV-K specific CAR and myc-FFLuc with SB11 transposase. The SB vector with myc-ffLuc gene has a neomycin resistance gene connected by a linker ( Figure 27A These HERV-K specific CAR-ffLuc + T cells with HERV-K-specific CAR + T cells have similar growth kinetics, and their tumor killing capacity and specificity are comparable ( Figure 27B ,C).

[0306] A metastatic melanoma model was developed in which the A375-super metastatic (A375-SM) cell line was infused through the tail vein of NSG mice. These cells migrated into the lungs and metastasized to the liver. To non-invasively visualize the reduction of tumor mass, tumor cells were transduced with a lentiviral vector carrying the mKate-rRLuc gene and sorted using the mKate marker to obtain a pure cell population ( Figure 21A ). After 1 week of tumor cell implantation, 20 million CARs were injected into the CAR T cells on days 8, 15, and 22. + T cells were infused intravenously along with IL-2 (intraperitoneally) twice a week for 3 weeks. Bioluminescence imaging (BLI) was used to assess rRLuc activity in each group of mice. The group of mice with tumor cells alone had significantly higher levels of rRLuc activity on day 25 compared to the group receiving HERV-KCAR. + The group of mice with tumor cells had significantly higher luciferase activity ( Figure 21A , B). In addition, mice with isolated tumors became moribund on day 28 due to high metastasis of tumor to liver, whereas mice receiving CAR + The mice in the T cell group showed healthy appetite and activity. Ex vivo imaging of mKate on tumor cells showed that mice with isolated tumors had more prominent tumor colonies in the liver than the treated group ( Figure 21C ). Pathological examination provided the observation that the tumor group had a significantly higher metastatic population in the liver compared to the treatment group, suggesting that HERV-K CAR + The role of T cells in reducing tumor growth and metastasis ( Figure 21D ).

[0307] Discussion. These results show that HERV-K-specific CAR + T cells were able to successfully target viral glycoproteins and kill HERV-K in an antigen-specific manner in vitro + tumor cells and reduced melanoma tumor growth and metastasis in vivo.

[0308] HERV-K env is significantly upregulated during melanoma metastasis and is present only on tumor cells and not on adjacent normal melanocytes. In melanoma, HERV-K env protein is associated with activation of the MEK-ERK and p16INK4A–CDK4 pathways, which are associated with tumor progression (Li et al., 2010). HERV-K molecular mimicry also leads to decreased glutathione peroxidase levels, which in turn increases reactive oxygen species in tissues, contributing to melanomagenesis (Krone et al., 2005). Abnormal levels of HERV-K env expression appear to be a triggering factor involved in melanoma onset, leading to morphological and cellular modifications that contribute to tumor progression (Serafino et al., 2009). Modulation of HERV-K viral transcription leads to increased inflammation and morphological differentiation of melanocytes (Sciamanna et al., 2005). Tissue microarrays demonstrate elevated levels of antigen expression associated with melanoma progression, whereas normal human tissues have baseline levels of HERV-K expression or are absent.

[0309] The immunogenic nature of melanoma makes it an attractive model and target for developing efficient T cell-based therapies. One of the main limitations of T cell-based therapies is the HLA-based restriction of TCR, which limits antigen recognition (Garrido et al., 1997). Genetically modified T cells overcome this limitation by expressing tumor antigen-specific receptors and confer non-HLA-based antigen recognition (Gross et al., 1989). T cell modification can be produced by using viral or non-viral vectors. The "Sleeping Beauty" vector is a non-viral approach to introduce genes into T cells. One of the main advantages of the "Sleeping Beauty" system over other viral transduction methods is that it is genetically safe, efficient and not expensive without retroviral transduction (Maiti et al., 2013). These CARs are cultured using IL-2 and IL-21. + T cells to produce enough cells for infusion purposes. This has led to the use of CD19 CARs against B-lineage malignancies. + Initial clinical trials of T cells. Make HERV-K specific CAR + T cells with clinical-grade CD19 CAR + T cells grow in a similar way. These HERV-K specific CARs + T cells are primarily of the effector memory phenotype, which are well suited to target and kill tumor cells.

[0310] It is known that multiple factors such as cytokines, hormones and chemicals regulate HERV-K levels in cancer processes (Taruscio and Mantovani, 2004). HERV-K env antigens are known to circulate between the cell membrane and the cytoplasm and, in certain tumor states, are also separated from cell surface budding. Therefore, adoptive T cell therapy may be a favorable treatment strategy that only requires transient antigen expression on the cell surface.

[0311] HERV-K env antigens have also been found to be associated with breast cancer, ovarian cancer, prostate cancer, lymphoma, teratocarcinoma, autoimmune diseases such as multiple sclerosis, and infectious diseases such as HIV (Wang-Johanning et al., 2003; Contreras-Galindo et al., 2008; Jones et al., 2012; Ono, 1986; Wang-Johanning et al., 2007). Therefore, targeting env antigens using adoptive T cell therapy may be a therapeutic option for a variety of disease states. Consistent with this hypothesis, HERV-K env can be targeted by HERV-K CAR + T cells successfully and selectively targeted the CARs. + T cells were associated with tumor regression and reduced metastasis, demonstrating the anti-tumor activity of these cells.

[0312] Example 7 - Generation of T cells with long-lived in vivo potential for minimal residual disease immunotherapy using membrane-bound cytokines

[0313] The generation and expression of mIL15. The mIL15 construct (Figure 28) utilizes serine-glycine linker to fuse IL-15cDNA sequence (NM_000585.4) with full-length IL-15Rα (NM_002189.3). The signal peptide of IL-15 and IL-15Rα is omitted and IgE signal peptide (gb|AA B59424.1) is used for mIL15 construct. The construct will be produced as membrane-bound, but it is also present in the IL-15 in the case of IL-15Rα similar to the above-mentioned trans-presentation model. The DNA plasmid is analyzed by GeneArt (Regensburg, Germany) and subsequently subcloned into "Sleeping Beauty" plasmid (non-viral gene transfer method). With CAR plasmid (specific for CD19), primary human T cells are co-electroporated with or without mIL15 plasmid and SB-11 transposon. Propagation and expansion of genetically modified T cells by expressing 41BBL and CD86 co-stimulatory molecules CD19 + The weekly stimulation of K562 artificial antigen presenting cell (aAPC) variants is achieved. The CAR used is a second generation CAR containing CD3ζ and CD28 signaling cytoplasmic domains. The presence of CD19 on aAPC allows the selective production of antigen-specific T cells, while costimulatory molecules promote in vitro expansion. The mIL15 molecule can be stably co-expressed with the CAR by modified T cells, and the co-expressing T cells represent the majority of the population (Figure 29). In addition, for T cells modified with mIL15-CAR, the total CAR expression in the modified T cells reaches greater than 90% (Figure 29).

[0314] Functionality of mIL15. Signaling by the IL-15 receptor complex primarily induces phosphorylation of signal transducer and activator of transcription 5. By using phosflow to visualize phosphorylated STAT5 (pSTAT5), it can be determined whether mIL15 can induce cytokine signaling pathways. The level of pSTAT5 is increased in CARs supplemented with these cytokines. + mIL15 is elevated in T cells, and these levels are abolished by serum and cytokine starvation. + CAR + pSTAT5 levels in T cells were maintained ( Figure 30 These data indicate that mIL15 is functional and activates the pSTAT5 portion of the cytokine signaling pathway.

[0315] Clinically significant numbers of mIL15 + CAR + When using CAR to redirect T cell specificity, the focus of ex vivo expansion is on driving the CAR + T cell populations, in this case with or without mIL15 CAR. Standard CAR grown with soluble IL-2 and IL-21 + T cells, but mIL15 + CAR + Soluble IL-21 was provided to T cells to exploit the reported synergistic effect between IL-15 and IL-21. mIL15 supplemented with IL-21 + CAR + T cells showed promise alongside standard CAR + T cells and control CARs administered IL-15 and IL-21 + T cells compared with the expression (P = 0.53, two-way ANOVA; Figure 31 ). mIL15 + CAR + Ex vivo expansion of T cells yields clinically significant numbers of cells.

[0316] Evaluation of mIL15 + CAR + Phenotype and functionality of T cells. Ex vivo expanded mIL15 + CAR + T cell phenotype and CAR + T cells were very similar, except for IL-7Ra expression. The general phenotype of cells representing the infused product at this time point was predominantly CD8 with moderate to high expression of the activation markers CD45RO and CD25. + (Cytotoxic) T cells. There was variable low to moderate expression of T cell memory-associated markers (CD62L, CCR7, CD27, and CD28) for both T cell populations. Figure 32A ). After ex vivo expansion of the modified T cells, the T cells must retain their redirected T cell specificity and lytic function. A chromium release assay was performed to assess the function of the T cells in the amplified product. + and CD19 - EL4 targets and modified T cells were plated at different effector to target ratios. At all effector to target ratios, mIL15 + CAR + T cells and CARs + T cells all display CD19 +Specific lysis of tumor targets (P < 0.001, two-way ANOVA, n = 3) and both cells were sensitive to the CD19 + There was no difference between the specific lysis of tumor targets (P>0.05, two-way ANOVA). + CAR + T cells on CD19 + Target lysis is specific and associated with CD19 - Background cleavage of the target was significantly different (P < 0.001, two-way ANOVA) ( Figure 32B ). mIL15 + CAR + T cells retain their redirected specificity and lytic capacity.

[0317] Specific mIL15 + CAR + To evaluate the long-term persistence of T cell subsets in vitro, mIL-15 T cells were cultured for a long time to stimulate and expand the four aAPCs. + / - CAR + T cells without further antigen restimulation. + T cells received IL-2, IL-15, or no cytokine supplementation, while mIL15 + CAR + T cells did not receive exogenous cytokines. As expected, CARs that did not receive cytokine supplementation + T cells do not persist. mIL15 + CAR + T cells and CARs that receive IL-2 or IL-15 + T cells compared to unsupplemented CAR + T cells had a significantly greater relative fold expansion (P < 0.0001, repeated measures ANOVA, n = 3; Figure 33A ). mIL15 close to 0 + CAR + The maintenance of relative T cell expansion also suggests that these modified cells do not grow in an unlimited manner. To be beneficial for clinical applications, CAR + T cells must remain responsive to antigens. Therefore, use: no target, CD19 - EL-4, CD19 + EL-4, CD19 +These T cells were stimulated with Nalm-6 (a human leukemia cell line) 75+ days after antigen encounter or were assessed for lymphocyte activation cocktail (LAC) and interferon gamma (IFNg) production by intracellular cytokine staining 6 hours after incubation with target. + T cell-like, mIL15 + CAR + T cells also respond to CD19 + targets and LAC to produce IFNg ( Figure 33B ). In another assay, these 75-day withdrawal T cells were stimulated with aAPC and supplemented with IL-21. + T cells were provided with IL-2 and IL-21, IL-15 cultured T cells received IL-15 and IL-21, and mIL15 + CAR + T cells were supplemented with IL-21 only. T cell viability was assessed by annexin V staining 8 days after aAPC stimulation. mIL15 + CAR + T cells have the largest population of viable cells (Annexin V neg )( Figure 33C ) and showed resistance to activation-induced cell death.

[0318] Persistent mIL15 + CAR + In characterizing the long-term persistence of mIL15 + CAR + T cells, the present inventors hypothesized that persistent mIL15 + CAR + T cells may display characteristics associated with less differentiated T cell subsets, as these cell subsets are known for their long life potential. + CAR + During in vivo culture of T cells, the inventors evaluated whether these T cells had molecular programming towards a less differentiated state that could generate cells with a sustained advantage. + and mIL15 + CAR +T cells (withdrawal of assay T cell input) were analyzed for multiplexed digital gene expression profiles. Standardized mRNA counts were analyzed using a statistical program based on negative binomial distribution (Lohse et al., Nucleic Acids Res. (2012) 40, W622-7). If the difference mRNA counts were greater than 2 times (P < 0.05, FDR q < 0.05), the gene was considered to be significantly differentially expressed. Using these criteria, only 5 genes were considered to be differentially expressed, thereby indicating that there was no culture or molecular programming advantage provided by mIL15.

[0319] The present inventors then characterized the long-term persistence of mIL15 + CAR + T cells. First, their phenotype was assessed using CD45RA and CCR7 markers to phenotypically describe their differentiation state. These markers characterize the differentiation state of T cells as CD45RA + CCR7 + <CD45RA - CCR7 + <CD45RA - CCR7 - <CD45RA + CCR7, representing the cells from the lowest to the highest differentiation state. + CAR + T cells were compared with their counterparts at the beginning of the experiment (Stim4mIL15 + CAR + T cells), persistent mIL15 + CAR + T cell cultures have increased CD45RA + CCR7 + and CD45RA + CCR7 - The proportion of T cell subsets (***P<0.001, two-way repeated measures ANOVA, n=7; Figure 34). + CAR + CCR7 was significantly enhanced in T cells compared to CAR T cells. neg and CCR7 + mIL15 subpopulation activity after antigen withdrawal + CAR + T cells, CAR receiving IL-2 (50U / ml) + T cells and CAR receiving soluble IL-15 (5 ng / ml) +T cells were assessed by Annexin V staining. It was found that regardless of the type of cytokine stimulation (IL-2, IL-15 complex or mIL15), CCR7 neg T cells showed equal frequencies of viable cells. In contrast, CCR7 + T cells had significantly higher viability than CAR T cells that received IL-2 or IL-15 complexes (both P < 0.05; Figure 34). These data indicate that mIL15 is sufficient to support CCR7 + Phenotype, the CCR7 + phenotype, thereby contributing to the maintenance of a less differentiated CD45RA + CCR7 + T cell subsets. mIL15 + CAR + The ability of T cells to promote the persistence of less differentiated T cells is a desirable phenotype for adoptive therapy and appears to corroborate other studies reporting that long-lived T cell subsets have a less differentiated phenotype. In addition, the survival of highly differentiated subsets was also observed, likely supported by constitutive IL-15 signaling.

[0320] Long-term persistence of mIL15 + CAR + T cells displayed some molecular markers associated with less differentiated T cell subsets. The gene expression patterns of the T cells were analyzed using the nCounter analysis system and the production of mIL-15 from stimulation 4 + CAR + T cells survived until withdrawal of mIL15 on day 75. + CAR + Heatmap of hierarchical clustering of differential expression between T cells (>2-fold cutoff, P<0.05, FDR q<0.05). These studies identified 108 significantly differentially expressed genes (>2-fold cutoff, P<0.05, FDR q<0.05). Gene ontology classification was assessed using DAVID functional annotation. The functional classification of differentially expressed genes can be divided into broad categories: T cell activation, differentiation, proliferation, and apoptosis. That is, in mIL15 + CAR + There are more down-regulated genes in T cells involved in the positive regulation of differentiation, regulation of apoptosis and induction of apoptosis. + CAR + A greater number of genes were upregulated in T cells involved in differentiation and negative regulation of the Wnt signaling pathway (Figure 36). This suggests that persistent mIL15 + CAR +The molecular signature of T cells is not as good as that of mIL15 at stimulation 4 + CAR + The T cells (T cells at the beginning of the experiment) were highly differentiated.

[0321] The expression of selected genes was verified by flow cytometry. The expression of transcription factors associated with a less differentiated state (Tcf-7) and the acquisition of effector functions / differentiation state (Blimp-1 and Tbet) indicated that persistent mIL-15 + CAR + T cells display a balance of transcription factors associated with less differentiated cells. This is characterized by greater expression of Tcf-7 and lower expression of Blimp-1 and Tbet ( Figure 37 ). The cell surface markers IL-7Ra and CCR7 were evaluated because they are characteristically expressed by less differentiated T cell subsets. + CAR + T cells have increased expression of these markers associated with long-lived T cell subsets (Figure 38). An additional measure of the level of T cell differentiation is the ability of less differentiated T cells to produce IL-2. mIL-15 was expressed in culture from withdrawal cultures of cells stimulated for 4 or 75 days. + CAR + T cells were mock treated or treated with LAC for 6 hours, and their IL-2 production was assessed by intracellular cytokine staining. Stimulation of 4 T cells failed to produce IL-2, while withdrawal T cells that lasted for 75 days acquired the ability to produce IL-2 (Figure 39). Together, these results indicate that although CARs expanded ex vivo + T cells or mIL15 + CAR + There were no identifiable differences between T cells, but the resulting long-term persistence of mIL15 + CAR + The T cells displayed characteristics associated with less differentiated T cell subsets that exhibit long-term survival in vivo.

[0322] mIL15 + CAR + In vivo persistence and antitumor efficacy of T cells in a high tumor burden model. To evaluate in vivo persistence, mIL15 was co-modified + CAR + T cells and CARs + T cells were expressed firefly luciferase (ffLuc) to enable longitudinal monitoring of T cells in vivo using bioluminescence imaging (BLI). +) 20×10 6 CAR + These modified T cells were adoptively transferred by T cell infusion. The mice were sacrificed 14 days later. + T cells persisted, but mIL15 was observed by BLI + CAR + T cells persisted throughout the 11-day imaging period ( Figure 40B Bone marrow, spleen, and peripheral blood were collected and assessed for the presence of human T cells by flow cytometry (using human CD3 as a marker and gating to exclude murine lymphocytes). + CAR + Significant CD3 T cell expression was detected in the bone marrow (0.49-2.17%, P = 0.0001, unpaired t-test, n = 5), spleen (1.15-12.38%, P < 0.0001, unpaired t-test, n = 5), and peripheral blood (58.39-92.60%, P < 0.0001, unpaired t-test, n = 5) of mice. + T cells ( Figure 40C ). In the + T cell treatment group ( Figure 40C CD3 was not detected in any tissue evaluated in samples from the 24-hour ... + In this model, mIL15 + CAR + T cells in peripheral blood show tumor control ( Figure 40D ), but complete tumor clearance was not observed. These data suggest that despite the ubiquitous presence of tumor antigens, CAR + T cells are not persistent enough in vivo, but mIL15 + CAR + T cells are present in significant levels throughout the body.

[0323] mIL15 + CAR + In vivo persistence and antitumor efficacy of T cells in a low tumor burden model. The present inventors then evaluated T cell engraftment and antitumor activity in a prophylactic model with low tumor burden. + / - CAR + ffLuc + ) were infused into NSG mice (no exogenous cytokines) and allowed to engraft for 6 days, followed by infusion of Nalm-6 modified with Renilla luciferase (rLuc). The mice underwent BLI over the course of 30 days. In this preventive model, mIL15 was observed+ CAR + T cells persisted throughout the experiment and prevented tumor engraftment, which was significantly better than CAR + The T cell and no T cell treatment groups had significant anti-tumor effects (P < 0.0001, one-way ANOVA, n = 4-5) ( Figure 41C -D). Analysis of organs and peripheral blood by flow cytometry in mIL15 + CAR + Human CD3 was detected in the T cell treatment group + T cells, but interestingly they are only found in the bone marrow ( Figure 41E ), and may represent preferential homing or survival after tumor encounter. In similar experiments, survival was tested, and mIL15 + CAR + T cell-treated mice showed significant differences compared to mice without T cell treatment or CAR + T cell-treated mice had significantly improved survival [P = 0.045 (mIL15 + CAR + T cells vs. CAR + T cells, log-rank Mantel-Cox test, n=7-8; Figure 41).

[0324] mIL15 + CAR + In order to assess the in vivo persistence of T cells in the absence of CAR activation, + CAR + Can T cells persist in vivo for a long time without CAR signaling? + / - CAR + ffLuc + ) were infused into mice (without tumors or exogenous cytokines) and monitored for up to 47 days. Testing T cells in this manner will also clarify whether the persistent T cells show unrestricted growth or whether they maintain their population in a homeostatic manner. No CAR + T cells persist, however mIL15 + CAR + T cells exhibit sustained persistence in the absence of exogenous cytokines and antigens ( Figure 42B This was further confirmed by flow cytometry of CD3-stained cells isolated from bone marrow, spleen, and peripheral blood ( Figure 43 ). To assess longitudinal T cell persistence, flux values ​​indicated mIL15 + CAR +T cell levels appear to remain stable or decline slowly over time, but this does not indicate uncontrolled expansion. Figure 42D These persisting T cells were collected and expanded ex vivo in a manner similar to that previously described for the ex vivo expansion of genetically modified T cells. These cells were capable of antigen-specific activation, as measured by interferon gamma production ( Figure 42E The in vivo data showed that mIL15 + CAR + Enhanced persistence of T cells in a CAR-independent and homeostatic manner while retaining their antigen-specific responsiveness.

[0325] mIL15 + CAR + Flow cytometric analysis was used to further characterize the cell proliferation and memory dynamics of T cells. + CAR + T cells (results are shown in Figure 35). Long-term persistence of mIL15 + CAR + T cells were maintained in culture at a low turnover rate, as indicated by a minimum PKH dilution exceeding 10 days in samples that had been cultured consistently for more than a year. Furthermore, dividing cells appeared to be primarily CCR7 - These data suggest that, as opposed to unrestricted autonomous proliferation or growth, T cells maintain their homeostasis ( Figure 35A Activation of mIL15 in continuous long-term culture (1.5 to 2.45 years) using aAPC + CAR + T cells were phenotyped and submitted for karyotyping. Phenotyping showed that these T cells expressed mIL15, and karyotyping results of all submitted donors showed normal metaphase chromosome spreads ( Figure 35B -C). Figure 35D Showing the dynamics of memory after stimulation with K562 aAPC: mIL15 during stimulation vs. withdrawal + CAR conditioning: For these studies, CAR T cells received only IL-21 for the first 9 days of stimulation and then only IL2 during Ag withdrawal conditioning; mIL5 + CAR T cells were exposed to IL-21 during the first 9 days, and then no exogenous cytokines were provided during the withdrawal condition. +There was no difference in CCR7 and IL-7Ra expression between CAR T cells at day 19 after stimulation 1. Only mIL15-modified T cells showed an increase in CCR7 and IL-7Ra expression over time away from antigen exposure (day 29 vs. day 19). CAR expression increased to approximately 80% in the absence of antigen exposure (51% for PB522 and 53% for PB273 at day 9 after stimulation 1). Figure 35E Display of CAR and mIL15 + Memory kinetics of CAR T cells after 1 vs. 2 stimulations. Conditions during withdrawal: CAR T cells received only IL2 during Ag withdrawal, which was established 10 days after stimulation; mIL15 + CAR T cells did not receive exogenous cytokines during the withdrawal process. Comparison of CCR7 and IL-7Ra expression at similar time points after 1 stimulation and 2 stimulations showed that the expression of CCR7 and IL-7Ra at 19 days after 1 stimulation with mIL-15 + There was no difference in memory between CARs. In stimulation 2, at the same time point as stimulation 1, CAR T cells with 2 stimulations had much less CCR7 and almost no IL-7Ra compared to their counterparts with stimulation 1. mIL15 with 2 stimulations + CAR T cells also had less memory but retained more "memory" than CAR-only T cells, in which mIL15 + The CAR T cells had some CCR7 left (but IL-7Ra was almost completely gone).

[0326] Example 8 - Generation of minimally manipulated T cells using mRNA encoding SB transposase

[0327] Studies were conducted to determine whether electroporation using SB transposase provided as mRNA could further enhance the production of CAR T using the SB transposase system. For these studies, cells were electroporated using a plasmid encoding a CAR flanked by transposon repeats and mRNA encoding a transposase (e.g., SB11 or 100x) as described above. The mRNA used for the studies was m7GTP capped and contained a polyadenylation tail. Figure 43 A schematic diagram showing the protocol for CAR T cell generation using the mRNA SB transposase provided as mRNA is shown in For these studies, cells from donor #0 were electroporated with SB11 mRNA, while study cells from donor #1 were electroporated with 100x mRNA.

[0328] Cells generated from donor #0 were characterized by flow cytometry analysis for CAR expression and cell proliferation. Figure 44AAs shown in Figure 2, the percentage of T cells stably expressing CAR increased significantly from day 9 (8.3%) to day 16 (66.2%). T cells grew rapidly and expanded 20-fold by day 16 after transfection ( Figure 44B ). Figure 44D Further studies shown in the were used to assess the number of central memory T cells (Tcm) at day 9 (left panel) and day 17 (right panel) after electroporation. These results show that although the number of less differentiated central memory T cells (Tcm) decreased from day 9 to day 17, it still remained relatively high (27%). Subsequently, a chromium release assay was used to determine the cytotoxic activity of the cells. Figure 44C As shown in , the generated CART cells provide CD-19-specific cytotoxicity against target cells, whereas essentially no cytotoxic activity is seen against unmodified T cells.

[0329] Cells generated from donor #1 were also studied. Figure 45A As shown in , the CAR T cells generated from this donor also provided CD-19-specific toxicity against target T cells, and essentially no cytotoxic activity was seen against unmodified T cells (right graph). In addition, the unmodified T cells killed CD19-positive target cells with similar efficiency on days 9 and 15, despite the different numbers of CAR-positive cells. Figure 45B Results from studies evaluating CAR copy number and expression are shown in Figure 5. These results demonstrate that CAR DNA copy number decreased from 1.5 to 0.9 from day 15 to day 22 and remained stable after this time point.

[0330] Finally, flow cytometry was used to assess cell viability after electroporation. Figure 46 As shown in , after electroporation with DNA / mRNA, the total number of cells first (days 1 and 2) decreased, and then the cells began to grow. According to cell counter counting, the number of cells decreased by 59%-76% on day 2 after electroporation (viability 24-41%).

[0331] Therefore, the above data show that by using the CAR production technology of the mRNA of application encoding transposase, the target-specific cytotoxic T cells of effective number can be produced in an extremely short period of time and with minimum operation. Although the efficiency of mRNA electroporation is donor-dependent, SB100x transposase seems to be more efficient in CAR production. Interestingly, even after electroporation, the cells of lower percentages express CAR in the early stage (9th day), but the efficiency of total cell colony killing targeted tumor cells is almost as high as that from the cell colony in the later stage (15-16 days), and comprises more central memory (lower degree of differentiation) cells. It is also confirmed that SB11 and SB100x encoding mRNA provide the integration of about one copy of the CAR encoding gene per cell genome under the conditions of the test.

[0332] ***

[0333] In light of the present disclosure, all methods disclosed and claimed herein can be prepared and implemented without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the methods described herein and the steps of the methods or the order of the steps of the methods can be varied without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can replace the reagents described herein and still achieve the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the claims.

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Sequence Listing <110> BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM <120> Human applications of engineered chimeric antigen receptor (CAR) T cells <130> 157911F1 <140> unknown <141> 2014-05-14 <150> 61 / 823,253 <151> 2013-05-14 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 711 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 1 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Leu Cys Glu Leu Pro His 1 5 10 15 Pro Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Gln Thr Thr 20 25 30 Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg 35 40 45 Ala Ser Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys 50 55 60 Pro Asp Gly Thr Val Lys Leu Leu Leu Ile Tyr His Thr Ser Arg Leu 65 70 75 80 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Gly Ser Gly Thr 85 90 95 Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr 100 105 110 Tyr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly 115 120 125 Gly Thr Lys Leu Glu Ile Ile Thr Gly Ser Thr Ser Gly Ser Gly Lys 130 135 140 Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Glu Val Lys Leu Gln 145 150 155 160 Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr 165 170 175 Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp 180 185 190 Ile Arg Gln Pro Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 195 200 205 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Ala Leu Lys Ser Arg 210 215 220 Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Lys 225 230 235 240 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 245 250 255 His Tyr Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 260 265 270 Gly Thr Ser Val Thr Val Ser Ser Glu Glu Ser Lys Tyr Gly Pro Pro 275 280 285 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Ser Val 290 295 300 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 305 310 315 320 Pro Glu Val Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 325 330 335 Glu Val Gln Phe Asn Trp Tyr Val Val Asp Gly Val Glu Val His Asn 340 345 350 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Thr Tyr Arg 355 360 365 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 385 390 395 400 Glu Lys Thr Ile Ser Lys Ala Ala Lys Gly Gln Pro Arg Glu Pro Gln 405 410 415 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Thr Lys Asn Gln 420 425 430 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 435 440 445 Val Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 465 470 475 480 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Glu Gly Asn Val Phe 485 490 495 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 500 505 510 Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu Val Val 515 520 525 Val Gly Gly Val Leu Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 530 535 540 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Arg Ser Arg Gly Gly His 545 550 555 560 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Arg 565 570 575 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 580 585 590 Ser Arg Val Lys Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 595 600 605 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Glu Leu Asn Leu Gly Arg Arg 610 615 620 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Pro Glu 625 630 635 640 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 645 650 655 Glu Leu Gln Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670 Met Lys Gly Glu Arg Arg Arg Gly Gly Lys Gly His Asp Gly Leu Tyr 675 680 685 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Ala Leu His 690 695 700 Met Gln Ala Leu Pro Pro Arg 705 71� <210> 2 <211> 2034 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide sequence <400> 2 atgctgctgc tggtgaccag cctgctgctg tgtgagctgc cccaccccgc ctttctgctg 60 atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120 gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180 aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240 It should be noted that there is a character "�" in the original text at line 21 which might be an encoding issue or an incorrect character. 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If this is not what you intended, please check and correct the original text.cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300 gagcaggagg acatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360 ggcggcggaa caaagctgga gatcaccggc agcacctccg gcagcggcaa gcctggcagc 420 ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaga gcggccctgg cctggtggcc 480 cccagccaga gcctgagcgt gacctgtacc gtgtccggcg tgtccctgcc cgactacggc 540 gtgtcctgga tccggcagcc ccctaggaag ggcctggagt ggctgggcgt gatctggggc 600 agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660 agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720 tactgtgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780 accagcgtga ccgtgtccag cgagagcaag tacggccctc cctgcccccc ttgccctgcc 840 cccgagttcc tgggcggacc cagcgtgttc ctgttcccc ccaagcccaa ggacaccctg 900 atgatcagcc ggacccccga ggtgacctgt gtggtggtgg acgtgtccca ggaggacccc 960 gaggtccagt tcaactggta cgtggacggc gtggaggtgc aaacgccaa gaccaagccc 1020 cgggaggagc agttcaatag cacctaccgg gtggtgtccg tgctgaccgt gctgcaccag 1080 gactggctga acggcaagga atacaagtgt aaggtgtcca acaagggcct gcccagcagc 1140 atcgagaaaa ccatcagcaa ggccaagggc cagcctcggg agccccaggt gtacaccctg 1200 ccccctagcc aaggaggat gaccaagaat caggtgtccc tgacctgcct ggtgaagggc 1260 1320 aagaccacccc cccctgtgct ggacagcgac ggcagcttct tcctgtacag caggctgacc 1380 gtggcaaga gccggtggca ggagggcaac gtctttagct gctccgtgat gcacgaggcc 1440 ctgcacaacc actacaccca gaagagcctg tccctgagcc tgggcaagat gttctgggtg 1500 ctggtcgtgg tgggtggcgt gctggcctgc tacagcctgc tggtgacagt ggccttcatc 1560 atcttttgg tgaggagcaa gcggagcaga ggcggccaca gcgactacat gaacatgacc 1620 ccccggaggc ctggccccac ccggaagcac taccagccct acgcccctcc caggacttc 1680 gccgcctacc ggagccgggt gaagttcagc cggagcgccg acgcccctgc ctaccagcag 1740 ggccagaacc agctgtacaa cgagctgaac ctgggccgga gggaggagta cgacgtgctg 1800 gacaagcgga gaggccggga ccctgagatg ggcggcaagc cccggagaaa gaaccctcag 1860 gagggcctgt ataacgaact gcagaaagac aagatggccg aggcctacag cgagatcggc 1920 atgaagggcg agcggcggag gggcaagggc cacgacggcc tgtaccaggg cctgagcacc 1980 gccaccaagg atacctacga cgccctgcac atgcaggccc tgccccccag atga 2034 <210> 3 <211> 4246 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide Sequence <400> 3 agttgaagtc ggaagtttac atacacttaa gttggagtca ttaaaactcg tttttcaact 60 actccacaaa tttcttgtta acaaacaata gttttggcaa gtcagttagg acatctactt 120 tgtgcatgac acaagtcatt tttccaacaa ttgtttacag acagattatt tcacttataa 180 ttcactgtat cacaattcca gtgggtcaga agtttacata cactaagttg actgtgcctt 240 taaacagctt ggaaaattcc agaaaatgat gtcatggctt tagaagcttc tgatagacta 300 attgacatca tttgagtcaa ttggaggtgt acctgtggat gtatttcaag gaattctgtg 360 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 420 aagcatcgag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 480 tgtgtgaatc gtaactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag 540 caaaataggc tgtccccagt gcaagtgcag gtgccagaac attctctat cgaaggatct 600 gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 660 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 720 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tggggagaa ccgtatataa 780 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga 840 agcttcgagg ggctcgcatc tctcttcac gcgcccgccg ccctacctga ggccgccatc 900 cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg 960 ccgtctaggt aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga 1020 gcctacctag actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg 1080 tctttgtttc gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctacct 1140 gagatcaccg gcgaaggagg cctatcatga agatctatcg attgtacagc tagccgccac 1200 catgctgctg ctggtgacca gcctgctgct gtgtgagctg ccccaccccg cctttctgct 1260 gatccccgac atccagatga cccagaccac ctccagcctg agcgccagcc tgggcgaccg 1320 ggtgaccatc agctgccggg ccagccagga catcagcaag tacctgaact ggtatcagca 1380 gaagcccgac ggcaccgtca agctgctgat ctaccacacc agccggctgc acagcggcgt 1440 gcccagccgg tttagcggca gcggctccgg caccgactac agcctgacca tctccaacct 1500 ggagcaggag gacatcgcca cctacttttg ccagcagggc aacacactgc cctacacctt 1560 tggcggcgga acaaagctgg agatcaccgg cagcacctcc ggcagcggca agcctggcag 1620 cggcgagggc agcaccaagg gcgaggtgaa gctgcaggag agcggccctg gcctggtggc 1680 ccccagccag agcctgagcg tgacctgtac cgtgtccggc gtgtccctgc ccgactacgg 1740 cgtgtcctgg atccggcagc cccctaggaa gggcctggag tggctgggcg tgatctgggg cagcgagacc acctactaca acagcgccct gaagagccgg ctgaccatca tcaaggacaa cagcaagagc caggtgttcc tgaagatgaa cagcctgcag accgacgaca ccgccatcta ctactgtgcc aagcactact actacggcgg cagctacgcc atggactact ggggccaggg caccagcgtg accgtgtcca gcgagagcaa gtacggccct ccctgccccc cttgccctgc ccccgagttc ctggggcggac ccgcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc cggaccccg aggtgacctg tgtggtggtg gacgtgtccc aggaggaccc 2160 cgaggtccag ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc ccgggaggag cagttcaata gcacctaccg ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg aacggcaagg aatacaagtg taaggtgtcc aacaagggcc tgcccagcag catcgagaaa accatcagca aggccaaggg ccagcctcgg gagccccagg tgtacaccct gcccctagc caagaggaga tgaccaagaa tcaggtgtcc ctgacctgcc tggtgaaggg 2460 cttctacccc agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta 2520 caagaccacc ccccctgtgc tggacagcga cggcagcttc ttcctgtaca gcaggctgac 2580 cgtggacaag agccggtggc aggagggcaa cgtctttagc tgctccgtga tgcacgaggc 2640 cctgcacaac cactacaccc agaagagcct gtccctgagc ctgggcaaga tgttctgggt 2700 gctggtcgtg gtgggtggcg tgctggcctg ctacagcctg ctggtgacag tggccttcat 2760 catcttttgg gtgaggagca agcggagcag aggcggccac agcgactaca tgaacatgac 2820 cccccggagg cctggcccca cccggaagca ctaccagccc tacgcccctc ccagggactt 2880 cgccgcctac cggagccggg tgaagttcag ccggagcgcc gacgcccctg cctaccagca 2940 gggccagaac cagctgtaca acgagctgaa cctgggccgg agggaggagt acgacgtgct 3000 ggacaagcgg agaggccggg accctgagat gggcggcaag ccccggagaa agaaccctca 3060 ggagggcctg tataacgaac tgcagaaaga caagatggcc gaggcctaca gcgagatcgg 3120 catgaagggc gagcggcgga ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac 3180 cgccaccaag gatacctacg acgccctgca catgcaggcc ctgcccccca gatgactacg 3240 acccgggtga tcagcgggat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc 3300 ctccccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 3360 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctgggg gtggggtggg 3420 gcaggcagc aaggggagg attgggaaga aatagcagg catgctgggg atgcggtggg 3480 ctctatgggt acccaggtgc tgaagaattg acccggttcc tcctgggcca gaagaagca 3540 ggcacatccc cttctctgtg acacccctg tccacgcccc tggttcttag ttccagcccc 3600 actcatagga cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact 3660 tggagcggtc tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg 3720 aagaaattaa agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt 3780 gagagaataa tgagagaaat catagaatta tcgggccgct gcattctagt tgtggtttgt 3840 ccaaactcat caatgtatct tatcatgtct ggatcccatc acaaagctct gacctcaatc 3900 ctatagaaag gaggaatgag ccaaaattca cccaacttat tgtgggaagc ttgtggaagg 3960 ctactcgaaa tgtttgaccc aagttaaaca atttaaaggc aatgctacca aatactaatt 4020 gagtgtatgt taacttctga cccactggga atgtgatgaa agaaataaaa gctgaaatga 4080 atcattctct ctactattat tctgatattt cacattctta aaataaagtg gtgatcctaa 4140 ctgaccttaa gacagggaat ctttactcgg attaaatgtc aggaattgtg aaaaagtgag 4200 tttaaatgta tttggctaag gtgtatgtaa acttccgact tcaact 4246 <210> 4 <211> 680 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 4 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Ala Gln Val Lys Leu Gln Gln Ser Gly 20 25 30 Pro Asp Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 35 40 45 Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser 50 55 60 His Gly Lys Ser Leu Glu Trp Ile Gly Arg Val Asn Pro Asn Ser Gly 65 70 75 80 Gly Thr Ser Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Val 85 90 95 Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser 100 105 110 Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Lys Gly Asn Tyr Phe 115 120 125 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155 160 Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Leu Gly Gln 165 170 175 Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser His Gly 180 185 190 Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys 195 200 205 Phe Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg 210 215 220 Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn Pro 225 230 235 240 Val Glu Thr Asp Asp Val Ala Ile Tyr Tyr Cys Gln Gln Ser Asn Glu 245 250 255 Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Glu Ser 260 265 270 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 275 280 285 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 290 295 300 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 305 310 315 320 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 335 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 370 375 380 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 385 390 395 400 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 405 410 415 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 420 425 430 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 450 455 460 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 465 470 475 480 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 485 490 495 Leu Gly Lys Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 500 505 510 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 515 520 525 Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro 530 535 540 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 545 550 555 560 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 565 570 575 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 580 585 590 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 595 600 605 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 610 615 620 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 625 630 635 640 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 645 650 655 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 660 665 670 His Met Gln Ala Leu Pro Pro Arg 675 680 <210> 5 <211> 4255 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide sequence <400> 5 agttgaagtc ggaagtttac atacacttaa gttggagtca ttaaaactcg tttttcaact 60 actccacaaa tttcttgtta acaaacaata gttttggcaa gtcagttagg acatctactt 120 tgtgcatgac acaagtcatt tttccaacaa ttgtttacag acagattatt tcacttataa 180 ttcactgtat cacaattcca gtgggtcaga agtttacata cactaagttg actgtgcctt 240 taaacagctt ggaaaattcc agaaaatgat gtcatggctt tagaagcttc tgatagacta 300 attgacatca tttgagtcaa ttggaggtgt acctgtggat gtatttcaag gaattctgtg 360 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 420 aagcatcgag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 480 tgtgtgaatc gtaactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag 540 caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctat cgaaggatct 600 gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 660 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 720 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 780 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga 840 agcttcgagg ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc 900 cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg 960 ccgtctaggt aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga 1020 gcctacctag actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg 1080 tctttgtttc gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctacct 1140 gagatcaccg gcgaaggagg cctatcatga agatctatcg attgtacagc tagccgccac 1200 catgctgctg ctggtgacca gcctgctgct gtgtgagctg ccccaccccg cctttctgct 1260 gatccccatg gcccaggtga agctgcagca gagcggccct gatctggtga agcctggcgc 1320 cagcgtgaag atcagctgca aggccagcgg ctacagcttc accggctact acatgcactg 1380 ggtgaaacag agccacggca agagcctgga atggatcggc agagtgaacc ccaatagcgg 1440 cggcaccagc tacaaccaga agttcaagga caaggccatc ctgaccgtgg acaagagcag 1500 cagcaccgcc tacatggaac tgcggagcct gaccagcgag gacagcgccg tgtactactg 1560 cgcccggtcc aagggcaact acttctacgc catggactac tggggccagg gcaccaccgt 1620 gaccgtgtct agcagcggcg gaggaagcgg agggggagga tctggcggag gcggcagcga 1680 tatcgagctg acccagagcc ctagcagcct ggccgtgtca ctgggccaga gagccaccat 1740 cagctgcaga gcctccgaga gcgtggatag ccacggcacc agcctgatgc actggtatca 1800 gcagaagccc ggccagcccc ccaagttcct gatctaccgg gccagcaacc tggaaagcgg 1860 catccccgcc agattttccg gcagcggcag cagaaccgac ttcaccctga ccatcaaccc 1920 cgtggagaca gacgacgtgg ccatctacta ctgccagcag agcaacgagg accctcccac 1980 ctttggcgga ggcaccaagc tggaactgaa ggagagcaag tacggccctc cctgcccccc 2040 ttgccctgcc cccgagttcc tgggcggacc cagcgtgttc ctgttcccc ccaagcccaa 2100 ggacaccctg atgatcagcc ggacccccga ggtgacctgt gtggtggtgg acgtgtccca 2160 ggaggacccc gaggtccagt tcaactggta cgtggacggc gtggaggtgc aaacgccaa 2220 gaccaagccc cgggaggagc agttcaatag cacctaccgg gtggtgtccg tgctgaccgt 2280 gctgcaccag gactggctga acggcaagga atacaagtgt aaggtgtcca acaagggcct 2340 gcccagcagc atcgagaaaa ccatcagcaa ggccaagggc cagcctcggg agccccaggt 2400 gtacaccctg ccccctagcc aagaggagat gaccaagaat caggtgtccc tgacctgcct 2460 ggtgaagggc ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga 2520 gaacactac aagaccacccc cccctgtgct ggacagcgac ggcagcttct tcctgtacag 2580 caggctgacc gtggcaaga gccggtggca ggagggcaac gtctttagct gctccgtgat 2640 gcacgaggcc ctgcacaacc actacaccca gaagagcctg tccctgagcc tgggcaagat 2700 gttctgggtg ctggtcgtgg tgggtggcgt gctggcctgc tacagcctgc tggtgacagt 2760 ggccttcatc atcttttggg tgaggagcaa gcggagcaga ggcggccaca gcgactacat 2820 gaacatgacc ccccggaggc ctggccccac ccggaagcac taccagccct acgcccctcc 2880 cagggacttc gccgcctacc ggagccgggt gaagttcagc cggagcgccg acgcccctgc 2940 ctaccagcag ggccagaacc agctgtacaa cgagctgaac ctgggccgga gggaggagta 3000 cgacgtgctg gacaagcgga gaggccggga ccctgagatg ggcggcaagc cccggagaaa 3060 gaaccctcag gagggcctgt ataacgaact gcagaaagac aagatggccg aggcctacag 3120 cgagatcggc atgaagggcg agcggcggag gggcaagggc cacgacggcc tgtaccaggg 3180 cctgagcacc gccaccaagg atacctacga cgccctgcac atgcaggccc tgccccccag 3240 atgactacga cccgggtgat cagcgggatc tgctgtgcct tctagttgcc agccatctgt 3300 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 3360 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 3420 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 3480 tgcggtgggc tctatgggta cccaggtgct gagaattga cccggttcct cctgggccag 3540 aaagaagcag gcacatcccc ttctgtga cacaccctgt cacagccct gttcttagt 3600 tccagcccca ctcataggac actcatagct caggagggct ccgccttca tcccaccccc 3660 taaagtactt ggagcggtct ctccctccct catcagccca ccaaccaa cctagcctcc 3720 aagagtggga agaatttaaa gcaagatagg ctattaagtg cagagggaga gaaatgcct 3780 ccacatgtg aggagtaat gagagaatc attagaatt cggggccgctg cattctagtt 3840 gtggtttgtc caaccatc atgtatctt atcatgtctg gatcccatca caagctctg 3900 acctcaatcc tatagaagg aggaatgagc caaattcac ccaacttatt gtgggaagct 3960 tgtggaaggc tactcgaat gtttgaccca agttaaacaa tttaaaggca atgctaccaa 4020 atactaattg agtgtatgtt aacttctgac ccactgggaa tgtgatgaaa gaaayaaag 4080 ctgaaatgaa tcattctctc tactattatt ctgatatttc acatcttta aaaagtgg 4140 tgatcctaac tgaccttaag acaggaatc tttactcgga ttaatgtca ggaattgtga 4200 aaaagtgagt ttaaatgtat ttggctaagg tgtatgtaaa cttccgactt caact 4255 <210> 6 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 6 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp 20 25 30 Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp 35 40 45 Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu 50 55 60 Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr 65 70 75 80 Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly 85 90 95 Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys 100 105 110 Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe 115 120 125 Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Ile Thr 145 150 155 160 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 165 170 175 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 180 185 190 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 195 200 205 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 210 215 220 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr 225 230 235 240 Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu 245 250 255 Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 260 265 270 Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr 275 280 285 Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser 290 295 300 Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln 305 310 315 320 Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 325 330 335 Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu 340 345 350 Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu 355 360 365 Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg 370 375 380 Asp Glu Asp Leu Glu Asn Cys Ser His His Leu Ser Arg Met Asp Tyr 385 390 395 400 Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr 405 410 415 Lys Asp Asp Asp Asp Lys 420 <210> 7 <211> 3499 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide sequence <400> 7 agttgaagtc ggaagtttac atacacttaa gttggagtca ttaaaactcg tttttcaact 60 actccacaaa tttcttgtta acaaacaata gttttggcaa gtcagttagg acatctactt 120 tgtgcatgac acaagtcatt tttccaacaa ttgtttacag acagattatt tcacttataa 180 ttcactgtat cacaattcca gtgggtcaga agtttacata cactaagttg actgtgcctt 240 taaacagctt ggaaaattcc agaaaatgat gtcatggctt tagaagcttc tgatagacta 300 attgacatca tttgagtcaa ttggaggtgt acctgtggat gtatttcaag gaattctgtg 360 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 420 aagcatcgag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 480 tgtgtgaatc gtaactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag 540 caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctat cgaaggatct 600 gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 660 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 720 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 780 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga 840 agcttcgagg ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc 900 cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg 960 ccgtctaggt aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga 1020 gcctacctag actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg 1080 tctttgtttc gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctacct 1140 gagatcaccg gcgaaggagg cctatcatga agatctatcg attgtacagc tagccgccac 1200 catggattgg acctggattc tgtttctggt ggccgctgcc acaagagtgc acagcaactg 1260 ggtgaatgtg atcagcgacc tgaagaagat cgaggatctg atccagagca tgcacattga 1320 tgccaccctg tacacagaat ctgatgtgca ccctagctgt aaagtgaccg ccatgaagtg 1380 ttttctgctg gagctgcagg tgatttctt ggaagcgga gatgctcta tccacgacac 1440 agtggagaat ctgatcatcc tggccaacaa tagctgagc agcaatggca atgtgacaga 1500 gtctggctgt aaggagtgtg aggagctgga ggagagaac atcaggagt ttctgcagag 1560 ctttgtgcac atcgtgcaga tgttcatca tacaagctct ggcggaggat ctggaggagg 1620 cggatctgga gggaggca gtggaggcgg aggatctggc ggaggatctc tgcagattac 1680 atgccctcct ccaatgtctg tggagcacgc cgatatttgg gtgaagtcct acagcctgta 1740 cagcagagag agatacatct gcacagcgg ctttagaga aaggccggca cctcttctct 1800 gagagagtgc gtgctgaata aggccacaa tgtggcccac tggacacac ctagcctgaa 1860 gtgcattaga gatcctgccc tggtccacca gaggcctgcc cctcatcta cagtgacac 1920 agccggagtg acacctcagc ctgaatctct gagccctct ggaaagaac ctgccgccag 1980 ctctcctagc tctataata ccgccgccac aacagccgcc attgtgcctg gatctcagct 2040 gatgcctagc aagtctccta gcacaggcac aacagagatc agcagccacg aatctctca 2100 cggaacacct tctcagacca ccgccaagaa ttgggagctg acagcctctg cctctcacca 2160 gcctccagga gtgtatcctc agggccactc tgatacaaca gtggccatca gcacatctac 2220 agtgctgctg tgtggactgt ctgccgtgtc tctgctggcc tgttacctga agtctagaca 2280 gacacctcct ctggcctctg tggagatgga ggccatggaa gccctgcctg tgacatgggg 2340 aacaagcagc agagatgagg acctggagaa ttgttctcac cacctgtcgc gaatggacta 2400 caaggacgat gacgacaagg attataaaga tgatgatgat aaagattata aagacgacga 2460 tgataagtcg cgatgatgat gactcgagac tagtcccggg tgatcagcgg gatctgctgt 2520 gccttttagt tgccagccat ctgttgtttg cccctcccc gtgcccttcct tgaccctgga 2580 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 2640 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2700 agacaatagc aggcatgctg gggatgcggt gggctctatg ggtacccagg tgctgaagaa 2760 ttgacccggt tcctcctggg ccagaaagaa gcaggcacat ccccttctct gtgacacacc 2820 ctgtccacgc ccctggttct tagttccagc cccactcata ggacactcat agctcaggag 2880 ggctccgcct tcaatcccac ccgctaagt acttggagcg gtctctccct ccctcatcag 2940 cccaccaac caacctagc ctccaagagt gggaagaat taaagcaaga taggtatta 3000 agtgcagagg gagagaaaat gcctccaaca tgtgaggaag taatgagaga atcatagaa 3060 ttatcgggcc gctgcattct agttgtggtt tgtccaact catcaatgta tcttatcatg 3120 tctggatccc atcacaagc tctgacctca atcctataga aaggaggaat gagccaaaat 3180 tcacccact tattgtgga agcttgtgga aggctactcg aaatgtttga cccagttaa 3240 acaatttaaa ggcaatgcta ccaaatacta attgagtgta tgttaacttc tgacccactg 3300 ggaatgtgat gaagaaata aaagctgaaa tgaatcattc tcttactat tattctgata 3360 ttcacattc ttaaataaa gtggtgatcc taactgact taagacaggg aatctttact 3420 cggattaat gtcaggaatt gtgaaaaagt gagtttaaat gtatttggct aaggtgtag 3480 taaacttccg acttcaact 3499 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 8 cagcgacggc agcttctt 18 <210> 9 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 9 tgcatcacgg agctaaa 17 <210> 10 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 10 agagccggtg gcagg 15 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 11 atgggaaaat caaaagaaat c 21 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 12 ctagtatttg gtagcattgc 20 <210> 13 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 13 tctccagaac atcatccctg ccac 24 <210> 14 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 14 tgggccatga ggtccaccac cctg 24 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 15 gagggcaacg tctttagctg 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 16 gatgatgaag gccactgtca 20 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 17 agatgttctg ggtgctggtc 20

Claims

1. A method for producing an engineered cell, the method comprising: with (a) DNA encoding (i) a chimeric antigen receptor (CAR) and (ii) a membrane-bound fusion protein; and (b) electroporating the cell sample with DNA encoding a transposase, wherein the membrane-bound fusion protein comprises: (i) a first polypeptide consisting of amino acids 19-132 of SEQ ID NO:6; and (ii) a second polypeptide consisting of amino acids 159-395 of SEQ ID NO:

6.

2. The method of claim 1, further comprising culturing the engineered cell population ex vivo for no more than 2 days. 3 . The method of claim 1 , wherein the transposase is salmonid Tc1-like transposase (SB) or piggyBac transposase. The method of claim 3 , wherein the SB transposase is SB10, SB11 or SB100x transposase. The method of claim 3 , wherein the transposase is SB11 transposase.

6. The method of any one of claims 1-5, wherein the CAR is capable of binding to a cancer cell antigen.

7. The method of claim 6, wherein the cancer cell antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma-associated antigen, HER2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα chain, IL-11Rκ chain, IL-11Rλ chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, or HER1-HER2 in combination.

8. The method of claim 6, wherein the cancer cell antigen is CD19, ROR1, CD22, CD33, or mesothelin.

9. The method of claim 6, wherein the cancer cell antigen is CD19.

10. The method of claim 6, wherein the cancer cell antigen is ROR1.

11. The method of claim 6, wherein the cancer cell antigen is CD22.

12. The method of claim 6, wherein the cancer cell antigen is CD33.

13. The method of claim 6, wherein the cancer cell antigen is mesothelin.

14. The method of claim 1, wherein the cell sample is obtained by apheresis.

15. The method of claim 1, wherein the cell sample is a peripheral blood sample from a subject.

16. The method of claim 1, wherein the cell sample is an umbilical cord blood sample from the subject.

17. The method of claim 1, wherein the sample is a cryopreserved sample.

18. The method of claim 1, wherein the cell sample is a T cell, a T cell progenitor cell, or a NK cell. The method of claim 18 , wherein the cell sample is T cells or T cell progenitor cells.

20. The method of claim 18, wherein the cell sample is T cells.

21. The method of claim 19, wherein the T cells or T cell progenitors are allogeneic to the subject from which the T cells or T cell progenitors were obtained.

22. The method of any one of claims 18 to 21, wherein the expression of endogenous T cell receptor and / or endogenous HLA by the T cell or T cell progenitor is inactivated.

23. A method of producing an engineered cell population, comprising: The cell sample is transfected with a viral vector comprising DNA encoding a chimeric antigen receptor (CAR) and a membrane-bound fusion protein, wherein the membrane-bound fusion protein comprises a polypeptide consisting of amino acids 19-132 of SEQ ID NO:6; and a polypeptide consisting of amino acids 159-395 of SEQ ID NO:

6.

24. The method of claim 23, wherein the viral vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a lentiviral vector.

25. The method of claim 1, wherein the membrane-bound fusion protein comprises amino acids 1-395 of SEQ ID NO:

6.

26. The method of claim 1, wherein the membrane-bound fusion protein comprises amino acids 19-395 of SEQ ID NO:

6.

27. The method according to claim 1, wherein the first polypeptide and the second polypeptide are linked via a linker.

28. The method according to claim 27, wherein the linker is a flexible linker.

29. The method of claim 1, wherein the membrane-bound fusion protein comprises SEQ ID NO:

6.

30. The method of claim 1, wherein the membrane-bound fusion protein consists of SEQ ID NO:

6.

31. An engineered cell comprising (a) a CAR and (b) a membrane-bound fusion protein, wherein the membrane-bound fusion protein comprises a polypeptide consisting of amino acids 19-132 of SEQ ID NO:6; and a polypeptide consisting of amino acids 159-395 of SEQ ID NO:

6.

32. The engineered cell of claim 31, wherein the engineered cell further comprises a salmonid-type Tc1-like transposase (SB).

33. The engineered cell of claim 31 , wherein the SB transposase is SB10, SB11 or SB100x transposase.

34. The engineered cell of claim 31 , wherein the transposase is SB11 transposase.

35. The engineered cell of claim 31, wherein the engineered cell is a T cell, a T cell progenitor cell, or a NK cell.

36. The engineered cell of claim 35, wherein the engineered cell is a T cell or a T cell progenitor cell.

37. The cell of claim 35, wherein the engineered cell is a T cell.

38. The engineered cell of claim 31, wherein the artificial T cell receptor is targeted to a cancer cell antigen.

39. The cell of claim 38, wherein the cancer cell antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma-associated antigen, HER2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, or HER1-HER2 in combination.

40. The cell of claim 39, wherein the cancer cell antigen is CD19, ROR1, CD22, CD33, or mesothelin.

41. The cell of claim 39, wherein the cancer cell antigen is CD19.

42. The cell of claim 39, wherein the cancer cell antigen is ROR1.

43. The cell of claim 39, wherein the cancer cell antigen is CD22.

44. The cell of claim 39, wherein the cancer cell antigen is CD33.

45. The cell of claim 39, wherein the cancer cell antigen is mesothelin.

46. ​​The cell of claim 31 , wherein the membrane-bound fusion protein comprises amino acids 1-395 of SEQ ID NO:

6.

47. The cell of claim 31 , wherein the membrane-bound fusion protein comprises SEQ ID NO:

6.

48. The cell of claim 31 , wherein the membrane-bound fusion protein consists of SEQ ID NO:

6.

49. Use of the cell according to any one of claims 31 to 48 for the preparation of a medicament for treating cancer.

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