Constitutively active cytokine receptors for cell therapy

Abstract

To provide methods and compositions for enhancing expansion of immune cells for immunotherapy.SOLUTION: In particular embodiments, immune cells, such as T-cells, express a constitutively active cytokine receptor in which the transmembrane domain and the endo domain are able to provide an activating signal separately from any input to the corresponding exo domain to which they are operably linked. In specific embodiments, the transmembrane domain and the endo domain derived from IL-7Rα are utilized with the exo domain of CD34.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a joint venture with U.S. Provisional Patent Application No. 62 / 380,021, filed August 26, 2016. which is incorporated herein by reference in its entirety.

[0002] Embodiments of the present disclosure relate to at least cell biology, molecular biology, immunology, and at least Includes areas of medicine including cancer medicine. [Background technology]

[0003] Cellular therapy with antigen-specific T cells has shown promise in preclinical models and early clinical trials. Although it has been shown to be effective, few patients with significant disease have been cured. The lack of T cell proliferation is due to several factors, including limited in vivo T cell expansion after infusion. Lymphodepletion prior to T cell transfer with chemotherapy and / or radiation therapy Although patients greatly enhance T cell proliferation in vivo, these drugs are undesirable There is a need for a method to enable T cell proliferation without such toxic substances. It has been done.

[0004] The present disclosure provides methods and compositions for safely enhancing T cell proliferation in vivo. This invention meets a need in the art for Summary of the Invention [Problem to be solved by the invention]

[0005] Embodiments of the present disclosure include methods for expanding and / or proliferating immune cells, including in vivo after injection. In certain cases, the T cells are capable of reproducing certain constitutively. and expressing active cytokine receptor molecules and their use as treatments for medical conditions including cancer. Promotes in vivo T cell expansion for use in the treatment of cancer. Constitutively active cytokine receptors In certain embodiments, at least one endodomain, a transmembrane domain, and Contains at least one exodomain.

[0006] In a first aspect, provided herein is a method for detecting the binding of a cytokine. Engineered cytokine receptors that homodimerize and promote downstream signaling without the need for A common feature of such engineered cytokine receptors is that they In the absence of binding of the corresponding cytokine (e.g., to the transmembrane domain), homodimerization or and to contain one or more mutations that result in or enhance a signal. Thus, with regard to cytokine signaling, they are structurally It is genetically active (i.e., the mutation is a "gain-of-function" mutation).

[0007] In certain cases, the engineered cytokine receptor polypeptide comprises a transmembrane domain of the receptor. However, external signals (e.g., cytokines) activate the transmembrane and endodomains. One or more of the nucleotides that allow the receptor to homodimerize, so that the nucleotides are not required for homodimerization. In at least some cases, the transmembrane One or more mutations in the domains may bring the signal closer to the desired target molecule on which it acts. The transmembrane domain / endodomain provides a structural configuration that positions the molecule relative to the target domain. In certain instances, mutations in the transmembrane domain allow for the formation of disulfide bridges Transmembrane domain amines for binding to the polypeptide (thereby promoting homodimerization of the polypeptide) is or contains an insertion of at least one cysteine ​​in the amino acid sequence, and and / or the insertion of at least one proline to induce a conformational change ( For example, the molecule may be kinked, twisted, or rotated around an axis passing through the receptor molecule. Mutations in both of these have structural and chemical implications on the properties of the molecule and therefore its signaling. It has an impact on the

[0008] In certain embodiments, the transmembrane domain corresponds to a wild-type IL-7 cytokine receptor alpha IL-7 cytokine receptor alpha containing one or more mutations relative to the transmembrane domain In certain embodiments, the transmembrane domain is an IL-7 cytokine receptor. If it is from receptor alpha, the mutation is in the sequence LLTISILSFFSVALLVILACVLW (SEQ ID NO: 25). In certain aspects, the mutation introduces at least one cysteine ​​into the transmembrane domain, and and / or the mutation introduces a proline into the transmembrane domain. Therefore, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length.

[0009] In certain embodiments of the transmembrane domain, the domain self-dimerizes and in certain cases Thus, the transmembrane domain is an integral membrane domain of one or more cytokine receptor endodomains of the present disclosure. The transmembrane domain is a transmembrane domain or a self-activating derivative thereof. The transmembrane domain may contain one or more mutations compared to the wild-type transmembrane domain, Mutations in the ribosomal protein allow the receptor to homodimerize. In certain cases, the mutations The difference is that the receptor can homodimerize through its transmembrane and endodomain components. In certain embodiments, for example, a Janus kinase comprises an endodomain / polypeptide. The mutation or mutations are transmembrane and endonucleases so that the peptide can be phosphorylated and activated. The do domains may be structurally oriented (e.g., twisted). Alternatively, the multiple mutations may collectively organize the transmembrane domain and the endodomain in a helical fashion. This allows the wire to be twisted in an arbitrary direction.

[0010] In certain embodiments, the engineered cytokine receptor polypeptide comprises at least one An endodomain, at least one transmembrane domain, and at least one exodomain. In some embodiments, the nucleic acid comprises a nucleic acid sequence that is a nucleotide ... The endodomain is wild type, i.e., functional with respect to cytokine signaling. In a specific embodiment, the engineered cytokine receptor polypeptide is (wild type) exodomain, and in this embodiment the polypeptide corresponds to In another specific embodiment, the ribozyme homodimerizes in the presence or absence of ribozymes and transmits a signal. In this paper, we present a method for determining whether engineered cytokine receptor polypeptides can be used to identify wild-type cytokine receptors. In a more specific embodiment, the exodomain is a non-type exodomain. The main aim is to detect ligands that are normally detrimental to the cell in which the receptor resides (e.g., ligands that normally downregulate cells). downregulates the expression of cellular stimuli, induces cellular anergy, or induces cellular apoptosis. For example, such a non-wild-type exodomain binds to a ligand that induces cis-repression. For example, it may be a decoy receptor for a checkpoint protein, or a decoy For example, the exodomain can act as a receptor for the checkpoint protein PD In some cases, the non-wild-type exodomain comprises a receptor for exodomain 1. When bound to the polypeptide, the polypeptide destroys cells containing the engineered cytokine receptor polypeptide. In another particular embodiment, the engineered cytokine is a target for an antibody to target the cytokine. In certain embodiments, the receptor polypeptide lacks an exodomain. Interleukins (e.g., IL-7, IL-21, IL-23, or IL-12) be.

[0011] In certain aspects of the present disclosure, the constitutively active cytokine receptor is a constitutive IL-7 In certain embodiments, the IL-7Rα receptor is activated by a cleavage in the transmembrane domain of the IL-7Rα. One or more mutations or modifications that induce signal transduction in the homodimerized state of the receptor. In certain embodiments, the interleukin is IL-7. In the present invention, the engineered cytokine receptor polypeptide is an engineered IL-7 receptor polypeptide. It is a lipid peptide.

[0012] In other embodiments, a nucleic acid encoding such an engineered cytokine receptor polypeptide is provided. Polynucleotides which contain such polynucleotides, cells which contain such polypeptides, and cells expressing the chimeric antigen receptor (e.g., cells further expressing a chimeric antigen receptor), and diseases (e.g., For example, the use of such cells to treat cancer (e.g., hematological cancer or solid tumors) is disclosed herein. Provided in the specification.

[0013] In one embodiment, a polynucleotide encoding an engineered cytokine receptor polypeptide is and the polypeptide comprises the following operably linked components: a) one or more cytokine receptor endodomains; b) A transmembrane domain containing one or more mutations that promote homodimerization of the engineered receptor. Main; and c) the endogenous nature of the corresponding cytokine receptor endodomain or domains in component a) one or more extracellular domains that are not extracellular domains, In certain embodiments, such polynucleotides are not derived from the IL-1 receptor. There are one or more cells comprising

[0014] In certain embodiments, one or more cytokine receptor endodomains are Induce signal transduction through the STAT5 pathway or the STAT3 pathway in cells. In this case, the cytokine receptor endodomain is IL-7 receptor α, IL-21 receptor α, receptor α, CD122, IL-23 receptor α, IL-12 receptor α, or a combination thereof It comes from the word "wase."

[0015] In one embodiment, the extracellular domain is globular. The extracellular domain has signaling activity. The extracellular domain may be a decoy receptor lacking the extracellular domain. The extracellular domain may be a target for cytotoxic antibodies. In certain cases, the extracellular domain comprises at least 70 amino acids and / or at least 200 The length of the extracellular domain is 70-2000 amino acids, 100-1 000 amino acids, 500-2000 amino acids, 50-500 amino acids, 100-750 amino acids The amino acid sequence may be 200 to 2000 amino acids, or 500 to 2000 amino acids. In some cases, the extracellular domain is the extracellular domain of CD34. The extracellular domain is involved in the expression of CD30, HER2, EGFR, CD19, CD34, and TGF-β receptors. IL-4 receptor, IL-13 receptor α1 and α2, IL-8 receptor, IL-10 receptor Condition, PD-1, LAG3, TIGIT, CTLA4, FAS, CD19, CD27, C D28, CD52, CD134, CD137, HER2, EGFR, NGFR, or other It comes from a combination of these.

[0016] In certain embodiments, the cells expressing a constitutively active cytokine receptor are immune cells. The cells are T cells, NK cells, NKT cells, αβ cells, γδT cells, and mucosal associated immune cells. may be a myelin-associated T cell (MAIT T cell), an innate lymphoid cell, a stem cell, or a progenitor cell. In certain embodiments, the cells comprise a non-natural molecule that confers antigenic specificity to the cells. The cells express at least one additional engineered receptor, e.g., another constitutively active site. kine receptor, chimeric antigen receptor, recombinant T cell receptor, bispecific T cell receptor (BiTA) TE or T cell ENG), dual affinity retargeting (DART) proteins, or a combination thereof.

[0017] In some embodiments involving cells expressing constitutively active cytokine receptors, In one embodiment, the cell comprises a polynucleotide that expresses a receptor polypeptide. The peptide may be further provided as an expression vector, such as a viral or non-viral vector. Viral vectors can be defined as follows: retrovirus, lentivirus, adenovirus, The vector may be a viral vector, such as a virion vector, a serotype vector, or an adenovirus vector. For example, it may be a plasmid.

[0018] In one embodiment, there is a plurality of cells of the present disclosure that express a constitutively active cytokine receptor. The cells are T cells, NK cells, NKT cells, αβT cells, γδT cells, and mucosal-associated invariant MAIT T cells, innate lymphoid cells, stem cells, progenitor cells, or immune cells The antibody may comprise a mixture of one or more of the following immune effector cells:

[0019] In one embodiment, expressing a polynucleotide in a cell encompassed by the present disclosure. There are several methods to expand cell populations, including in vitro or in vivo (e.g., a mammal, including a human). In certain embodiments, the human may include cells Upon binding to the ectodomain, one or more inhibitors of the ectodomain target the cell for destruction. An effective amount of the inhibitor is provided. The inhibitor can be one or more antibodies of any type. In certain embodiments, the human is in need of treatment and a therapeutically effective amount of cells is provided to the human. In certain aspects, the human has cancer, an infectious disease, an autoimmune disease, an immune deficiency, or , or before or after solid organ or stem cell transplantation.

[0020] In certain embodiments, the constitutively active cytokine receptor is a constitutively active cytokine. by binding one or more molecules that are detrimental to cells expressing synaptic receptors. The exodomain is used as a ligand trap or a ligand-trap. The markers are those that normally activate signaling pathways to shut down T cells, for example in the case of immunosuppression. In some such cases, the exodomain may be immunosuppressive. Although the cytoplasmic domain binds harmful ligands as a decoy receptor, the transmembrane / endodomain is still independent. In some embodiments, normal As in other environments, decoy receptors prevent their corresponding ligands from suppressing T cells. The exodomain of the decoy receptor can, for example, bind to an inhibitory cytokine. Examples of harmful ligands that the carp receptor can bind include TGF-β, PD-L1, IL-4, In some embodiments, the decoy receptor includes IL-13, IL-8, and IL-10. The receptor exodomain is expressed by T cells, including LAG3, TIGIT, CTLA4, and FAS. It is one of the normally expressed inhibitory receptors.

[0021] The above is a summary of the features and advantages of the present invention so that the following detailed description of the invention may be better understood. The technical advantages have been outlined rather broadly. Additional features and advantages are described below. The concepts and specific embodiments disclosed are in accordance with the present invention. It may be readily utilized as a basis for modifying or designing other structures to carry out the same purpose. It should be understood by those skilled in the art that the present invention may be modified in accordance with the principles set forth in the appended claims. Such equivalent constructions do not depart from the spirit and scope of the invention as defined above. It should be recognized that the novel features believed characteristic of the present invention are in accordance with its construction and method of operation. Both the invention and the related art, together with further objects and advantages thereof, when considered in conjunction with the accompanying drawings. The present invention will be better understood from the following description. However, each of the drawings is by way of example and not of limitation. It is expressly intended that these figures are provided for illustrative purposes only and are not intended as a definition of the limits of the invention. This should be clearly understood. [Brief description of the drawings]

[0022] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0023] [Figure 1] Scheme of normal IL-7 cytokine / receptor signaling.

[0024] [Diagram 2] Scheme of signal transduction in the IL7RP2 receptor.

[0025] [Diagram 3] Scheme of Δ34 IL7RP2 protein signaling.

[0026] [Figure 4] Figures 4A and 4B: STAT5 is constitutively active in IL7RP2-transduced T cells. Figure 4A shows data from a representative donor, and Figure 4B shows the average results of three donors.

[0027] [Diagram 5] IL7RP2-transduced antigen-specific T cells have a higher proliferative capacity than unmodified antigen-specific T cells following antigen-specific stimulation.

[0028] [Figure 6] Figures 6A, 6B, and 6C: GD2.CAR T cells co-expressing Δ34.IL7RP2 have greater proliferation and antitumor effects than GD2.CAR T cells in vivo. GD2.CAR-Δ34.IL7RP2 T cells significantly proliferated in vivo and had a longer duration of T cells at the tumor site compared to GD2.CAR T cells (Figure 6A). LAN-1 tumors grew in mice administered GD2.CAR T cells, and tumors disappeared in GD2.CAR-Δ34.IL7RP2 T cells (Figure 6B). This resulted in a significant survival advantage for mice administered GD2.CAR-Δ34.IL7RP2 T cells (Figure 6C).

[0029] [Figure 7] Δ34.IL7RP2-transduced T cells have limited persistence in the absence of antigenic stimulation.

[0030] [Figure 8] Δ34.IL7RP2 prolongs the cytotoxic potential and proliferation of EphA2.CAR T cells.

[0031] [Figure 9] Δ34.IL7RP2 enhances the anti-glioma activity of EphA2.CAR T cells in vivo.

[0032] [Figure 10] Figures 10A, 10B and 10C: The Δ34.IL7RP2 protein can be fused to the IL-21Rα cytoplasmic domain to generate a constitutively active combinatorial cytokine receptor. Schematic of activation of Δ34.IL7RP2-linker-IL21Rα (Figure 10A). Constitutive activation of STAT5 by Δ34.IL7RP2 and Δ34.IL7RP2-linker-IL21Rα T cells (Figure 10B), and constitutive activation of STAT3 by Δ34.IL7RP2-linker-IL21Rα alone (Figure 10C).

[0033] [Figure 11] IL7RP2 enhances antigen-mediated proliferation of NK cells.

[0034] [Figure 12] Illustrative of one embodiment in which a constitutively active cytokine receptor is composed of two exodomains.

[0035] [Figure 13] Illustrated embodiment in which a constitutively active cytokine receptor utilizes the exodomain as a decoy receptor to bind a ligand that is harmful or potentially harmful to a cell expressing the receptor.

[0036] [Figure 14] 14A-14I: Constitutive signaling from C7R activates STAT5 in T cells but does not support autonomous cell proliferation. (14A) Schematic comparison of IL-7 bound to the native IL-7 receptor consisting of heterodimerized IL7Rα and γc compared to engineered C7R homodimerized receptor. (14B, 14C) Transduction efficiency of Δ34 and C7R (representing 3) in (14B) CD4 and (c) CD8 T cells versus non-transduced (NT) cells. (14D, 14E) Representative flow cytometry comparison of phosphorylated STAT5 (pSTAT5) in (14D) CD4 and (14E) CD8 T cells transduced with Δ34 or C7R. Cells were cultured without IL-15 and IL-7 for 24-72 hours prior to analysis. (14F, 14G) Mean pSTAT5 MFI values ​​when repeating the experiments in 14D and 14E with multiple donors. (14H,14I) Quantification of in vitro persistence of Δ34- or C7R-transduced (14H)CD4 or (14I)CD8 T cells cultured in complete cell culture medium without cytokines, starting 9-12 days after PBMC activation, without further antigenic stimulation. Viable cells were counted weekly using the trypan blue exclusion method. The x-axis indicates the number of days after IL-15 and IL-7 were removed from the culture medium. Area under the curve (AUC) values ​​were compared with two-tailed t-test: 10.5 ± 0.6616 (CD8 Δ34), 56.37 ± 7.972 (CD8 C7R), p<0.05; 10.22 ± 1.694 (CD4 Δ34) and 31.36 ± 2.590 (CD4 C7R), p<0.05. *P<0.05, **P<0.01, ***P<0.001 (two-tailed paired t-test, 14F-14I). Graphs 14F-14I represent the mean±SEM (n=3) from different donors.

[0037] [Figure 15]Figures 15A-15I: C7R enhances GD2-CAR T cell activity during continuous tumor challenge. (15A) Cytokines secreted by GD2-CAR T cells or GD2-CAR.C7R T cells after 24 hours of co-culture with LAN-1 tumor cells were determined by ELISA. (15B) 4-hour luciferase-based cytotoxicity assay of T cells killing LAN-1 tumor cells. (15C) Continuous co-culture schematic. The first co-culture (CC1) was initiated in the presence of 1x106 GD2-CAR or GD2-CAR.C7R T cells with 0.5x106 LAN-1 GFP-FFluc tumor cells for 7 days in the absence of IL-15 or IL-7. For the second and third co-cultures (CC2 and CC3), T cells were harvested from the previous co-culture and then re-seeded in new culture medium with fresh tumor cells at the same 2:1 E:T ratio. (15D) Cumulative proliferation of GD2-CAR or GD2-CAR.C7R T cells during serial co-culture. Arrows indicate the time points of T cell restimulation with tumor cells. (15E) LAN-1 tumor cells remaining after CC3 with GD2-CAR and GD2-CAR.C7R T cells, respectively. (15F, 15G) For proliferation analysis, GD2-CAR and GD2-CAR.C7R T cells collected at the end of CC1 were labeled with Cell Trace Violet before rechallenge during CC2. (15G) The experiment in F was repeated with multiple donors and mitotic indices were compiled from GD2-CAR and GD2-CAR.C7R proliferation histograms. (15H) For survival analysis, GD2-CAR and GD2-CAR.C7R T cells were stained with Annexin V and 7-AAD after two serial tumor challenges with LAN-1 tumor cells. Bar graphs show the frequency of T cell staining positive for Annexin V, 7-AAD, both, or neither. Mean comparisons for Annexin V(+)7-AAD(-) and Annexin V(-)7-AAD(+) were ns. After (15I)CC2 completion, tumors were labeled with GD2-specific antibody and magnetically separated from CAR T cells.Total RNA was isolated from T cells and then gene expression analysis was performed using Human Immunology Panel Version 2 and nCounter Analysis System (Nanostring). The displayed heatmap indicates genes with log2 fold change (GD2-CAR.C7R / GD2-CAR) with P value less than 0.02. Data were generated from 5 donors (10 paired samples). *P<0.05, **P<0.01 (2-tailed paired t-test, 15A, 15B, 15D, 15E, 15G, 15H). Graphs 15A, 15B, 15C, 15E, 15G, 15H represent the mean ± SEM (n=6, 15A, 15D, 15E; n=3, 15G, 15H) from different donors.

[0038] [Figure 16]Figures 16A-16G: C7R enhances adoptive T cell immunotherapy for metastatic and intracranial malignancies. (16A) and (16B) 1x106 CHLA-255 FFluc cells were injected intravenously into female NSG mice, followed 7 days later by 1x106 T cells expressing an irrelevant CAR, GD2-CARΔ.C7R, GD2-CAR, or GD2-CAR.C7R. (16A) Representative bioluminescence images of neuroblastoma growth over time. (16B) Kaplan-Meier survival analysis of CHLA-255 FFluc challenged mice. (16C,16D) To track T cell migration and persistence, in parallel studies, 1x106 CHLA-255 cells were injected intravenously into NSG mice, followed 7 days later by 1x106 GFP-FFluc T cells co-expressing GD2-CAR or GD2-CAR.C7R. (16C) Sequential bioluminescence imaging of T cells. (16D) Quantified bioluminescence signal of T cells over time. (16E) 1×105 U373 GFP-FFluc cells were injected intracranially into male SCID mice. Seven days later, 1×104 T cells expressing EphA2-CARΔ.C7R, EphA2-CAR.C7R, EphA2-CAR, or EphA2-CAR.C7R were injected intracranially into the tumor. Quantified U373 GFP-FFluc bioluminescence from each treatment group is shown over time. (16F) Kaplan-Meier survival analysis of U373 GFP-FFluc challenged mice after treatment with T cells. *P<0.05, **P<0.01 (Welch's t test, D; paired two-tailed t test, 16G). Five mice were used for all groups. Graph D represents the mean ± SEM from experimental replicates. In the experiment shown in D, one mouse in the GD2-CAR.C7R group died during imaging on day 5 for reasons unrelated to tumor burden, so the mean bioluminescence signal in GD2-CAR.C7R on days 9–16 is calculated from n = 4.

[0039] [Figure 17]Figures 17A-17D: C7R-CAR T cells can be deleted using an iC9 suicide switch. (17A) T cells double transduced with GD2-CAR.C7R and iC9-CD19t vectors were selected for iC9 expression using CD19-specific Miltenyi beads. Cells were then incubated with AP20187 in complete culture medium for 24 hours and then stained with Annexin V and 7-AAD. Bar graphs show the relative frequency of T cells staining positive for Annexin V, 7-AAD, both, or neither. Comparisons of Annexin V(+)7-AAD(-) and Annexin V(-)7-AAD(+) were ns. (17B, 17C) LAN-1 tumors were established subcutaneously in NSG mice 8 days after intravenous injection of 1×106 T cells transduced with GFP-FFluc and GD2-CARΔ.C7R, GD2-CAR.C7R, or GD2-CAR.C7R+iC9-CD19t. GD2-CARΔ.C7R was used as the same control in 17B and 17C. Tumor volumes were measured over time. Two mice in the GD2-CARΔ.C7R group were euthanized after 21 days due to tumor burden, and tumor sizes of the remaining three mice were compared to the GD2-CAR.C7R and GD2-CAR.C7R+iC9-CD19t groups on day 24. Mean tumor volumes 32 days after T cell injection: 236±11 mm3 for GD2-CAR.C7R and 196±18 mm3 for GD2-CAR.C7R+iC9-ΔCD19t, ns (p=0.1857). (17D) Bioluminescence signal of GD2-CAR.C7R T cells (with or without iC9-CD19T) from tumor sites was quantified over time. Red arrows indicate the start of AP20187 administration every 24 hours on day 28 for a total of three administrations. *P<0.05, **P<0.01 (two-tailed t-test, 17A, 17D; Welch's t-test, 17B, 17C). n=5 mice per group. Graph in 17A represents the mean±SEM (n=3) from different donors.

[0040] [Figure 18]Figures 18A-18D: CD34-IL7R* (C7R) is stably expressed in T cells and enhances constitutive STAT5 activation compared to IL7R*. (18A) Schematic of IL7R* fusion constructs and Δ34. All IL7R* constructs contain a modified IL-7Rα transmembrane domain (insertion of cysteine, proline, and threonine between Thr244 and Ile245 in IL-7Rα) and an unmodified IL-7Rα endodomain. The Δ34 construct contains the ectodomain and transmembrane of CD34 but retains only a portion of the endodomain. (18B) Transduced cells were stained with anti-CD34 antibody specific for QBEND10 (detecting the CD34 epitope used in Q8) to compare the transduction efficiency of the constructs in a compared to NT cells. Due to background expression of IL-7Rα on cultured NT cells, IL7R* transduction was indirectly detected by the Q8 tag in the bicistronic vector. Differences in transduction efficiency were ns when comparing Δ34 with IL7R* (p = 0.2951) and IL7R* with C7R (p = 0.1736). (18C) MFI of the different constructs were compared from B. MFI comparisons between Δ34 and IL7R* and between Δ34 and C7R were ns. (18D) Comparison of STAT5 phosphorylation by T cells was performed. Cells were cultured without IL-15 and IL-7 for 24-72 hours before analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (two-tailed t-test, 18B-18D). Graphs 18B-18D represent the mean (n=3) ± SEM from different donors.

[0041] [Figure 19] Figures 19A-19B: Efficient retroviral transduction of C7R and Δ34 in both CD4 and CD8 selected T cells. CD34 expression was assessed by flow cytometry to detect C7R and Δ34 transduction in (19A) CD4 and (19B) CD8 T cells compared to NT T cell controls. Differences were ns when compared with two-tailed t-tests (n=3).

[0042] [Figure 20] Figures 20A-20D: C7R signaling does not change the phenotypic composition of GD2-CAR T cells during culture. (20A) Scheme of GD2-CAR and GD2-CAR.C7R vectors. The GD2-CAR construct is composed of 14g2a scFv, CD8 extracellular spacer, CD8 transmembrane domain, and 41BB ζ endodomain followed by IRES and truncated NGFR. The GD2-CAR.C7R construct is identical except that the IRES and truncated NGFR are replaced with the 2A sequence followed by C7R. (20B, 20C) Comparison of memory phenotype of T cells expressing GD2-CAR or GD2-CAR.C7R. Bar graphs show the relative frequency of (20B) CD4 T cells and (20C) CD8 T cells that are CD45RO-CCR7- (terminal effector-like, TE), CD45RO+CCR7- (effector memory, EM), CD45RO+CCR7+ (central memory, CM), or CD45RO-CCR7+ (naive). Differences between GD2-CAR and GD2-CAR.C7R were ns for all four memory subpopulations of CD4 and CD8 T cells. (20D) CD4 and CD8 percentages were compared between GD2-CAR and GD2-CAR.C7R T cells. Differences were ns. Graphs 20B-20D represent the mean ± SEM (n=3) and populations were compared using a two-tailed t-test.

[0043] [Figure 21] Figures 21A-21B: C7R does not significantly increase intracranial EphA2-CAR T cell proliferation against U373 tumors. (21A,21B) To track T cells, 1x105 U373 cells were intracranially injected into SCID mice, and 7 days later, 1x104 T cells expressing EphA2-CAR or EphA2-CAR.C7R, co3 expressing GFP-FFluc were intracranially injected. (21A) Bioluminescence images were collected over time and quantified. (21B) AUC analysis was performed and found to be ns between both groups (two-tailed t-test). n=5 mice per group.

[0044] [Figure 22] Differential gene expression in GD2-CAR.C7R T cells compared to GD2-CAR T cells. After completion of CC2, tumors were labeled with GD2-specific antibodies and magnetically separated from CAR T cells. Total RNA was isolated from T cells, followed by gene expression analysis using Human Immunology Panel Version 2 and nCounter Analysis System (Nanostring). The displayed table shows the fold change of genes (GD2-CAR.C7R / GD2-CAR) with P-values ​​less than 0.02. Data generated from 5 donors (10 paired samples).

[0045] [Figure 23] Figures 23A-23B: Treatment of LCL tumors with 34.IL7RP2-EBVST. In Figure 23A, there is luciferase imaging in mice treated with 34.IL7RP2-EBVST. Figure 23B shows measurements of tumor growth in 34.IL7RP2-EBVST-treated mice.

[0046] The scope of the present application is to include any process, machine, manufacture, composition of matter, means, or methods described in the specification. and is not intended to be limited to the particular embodiments of the steps. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Following long-standing patent law practice, the terms "a" and "an" are used in conjunction with the claims. When used herein with the word "comprising," which includes "one or more Some embodiments of the present disclosure include one or more elements of the present disclosure, may consist of or consist essentially of method steps and / or methods Any of the methods or compositions described herein may be used in combination with any of the methods or compositions described herein. It is contemplated that the subject invention may be used in connection with any other method or composition.

[0048] As used herein, the term "cytokine" refers to the action of the immune system against inflammation and infection. These are cell signaling molecules that regulate responses and aid in intercellular communication in the immune response. Examples include: These include chemokines, interferons, interleukins, lymphokines, and tumor Necrotic factor is one example.

[0049] I. General embodiment

[0050] The present disclosure relates to methods of administering cellular immunotherapeutic agents to individuals in need thereof, including in vivo after delivery to such individuals. The present invention relates to an improvement of cellular immunotherapeutic agents for enhancing the proliferation of cytokines. at least one endodomain of a phosphodiesterase receptor, a transmembrane domain, and at least one Utilizing a constitutively active cytokine receptor composed of multiple exodomain-containing components However, in some cases, the exodomain is missing in the receptor. Constitutively active cytokine receptor is the endodomain of the IL-7 cytokine receptor α chain Antigens genetically engineered to express non-natural chimeric IL7R-containing receptors Specific T cells, in contrast to unmodified T cells, have been shown to be highly potent in cell culture studies and animal models. showed vigorous proliferation.

[0051] II. Constitutively active cytokine receptor molecules

[0052] In certain embodiments, constitutively active cytokine receptors (protein and / or nuclear In acid form), methods of making them, and methods of using them are encompassed by this disclosure. The receptor comprises at least one exodomain, a transmembrane domain, and at least It contains one endodomain, the endodomain is derived from a cytokine receptor, and the exodomain is The domain may be derived from the same or different molecule as the transmembrane and endodomain components. In the present study, the transmembrane domain and exodomain are derived from the same molecule, whereas the endodomain are derived from different molecules, but the molecules have transmembrane and / or endodomains that are twisted together. The receptor remains constitutively active because it contains a mutation that

[0053] In certain cases, constitutively active cytokine receptors are expressed in their transmembrane domains. and / or the endodomain components are linked to the exodomain to which they are operably linked. The activating signal is transmitted without receiving a corresponding signal from the In particular embodiments of the receptor, the receptor is constitutively active. There is no need for a ligand for the constitutively active cytokine of the present disclosure. The transmembrane domains and / or endodomains of the receptors may be expressed in a non-natural manner or In certain circumstances, the exodomain can homodimerize, thereby signaling It is possible for the activating signal to remain in a state where it can be transmitted to the corresponding entity downstream in the signaling pathway. In certain embodiments, the constitutively active cytokine receptor can be configured to The transmembrane / endodomains of the cytoplasm are bound to the exodomain to which they are operably linked. It independently confers a positive cytokine signal in the absence of cytokine binding.

[0054] In certain embodiments, certain constitutively active cytokine receptors are used in the methods of the present disclosure. It is used in.

[0055] The dCD34.IL7RP2 cDNA sequence is provided in SEQ ID NO:2, and the corresponding protein The gene sequence is provided in SEQ ID NO: 3. The construct dCD34.IL7RP2 contains the CD34 exon. the entire domain; a cysteine, proline, threonine (CPT) insertion; and normal IL7 R endodomain.

[0056] The IL7RP2 cDNA sequence is provided in SEQ ID NO:4, and the corresponding protein sequence is provided in SEQ ID NO:5. Construct IL7RP2 contains the IL7R exodomain; CPT insertion. a normal IL7R transmembrane domain; and a normal IL7R endodomain.

[0057] The Q8E.IL7RP2 cDNA sequence is provided in SEQ ID NO:6, and the corresponding protein sequence is The sequence is provided in SEQ ID NO: 7. Construct Q8E.IL7RP2 contains the CD34 extracellular domain plus a fragment of the CD8 stalk; IL7R transmembrane domain with a CPT insertion; and and a normal IL7R endodomain.

[0058] The 19AA.IL7RP2 cDNA sequence is provided in SEQ ID NO:8, and the corresponding protein The sequence is provided in SEQ ID NO: 9. The 19AA.IL7RP2 construct contains (IL7R transmembrane domain (derived from the last 19 amino acids in the IL7R ectodomain before the start of the main domain) 19 amino acid residues of the 7R exodomain;IL7R transmembrane domain with a CPT insertion; and the normal IL7R endodomain.

[0059] A. Exodomain In certain embodiments, one, two, or more constitutively active cytokine receptors are In certain embodiments, the exodomain binds to a ligand. However, the signal itself may not be transmitted, for example for structural or other reasons. An exodomain may, in certain embodiments, comprise its corresponding transmembrane and / or end domain. The end domain may or may not be derived from the same naturally occurring molecule as the end domain. The exodomain is derived from the same naturally occurring molecule as the endodomain to which it is operably linked. It may or may not be the case.

[0060] In some cases, the exodomain is the IL7 receptor exodomain and the CD34 exodomain. The globular morphology resembles the three-dimensional geometry of the domain. One feature that can be seen is that at least some of the data indicate that certain exodomains are highly Allows protein expression and concomitant high signal activation (e.g., pSTAT5 activation) This may be due to the fact that it provides protein stabilization.

[0061] In some cases, exodomains can stabilize proteins and increase signaling. Contains hyperglycosylation.

[0062] In some embodiments, the exodomain allows for identification of transduced cells. For example, if the exodomain is not normally expressed on T cells, the exodomain may For example, transduced cells can be identified by gating out during flow cytometry analysis. This allows cells to be identified and also, for example, magnetically selected for enrichment. (e.g., using magnetic beads coupled to the corresponding antibodies).

[0063] In some embodiments, a constitutively active cytokine receptor comprises a constitutively active site. A sink or ligator, such as the binding of one or more molecules that are detrimental to cells expressing kine receptors. In one embodiment, the ligand utilizes an exodomain that confers end-trapping function. For example, it normally activates signaling pathways to shut down T cells (immunosuppression), so it is immunosuppressive. In some such cases, the exodomain acts as a decoy receptor. Although it binds harmful ligands, the transmembrane / endodomain still independently binds to the positive site In certain embodiments, this is the case under normal circumstances. In this way, it prevents the ligand corresponding to the decoy receptor from suppressing T cells. In this case, the decoy receptor exodomain can bind to, for example, an inhibitory cytokine. Examples of harmful ligands include TGF-β, PD-L1, IL-4, IL-13, IL In addition, the decoy receptor exodomains include LAG3, The expression of inhibitory receptors normally expressed by T cells, such as TIGIT, CTLA4, and FAS, In certain embodiments, the receptor may encode a structural domain, but is not limited to a structural domain. They signal genetically and signaling is further increased in the presence of a specified trigger. In another embodiment, the exodomain is, for example, an IL-4 cytokine receptor or an IL- 13 Cytokine receptor-derived proteins, including those not derived from cytokine receptors In some cases, the exodomain may or may not comprise an antibody such as an scFv. It's okay.

[0064] In certain aspects, the exodomain is a target for the destruction of the cell. The xodomain directly or indirectly induces apoptosis in cells expressing the receptor. For example, the exodomain can be used as a target for a molecule. It can be targeted with a corresponding antibody to which it binds, resulting in the ultimate destruction of the cell.

[0065] Constitutively active receptors contain exodomains that act as decoy receptors for inhibitory ligands. In some cases, they also have a suicide targeting domain, but the decoy receptor exodomain and the suicide targeting domain are In some cases, the exodomain that targets killing is exactly the same.

[0066] In certain embodiments, the exodomain is selected from the group consisting of CD30, HER2, EGFR, CD19, C D34, CD34, TGF-β receptor, IL-4 receptor, IL-13 receptor α1 and α 2. IL-8 receptor, IL-10 receptor, PD-1, LAG3, TIGIT, CTLA4 , FAS, CD19, CD27, CD28, CD52, CD134, CD137, HER 2, EGFR, or NGFR extracellular domains. Clonal antibodies or derivatives thereof (e.g., but not limited to, scFV), or It may include a merization domain (for example, but not limited to, FKBP).

[0067] In certain cases, the exodomain is a podocalyxin (podocalyxin-like protein) PODXL (also known as PCLP1); Thrombocytopenic Glucocorticoids (PODXL) ;gp135;GCTM2;TRA-1-60;TRA-1-81;or endoglyca podocalyxin-like protein 2 (also known as PODXL2 or PCLP2) Includes.

[0068] In some embodiments, the exodomain component of the receptor is derived from a naturally occurring molecule. , wild-type, but in other cases the exodomain is different from the corresponding wild-type naturally occurring molecule. In comparison, one or more mutations may be included, for example, to further stabilize the receptor. The exodomain may contain one or more mutations compared to the corresponding wild-type sequence. If so, the mutant version has a sequence similar to that of the corresponding wild-type protein or nucleic acid sequence. For example, at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% It is one.

[0069] In some embodiments, the extracellular domain is of a certain length. The length of the extracellular domain is 10-275 amino acids, 10-225 amino acids, and 10-20 0 amino acids, 10-175 amino acids, 10-150 amino acids, 10-125 amino acids, 1 0-100 amino acids, 10-50 amino acids, 50-250 amino acids, 50-225 amino acids Acid, 50-200 amino acids, 50-175 amino acids, 50-150 amino acids, 50-10 0 amino acids, 90-250 amino acids, 90-225 amino acids, 90-200 amino acids, 9 0-175 amino acids, 90-150 amino acids, 90-125 amino acids, 90-100 amino acids 100-250 amino acids, 100-225 amino acids, 100-200 amino acids, 1 00-175 amino acids, 100-150 amino acids, 100-125 amino acids, 125-2 50 amino acids, 125-225 amino acids, 125-200 amino acids, 125-175 amino acids 125-150 amino acids, 150-250 amino acids, 150-225 amino acids, 1 50-200 amino acids, 150-175 amino acids, 175-250 amino acids, 175-2 25 amino acids, 175-200 amino acids, 200-250 amino acids, 200-225 amino acids It is an acid.

[0070] In an alternative embodiment, the receptor lacks the exodomain.

[0071] B. Transmembrane domain In certain embodiments, a constitutively active cytokine receptor comprises an exodomain and an end domain. The transmembrane domain comprises a transmembrane domain operably linked to a transmembrane domain. It may or may not be derived from the same naturally occurring molecule as the endodomain to which it is operably linked. In embodiments of the present invention, the transmembrane is the same naturally occurring molecule as the exodomain to which it is operably linked. The transmembrane domain requires a mutation that causes a twist in the molecule, In certain embodiments, the transmembrane domain is not naturally occurring. , compared to its corresponding wild-type molecule, allowing the membrane spanning to be autoactive. As used herein, the term "autoactivation" refers to the ability of a gene to produce an autoactivating enzyme. The exodomain to which it is operably linked, together with the endodomain to which it is operably linked. A transmembrane domain refers to a membrane-spanning domain that transmits a signal to a cell in the absence of a corresponding activating signal from the In certain embodiments, the transmembrane domain is one that causes or promotes homodimerization. This includes the above mutations.

[0072] In certain cases, the transmembrane domain confers a functional configuration to the receptor, allowing autoactivation. and allowing downstream endodomains to be aligned relative to one another in a manner that directs, for example, signal transduction. For example, Janus kinases associated with endodomains can interact with each other. In certain aspects, the transmembrane domains allow the proteins to interact with each other and cross-activate each other. The transmembrane and endodomains do not naturally function in a non-transient manner. The mutants contain one or more mutations that allow them to act together in a In some aspects, the mutation artificially homodimerizes the transmembrane and ectodomains. For example, the transmembrane domains each comprise a transmembrane domain and at least one or more that allow homodimerization of two separate molecules that both contain one endodomain The present invention may include mutations in the nucleotide sequence of ... In embodiments, one or more mutations in the transmembrane domain are, for example, a receptor homodimer. Induce structural twisting of the transmembrane and endodomains of the immersion complex to facilitate autoactivation. In certain embodiments, one or more mutations in the transmembrane domain form a helical structure. is the helical orientation of part or all of the receptor molecule (or one or more of its components) around a perpendicular axis. Specific mutations induce a circular twist in the transmembrane and endodomains that renders them self-active. Whether or not the induction of a structural configuration that activates the growth factor can be determined by conventional methods (e.g., the non-activation of growth factors). In the presence of STAT3 or ST, cells bearing the particular receptor being tested are grown. This can be determined using a method that assays for AT5 phosphorylation.

[0073] In certain embodiments, a transmembrane domain having one or more such gain-of-function mutations Moieties may be used in the methods and compositions of the disclosure. Mutations include, for example, substitutions, insertions, In certain embodiments, one or more mutations may be present, such as a deletion, a deletion, or a combination thereof. The differences include the inclusion of at least one cysteine ​​and / or at least one proline. In some cases, the transmembrane domain is modified by a gain-of-function mutation in the transmembrane domain. In at least some cases, the present invention utilizes mutations identified in tumor patients to The mutant version contains cystadenomas that induce disulfide bond formation in the transmembrane domain. However, in other embodiments, the one or more mutations include a cysteine ​​insertion. For example, a mutation may be present that results in a loss of a transmembrane domain compared to the native version of the transmembrane domain. To conformationally change and thereby enable induction of signal transduction, It still transmits a signal and does not have the cysteine ​​insertion that is constitutively active (thus For example, a transmembrane derivative (without disulfide bonds) can be used. The insertion of an amino acid such as phosphorylation creates a "kink" in the transmembrane domain, which Induce null transmission (e.g., Shochat et al., 2011, J Exp Med. 2011 May 9;208(5):901-8; See Zenatti et al., 2011, Nat Genet. 2011 Sep 4;43(10):932-9.

[0074] In certain embodiments, the transmembrane domain is derived from the IL-7Rα receptor, and the transmembrane domain The mutation in is in the sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 1). In the present specification, the mutation is one or more cysteines and / or one The mutation is or contains an insertion of one or more prolines into the receptor. In some cases, the mutations are cysteine, proline, The insertion of a trimeric peptide of threonine (CPT) into the transmembrane domain. The mutations are -S at cysteine ​​residues in two molecules (e.g., the two IL7RP2 receptor α chains). It allows disulfide bond formation between H (thiol) groups to form homodimers between them. (In certain embodiments, the proline immediately following the cysteine ​​aligns the homodimer in the correct orientation. In certain embodiments, the threonine in the CPT insertion is not a threonine but an alternative and, at least in certain cases, the other amino acid is cysteine ​​or proline. or not cysteine ​​or proline.

[0075] In an embodiment in which one or more amino acids are inserted into SEQ ID NO:1 for use in a receptor, In certain embodiments, the insertion may be between any two amino acids of SEQ ID NO:1. The insertion is at the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth position of the sequence number. , 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th , after the 21st, 22nd, 23rd or 24th amino acid.

[0076] Used in the exemplary construct described herein (Δ34.IL7RP2) An example of a mutated TM sequence (where the sequence is mutated by the addition of the underlined sequence below) is It is as follows. PILL CPT ISILSFFSVALLVILACVLW (SEQ ID NO:2) Mutated TM sequences in other constitutively active IL-7 receptors. 1. PIL NPC LTISILSFFSVALLVIL ACVLW (SEQ ID NO:3) 2.P T.C. LTISILSFFSVALLVILACVLW (SEQ ID NO: 4) 3.P SANCGAISILSFFSVAL LVILACVLW (SEQ ID NO:5) 4. PILL VSCP TISILSFFSVALLVILACVLW (SEQ ID NO:6) 5. PILL I ISI QW LSFFSVALLVILACVLW (SEQ ID NO:7)6. NSPSC LTISILSFFSVALLVILACVLW (SEQ ID NO:8) 7.P C L E.G. LTISILSFFSVALLVILACVLW (SEQ ID NO: 9) 8. PILLTISILSFF WN LLVILACVLW (SEQ ID NO: 10) 9. R.F.C.P.H. ISILSFFSVALLVILACVLW (SEQ ID NO:11) 10. IKC ILSFFSVALLVILACVLW (SEQ ID NO:1 2) 11. PI FHPFNCGP ISILSFFSVALLVILACVLW (SEQ ID NO: 13) 12. PILL MCP TISILSFFSVALLVILA CVLW (SEQ ID NO: 14) 13. PILLTISILSFFS GPSL ALLVILACVLW (SEQ ID NO: 15) 14. PIL RLGCV T.I. SILSFFSVALLVILACVLW (SEQ ID NO: 16) 15. PI PQGGC ILSFFSVALLVILACVLW (SEQ ID NO:17)16 . LQSC ILSFFSVALLVILACVLW (SEQ ID NO: 18) 17. PI FPHQHC TISILSFFSVALLVILACVLW (sequence number No. 19) 18. PILLTIS KCH LSFFSVALLVILACVLW (SEQ ID NO:20) 19. PILLT CHL ISILSFFSVALLVIL ACVLW (SEQ ID NO:21) 20. PI FSCGPLTISILSFFSVALLVILACVLW (SEQ ID NO:22) 21. PILL PPCL TISILSFFSVALLVILACVLW (SEQ ID NO:23) 22. PILLT PPVCSVT ISILSFFSVALLVILACVLW (sequence number No. 24)

[0077] In certain embodiments, one or more transmembrane domain mutations are in the dimerized alpha chain (e.g. The structure of the IL-7Rα was modified to bring the bound Janus kinase into close proximity. This structural modification orients the protein in a manner that allows for cross-phosphorylation and activation; In certain embodiments, this is due to a twist in the dimerization chain (Shochat et al., 2011; Durum, 2014).

[0078] In certain embodiments, a transmembrane domain has one or more amino acids different from its corresponding wild-type component. Contains a mutation and has a certain percent identity compared to its corresponding wild-type component In certain cases, the transmembrane domain has a similar affinity to the corresponding wild-type transmembrane domain. At least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94 , 95, 96, 97, 98, or 99% identical.

[0079] The transmembrane can be of any suitable length, but in certain embodiments, , 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length .

[0080] C. Endodomain Constitutively active cytokine receptors contain one or more endodomains. The domain may or may not be derived from the same molecule as the transmembrane domain. In one embodiment, the endodomain is a cytokine receptor, including an immunostimulatory cytokine receptor. In certain embodiments, the cytokine receptor is STAT5, STAT3 and the like. Immunostimulatory cytotoxicity factors useful for the receptors of the present disclosure include: Examples of tokine endodomains include IL-7Ra receptor α, CD122 (IL- IL-2 and IL-15 common receptor β), IL-21 receptor α, IL-23 receptor α, and and IL-12 receptor alpha, and IL-6 receptor.

[0081] In some embodiments, the endodomain is selected based on the desired downstream pathway. For example, the endodomains of JAK1, STAT5, STAT4, JAK3, and STAT3 are The desired signal to be transmitted over the network may be selected based on the desire for the signal to be transmitted over the network.

[0082] In some cases, the signal transduction pathway includes STAT5. STAT5 is an immunostimulatory site. It is a major downstream signaling node for the kinases IL-15 and IL-7, both of which are immune It is known that these antibodies are useful for activating T cells in therapeutic settings. The publication bypasses cytokine and cytokine receptor interactions and directly activates STAT5. Activation (with a constitutively active STAT5 mutant) enhances CD8 T cell function by Increased persistence in vivo and antitumor activity in preclinical models The same concept has already been demonstrated in the downstream of IL-12. Activates STAT proteins downstream of other known immune stimulatory cytokines, such as STAT4. This can be extended to sexualize.

[0083] In certain embodiments, the endodomain comprises an endodomain derived from the IL7 receptor alpha chain, It may or may not contain one or more mutations. In certain cases, it may A transmembrane domain containing a mutation that allows it to cause or promote homodimerization is operably linked to

[0084] In some embodiments, the endodomain is of a particular length. So, the length of the endodomain is 70 to 250 amino acids, 70 to 225 amino acids, and 70 to 200 amino acids, 70-175 amino acids, 70-150 amino acids, 70-125 amino acids , 70-100 amino acids, 80-250 amino acids, 80-225 amino acids, 80-200 Amino acids, 80-175 amino acids, 80-150 amino acids, 80-100 amino acids, 90 ~250 amino acids, 90~225 amino acids, 90~200 amino acids, 90~175 amino acids Acid, 90-150 amino acids, 90-125 amino acids, 90-100 amino acids, 100-2 50 amino acids, 100-225 amino acids, 100-200 amino acids, 100-175 amino acids 100-150 amino acids, 100-125 amino acids, 125-250 amino acids, 1 25-225 amino acids, 125-200 amino acids, 125-175 amino acids, 125-1 50 amino acids, 150-250 amino acids, 150-225 amino acids, 150-200 amino acids 150-175 amino acids, 175-250 amino acids, 175-225 amino acids, 1 75 to 200 amino acids, 200 to 250 amino acids, 200 to 225 amino acids, etc. In certain embodiments, these fragments of a particular length retain signaling activity. Hold.

[0085] III. Cells containing engineered cytokine receptors

[0086] Additionally, the pharmaceutical compositions of the present disclosure may be transformed or fused with a vector encoding the engineered receptor. A host cell expressing a constitutively active cytokine receptor, such as a host cell transfected with The host cell is envisioned to include a host cell. The host cell can be transformed with a vector encoding the receptor. The engineered receptor can be produced by introducing a nucleic acid molecule encoding the engineered receptor into a host cell. Alternatively, the vector may be integrated into the host genome or maintained extrachromosomally.

[0087] The host cell can be any prokaryotic or eukaryotic cell, but in certain embodiments is a eukaryotic cell. In certain embodiments, the host cell is a bacterial, insect, fungal, plant or animal cell. The host cell may be a mammalian cell, more preferably a human cell or a human cell line. Particularly preferred host cells are immune cells (e.g., T cells, NK cells, or includes NKT cells).

[0088] In one embodiment, the host cell is a T cell that contains an engineered cytokine receptor. In some cases, cells expressing engineered cytokine receptors may express engineered T cell receptors. Naturally occurring T cell receptors are divided into two subtypes: Each T cell contains two subunits (α and β subunits), It is a unique protein produced by a recombination event in the genome. The rallies can be screened for their selectivity for a particular target antigen. The engineered TCRs are then selected and cloned into populations of T cells to be used in adoptive immunotherapy. and / or subsequently introduced target antigens, In some cases, it refers to a TCR that expresses an engineered cytokine receptor. The cells that express chimeric antigen receptors (CARs) In contrast to engineered TCRs, CARs express MHC-independent In certain embodiments, the CAR is engineered to bind to a target antigen. However, extracellular binding domains including antibodies or antigen-binding fragments thereof, such as scFvs a transmembrane domain; one or more intracellular costimulatory signaling domains and a primary signal transduction domain Includes reachable domains.

[0089] Therapeutic Uses of Host T Cells Expressing the IV Receptor

[0090] The methods of treating cancer provided herein include administering to a subject an effective amount of a T cell expressing a cancer-specific CAR. and administering the CAR T cells to an individual having said cancer, wherein said CAR T cells are constitutively active. In a more specific embodiment, the cancer further expresses a cytokine receptor. In another specific embodiment, the CAR T cells target the antigen GD2. In a more specific embodiment, the constitutively active cytokine receptor is an IL-7 receptor. The transmembrane domain is derived from the cytokine receptor homodimer. Contains one or more mutations that promote somatization.

[0091] In certain methods of treating cancer, the cells are provided with a transmembrane domain having one or more mutations. In certain embodiments, the T cell comprises a cytokine receptor having a transmembrane domain having the sequence In certain embodiments, the transmembrane domain in T cells for treatment includes a sequence of The gene includes one of the sequences shown in SEQ ID NO:2 to SEQ ID NO:24.

[0092] An effective amount of cells expressing a constitutively active cytokine receptor is administered to an individual in need thereof. In certain cases, an individual may have cancer, while in other cases the individual may have a non-cancerous disease. A therapeutically effective amount of cells is provided to an individual, and administered in one or more doses. In some cases, the cell therapy of the present disclosure may be provided to the individual at the same time. The therapeutic approach is provided to an individual in addition to one or more other types of treatment that may or may not be required.

[0093] For example, a patient with cancer, or a patient susceptible to cancer, or a patient suspected of having cancer may , can be treated as described herein. The cells may be administered to an individual and maintained for extended periods of time. One of a variety of administration routes may be utilized. In some embodiments, the genetically modified cells can be used to inhibit immune recognition. The compound is encapsulated in a pharmaceutical formulation and administered locally (eg, at the site of a tumor).

[0094] In various embodiments, the expression constructs, nucleic acid sequences, vectors, host cells and / or Pharmaceutical compositions containing them are useful for the prevention, treatment or amelioration of cancerous diseases (e.g., neoplastic diseases). In certain embodiments, pharmaceutical compositions comprising the cells of the present disclosure are used to treat, for example, solid tumors. The present invention may be particularly useful in preventing, ameliorating and / or treating cancer, including cancer involving tumors.

[0095] As used herein, "treatment" or "treating" refers to the alleviation of a disease or pathological condition. Any beneficial or desirable effect on a condition or pathology that is to be treated. Even a minimal decrease in one or more measurable markers of a disease or condition (e.g., cancer) Treatment may optionally include reducing or ameliorating the symptoms of a disease or condition, or preventing or alleviating the symptoms of a disease or condition. "Treatment" does not necessarily mean either the progression of a disease or condition, or the slowing of progression of a condition. It does not represent a complete eradication or cure of any of the associated symptoms.

[0096] As used herein, "prevent" and "prevented" and "preventing " and similar words are used to describe the prevention, inhibition, or occurrence or progression of a disease or condition (e.g., cancer). It also presents approaches to reduce the likelihood of recurrence and to prevent the onset or recurrence of a disease or condition. refers to delaying the onset or recurrence of symptoms of a disease or condition As used herein, "prevention" and similar words also refer to the prevention or amelioration of the onset of a disease or condition. Reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence Includes:

[0097] The methods and compositions described herein target a broad range of immune cells (e.g., αβ T cells, γδT cells, NK cells, NKT cells, mucosa-associated invariant T cells (MAIT T cells) ), innate lymphocytes, or a mixture thereof) Additionally, the receptors of the present invention may be expressed in stem and / or progenitor cells, These subsequently differentiate into the immune cells listed above. Furthermore, all of the immune cells listed above can be differentiated into the second Genetic modification (e.g., chimeric antigen receptor (CAR), bispecific T cell engager ( BiTE or T cell ENG), dual affinity retargeting (DART) proteins, or αβ T cell receptors), but are not limited to these. In certain embodiments, the cell is a CAR-expressing immune cell as defined above, or a tumor-expressing immune cell. Targeting antigen-specific immune cells (e.g., virus-specific T cells, or tumor antigen-specific In some cases, cells expressing constitutively active cytokine receptors also express CAR. CAR targets EphA2, EphA3, HER2 (ERBB2), GD2, and Glyp ican-3, 5T4, 8H9, α v β6 integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD 70, CD96, CD123, CD138, CD171, CEA, CLL-1, CSPG 4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, E RBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor α, G D2, GD3, HLA-AI MAGE A1, HLA-A2, IL13Ra2, KDR , Lambda, Lewis-Y, MCSP, mesothelin, Muc1, Muc16, NC AM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, RO R1, Sp17, TAG72, TEM8, Tn-O-glycopeptide, VEGRR2, cancer Fetal antigen, HMW-MAA, VEGF receptor, TSHR, CS-1, CMA, Tn A g, prostate-specific membrane antigen (PSMA), FLT3, CD44v6, KIT, interleukin Interleukin-11 receptor a (IL-11Ra), PRSS21, VEGFR2, CD24, blood Platelet-derived growth factor receptor-β (PDGFR-β), SSEA-4, ERBB2 (Her2 / neu), prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor , CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fuco sirGM1, sLe, GM3, TGS5, o-acetyl-GD2, folate receptor β, TEM 1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, U PK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2 , TARP, WT1, LAGE-la, MAGE-Al, legumain, HPV E6, E 7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, pr ostein, survivin and telomerase, PCTA-l / Galectin 8, MelanA / MART1, Ras mutants, hTERT, sarcoma translocation breakpoints, ML-IAP , ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor Condition, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS , SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE -1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GP C3, FCRL5, lGLL1, and / or fibronectin, tenascin, or in the extracellular matrix of tumors, such as oncofetal regions of necrotic areas and other tumor-associated antigens. Other exemplary antigens present in, or, for example, genomic analysis and / or differential expression of tumors. Actionable mutations such as those identified through the current study are present within the extracellular matrix of tumors. It may be against any tumor antigen, such as the exemplary antigens.

[0098] Also provided is a method of treating cancer in an individual, the cancer expressing a tumor antigen, comprising: (1) administering to the tumor (1) a chimeric antigen receptor that targets a tumor antigen; and (2) a constitutively active cytokine receptor. Provided herein are methods for administering to the individual immune cells expressing The constitutively active cytokine receptor is a constitutively active receptor as encompassed herein. In certain embodiments, the constitutively active site of the cytokine receptor may be any of the cytokine receptors. The interleukin-7 (IL-7) receptor endodomain and the cytokine Membrane-mediated homodimerization of cytokine receptors such that receptors are constitutively active In certain embodiments, the constitutively active cytokine receptor comprises an insulin receptor. Interleukin-21 (IL-21) receptor endodomain and cytokine receptors and a transmembrane domain that promotes homodimerization of the cytokine receptor so that it is functionally active. In certain embodiments, the constitutively active cytokine receptor is interleukin-2 3 (IL-23) receptor endodomain and the cytokine receptor as a constitutively active and a transmembrane domain that promotes homodimerization of cytokine receptors, such as In morphology, the constitutively active cytokine receptor is interleukin-12 (IL-12). The receptor endodomain and the cytokine receptor are constitutively active. and a transmembrane domain that promotes receptor homodimerization. In certain embodiments, the transmembrane The domain comprises any of SEQ ID NOs: 1 to 24. In this study, constitutively active cytokine receptors express the corresponding cytokine in their extracellular domains. It includes an extracellular domain that does not transmit a signal when bound. In this form, the constitutively active cytokine receptor contains the IL-7 receptor endodomain, The corresponding cytokine is IL-7. The genetically active cytokine receptors are expressed by the extracellular domain, which is derived from CD34. In another specific embodiment of any of the foregoing, a constitutively active cytokine receptor is The antibody comprises an extracellular domain that is an extracellular domain derived from PD-1 or B7.

[0099] In certain embodiments of any of the methods of treatment provided herein, the cancer is glioblastoma. In certain specific embodiments of the methods provided herein, the cancer is breast cancer, prostate cancer, , lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), brain cancer, colon cancer, head and neck cancer, skin cancer (e.g., melanoma), ovarian cancer, endometrial cancer, cervical cancer, kidney cancer, stomach cancer, small intestine cancer, liver cancer, Pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, or thyroid cancer.

[0100] In certain embodiments of any of the methods provided herein, the tumor antigen is GD2. In another specific embodiment of any of the methods provided herein, the tumor antigen is Eph In another specific embodiment of the methods provided herein, the tumor antigen is EphA2. A3, HER2(ERBB2), GD2, Glypican-3, 5T4, 8H9, αv β6 integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD96, CD123, CD1 38, CD171, CEA, CLL-1, CSPG4, EGFR, EGFRvIII, E GP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor α, GD2, GD3, HLA-AI MAG E A1, HLA-A2, IL13Ra2, KDR, Lambda, Lewis-Y, M CSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand , NY-ESO-1, PRAME, PSCA, PSC1, ROR1, Sp17, TAG7 2, TEM8, Tn-O-glycopeptide, VEGFR2, carcinoembryonic antigen, HMW-MA A, VEGF receptor, TSHR, CS-1, CMA, Tn Ag, prostate-specific membrane antigen ( PSMA), FLT3, CD44v6, KIT, interleukin-11 receptor a (IL- l lRa), PRSS21, VEGFR2, CD24, platelet-derived growth factor receptor -β (PDGFR-β), SSEA-4, ERBB2 (Her2 / neu), Prostar Ze, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2 , gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TE M7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, AL K, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVC R1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT 1. LAGE-la, MAGE-Al, legumain, HPV E6, E7, MAGE A l, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1 , MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, Vivin and telomerase, PCTA-l / Galectin 8, MelanA / M ART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG(TM PRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human Terrorism merase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp 70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL 5, or lGLL1.

[0101] In some embodiments, the constitutively active cytokine receptor expressing cells are non-cancer In addition, lymphocyte therapy is therapeutic for other diseases, including infectious diseases, autoimmune diseases, and solid tumors. Medical conditions involving other diseases, including but not limited to complications following organ and stem cell transplants In such cases, T cells may have enhanced expansion. It is desirable to have a constitutively active cytokine receptor molecule for cancer indications. The cells may be specifically modified to target non-malignant indications, although similar to or similar to those described above. In embodiments in which the individual has an autoimmune disease, for example, the cells may react with, for example, Using autoreactive T cells (as in OPEXA Therapeutics; The Woodlands, TX) They may be broadly specific for autoreactive T cells via their TCR.

[0102] In certain embodiments, the present disclosure is directed to a method for treating a variety of conditions, including but not limited to the following: Expression constructs, nucleic acid molecules, and / or vectors that can be administered either in a In at least some embodiments, the composition is administered together with a pharma- ceutically acceptable carrier or excipient. The present invention also contemplates cells having expression constructs, nucleic acid molecules, and / or vectors that can be administered to the subject. In certain embodiments, the nucleic acid molecule or vector is stably integrated into the genome of the cell prior to administration of the cell. In certain embodiments, the nucleic acid sequence may be specific to a particular cell or tissue, Viral vectors that persist in cells may be used. Suitable pharmaceutical carriers and excipients are described in The compositions prepared according to the present disclosure are well known in the art for the prevention and treatment of the above identified diseases. It may be used for the prevention or treatment or delay.

[0103] Further disclosed herein is a method for preventing, treating, or ameliorating cancerous (including tumor) diseases. and administering to a subject in need thereof constitutively active cytokine receptors, the proteins encoding them, to said subject. The method comprises administering to the subject an effective amount of cells comprising a nucleic acid sequence(s) encoding the nucleic acid sequence(s) of the present invention. As contemplated herein and / or produced by the methods contemplated herein The present invention relates to a method comprising administering

[0104] Exemplary possible indications for administration of compositions of engineered immune cells include cancerous diseases (breast cancer, prostate cancer, Adenocarcinoma, lung cancer, brain cancer, colon cancer, head and neck cancer, skin cancer, ovarian cancer, endometrial cancer, cervical cancer, kidney cancer Cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer and thyroid cancer Adenocarcinoma of the blood and T-cells, B-cells, NK-cells and myeloid cells or their precursors Exemplary indications for administration of the compositions of cells include, for example, receptor expression Cancerous diseases, including any malignant tumors whose cells express the antigen targeted by the current cells. In addition, it includes malignant tumors that aberrantly express other tumor antigens, which can also be targeted. Administration of the compositions of the present disclosure can be used, for example, to treat minimal residual disease, early stage cancer, advanced cancer, and / or The present invention is useful for treating all stages and types of cancer, including early stage, metastatic and / or refractory cancers.

[0105] The present disclosure relates to other compounds that act via immune cells, e.g., bispecific antibody constructs, targeting Further included are protocols for co-administration of the compounds of the present invention with toxins or other compounds. The clinical regimen for is intended to include co-administration of the drug simultaneously, prior to and / or after administration of other components. Specific combination therapies may include chemotherapy, radiation therapy, surgery, hormone therapy, or includes other types of immunotherapy.

[0106] Embodiments include one or more of the immune cells described herein, the nucleic acid sequences described herein, The present invention also relates to kits comprising the vectors described herein and / or the hosts described herein. In addition, the kits of the present disclosure may be administered alone or to an individual in need of a medical treatment or intervention. It is contemplated that the pharmaceutical compositions described herein above may be used in combination with additional agents that may be will be done.

[0107] The immune cells (e.g., T cells or NK cells) modified with the construct(s) are then The cells can be grown in culture under selective conditions (optionally only), and then Cells selected as having the construct are, for example, isolated from the host cell by infecting the construct (singular or amplify and further analyze using polymerase chain reaction to determine the presence of After modified host cells have been identified, they can then be They can be used in a variety of ways, e.g., expanded in culture or introduced into a host organism. do.

[0108] Depending on the nature of the cells, cells can be introduced into a host organism (e.g., a mammal) in a variety of ways. The cells may be introduced at the site of a tumor, but in an alternative embodiment, the cells are administered to the cancer. The number of cells used can be many. The situation, the purpose of the transfer, the life span of the cells, the protocol used, e.g., number of doses, the proliferation potential of the cells The amount of time that the cells can be cultured will depend on the strength of the culture, the stability of the recombinant construct, etc. The cells may be applied as a dispersion, generally The cells may be injected or delivered intravenously at or near the site of interest. The culture may be in a biologically acceptable medium.

[0109] Routes of administration include, for example, intravenous, subcutaneous, intraperitoneal, intramuscular, topical, or intradermal administration. In some cases, a specific dose of cells (e.g., 10 total cells) may be used. 4 ~10 11 of In a specific embodiment, 1×10 5cells / kg~1×10 9 Doses of cells / kg may be used. Specific doses include 1×10 6 cells / mL~1×10 8 Contains cells / mL.

[0110] Introduction of a vector does not necessarily result in integration in all cases. In some circumstances, transient maintenance of the introduced DNA may be sufficient. The cells are introduced into a host and then, after a period of time, the cells are transformed into homing cells at a particular site, e.g. It may have a short-term effect that may be turned on after the device has been powered on.

[0111] This system allows for the determination of the efficiency of expression, the activity of the expressed product, and the specific needs of the patient (which varies with time and environment). (both may vary), cell activity as a result of cell loss or expression activity of individual cells It should be understood that the results may be subject to variables such as the rate of loss. It is expected that each physician will monitor each patient for appropriate dosage for the individual patient. and such practices of monitoring patients are routine in the art.

[0112] V Vector General

[0113] The vectors of the present disclosure produce a constitutively active cytokine receptor and at least In some cases, it may be used for recombinant engineering to achieve expression.

[0114] The term "vector" refers to a vector in which a nucleic acid sequence is introduced into a cell where it can be replicated by the cell. The term "exogenous nucleic acid sequence" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted. The vector may be "endogenous," meaning that it is foreign to the cell into which it is introduced; is a position in the host cell nucleic acid where the sequence is homologous to a sequence in the cell, but where the sequence is not normally found Vectors include plasmids, cosmids, transposons, viruses, etc. (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g. For example, YACs. Those who are likely to be well equipped (e.g., Maniatis et al., 1988 and Ausubel et al., 1994 (both of which are incorporated herein by reference). (see section 1.1.2.2).

[0115] The term "expression vector" refers to any type of vector that contains a nucleic acid encoding an RNA that can be transcribed. In some cases, the RNA molecule is then transformed into a protein, polypeptide, or In other cases, these sequences are translated into, for example, antisense oligonucleotides or peptides. The expression vector is not translated in the production of a gene molecule or a ribozyme. Nucleic acid necessary for the transcription and optionally translation of an operably linked coding sequence in In addition to the control sequences that govern transcription and translation, Thus, vectors and expression vectors may serve other functions as well and may be used in the manufacture of nucleic acid sequences as described below. It may contain columns.

[0116] A "promoter" is a regulatory sequence that is a region of a nucleic acid sequence at which the initiation and rate of transcription are controlled. It is a sequence that requires RNA polymerase and Can contain genetic elements to which other regulatory proteins and molecules, such as transcription factors, can bind. "Operably located," "operably linked," "under control," and and "under transcriptional control" refers to a promoter that controls the initiation and / or regulation of transcription of that sequence. is in the correct functional location and / or orientation with respect to the nucleic acid sequence to control expression This means that...

[0117] A promoter generally contains a sequence that functions to position the start site of RNA synthesis. The best known example of this is the TATA box, but several mutants lacking the TATA box Some promoters, such as mammalian terminal deoxynucleotidyl transferase In the promoters of genes and in the promoters of SV40 late genes, above the start site itself Overlapping separate elements help to fix the start position. These regulate the frequency of transcription initiation. Typically, they occur 30–110 bp upstream of the initiation site. Although most promoters contain functional elements downstream of the initiation site, It has been shown that in order to place a coding sequence "under the control" of a promoter, a transcription lead is The 5' end of the transcription start site of the transcription frame is placed "downstream" (i.e., The "upstream" promoter stimulates transcription of DNA. and promotes expression of the encoded RNA.

[0118] The spacing between promoter elements is often flexible, resulting in elements that are easily spaced apart from one another. Promoter function is maintained when the promoter is inverted or moved relative to the Depending on the target, individual elements function either cooperatively or independently to activate transcription. A promoter may function to mediate transcriptional activation of a nucleic acid sequence. These may or may not be used in combination with an "enhancer," which refers to a gene-acting regulatory sequence.

[0119] A promoter is a 5'-non-coding region located upstream of a coding segment and / or exon. The nucleic acid sequence may be isolated by isolating the nucleic acid sequence. Such a promoter may be referred to as "endogenous." Similarly, an enhancer may be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence Alternatively, the coding nucleic acid segment can be coupled to a recombinant or heterologous promoter (which can be its natural (a promoter that is not normally associated with the nucleic acid sequence in its natural environment) Certain advantages may be obtained by using recombinant or heterologous enhancers. It also refers to enhancers that are not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may be promoters or enhancers of other genes, and any other viral or prokaryotic or eukaryotic promoter isolated therefrom. nucleotide sequences or enhancers, as well as "naturally occurring" i.e., sequences of different transcriptional regulatory regions. Promoters that do not contain differential elements and / or contain mutations that alter expression or enhancers.

[0120] Of course, the expression vector may be expressed in a variety of different ways depending on the organelle, cell type, tissue, organ, or organism selected for expression. A promoter and / or enhancer that effectively directs expression of a DNA segment in It is important to use a gene encoding a nucleotide sequence that is highly specific for the expression of a protein. Know the use of promoter, enhancer, and cell type combinations for See, for example, Sambrook et al., 1989, incorporated herein by reference. The promoters used are suitable for large-scale production of recombinant proteins and / or peptides. To direct high level expression of an introduced DNA segment, which is advantageous in Under the appropriate conditions, the promoter may be constitutive, tissue-specific, inducible, and / or useful. The motor can be heterologous or endogenous.

[0121] Characterize the identity of tissue-specific promoters or elements, as well as their activity Assays for detecting hypoxia-specific regulatory elements are well known to those of skill in the art. Environment-specific promoters or elements such as transcription factors are utilized. Specific and / or activation specific promoters can be used, e.g., activation activated T cell elements, NFAT (lineage restricted activation), early growth response genes (activation), Liver X receptor response element (activation), hypoxia response element (environment), etc. do.

[0122] A vector contains a multiple cloning site (mult), which is a nucleic acid region that contains multiple restriction enzyme sites. A vector may contain a multiple cloning site (MCS), any of which may be Restriction enzyme digestion may be used in conjunction with standard recombinant techniques to digest a nucleic acid sequence. refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at a specific location on the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those of skill in the art. Often, the vector is provided with a nucleic acid sequence that allows exogenous sequences to be ligated into the vector. To achieve this, the DNA is linearized or fragmented using a restriction enzyme that cuts within the MCS. "Ligation" refers to the ligation of two nucleic acid fragments, which may or may not be adjacent to each other. The process of forming phosphodiester bonds between fragments. Restriction enzymes and ligation Techniques involving the transcription reaction are well known to those skilled in the art of recombinant technology.

[0123] Splicing sites, termination signals, origins of replication, and selectable markers may also be used. do.

[0124] In certain embodiments, the plasmid vector is used to transform a host cell. Generally, a plasmid containing replicon and control sequences derived from a species compatible with the host cell is used. Plasmid vectors are used in combination with these hosts. Vectors are usually The recombinant vector has a gene encoding a genomic DNA sequence, and a marking sequence which is capable of providing phenotypic selection in transformed cells.

[0125] Additionally, phage vectors containing replicon and control sequences that are compatible with the host microorganism. can be used as a transformation vector in connection with these hosts. For example, λ phage GEMTM11 is used to transform host cells (e.g., E. coli LE392). It can be utilized in generating recombinant phage vectors which can be used.

[0126] The bacterial host cells, e.g., E. coli, containing the expression vector are cultured in any number of suitable media, e.g., L. The expression of a recombinant protein in a particular vector is understood by those of skill in the art. by contacting the host cell with an agent specific for a particular promoter, For example, by adding IPTG to the medium or by increasing the incubation time. This can be induced by switching to a lower temperature.

[0127] D. Viral Vectors Certain viruses infect cells through receptor-mediated endocytosis or enter cells. They invade the host cell and integrate into the host cell genome, transfecting the viral genome stably and efficiently. Their ability to express make them attractive candidates for introducing foreign nucleic acids into cells (e.g., mammalian cells). A component of the present disclosure may be a viral vector encoding heparanase. Non-limiting examples of viral vectors that can be used to deliver the nucleic acids of the present disclosure include: It is described in.

[0128] 1. Adenovirus Vector A particular method for delivery of nucleic acids involves the use of adenovirus expression vectors. Viral vectors are known to have a low ability to integrate into genomic DNA. However, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. An "adenovirus expression vector" is one that (a) supports packaging of a construct; and (b) ultimately expressing the tissue- or cell-specific construct cloned therein. The term "adenovirus construct" refers to a construct that contains sufficient adenovirus sequences to Knowledge of the genetic organization of adenoviruses, double-stranded DNA viruses, allows the identification of foreign sequences up to 7 kb. allows replacement of large pieces of adenovirus DNA by sequences (Grunhaus a nd Horwitz, 1992).

[0129] 2. AAV Vectors Nucleic acids can be introduced into cells using adenovirus-assisted transfection. Increased transfection efficiency in cell lines using the adenovirus conjugation system It has been reported (Kelleher and Vos, 1994; Cotten t al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is highly For use in cells of the present disclosure, the vectors have high frequency of integration and can infect non-dividing cells. and thus, for example, in tissue culture (Muzyczka, 1999). 92) or in vivo. AAV is useful for delivering genes to mammalian cells. , with a broad host range for infectivity ( Tratschin et al., 1984 ; Laughlin et al, 1986; Lebkowski et al, 19 88; McLaughlin et al., 1988). Generation and characterization of rAAV vectors For further details regarding use, see U.S. Patents 5,139,941 and 4,797,368. and, each of which is incorporated herein by reference.

[0130] 3. Retroviral Vectors Retroviruses integrate their genes into the host genome, transferring large amounts of foreign genetic material. It can infect a wide range of species and cell types and is packaged in specialized cell lines. These capabilities make them useful as delivery vectors (Miller, 1992).

[0131] To construct a heparanase retroviral vector, a nucleic acid (e.g., heparanase (a nucleic acid encoding part or all of the viral genome) in place of a specific viral sequence To produce virions, the gag and pol genes are inserted into the , and a package containing the env gene but without the LTRs and packaging components To construct a recombinant plasmid containing the cDNA, a recombinant cell line was constructed (Mann et al., 1983). The smid is coupled to the retroviral LTR and packaging sequences (e.g., calcium phosphate When introduced into a particular cell line (by precipitation with lysine), the packaging sequence is This allows the RNA transcripts of the mide to be packaged into viral particles, which then The virus particles are secreted into the culture medium (Nicolas and Rubenstein, 1999). 88; Temin, 1986; Mann et al., 1983). The medium containing the retroviral vector is collected, optionally concentrated, and used for gene transfer. Viral vectors are capable of infecting a wide variety of cell types. However, integration and stability are not possible. Effective expression requires the division of host cells (Paskind et al., 1975).

[0132] Lentiviruses are complex retroviruses that express the common retroviral gene gag , pol, and env, as well as other genes that have regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., Naldini et al., 1996 Zuffery et al., 1997; Blomer et al., 1997; U.S. Pat. No. 6,013 (See, e.g., 516 and 5,994,136.) Some examples of lentiviruses These include the human immunodeficiency viruses HIV-1, HIV-2 and the simian immunodeficiency virus SIV Lentiviral vectors are capable of attenuating HIV virulence genes multiple times. For example, the genes env, vif, vpr, vpu and nef are deleted, making the animal biosafe.

[0133] Recombinant lentiviral vectors can infect non-dividing cells and are capable of infecting cells in vivo and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, the packaging functions, namely gag, pol and env, and rev and A suitable host cell is transfected with two or more vectors carrying the tat gene and the tat gene. A recombinant lentivirus capable of infecting mitotic cells is described in U.S. Pat. No. 5,994,136. The envelope proteins and the specific cell type Recombinant viruses can be targeted by antibodies or specific ligands to target receptors. The sequence of interest (including the regulatory region) can be targeted to, for example, a particular target cell. It is inserted into a viral vector together with another gene encoding a ligand for the above receptor. This then makes the vector target specific.

[0134] 4. Other viral vectors Other viral vectors may be used as vaccine constructs in the present disclosure. Nia viruses (Ridgeway, 1988; Baichwal and Sugde n, 1986; Coupar et al, 1988), Sindbis virus, site Vectors derived from viruses such as megalovirus and herpes simplex virus are used They offer several attractive features to a variety of mammalian cells ( Friedmann, 1989; Ridgeway, 1988; Baichwal et al. and Sugden, 1986; Coupar et al., 1988; Horwich et al., 19 90).

[0135] E. Vector Delivery and Cell Transformation Suitable methods for nucleic acid delivery for the transfection or transformation of cells include: Such methods include, but are not limited to, ex vivo transfection. This includes direct delivery of DNA by transfection, injection, etc., using techniques known in the art. Through use, cells can be stably or transiently transformed.

[0136] F.Ex Vivo Conversion Transfect eukaryotic cells and tissues removed from organisms in an ex vivo environment Methods for inducing heparanase of the present disclosure are known to those of skill in the art. Or other nucleic acids can be used to extract and transfect ex vivo. In certain aspects, the transplanted cells or tissues are placed in an organism. In a preferred aspect, the nucleic acid is expressed in the transplanted cells.

[0137] VI Combination Therapy

[0138] In certain embodiments of the present disclosure, the methods of the present disclosure for clinical aspects (e.g., constitutive Constitutively active immune cells, such as immune cells (e.g., T cells) that express constitutively active cytokine receptors. Administration of the cytokine receptor to an individual having cells expressing the cytokine receptor may be used to treat a medical condition in the individual. and one or more other drugs (e.g., drugs for hyperproliferative disorders, including anti-cancer drugs) that are effective in the treatment of An "anti-cancer" agent may, for example, kill cancer cells, induce apoptosis in cancer cells, or Induce cytosis, reduce the rate of cancer cell proliferation, and reduce the incidence or number of metastases. reducing, decreasing tumor size, inhibiting tumor growth, tumor or cancer Reducing blood supply to cells and promoting an immune response against cancer cells or tumors , preventing or inhibiting the progression of cancer, or increasing the longevity of a subject with cancer. More generally, these and another composition are provided in a combined amount effective to kill or inhibit the proliferation of the cells. This process involves contacting cancer cells with an expression construct and a drug or multiple factors simultaneously. This may include treating the cells with a single composition or pharmacological formulation that includes both agents. contacting the cells with two different compositions or formulations, either simultaneously or by contacting the cells with two different compositions or formulations This can be accomplished by transfecting one composition with the expression construct and the other with the The article includes a second agent.

[0139] In embodiments of the disclosure in which an individual with cancer requires combination therapy, one of the following: One or more of the following may be provided to an individual in addition to the therapeutic cells of the present disclosure: chemotherapy or other drugs, t Pathogen-associated molecular patterns (PAMPs) such as oll-like receptor (TLR) ligands, immunotherapy chemotherapy, radiation therapy, surgery, hormone therapy, and combinations thereof. Immunotherapy is offered to individuals. When provided, the immunotherapy is a subset of constitutively active cytokine receptor expressing cells. In some cases, cells expressing constitutively active cytokine receptors may be used to treat tumors. Other receptors that themselves provide a treatment for an individual, such as binding to an antigen or viral antigen. Includes receptors or molecules.

[0140] Tumor cell resistance to chemotherapy and radiotherapy agents is a major problem in clinical oncology. One of the goals of current cancer research is to combine chemotherapy and radiation therapy with other therapies to Finding ways to improve the effectiveness of chemotherapy and radiation therapy by combining In the context of this disclosure, cell therapy includes other pro-apoptotic or cell cycle modulating agents. In addition to the therapeutic agent, it may also be used in conjunction with chemotherapy, radiotherapy, or immunotherapy. It is intended that this may be achieved.

[0141] The disclosed methods and compositions can be administered minutes to days prior to or after one or more other drug treatments. The intervals may range from several weeks. The other agent and the agent of the present disclosure may be administered separately to the individual. In certain embodiments, the method generally ensures that a significant period of time does not expire between the times of each delivery, As a result, the drugs and treatments of the invention may still exert a beneficial combined effect on the cells. In such cases, they are preferably within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other. It is believed that cells can be contacted in both ways within a short period of time. It may be desirable to significantly extend the period for administration of the (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) Pass.

[0142] It is anticipated that treatment cycles will be repeated as necessary. It is also contemplated that surgical intervention may be applied in combination with the cell therapy of the present invention.

[0143] Another treatment may be provided in conjunction with the cells of the present disclosure, and multiple administrations of one or both may be required. When stated, administration of the agents may be by different routes of administration and at different times.

[0144] A. Chemotherapy Cancer treatments also include a variety of combinations, for example, using both chemical and radiation-based therapies. Examples of chemotherapy that can be used with the therapeutic cells of the present disclosure include, for example, , Acivicin; Aclarubicin; Acodazole hydrochloride; Acronine; Adzelesin; Aldehyde threukin; altretamine; ambomycin; amethanthrone acetate; amsacrine; Nastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide dimesylate; Bizelesin; Bleomycin sulfate; Brequinarna Thorium; Bropirimine; Busulfan; Cactinomycin; Calsterone; Caracemide ;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzelesin; Cedefingol; Celecoxib (COX-2 inhibitor); Chlorambucil; Cilolemycin ;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytosine Rabin; Dacarbazine; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Dexphos Lumaplatin; Desaguanine; Desaguanine mesylate; Diaziquone; Docetaxel; Docetaxel Doxorubicin; Doxorubicin hydrochloride; Droloxifene; Droloxifene citrate; Do Lomostanolone propionate;Duazomycin;Edatrexate;Eflomitin salts Salt; Elsamitrucin; Enloplatin; Enpromate; Epipropidin; Epirubi esorubicin hydrochloride; elbrozole; esorubicin hydrochloride; estranustine; estramustine Sodium phosphate, estramustine. Sodium phosphate of estramustine. Ethanidazo etoposide; etoposide phosphate; etopine; fadrozole hydrochloride; faza Rabin; Fenretinide; Floxuridine; Fludarabine phosphate; Fluorouracil fluorocitabine; fosquidone; fostricin sodium; gemcitabine; gemcitabine Tabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Irumofosine ;Iproplatin;Irinotecan;Irinotecan hydrochloride;Lanreotide acetate;Letrozole ;Leuprolide acetate;Liarozole hydrochloride;Lometrexol sodium;Lomustine; Loxoxantrone hydrochloride; Mesoprocol; Maytansine; Mechlorethamine hydrochloride; Acetic acid Megestrol; Melengestrol acetate; Melphalan; Menogaril; Mercaptopril methotrexate; methotrexate sodium; metoprine; metuledep; mitigating Nectomycin; Mitocalcin; Mitochromin; Mitogillin; Mitomarcin; Mitomycin ;Mitospar;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegasparagase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobro Mann; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Pol Fimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Pi Puromycin; Puromycin hydrochloride; Pyrazofurin; Ribopurin; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Spa Rusomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptomycin Tonigrin;Streptozocin;Sulfofenur;Tallysomycin;Tecogalactanthate ;Taxotere;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Te Roxiron;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin; Tirapazamine; Toremifene citrate; Trestron acetate; Triciribine phosphatase Trimetrexate; Trimetrexate glucuronate; Triptorelin; Triptorelin hydrochloride ubrozole; uracil mustard; uredepa; vapreotide; verteporfin; sulfur Vinblastine acid; Vincristine sulfate; Vindesine; Vindesine sulfate; Binepidine sulfate ;vinorusinate sulfate;vinorelbine sulfate;vinorelbine tartrate;vinrocidine sulfate; Vinzolidine sulfate;Vorozole;Zeniplatin;Zinostatin;Zorubicin hydrochloride;20- Epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; acyl Larubicin; Acylfulvene; Adecipenol; Adzelesin; Aldesleukin; AL L-TK antagonists; altretamine; ambamustine; amidox; amifostine Aminolevulinic acid; Amrubicin; Amsacrine; Anagrelide; Anastrozole ;Andrographolide;Angiogenesis inhibitor;Antagonist D;Antagonist G;Antarellic Dorsal morphogenetic protein-1; Antiandrogen, prostate cancer; Antiestrogen; Anti Neoplaston;Antisense oligonucleotide;Aphidicolin glycinate;Apo apurinic acid;ara-C DP-DL-PTBA;Arginine deaminase;Aslaculin;Atamestane;Atri Mustine; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azaacet Lon; Azatoxin; Azatyrosine; Baccatin III derivatives; Balanol; Batimastat BCR / ABL antagonists; benzochlorines; benzoylstaurosporines; betalactones Tam derivatives; β-arretin; betaclamycin B; betulinic acid; bFGF inhibitors; bicalc Bisanthrene; Bisaziridinylspermine; Bisnafide; Bistren A; Bis Zelesin; Breflate; Bropirimine budotitanium; Buthionine sulfoximine; Calcium Potriol; Calphostin C; Camptothecin derivatives; Capecitabine; Carboxamide -Amino-triazole;Carboxyamidotriazole;CaRest M3;CAR N 700; Cartilage-derived inhibitor; Carzelesin; Casein kinase inhibitor (ICOS); Stanospermine; Cecropin B; Cetrorelix; Chlorin; Chloroquinoxalins Sulfonamide; Cicaprost; Cisporphyrin; Cladribine; Clomiphene analogues; Clotrimazole; Collismycin A; Collismycin B; Combretastatin A4; humbretastatin analogues; conagenin; clambecidin 816; crisnatol; crisnatol Cryptophycin 8; Cryptophycin A derivatives; Curacin A; Cyclopentaanthraquinone cycloplatam;cytarabine octophosphate;cytolytic factor;cytarabine tostatin; dacliximab; decitabine; dehydrogenase B; deslorelin; dextran; Samethasone; Dexphosphamide; Dexrazoxane; Dexverapamil; Diaziquo Didemnin B; Didox; Diethylnorspermine; Dihydro-5-azacytidine dihydrotaxol;9-dioxamycin;diphenylspiromustine;docetamine Cel; Docosanol; Dolasetron; Doxifluridine; Doxorubicin; Droloxif phenanthrene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelph Osin; Edrecolomab; Eflunitine; Elemene; Emiteful; Epirubicin; Epis teride; estramustine analogues; estrogen agonists; estrogen antagonists; etha Nidazole; Etoposide phosphate; Exemestane; Fadrozole; Fazarabine ;Fenretinide;Filgrastim;Finasteride;Flavopiridol;Freseras tin; fluasterone; fludarabine; fluorodaunorhysine hydrochloride; forphenime Formestane; Fostriecin; Fotemustine; Gadolinium texaphylline gallium nitrate;galocitabine;ganirelix;gelatinase inhibitors;gemcitabine; Glutathione inhibitors;Hepsulfame;Heregulin;Hexamethylenebisacetamide; Hypericin; Ibandronic acid; Idarubicin; Idoxifene; Idramanton; Irumo fosine; ilomastat; imatinib (e.g., GLEEVEC); imiquimod; immunostimulant Insulin-like growth factor-1 receptor inhibitor; Interferon agonist; Insulin Interferon; Interleukin; Iobenguane; Iododoxorubicin; Ipomoea Anol;4-Iropract;Irglagladine;Isobengazole;Isohomohalicon Dorin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N Triacetate Tate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolus tatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprorelin do+estrogen+progesterone; leuprorelin; levamisole; liarozole; Linear polyamine analogues; Lipophilic disaccharide peptides; Lipophilic platinum compounds; Lysoclinamide 7; Baplatin; Lonbricin; Lometrexol; Lonidamine; Rosoxantrone; Loxoxol Livin;Lurtotecan;Lutetium;Texaphyrin;Rhizophyllin;Lytic peptide;Ma Itansine; Mannostatin A; Marimastat; Masoprocol; Maspin; Matrila Isin inhibitors;Matrix metalloproteinase inhibitors;Menogaril;Marvalon;Me Tereline;Methioninase;Metoclopramide;MIF inhibitor;Mifepristone;Mir Tefosin; Millimostim; Mitoguazone; Mitolactol; Mitomycin analogues; Mitomycin Tonafide;Mitotoxin Fibroblast Growth Factor-Saporin;Mitoxantrone;Mopha Lotene; Molgramostim; Erbitux; Human chorionic gonadotropin; Monophosphorylation Lulipid A + Myobacterium cell wall sk; Mopidamol; Mustard anticancer drug; Mica Peroxide B; Mycobacterial cell wall extract; Myriaporone; N-acetylglutaminone ;N-substituted benzamides;Nafarelin;Naglestip;Naloxone + Pentazocine ;Napavine;Nafterpin;Nartograstim;Nedaplatin;Nemorubicin:Nelidro Nitric acid; Nilutamide; Nisamycin; Nitric oxide modulators; Nitroxide antioxidants ;Nitrulline;Oblimasen (GENASENSE®);O6-Benzylglucose Anin;Octreotide;Oxenon;Oligonucleotide;Onapristone;Ondan ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; Satelon; Oxaliplatin; Oxaunomycin; Paclitaxel; Paclitaxel derivatives analogues;paclitaxel derivatives;palauamine;palmitoyl rhizoxin;pamidronic acid; Panaxytriol;Panomyphen;Parabactin;Pazeliptin;Pegaspargase; Perdecin; Pentosan polysulfate sodium; Pentostatin; Pentrozole; Perf Lubron; Perfosfamide; Perillyl alcohol; Phenazinomycin; Phenylactate le;phosphatase inhibitors;picibanil;pilocarpine hydrochloride;pirarubicin;piritre Plasminogen activator inhibitors; Plasminogen complexes Platinum compounds;Platinum-triamine complexes;Porfimer sodium;Porfiromycin ;Prednisone;Propyl bis-acridone;Prostaglandin J2;Proteasome inhibitors;Protein A-based immunomodulators;Protein kinase C inhibitors;Microalgae;Ta Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; -Purines; Pyrazoloacridines; Pyridoxylated hemoglobin polyoxyethylene conjugates ;raf antagonist;raltitrexed;ramosetron;ras farnesyl protein tra Ras inhibitor; ras-GAP inhibitor; demethylated reteliptin ;Rhenium Re186 etidronate;Rhizoxin;Ribozyme;RII retinamide;Ro Hitsucine; Romurtide; Roxinimex; Rubidinone B1; Ruboxyl; Safingo le;Saintpin;SarCNU;Sarcophytol A;Sargramostim;Sdi1mi metic; semustine; senescence induction inhibitor 1; sense oligonucleotide; signal transduction Inhibitors; Schizofuran; Sobuzoxane; Sodium borocaptate; Sodium phenylacetate Mu;sol Verol; Somatomedin binding protein; Sonermin; Sparfosic acid; Spicamycin D;spiromustine;splenopentin;spongistatin 1;squalamine;stypia amide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal peptide antagonist Nist; Sradista; Suramin; Swainsonine; Talimustine; Tamoxifen Methionine Ojido; Tauromustine; Tazarotene; Tecogalan sodium; Tegafur; Terlapiri Lithium; Telomerase inhibitors; Temoporfin; Teniposide; Tetrachlorodecaoxide ;Tetrazomine;Thaliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin Mimetics; Thymalfasin; Thymopoietin receptor agonist; Thymotrin; Thyroid stimulator Hormones;Stannane ethyl etiopurpurine;Tirapazamine;Titanocene dichloride;Topsen tin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine ;Trimetrexate;Triptorelin;Tropisetron;Trosteride;Tyrosinequinone Tyrphostin; UBC inhibitor; Ubenimex; Urogenital growth inhibitor Urokinase receptor antagonist; Vapreotide; Variolin B; Veraresol; Veramine ;Verdin;Verteporfin;Vinorelbine;Vinxartin;Vitaxin;Borozo zinostatin; zinostatin stimalamer, or any analog or derivative variant of the foregoing.

[0145] B. Radiation therapy Other agents that cause DNA damage and have been used extensively include gamma radiation, X-rays, and These include what is commonly known as directed delivery of radioisotopes to tumor cells and / or tumor cells. Other types of DNA damaging agents, such as microwave and UV radiation, are also contemplated. Most likely, all of these factors are related to DNA, to the precursors of DNA, to D Widespread damage to NA replication and repair, and to chromosome assembly and maintenance The dose range for X-rays is 50 to 200 roentgens for a prolonged period (3 to 4 weeks). The range is from a daily dose of 1000 to a single dose of 2,000 to 6,000 roentgens. Dose ranges for vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, , and depends on uptake by the neoplastic cells.

[0146] The terms "contact" and "expose" as applied to cells include therapeutic agent constructs and chemotherapeutic agents. or a process in which the radiotherapeutic agent is delivered to or directly apposed to the target cell. The term is used herein to describe the process of achieving cell death or stasis. Both drugs combine to produce effective results in either killing cells or preventing them from dividing. The combined amount is delivered to the cells.

[0147] C. Immunotherapy In one embodiment, immunotherapy other than constitutively active cytokine receptor-specific expressing cells is Such treatments may be used in conjunction with the methods and compositions of the present disclosure. It's not necessary.

[0148] Immunotherapy generally involves the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can, for example, target certain markers on the surface of tumor cells. The antibody may be specific for the target protein. The antibody alone may act as an effector of therapy. Or they can recruit other cells to actually carry out cell killing. Substances or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, 100-day cough Toxins and the like) and simply serve as targeting agents. The effectors then bind surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include T cells, cytotoxic T cells, , NKT cells, NK cells, dendritic cells or macrophages.

[0149] Therefore, immunotherapy can be used as part of a combination therapy in conjunction with this cell therapy. General approaches for combination therapy are discussed below. In general, tumor cells Apoptosis is a process that is sensitive to the expression of certain markers that are not present in most other cells. There are many tumor markers, any of which may be used in the present disclosure. Common tumor markers include prostate specific antigen, urinary Medium tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMF G, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, lamini ERb receptor, erb B, p155, melanoma-associated antigen (MAGE), preferential onset of melanoma Present antigen (PRAME), survivin, CD19, CD20, CD22, kappa light chain, CD30 , CD33, CD123, CD38, ROR1, ErbB2, ErbB3 / 4, ErbB Dimer, EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TA G72, PSMA, NKG2D ligand, B7-H6, IL-13 receptor a2, MUC1 , MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y , CLL-1, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 N Y-ESO-1, PSCA, folate receptor-α, CD44v6, CD44v7 / 8, avb 6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2 D ligand, CD44v6, dual antigen, universal, EphA2, EphA3, HER 2(ERBB2), GD2, glypican-3, 5T4, 8H9, α v β6 integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6, CAIX, CA9, CD19, C D20, CD22, kappa light chain, CD44, CD44v6, CD44v7 / 8, CD70 , CD96, CD123, CD138, CD171, CEA, CLL-1, CSPG4, EGFR, EGFRvIII, EPCAM, ERBB3, ERBB4, FAP, FAR, FBP, fetal AchR, IL11Ra, KDR, Lambda, MCSP, NCAM, P SC1, PSMA, ROR1, Sp17, survivin, TAG72, TEM1, TEM8 , Tn-O-glycopeptide, VEGRR2, and HMW-MAA.

[0150] Another type of immunotherapy uses PAMPs, which are molecules that activate the innate immune system. The tumor can then be infused with the constitutively active cytokine receptor of the present invention. It recruits and activates adaptive immunity, including specific expressing cells.

[0151] D. Genes In yet another embodiment, the second line treatment is a therapeutic polynucleotide comprising It is a gene therapy administered before, after, or simultaneously with the induction of cell proliferation. Various expression products, including inhibitors or regulators of programmed cell death, are disclosed herein. is included in.

[0152] E.Surgery Nearly 60% of individuals with cancer undergo some type of surgical procedure, which may be preventative, Includes diagnostic or staging, curative and palliative surgical procedures. Curative surgery includes: The disclosed treatments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or or it is a cancer treatment that may be used in conjunction with other therapies, such as alternative therapies.

[0153] Curative surgical procedures include resection, in which all or part of the cancerous tissue is physically removed. Tumor resection refers to the removal, excision, and / or destruction of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, This disclosure includes methods for treating superficial cancers, pre-cancer, and osteoarthritis. It is further contemplated that such treatment may be used in conjunction with removal of normal tissue, or concomitant amounts of normal tissue.

[0154] Upon removal of any part of the cancerous cells, tissue or tumor, a cavity is created in the body. Treatment can include perfusion, direct injection or administration of additional anti-cancer therapy to the area. Such treatments can be achieved by topical application. , 5, 6, 7 days, or 1, 2, 3, 4, and 5 weeks, or 1, 2, 3, 4, These treatments may be repeated every 5, 6, 7, 8, 9, 10, 11 or 12 months. The treatments may similarly vary in dosage.

[0155] VII. Pharmaceutical Compositions

[0156] According to the present disclosure, the term "pharmaceutical composition" refers to a composition for administration to an individual. In certain aspects of the invention, the pharmaceutical composition comprises one or more constitutively active cytokine receptors. In a preferred embodiment, the pharmaceutical composition is administered parenterally, Compositions for percutaneous, intraluminal, intraarterial, intrathecal or intravenous administration, or for direct injection into the cancer In particular, the pharmaceutical compositions are intended to be administered to an individual by infusion or injection. It is envisaged that the administration of the suitable compositions may be by different methods, for example, intravenously, dermally, Administration can be by intravenous, intraperitoneal, intramuscular, topical or intradermal route.

[0157] The pharmaceutical compositions of the present disclosure may further comprise a pharma- ceutically acceptable carrier. Examples of such are well known in the art and include phosphate buffered saline, water, oil / water emulsions, etc. Compositions containing such carriers include emulsions, various types of wetting agents, sterilizing solutions, etc. The products can be formulated by well-known conventional methods. These pharmaceutical compositions are The compound can be administered to a subject in a suitable dose.

[0158] Dosing regimens will be determined by the attending physician and clinical factors. As such, the dosage for any one patient will vary depending on the patient's size, body surface area, age, and dosage. The specific compound being administered, the gender, time and route of administration, general health status, and any concurrent This depends on many factors, including other drugs being administered. The preferred dosage is 1×10 7 / m 2 ~1×10 10 / m 2 Progress should be monitored by regular evaluation. Constitutively active cytokine receptor-modified cells (e.g., T cells) can be injected intravenously. The dose may be 1×10 7 / m 2 ~1×10 10 / m 2 It is.

[0159] The compositions of the present disclosure may be administered locally or systemically. Administration is generally parenteral (e.g. DNA can also be delivered directly to a target site (e.g., an internal or external target site). by biolistic delivery to the site or by catheter to a site in the artery In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and even more preferably In a preferred embodiment, it is administered intravenously. An example of a preparation for parenteral administration is a sterile Examples of non-aqueous solvents include aqueous or non-aqueous solutions, suspensions, and emulsions. ethylene glycol, polyethylene glycol, vegetable oils such as olive oil, and oleic acid ethylene glycol Aqueous carriers include water, alcoholic / aqueous solvents, and injectable organic esters such as ethylhexyl. Parenteral vehicles include solutions, emulsions or suspensions, including saline and buffered media. Sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Intravenous vehicles include lactated Ringer's or fixed oils. These include dietary supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), etc. Preservatives, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like, may also be present. and other additives may be present. Additionally, the pharmaceutical compositions of the present disclosure may include a proteinaceous carrier (e.g., serum albumin or immunoglobulin, preferably of human origin) The pharmaceutical compositions of the present disclosure may comprise a constitutively active cytokine receptor construct or In addition to the encoding nucleic acid molecule or vector, depending on the intended use of the pharmaceutical composition, It is envisioned that the composition may contain additional biologically active agents.

[0160] Any of the compositions described herein may be used in combination with any of the compositions described herein that are directed to cells expressing constitutively active cytokine receptors. In a non-limiting example, the present invention can be used in cell therapy. one or more constitutively active cytokine receptor-directed immune cells for use in The present invention relates to a method for producing one or more cells for use in cell therapy that harbor a recombinant expression vector. The kit components may be provided in suitable container means.

[0161] Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or The method includes disposing the components in a vessel or other container, preferably with suitable aliquots. If there is more than one component in the kit, the kit generally includes a second, third, or other The present invention also includes additional containers in which additional ingredients may be separately placed. The kit may also contain various combinations of Such containers include a means for containing the ingredients in close confinement for commercial sale. The container may comprise an injection or blow molded plastic container into which the desired vials are held.

[0162] When kit components are provided in one and / or more liquid solutions, the liquid solutions are water-soluble. In some cases, the container means may itself be a syringe or a container for administering the agent to a patient. , a pipette, and / or other similar device from which the preparation is delivered to an infection site of the body. applied to the area, injected into the animal, and / or further applied to other components of the kit, and and / or mixed with

[0163] In certain embodiments, the cells used in cell therapy are provided in a kit, and optionally The cells are essentially the only component of the kit. The kit includes a kit for producing the desired cells. In certain embodiments, the reagents and materials may include a desired sequence, nucleic acid sequence, primers for amplifying nucleotides, appropriate buffers or buffering reagents, salts, etc.; And in some cases, the reagent comprises an engager molecule as described herein. Vectors and / or DNA encoding and / or regulatory elements therefor Includes.

[0164] In certain embodiments, one or more devices suitable for extracting one or more samples from an individual are provided. The device may be a syringe, a scalpel, or the like.

[0165] In certain embodiments, the kit comprises a cell therapy of the present disclosure and a chemotherapy in which the cells are immunogenic. In some cases, the kits may also include, in addition to the cell therapy embodiments, methods for treating, e.g., chemotherapy, Also includes a second cancer therapy, such as hormonal therapy, and / or immunotherapy. , can be tailored to the particular cancer for the individual, and each second Including cancer treatment. EXAMPLES

[0166] The following examples are included to demonstrate preferred embodiments of the disclosure. The present inventors have determined that the techniques disclosed in the above publication work well in the practice of the present disclosure. represents a discovered technique and is therefore believed to constitute a preferred mode for its practice. However, it should be understood by those skilled in the art that in light of this disclosure, Therefore, modifications may be made in the specific embodiments disclosed without departing from the spirit and scope of the disclosure. It is understood that many changes can be made in the present invention and still obtain the same or similar results. It should be. EXAMPLES

[0167] [Constitutively active cytokine receptors for cell therapy] This example demonstrates the generation and activity of specific embodiments of constitutively active cytokine receptors. The present disclosure demonstrates that the present invention provides novel polypeptides that are non-naturally occurring and that result in altered signaling properties compared to wild-type. FIG. 1 shows a normal IL-7 receptor and a constitutively active cytokine receptor that has been modified by This is an example of what happens in cytokine / receptor signaling. At rest, IL-7 receptor The interleukin-7 receptor α (IL-7Rα) protein remains separate from the γ-chain receptor (γc) protein. This changes when extracellular IL-7 binds to the extracellular domains of both receptors, resulting in IL- Induces heterodimerization of 7Rα and γc, activating JAK1 / JAK3 signaling Activates STAT5 phosphorylation. STAT5 is activated by the IL-7 receptor The IL7RP2 receptor is a major downstream signaling node. The transmembrane domain of the IL7RP2 monomer protein was expressed independently of extracellular IL-7. The cysteine ​​residues of this monomer form disulfide bonds with the cysteine ​​residues of other IL7RP2 monomers. This leads to JAK1 / JAK1 signaling, which results in STAT5 phosphorylation. It induces the formation of a homodimeric protein that constitutively activates

[0168] Thus, the present disclosure in some cases relates to specific extracellular domains and specific transmembrane domains. The present invention encompasses embodiments of engineered cytokine receptors having domains, such that the engineered The receptors that are expressed are constitutively active even in the absence of the initial signal for the receptor. Figure 3 shows signal transduction in the specific example of the Δ34.IL7RP2 protein. The extracellular domain of the L7RP2 receptor was replaced with the extracellular domain of CD34 to create Δ34.I L7RP2, which inhibits sensitivity to extracellular cytokines such as IL-7. while keeping the transmembrane and cytoplasmic domains of the protein intact. Figure 4 shows that STAT5 activates IL7RP2-transduced T cells. Δ34.IL7RP2 (SFGΔ34.IL7 A retroviral vector carrying an expression cassette encoding the RP2 was constructed. 3 / CD28-activated T cells were transduced with RD114 pseudotyped SFG△34.IL7RP2 retrovirus. As a negative control, T cells were transduced with avian tyrosine kinase inhibitors (AVKs) expressing the extracellular domain. A nonsignaling domain expressing the transmembrane and transmembrane domains and part of the cytoplasmic domain of CD34. The SFG.Δ34 vector was transduced separately with the SFG.Δ34 vector. Representative FAs from one donor In the C data (Fig. 4A), constitutive STAT5 activation was observed in SFG.Δ34 (black). Compared to transduced T cells resting without cytokines for 24 h, Δ34.IL7R Higher phosphorylated STAT5 (pSTAT5) signaling in P2-transduced T cells (white). The mean results of n=3 donors are demonstrated for Δ34.IL7RP2 transduced The mean fluorescence intensity (MFI) of pSTAT5 in T cells was significantly higher than that of SFG.Δ34. We showed that this was the case (Figure 4B).

[0169] The modified cytokine receptors of the present disclosure also include chimeric antigen receptors (for example). Cells containing IL-1 maintain the ability to proliferate even in the absence of cytokines. 7RP2-transduced antigen-specific T cells were able to express naïve antigen-specific T cells after antigen-specific stimulation. It has been shown that it has a greater proliferation ability than cells. Varicella-zoster virus (VZV) specific Targeted T cells (VZVST) were transfected with a chimeric antigen receptor (CAR) specific for the solid tumor antigen GD2. The benefit of IL7RP2 in this system was evaluated. D28-ζ signaling domain (SFG.GD2.CAR.CD28ζ) or SFG .GD2.CAR.CD28ζ and SFG.IL7RP2-mOrange (mO) Both were genetically modified with a retroviral vector encoding GD2-CAR. T cell populations were repeatedly exposed to GD2+LAN-1 tumor cells in the absence of IL2. 7RP2-transduced GD2.CAR VZVST continued to proliferate, whereas unmodified GD2.CAR VZVST did not proliferate.

[0170] The constitutively active cytokine receptors of the present disclosure exhibit increased cytokine expression levels compared to cells lacking the receptor. Figure 6 shows that Δ34.IL7RP2-coexpressing GD 2. CAR T cells showed greater proliferation and progression in vivo than GD2. CAR T cells We show that Δ34.IL7RP2 has antitumor efficacy in vivo. To evaluate whether OKT3 / CD28-activated T cells can enhance T cell proliferation, , OX40.CD28ζ signaling domain (SFG.GD2.CAR.OX40.C D28.ζ) or GD2.CAR.OX40.CD28.ζ and Δ34.IL7RP2 GD2 with a bicistronic SFG vector (with 2A sequence between them) coexpressing Both T cell populations were transduced with a retroviral vector encoding a CAR. were transduced with a retroviral vector encoding firefly luciferase to produce a non-invasive The results of this study enabled quantitative bioluminescence imaging of 8-day-old GD2+ LAN-1 tumors on the left flank. NSG mice bearing the CAR-T cells were then injected with 2 million GD2.CAR T cells or 2 million GD2 .CAR-Δ34.IL7RP2 T cells were injected i.v. IL7RP2 T cells expanded significantly in vivo compared with GD2.CAR T cells The persistence of T cells at the tumor site was extended by increasing the number of GD2.CAR T cells in the LAN-1 tumor. Tumors grew in mice that received T cells, and GD2.CAR-Δ34.IL7RP2 T cells This was abolished in cells receiving GD2·CARΔ34·IL7RP2 T cells (Figure 6B). This resulted in a significantly enhanced survival advantage in mice receiving the treatment (Figure 6C).

[0171] Δ34.IL7RP2-transduced T cells have limited persistence in the absence of antigenic stimulation. Δ34.IL7RP2 mediates constitutive proliferation of T cells in the absence of antigen or cytokine stimulation To assess whether Δ34.IL7RP2-transduced T cells could induce The mice were cultured in complete culture medium lacking cytokine supplements or antigen stimulation (PBMC isolation and and 10 days after activation). As controls, Δ34-transduced T cells and non-transduced (NT) T cells were cultured in the same manner. Δ34.IL7RP2-transduced T cells were cultured for the first 14 h. The Δ34 and NT T cells expanded 1-fold over the course of 3 days, after which the cells shrank (Figure 7A). There was no expansion or contraction during the course of culture. was unable to induce constitutive cell proliferation in T cells, and the Δ34.IL7RP2 phenotype We demonstrate that transduced T cells still require antigen for proliferation.

[0172] Δ34.IL7RP2 prolongs the cytotoxic potential and proliferation of EphA2.CAR T cells Δ34.IL7RP2 targets EphA2-expressing targets via EphA2-CAR. To assess whether OKT3 / CD28 activity enhances proliferation of targeted T cells, The engineered T cells were then transduced with a CAR encoding EphA2.CAR carrying the 41BB.ζ signaling region, The A sequence confers a truncated CD19 molecule, ΔCD19 (SFG.EphA2.41BB.ζ- SFG.Eph A2.41BBζ and Δ34.IL7RP2 were co-expressed with the 2A sequence between them. Cistronic SFG vector (SFG.EphA2.41BB.ζ-2Α-ζ-2Α-Δ 34.IL7RP2) were transduced into EphA2-positive U373 glioblastoma cells. When subjected to a serial killing assay with weekly challenges with tumor cells (2 T cells:1 tumor cell ratio) , SFG.EphA2.41BB.ζ-2A-Δ34.IL7RP2 T cells are tumor cells were eliminated and could proliferate after eight stimulations (black), whereas SFG.EphA2.41 BB.ζ-2A-ΔCD19 T cells (gray) became dysfunctional after two rounds of stimulation (Figure 8).

[0173] Δ34.IL7RP2 inhibits antiglionosis of EphA2.CAR T cells in vivo Mice were injected with U373 cells expressing firefly luciferase, On day 7, EphA2.CAR T cells or EphA2.CAR.Δ34.IL7RP2 T cells were injected intratumorally, and tumor growth was followed by serial bioluminescence imaging. Tumors were identified in all four mice treated with EphA2·CAR·Δ34·IL7RP2 T cells. In contrast, after EphA2.CAR T cell therapy, Tumor regression was observed in only 4 / 5 mice within 4 weeks of T cell injection. All four responsive tumors recurred.

[0174] Combinatorial constitutively active cytokine receptors are encompassed within the embodiments of the present disclosure. For example, the Δ34.IL7RP2 protein is fused to the IL-21Rα cytoplasmic domain. Δ34.IL7 can be used to generate constitutively active combinatorial cytokine receptors. RP2 is fused at its C-terminus to the cytoplasmic domain of the IL-21 receptor, providing steric hindrance to STAT5 binding. There is a flexible linker between the two cytoplasmic domains that avoids the damage caused by Δ34.IL7RP2 Linker-IL21Rα was generated (FIG. 10A). To evaluate its STAT activation ability, T cells (NT, Δ34.IL7RP2-transduced T cells, or Δ34.IL7RP2-transduced T cells) IL21Rα-transduced T cells) were left without cytokines for 24 hours. .IL7RP2 and Δ34.IL7RP2-linker-IL21Rα T cells were Although it was able to constitutively activate T5 (Fig. 10B), the Δ34.IL7RP2-linker Only IL21Rα exhibited constitutive STAT3 activity (Fig. 10C). It is a key downstream signaling node activated by the L-21 receptor.

[0175] Cells other than T cells are adapted to express one or more constitutively active cytokine receptors. In FIG. 11, IL7RP2 enhances antigen-mediated proliferation of NK cells. NK cells were transfected with the IL7RP2-IRES-mOrange (IL7RP2.mO) vector. or the control vector IRES-mOrange (empty.mO). and then stimulated with irradiated K562 target cells for 7 days in the absence of cytokines. IL7RP2.mO NK cells showed an 8-fold increase in proliferation, whereas empty mO NK cells The cells were only multiplied by 4 times. EXAMPLES

[0176] Specific embodiments of the exodomain FIG. 12 shows that multiple exodomains are utilized in constitutively active cytokine receptors. A specific example is shown in which the PD1 exodomain is expressed in the ΔCD3 of Δ34.IL7RP2. 4 exodomain, and this particular PD1 exodomain is involved in PDL1 binding. It competes with normal PD1 (thereby acting as a decoy receptor) and inhibits PD1 immunosuppression In some cases, the receptor may reduce such exodomains to a single exodomain. As for the decoy receptor, the receptor is CD34 or one of its receptors. Other embodiments of the exodomain of the decoy receptor include the CT Includes LA4. EXAMPLES

[0177] Constitutive delivery of cytokine signaling to lymphocytes promotes enhanced antitumor efficacy [R] Adoptive lymphocyte therapy has been successful in treating leukemia and lymphoma, but has not been shown to be effective in treating solid tumors. The challenge that solid tumors pose to tumor-specific T cells is This can be understood by examining the three signaling paradigms required for cellular activation. Signal 1 (T cell receptor activation by antigen), signal 2 (costimulation), signal 3 (cytokine activation). These signals act together to promote adoptive transfer in vivo. Solid tumors present tumor antigens to T cells. Downregulation of MHCI molecules, impaired expression of costimulatory ligands, and immunosuppression It prevents the activation of T cells by 3-signal activation through mechanisms such as the production of cytokines. is the genetic modification of T cells to express chimeric antigen receptors (CARs), CAR provides signals 1 and 2 upon MHCI-independent antigen ligation, and I However, signal 3 does not contribute to this acetylcholinesterase. This defect is due to the IL-7 cytokine This was corrected by overexpression of a gene construct that constitutively delivered the signaling. Without clinical support, cytokine delivery strategies have not been shown to enhance CAR T cell immunity in vitro. It enhances antigen-dependent cytotoxicity, proliferation, and persistence in xenograft neuroblastoma and and glioblastoma mouse models (by way of example only) are recapitulated in vivo and in solid tumors. demonstrated significantly improved efficacy of signal 3-supplemented adoptive immunotherapy against AML. EXAMPLES

[0178] [Example 4]

[0179] [Constitutive signaling from engineered IL-7 receptor enhances tumor-redirected T cells Promotes sustained tumor elimination by

[0180] (Introduction)

[0181] Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells for refractory leukemia and lymphomas, but have not yet been able to extend these successes to solid tumors. Challenges remain to convert adoptively transferred T cells into , are required for sustained antitumor activity. Among these signals, CAR activation is mediated by signal 1 (T cell receptor (TCR) activation) and Signal 2 (costimulation) can be reproduced, but is poorly expressed in the tumor microenvironment. In xenograft tumor models, the positive signaling pathway derived from cytokines 3 cannot be maintained. In this study, signaling 3 was shown to enhance antitumor activity by inhibiting IL-2 without significant adverse effects. However, systemic administration of cytokines to cancer patients has not been Administration of 100 mg ... Alternative approaches, such as genetic modification of T cells, could be used to reduce neurotoxicity and the total secreted cytokines. Although there is a risk of serious adverse events, including cytokine release syndrome, due to accumulation in the body, T cells that overexpress kine receptors do not eliminate the need for exogenous cytokines.

[0182] The present example describes a signal transduction assay using a constitutively active IL-7 cytokine receptor (C7R) This novel chimeric receptor provides a strategy to selectively deliver IL-1 to T cells, thereby avoiding the above problems. The condition is induced by exogenous substances or by forced expression of transgenic cytokines. C provides signal 3 without the need for non-specific bystander T cell activation. The proliferation and survival of 7R-expressing CAR T cells remained antigen-dependent but was associated with tumor presence. Currently, these cells have potent antitumor activity in multiple model systems.

[0183] [C7R was engineered to constitutively activate STAT5 and to be unresponsive to extracellular ligands. [Can be]

[0184] In this example, we show that cytokines enhance the persistence of tumor-specific T cells by IL-7. The IL-7 receptor with a specific mutation inhibits the system function in the transmembrane domain. IL-7Rα homing, resulting in a gain of function due to insertion of amino acids and / or prolines Once homodimers are formed, JAK1 / JAK1 cross-linking occurs. Phosphorylation activates STAT5, a core signaling node downstream of IL-7.

[0185] This class of receptor completes the three-signal requirement for optimal CAR T cell activity. To discover whether STAT5 could generate a consistent signal for Constitutively active IL-7 receptor mutant (IL7R * ) was selected. This avoids further activation of the receptor by β-actin and provides a means to detect transduced cells. To achieve this, the native extracellular domain of the receptor was replaced with the ectodomain from CD34. Whether the size of the ectodomain is a factor in protein expression and function To find out whether the ectodomain of Q8 (65 amino acids) and CD34 (259 amino acids) The Q8 ectodomain is a CD34 domain mounted on a CD8 spacer. It contains an epitope that allows detection by the anti-CD34 antibody clone QBEND10. CD34-IL7R in healthy donor T cells * and Q8-IL7R * Retro of the construct Viral-mediated expression of Q8-IL7R * Poor expression of fusion proteins and suboptimal S TAT5 activation was observed (Figure 18). In contrast, CD34-IL7R * In T cells Thus, CD34-IL7R was robustly expressed and functionally active in * From now on, C7R (also referred to herein as Δ34.IL7RP2), and all subsequent was used for the study (Figure 14A).

[0186] To determine the relative effect of C7R on CD4 and CD8 T cells, antibody coating Using magnetic beads, two subpopulations are isolated, activated and transduced, and then expressed as T cell subpopulations. The subsets were cultured separately from each other. C7R binds to both CD4 and CD8 T cells. 14B, 14C and FIG. 19), and in T cells, the truncated CD Constitutive activation of STAT5 is greater than that of a control construct consisting of 34 (Δ34) molecules. (Quintarelli, 2007) (Figures 14D-14G). C7R does not promote antigen-independent proliferation of CD4 and CD8 T cells in vitro. C7R-transduced cells expressed more antigen and IgG than control cells in vitro (Fig. 14H, 14I). C7R population persisted significantly longer in the control and cytokine-depleted conditions, whereas C7R population declined by 14–21 days. The cells began to shrink and all cells had died by 70 days after the start of the persistence assay. R alone did not sustain autonomous T cell proliferation, a property important for the safety of CAR T cells. It was confirmed that this would not be allowed.

[0187] During a series of in vitro tumor cell challenges, C7R inhibited GD2-CAR T cells Promoting survival of

[0188] We investigated whether C7R could increase the antitumor efficacy of CAR T cells. To assess the effect of CD8α on the expression of GABAergic markers, GD2+ neuroblastoma cells were cultured using a cloning assay using the CD8α stalk and transmembrane domains. The 14g2a scFv bound to the 41BB.ζ signaling endodomain and T cells expressing the GD2-CAR containing the 14g2a-based GD 2-CAR T cells have safety profile in clinical trials treating patients with neuroblastoma Although complete responses have been achieved in selected patients, greater efficacy is needed. This is expected. Compare T cells expressing either of the cistronic constructs (GD2-CAR.C7R) and C7R regulates the memory subset composition or CD4 / CD8 partitioning of GD2-CAR T cells. C7R did not induce any significant difference in the percentage of LAN-1 Secretion of IFN-γ and TNF-α in GD2-CAR T cells after stimulation with tumors (Figure 15A), which increased T cell killing during a 4-hour cytotoxicity assay (Figure 15B). However, during the in vitro sequential co-culture killing assay The GD2 inhibitors were measured for their cytotoxicity and ability to sustain growth after repeated tumor challenge. -CAR.C7R T cells performed significantly better than GD2-CAR T cells ( (Figure 15C). GD2-CAR T cells failed by the third challenge and inhibited tumor cells They lost both the ability to spread and to eliminate (Figures 15D, 15E). In contrast, C7R-expressing GD2-CAR T cells responded to all three consecutive tumor challenges. Improved expansion of CD2-CAR.C7R T cells by increasing proliferation while decreasing thrombocytosis To determine the relative contribution of the β-amyloides to the β-amyloides, Cell Trace Viol was used after the first co-culture. Subsequent restimulation with tumor cells resulted in increased expression of GD2-CAR.C7R T cells. showed greater cell division than T cells expressing GD2-CAR alone (Figure 15F, 1 To assess whether C7R also reduces T cell apoptosis, we performed annexin-dependent V and 7-AAD staining was used after the second tumor restimulation. Analysis showed that Annexin V(+) / 7-AAD( +) showed a larger population of GD2-CAR T cells (Fig. 15H), indicating sequential tumor challenge These results show that the increased viability generated by C7R is consistent with previous studies. To further understand the molecular basis of Using the 2nd tumor restimulation, GD2-CAR and GD2-CAR.C7R T cells were Gene expression analysis was performed (Fig. 15I and Fig. 22). BCL2, which mediates tumor growth, is upregulated by C7R in GD2-CAR T cells It was one of the top genes in the study. D) involved in cytolytic granzyme A (GZMA) and proapoptotic FA There was downregulation of S and caspase 8 (CASP8) (Kramme et al., 2013). Thus, C7R promotes both proliferation and survival of GD2-CAR T cells. This increases the number of IL-1 receptors in the IL-1 receptors, enhancing their performance during successive encounters with tumor cells.

[0189] [C7R co-expression in CAR T cells enhances antitumor activity against xenograft tumor models. [Increase]

[0190] C7R-enhanced GD2-CAR T cells inhibit metastatic neuroblastoma in xenograft models The ability of the tumor-specific markers to eradicate glioma cells was tested. 5 Multidrug-resistant MYC-N non-amplified neuroblastoma engineered to express FFluc The cell line CHLA-255 (Heczey, 2014, Blood. 2014 Oct 3 0;124(18):2824-33) was administered intravenously to non-obese diabetic (NOD) Tumors were engrafted into severe / combined immunodeficient (SCID) IL-2rγ- / (NSG) mice. Treatment with low doses of GD2-CAR T cells one week after engraftment resulted in expression of an irrelevant CAR The median survival time was increased by one week compared to control mice treated with T cells expressing C7 T cells expressing R and a non-functional GD2-CAR with a truncated endodomain (GD Mice administered 2-CARΔ.C7R) showed identical survival rates to control mice (Figure 1 6A, 16B). In contrast, mice injected with GD2-CAR.C7R T cells showed no disease progression. The disease disappeared. T cells, but not CHLA-255 cells, were transduced with GFP-FFluc. In parallel experiments, there was no proliferation of GD2-CAR T cells at the dose-limited infusion. However, robust proliferation of GD2-CAR T cells was observed across a wide range of neuroblastoma metastatic sites. These results suggest that GD2-CA1 expression may be a signaling pathway that may be involved in the accumulation of T cell signals in the liver. RT cells were unable to persist in tumors in vivo but expressed C7R. Expressing GD2-CAR T cells proliferate, survive and mediate metastatic tumor clearance. It was shown that.

[0191] To investigate whether C7R could enhance the performance of other CAR T cells, , co-expressed with EphA2-CAR intended to treat glioblastoma. GFP-FFl U373 glioblastoma cells genetically modified with uc were injected intracranially into SCID mice. Seven days later, 10 4 EphA2-CAR T cells were administered intratumorally, based on previous experience. The dose was such that it was not possible to eradicate gliomas with the gliomas. and non-functional EphA2-CAR (EphaA2-CARΔ.C7R) Rapidly increased in mice treated with CAR T cells and received EphA2-CAR T cells No significant improvement in antitumor controls was observed in mice (Figures 16E, 16F In contrast, mice injected with a low, “stress” dose of EphA2-CAR T cells When they co-expressed C7R (EphA2-CAR.C7R), the tumors were completely These mice remained disease-free at the end of the experiment. When the experiment was repeated using EphA2-CAR T cells that were also transduced with the CAR alone Bioluminescence signals from T cells expressing IgG4 largely disappeared 4–6 days after injection. , EphA2-CAR.C7R T cells were observed in (extracranial) neuroblastoma models. Although EphA2-CAR and EphA2-CAR.C7R lacked significantly greater expansion than Larger T cells, as determined by area under the curve (AUC) comparison between T cells There was a trend toward increased vesicle persistence (Figure 21).

[0192] [GD2-CAR.C7R T cells efficiently transfected with iC9 after tumor clearance Can be deleted]

[0193] However, as an additional safety measure, clinically validated antibodies that can selectively deplete T cells are Co-expression of the inducible caspase 9 (iC9) suicide gene (Di Stasi et al., 2011) After dual transduction with iC9 and GD2-CAR.C7R, T Cells remain sensitive to iC9 signaling and dimerized AP20187 (CID) underwent apoptosis in vitro within 24 hours of exposure to the chemical inducer (Figure 17 A) iC9 efficiently inhibited GD2-CAR.C7R T cells in vivo after tumor regression. To simultaneously evaluate T cell activity and tumor growth, A subcutaneous LAN-1 neuroblastoma model was used. Tumor cells were injected into the left flank of NSG mice. After 8 days, 1×10 6 GD2-CAR.C7R T cells alone or with iC9 The control mice were given GD2-CARΔ and injected intravenously. All T cells were co-transfected with GFP-FFluc for in vivo visualization. Three weeks later, LAN-1 tumors appeared in euthanized control mice. GD2-CAR.C7R T cells and T cells co-expressing the iC9 vector were 2-CAR.C7R T cells demonstrated similar antitumor efficacy and in vivo expansion to T cells alone (Figures 17B and 17C). Modeling T cell deletion required to control toxicity after immunotherapy. To achieve this, we administered three doses of CID to mice starting 28 days after T cell infusion within the same experimental approach. There was a loss of T cell bioluminescence signal (average 93%) immediately after CID administration. (FIG. 17D), the signal remained at baseline after 2 weeks without tumor recurrence, at which point These results suggest that CAR T cells that co-express C7R can be used to treat , together with iC9, without compromising efficacy and without allowing selective T cell depletion. It was confirmed that it can be used for.

[0194] 34. Treatment of LCL tumors with IL7RP2-transduced EBV-specific T cells shows antitumor efficacy The results are shown in Fig. 1. LCL tumors were injected subcutaneously into the left dorsal flank of female NSG mice. After 8 days, 5 × 10 6 EBV-specific T cells (EBVST) or 34.IL7RP2-transduced EBVST (34.IL7RP2-EBVST), and co-expressing GFP-firefly luciferase Tumor-bearing mice were intravenously injected with GFP-Ffluc as a control. In FIG. 23A, imaging of luciferase signals in mice was performed. , 34.IL7RP2-EBVST was extended at the tumor site compared with EBVST alone Similarly, in FIG. 23B, the tumor growth was measured. reported that 34.IL7RP2 EBVST had superior antitumor efficacy compared with EBVST alone. This indicates that.

[0195] [Importance of specific embodiments]

[0196] Results show that signal 3 in T cell activation mediates the activity of CAR T cells against solid tumors. Significantly improved maintenance and constitutively active IL-7 receptor for augmentation of adoptive immunotherapy This study demonstrates that IL-1 can provide signal 3 for T cells to target the IL-1 receptors that they encounter in solid malignancies. Under the stress of repeated antigen exposure, which is likely to be a factor in the progression of the disease, GD2-CAR, which co-expresses C7R, Only C7R-enhanced CA T cells were able to undergo multiple rounds of expansion and retain antitumor activity. The superiority of RT cells was demonstrated for two different CAR and tumor models in vivo. Functional CAR T cells administered at ineffective doses were established when combined with C7R The ability to eradicate tumors may be maintained.

[0197] Gene expression analysis demonstrated C7R promotion of GD2-CAR T cells during repeated tumor cell challenge We found that progression survival correlated with increased BCL2 transcription and decreased expression of FAS and CASP8. This indicates that C7R exerts a broad anti-apoptotic effect in CAR T cells. These results suggest that GD2-CAR.C7 exerts a potent inhibitor of GABAergic inflammatory cytokines, which may reduce susceptibility to AICD. RT cells down-regulated TGF-βRII compared with GD2-CAR T cells alone Other potential benefits of C7R include the inhibition of TGs in the tumor microenvironment, as This includes resistance to immunosuppressants such as F-β (Pickup, 2013).

[0198] In certain embodiments, C7R mediates antigen-driven cytokine secretion by CAR T cells. Based on the observation that IFN-α only moderately enhances the severity of cytokine release syndrome, Furthermore, intraventricular IL-13Rα2 in patients with glioblastoma Low toxicity observed with successful treatment with CAR T cell infusion (Brown, 2016) demonstrated that C7R functionally activates EphA2-CAR T cells in our orthotopic glioblastoma model. Treatment of glioblastoma with the observation that the effect of IFN-γ on the progression of gliomas was enhanced by IFN-γ but did not substantially increase proliferation. When a combination strategy of CAR T cells and C7R was used in There was no evidence that C7R could induce antigen-independent proliferation, but The SG model has clear limitations for assessing the long-term fate of human T cells in vivo. However, as an additional safeguard against both concerns, dimerizable iC9-mediated safety Including the switch allows for the deletion of CAR T cells expressing C7R.

[0199] GD2-CAR T cells and E for metastatic neuroblastoma and orthotopic glioblastoma Considering the effective application of C7R to phA2-CAR T cells, the C7R molecule (simply As one example, the CAR T cells of the present invention are useful for enhancing many other CAR T cells. This same approach could be used to investigate whether native (Dudley, 2008) or transgenic TCR (Obenaus, 20 15) and those that utilize the properties of NK and NKT cells (P It enhances the antitumor activity of other adoptive cell therapies for cancer, including IL-19 (Ellegrini, 2009). Add.

[0200] [Examples of Materials and Methods]

[0201] (Generation of retroviral vectors)

[0202] pSFG.ΔCD34-IL7R *(pSFG.C7R): Between base pairs 731 and 732 Mutant IL-7Rα (IL7R*) with a TTGTCCCCAC insertion in cDNA encoding the gene was synthesized (Genscripto®) (Genscripto®, 2011). pSFG.C7R was digested with XhoI and MluI. SFG vector backbone, IL7R * cDNA and the full length extracellular domain of CD34 (Δ CD34) using IN-Fusion (Takara, Mountainview, CA) were generated by cloning.

[0203] pSFG.GD2-CAR, pSFG.GD2-CAR-2A-C7R(GD2-CA R.C7R), pSFG.GD2Δ-CAR-2A-C7R(GD2-CARΔ.C7R ): n-terminal leader peptide, GD2-specific 14g2A single chain variable fragment (scFv ), encoding the CD8 stalk and transmembrane domains, and the 41BB.ζ endodomain cDNA was synthesized (Biobasic, Marham, ON, Canada) and purified in-house. Ribosome entry site (IRES) and the SFG retroviral vector upstream of the truncated NGFR Cloning was performed by IN-Fusion (Takara) cloning into the vector. For pSFG.GD2-CAR-2A-C7R, GD2-CAR was cloned into the 2A site. The GD2Δ-SEQ ID NO:1 was subcloned into the SFG vector upstream of the C7R. For CAR-2A-C7R, 2A-C7R was used, as well as 14g2a scFv, short IgG1 Exodomain spacer, CD28 transmembrane domain, and truncated CD28 endodomain A non-functional GD2- It was cloned downstream of CAR.

[0204] pSFG.EphA2-CAR-2A-CD19t(EphA2-CAR), pSFG .EphA2-CAR-2A-C7R (EphA2-CAR.C7R), and pSFG .EphA2-CARΔ-2A-C7R(EphA2-CARΔ.C7R): pSFG .EphA2-CAR-2A-CD19t is an EphA2-specific 4H5 scFv (re f 25), CD8 extracellular spacer, CD8 transmembrane domain, 41BB.ζ endodomain EphA2-specific C domain consisting of the 2A sequence and a truncated CD19 molecule (CD19t) pSFG.EphA2-CAR-2A-C7R encodes 2A-CD19t AR. Generated by IN-Fusion (Takara) cloning to replace 2A-C7R with 2A-C7R For pSFG.EphA2-CARΔ-2A-C7R, 2A-C7R was inserted into the outer EphA2-specific 4H5scFv, CD8 extracellular spacer, CD8 transmembrane domain, and cloned downstream of non-functional EphA2-CAR, which is composed of a truncated endodomain. I tuned in.

[0205] pSFG.iC9-2A-CD19t: The vector was purchased elsewhere (Di Stasi, 2011) were prepared as previously described.

[0206] All restriction enzymes were obtained from New England Biolabs (Ipswich, MA). All cloned constructs were purchased from Seqwright (Houston, Texas). X).

[0207] (T cell generation)

[0208] Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained in accordance with the Declaration of Helsinki. With informed consent, Baylor College of Medici ne obtained under an IRB approved protocol. CD4 and CD8 T cell counts were assessed separately. In the case of PBMCs, CD4 or CD8 magnetic selection beads (Miltenyi, Aubur n, CA) and positively selected according to the manufacturer's instructions. T cell activation on day 0 For the study, bulk or selected T cells were cultured in 90% RPMI 1640 (Hyclo ne, Logan, UT), 10% fetal bovine serum (Hyclone), and 1% Gl Suspension in complete medium consisting of utamax (Gibco, Grand Island, NY) Then, OKT3 (CRL-8001, American Type Culture Collection) was used for activation. Culture Collection [ATCC], Manassas, VA) and CD2 8 antibody (BD Biosciences, San Jose, CA) IL-15 and IL-7 (Peprotech, Rocky H ill, NJ) was added 1 day after activation, and cells were retrovirally transduced on day 2. T cells were used for experiments starting 9-12 days after OKT3 and CD28 activation.

[0209] (Flow Cytometry)

[0210] Fluorochrome-conjugated antibodies were from Biolegend (San Diego, CA); CCR7, CD45RO, NGFR);Abnova, (Taiwan City, Taiwan; CD34); Thermo Fisher(Life Technologies, Frederick, MD;CD8); eBioscience(San Diego , CA; CD4); Beckman Coulter(Indianapolis, I N; CD3);BD Biosciosciences(CD8, CD4, CD3, C D34, Stat5(pY694), Annexin V, 7-AAD). For surface staining, cells were incubated with antibodies for 15 min at 4°C and stained as described by Beckman et al. Cells were acquired on a Coulter Galios (10,000 events) and analyzed using Flowj Analysis was performed using 10.0.7r2 (Tree Star, Ashland, OR). Proliferation analysis was performed using Flowjo 9.3.2 (Tree Star).

[0211] (Cytotoxicity Assay)

[0212] A 4-h luciferase-based cytotoxicity assay was performed using GFP-firefly luciferase ( We used the LAN-1 neuroblastoma cell line expressing GFP-FFluc) with minor modifications. The study was carried out according to a previously described protocol (Liu, 2013) with the following steps: 2 × 104 LAN-1 neuroblastoma cells were plated per well in a 96-well black plate (Co After 24 hours, CAR T cells were plated on a 3x100 plate (3x100, ... Various effector to target (E:T) ratios were added. The percentages were determined using a standard curve generated by serial dilutions of LAN-1 cells. The formula used to calculate cytotoxicity is as follows: The number of cells in assay wells was calculated as (number of cells in assay wells-number of cells in assay wells) / (number of cells in untreated wells).

[0213] (Serial tumor challenge test)

[0214] 0.5 × 106 LAs transduced with GD2-CAR or GD2-CAR.C7R N-1 cells and 1 × 106 T cells were cultured in fresh cultures without IL-15 and IL-7. After 7 days, the cells were analyzed by FACs or trituration. The cells were then harvested for either T cell quantification by vitreous blue exclusion. The cells were then re-plated with fresh LAN-1 cells in fresh cell culture medium at an E:T ratio of 2:1. At the end of the third coculture, T cells were were counted and the co-cultures were analyzed by FACS.

[0215] (Quantitative Flow Analysis)

[0216] To count the antibody-stained cells, after washing with PBS, 25 μL of counting beads (Life Technologies Technologies) and 2 μL of 7-AAD (to exclude dead cells) were added. Event acquisition was based on the collection of 3000 counting beads. There was.

[0217] (Analysis of cytokine production)

[0218] T cells expressing GD2-CAR or GD2-CAR.C7R were treated with cytokines LAN-1 was cultured in 24-well plates in complete culture medium without any added medium, using an E:T ratio of 1:4. The cells were cultured together. After 24 hours, the supernatant was collected. ELISA kit (R&D System IFN-γ and TNF-α release were measured using a 30-μg / mL ELISA kit (Minneapolis, MN). Quantitative analysis was performed.

[0219] (Phospho-STAT5 assay)

[0220] Transduced T cells were harvested and cultured at 0.5 × 106 cells / mL complete culture without cytokines. Resuspend in full medium and then place 0.5 x 10 cells per well in a 48-well tissue culture plate. After 24-72 hours, cells were harvested in FAC tubes and diluted with cold flow buffer ( Wash with 100 μL of Fix & Perm Reagen Add t A (Life Technologies) to the cells and vortex gently. After incubation at room temperature for 3 min, 3 mL of ice-cold methanol was added to the wells with constant vortexing. The tube was then incubated at 4°C for 10 minutes, after which it was The tubes were centrifuged and the methanol was discarded, followed by another washing step with cold running buffer. Then, 100 μL of Fix & Perm Reagent B (Life Technologies) and 5 μL of anti-STAT5 antibody were added to the cells. The cells were gently vortexed and then incubated at room temperature for 30 minutes in the dark. The cells were washed once more with cold flow buffer and then immediately analyzed.

[0221] (Cell Trace Violet Proliferation Assay)

[0222] After single co-culture with LAN-1 tumor cells, GD2-CAR T cells and GD2-CAR C7R T cells were purchased from Thermo Fisher according to the manufacturer's instructions. The T cells were then labeled with Cell Trace Violet using a kit. The cells were then rechallenged with tumor cells for 1 week before analysis. 7-AAD was added to eliminate dead cells. did.

[0223] (Apoptosis analysis)

[0224] Cells were incubated with Annexin V antibody and 7-AAD and analyzed by flow cytometry. For experiments with iC9, a chemical inducer of dimerization (CI D), AP20187 was purchased from Takara Clontech.

[0225] (cell line)

[0226] LAN-1 and U373 were purchased from ATCC, and LAN-1 GFP-FFluc and and U373 were used to generate GFP-FFluc. CHLA-255 FFluc were established and maintained as previously described (Liu, 2012, J Clin Invest.2012 Jun 1;122(6)): 2221-223). Enzyme-based assays (Lon za, Rockland, ME) to conduct routine mycoplasma surveillance. The cells were then subjected to STR profiling and authenticated within one year of the described experiment.

[0227] (Gene expression analysis)

[0228] Total RNA was analyzed using Qiazol reagent and the miRNeasy Micro Kit (Qia Gene expression analysis was performed using the Baylor Biosciences (Bayside, Germantown, MD). r College of Medicine Genomic and RNA Pr Immunology Panel version 2 of the OFIL Core ( NanoString, Seattle, WA) using nCounter Analy The NanoStructure System was used. The data were analyzed using the .

[0229] (In vivo experiment)

[0230] All animal studies were performed in accordance with the guidelines of the Baylor College of Medicine Institute. Titional Animal Care and Use Committee The protocol approved by was followed.

[0231] Subcutaneous neuroblastoma mouse model: 10-14 week old female NSG mice were inoculated with 100 μL of basal 2 million LAN-1 neuroblastoma cells in basement membrane Matrigel (Corning) were implanted subcutaneously in the dorsal left flank. After 8 days, the mice were cultured until the tumor mean and distribution of the groups were similar. Tumors were then divided into groups based on tumor size, as described above. Cells were injected intravenously (10–12 days after PBMC isolation). Tumor size was measured weekly using a caliper. The measurements were performed twice and the mice were treated with 150 mg / kg D-luciferin (Xenog Ten to fifteen minutes after intraperitoneal injection of the IVIS® system (IV IS, Xenogen Corporation, Alameda, CA). Bioluminescence signals from T cells were imaged at the same time points. Tumor diameter ≥ 15 mm Mice were euthanized if tumors were >10% of the mouse body weight or if tumors were >10% of the mouse body weight.

[0232] Metastatic neuroblastoma mouse model: 10-14 week old female NSG mice were inoculated with Firefly. One million CHLA-255 cells expressing ly-luciferase (CHLA- After 7 days, mice were intravenously injected with 1 million GD2-CAR T cells were injected intravenously (10-12 days after PBMC isolation). Cell proliferation or T cell proliferation was indirectly assessed by weekly bioluminescence imaging as described above. In tumor growth follow-up experiments, CHLA-255 FFluc luminescence was Mice groups were not standardized for tumor burden, as it was undetectable at the time of cell injection. .

[0233] Orthotopic glioblastoma mouse model: 105 U373 glioblastoma cells were inoculated into 8-week-old male ICR-SC mice. The cells were established intracranially in ID mice as previously described (Chow, 2013, Mol Ther. 2013). 13 Mar;21(3):629-637). Seven days after tumor engraftment, 104 T cells were injected intracranially directly into the tumor. Tumor growth or T cell proliferation was monitored weekly by bioluminescence imaging as described above. Mice in tumor growth experiments were normalized for tumor burden but not for variance. was not standardized.

[0234] (statistical analysis)

[0235] Graphs and statistics were generated using Prism 5.0 software for Windows (Graph The measurement data was created using the NIRS Pad Software Inc. (La Jolla, CA). Data are presented as mean ± standard error of the mean (SEM). Differences between means were analyzed using paired two-tailed t In mouse experiments, the change in tumor radiance from baseline at each time point was Changes were calculated and compared between groups using two-tailed paired t-tests or Welch's t-tests, where appropriate. Tumor means between groups were compared using one-way analysis of variance and Bartlett's test for equal variances, as appropriate. Survival was determined from the time of tumor cell injection and was compared with that of the control group. The data were analyzed by the Plan-Meier method, and the differences in survival rates between groups were evaluated by the log-rank test. Compared.

Claims

[Claim 1] The invention described herein.

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