Chimeric cytokine receptors

Abstract

To provide chimeric cytokine receptors.SOLUTION: The invention provides a chimeric cytokine receptor (CCR) comprising: (i) an exodomain that binds to a ligand selected from tumor secreted factors, chemokines and cell surface antigens; and (ii) a cytokine receptor endodomain. In a specific aspect, the chimeric cytokine receptor (CCR) comprises: (i) a first polypeptide comprising (a) a first antigen-binding domain that binds to a first epitope of the ligand, and (b) a first chain of the cytokine receptor endodomain; and (ii) a second polypeptide comprising (a) a second antigen-binding domain that binds to a second epitope of the ligand, and (b) a second chain of the cytokine receptor endodomain.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to chimeric cytokine receptors (CCRs) and cells expressing such chimeric cytokine receptors and optionally chimeric antigen receptors on their cell surface. [Background technology]

[0002] 2. Background of the Invention Chimeric antigen receptors (CARs) Several immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), bispecific T cell engagers and chimeric antigen receptors (CARs).

[0003] Chimeric antigen receptors are proteins that link the specificity of monoclonal antibodies (mAbs) to the effector functions of T cells. The usual form of a chimeric antigen receptor is that of a type I transmembrane domain protein with an antigen-recognizing amino terminus, a spacer, and a transmembrane domain all linked to a composite endodomain that transmits T cell survival and activation signals.

[0004] The most common form of these molecules is a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody that recognizes a target antigen, fused to a signaling endodomain via a spacer and a transmembrane domain. The above molecules result in the activation of T cells in response to the recognition of the target by the scFv. When a T cell expresses such a CAR, the T cell recognizes and kills a target cell that expresses the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer methods using such CAR-expressing T cells are currently in clinical trials for the treatment of various cancers. Summary of the Invention [Means for solving the problem]

[0005] CAR-Based Approaches to Treat Prostate Cancer Prostate cancer is the second most common cancer in men worldwide and the sixth leading cause of cancer-related death. Globally, there are approximately 1.1 million new cases and 300,000 deaths each year, constituting 4 percent of all cancer deaths. It is estimated that one in six men will be diagnosed with the disease during their lifetime.

[0006] Initial treatment for prostate cancer may consist of surgery, radiation or hormone therapy, or any combination of each. Hormone therapy consists of lowering levels of testosterone, the male hormone that promotes uncontrolled cell growth. Chemotherapy is usually left for advanced stages of cancer.

[0007] When prostate cancer grows despite the reduction of testosterone levels by hormone therapy, treatment options are limited. Usually, the cancer vaccine sipuleucel-T (Provenge®), a dendritic cell-based therapeutic cancer vaccine designed to induce an immune response targeting prostatic acid phosphatase (PAP) antigen, radiopharmaceuticals (e.g., radium-223 chloride), second-line hormone therapy (e.g., abiraterone or enzalutamide), and / or chemotherapy (docetaxel and cabazitaxel) are added to hormone therapy in sequence. Each of these treatments can slow the growth of the cancer and alleviate the symptoms caused by the disease for several months, but the disease eventually becomes resistant to them.

[0008] Preclinically, two antigens associated with prostate cancer have been targeted by CAR T cell-based therapy: prostate-specific membrane antigen (PSMA) and prostate stem cell antigen (PSCA).

[0009] Mice treated with PSCA CAR engineered T cells showed delayed tumor growth (Hillerdal et al. (2014) BMC Cancer 14:30; and Abate-Daga et al. (2014) 25:1003-1012). The cells showed high in vitro cytotoxicity, but in vivo, tumor growth was delayed but tumor-bearing mice were not cured.

[0010] This may be because CAR T cells struggle to overcome the harsh cancer microenvironment in vivo, and in particular, CAR T cells may not engraft and expand within the tumor bed of prostate cancer.

[0011] The persistence and activity of CAR T cells can be enhanced by administration of cytokines or by CAR T cells that constitutively produce cytokines. However, these approaches have limitations: systemic administration of cytokines can be toxic; constitutive production of cytokines can lead to uncontrolled proliferation and cancer (Nagarkatti et al. (1994) PNAS 91:7638-7642; Hassuneh et al. (1997) Blood 89:610-620).

[0012] Therefore, alternative CAR T cell approaches that promote T cell engraftment and expansion to counter the effects of the harsh tumor microenvironment are needed.

[0013] On-target off-tumour toxicity Cancers can lose specificity, so it is relatively rare for them to be effectively described by the presence of a single antigen.

[0014] Most cancers cannot be differentiated from normal tissues based on a single antigen. Therefore, significant "on-target off-tumor" toxicity occurs, whereby normal tissues are damaged by treatment. For example, rituximab targets CD20 to treat B-cell lymphoma, but depletes the entire normal B-cell compartment; targets CD52 to treat chronic lymphocytic leukemia, but depletes the entire lymphoid compartment; targets CD33 to treat acute myeloid leukemia, but damages the entire myeloid compartment, etc.

[0015] The predicted problem of "on-target off-tumor" toxicity is supported by clinical trials. For example, targeted approaches to ERBB2 have led to the deaths of patients with colon cancer that has metastasized to the lung and liver. ERBB2 is overexpressed in colon cancer in some patients, but is also expressed on several normal tissues, including the heart and normal vasculature.

[0016] Therefore, there is a need for improved cancer treatment approaches in which such "on-target off-tumor" toxicities are reduced or eliminated. The present invention provides, for example, the following items. (Item 1) an exodomain that binds to a ligand selected from a tumor-secreted factor, a chemokine, and a cell surface antigen; and Cytokine Receptor Endodomains A chimeric cytokine receptor (CCR). (Item 2) Two polypeptides: (i) (a) a first antigen-binding domain that binds to a first epitope of the ligand; (b) a first chain of the cytokine receptor endodomain. a first polypeptide comprising: (ii) (a) a second antigen-binding domain that binds to a second epitope of the ligand; (b) a second chain of the cytokine receptor endodomain. A second polypeptide comprising 2. The chimeric cytokine receptor of item 1, comprising: (Item 3) 3. The chimeric cytokine receptor of claim 2, wherein each of the first and second antigen-binding domains is a single-chain variable fragment (scFv). (Item 4) 3. The chimeric cytokine receptor of claim 2, wherein each of the first and second antigen-binding domains is a single domain binder (dAb). (Item 5) Two polypeptides: (i) (a) Heavy chain variable domain (V H ) (b) a first chain of the cytokine receptor endodomain. a first polypeptide comprising: (ii) (a) Light chain variable domain (V L ) (b) a second chain of the cytokine receptor endodomain. A second polypeptide comprising 2. The chimeric cytokine receptor of item 1, comprising: (Item 6) 6. The chimeric cytokine receptor according to item 5, wherein the first and second chains for said cytokine receptor endodomain are different and are selected from the alpha, beta and gamma chains of a type I cytokine receptor endodomain. (Item 7) 6. The chimeric cytokine receptor according to item 5, wherein the first and second chains for said cytokine receptor endodomain are the same and are selected from the alpha, beta and gamma chains of a type I cytokine receptor endodomain. (Item 8) The cytokine receptor endodomain (i) IL-2 receptor β chain endodomain (ii) the α-chain endodomain of the IL-7 receptor; or (iii) the α-chain endodomain of the IL-15 receptor; and / or (iv) Common γ chain receptor endodomain The chimeric cytokine receptor of any of the preceding items. (Item 9) The chimeric cytokine receptor of any of the preceding items, wherein the ligand is a tumor-secreted factor selected from prostate specific antigen (PSA), carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF) and CA125. (Item 10) 8. The chimeric cytokine receptor according to any one of items 1 to 7, wherein the ligand is a chemokine selected from CXCL12, CCL2, CCL4, CCL5 and CCL22. (Item 11) A cell comprising a chimeric cytokine receptor according to any of the preceding items. (Item 12) The cell of item 11, comprising a first chimeric cytokine receptor and a second chimeric cytokine receptor that bind to different epitopes on the same ligand. (Item 13) The cell described in item 12, wherein the first chimeric cytokine receptor comprises an alpha or beta chain of a type I cytokine receptor endodomain and the second chimeric cytokine receptor comprises a gamma chain of a type I cytokine receptor endodomain, such that when the first chimeric cytokine receptor and the second cytokine receptor bind to the ligand, combined signaling occurs via the alpha / beta and gamma chains of the type I cytokine receptor endodomain. (Item 14) 14. The cell according to any of items 11 to 13, which also comprises a chimeric antigen receptor. (Item 15) 15. The cell of item 14, wherein the chimeric antigen receptor binds to a tumor-associated cell surface antigen. (Item 16) 16. The cell of item 15, wherein the chimeric antigen receptor binds to a cell surface antigen associated with prostate cancer. (Item 17) 17. The cell of item 16, wherein the cell surface antigen is prostate stem cell antigen (PSCA) or prostate specific membrane antigen (PSMA). (Item 18) A nucleic acid sequence encoding the chimeric cytokine receptor (CCR) according to any one of items 1 to 10. (Item 19) A nucleic acid construct comprising a first nucleic acid sequence encoding a first CCR and a second nucleic acid sequence encoding a second CCR, the nucleic acid construct having the structure: AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 (where: AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR; spacer 1 is a nucleic acid sequence encoding the spacer of said first CCR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR; endo1 is a nucleic acid sequence encoding the endodomain of the first CCR; coexpr is a nucleic acid sequence that allows for coexpression of both CCRs, AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR; spacer2 is a nucleic acid sequence encoding the spacer of said second CCR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; endo2 is a nucleic acid sequence encoding the endodomain of the second CCR. A nucleic acid construct comprising: (Item 20) 20. The nucleic acid construct according to item 19, which also encodes a chimeric antigen receptor (CAR), the nucleic acid construct having the structure: (i) CCRAgB1-CCR spacer1-CCRTM1-CCR end1-coexpr1-CCRAgB2-CCR spacer2-CCRTM2-CCR end2-coexpr2-CARAgB-CAR spacer-CARTM-CAR end; (ii) CCRAgB1-CCR spacer1-CCRTM1-CCR endo1-coexpr1-CARAgB-CAR spacer-CARTM-CAR endo-coexpr2-CCRAgB2-CCR spacer2-CCRTM2-CCR endo2; or (iii) CARAgB-CAR spacer-CARTM-CAR endo-coexpr1-CCRAgB1-CCR spacer1-CCRTM1-CCR endo1-coexpr2-CCRAgB2-CCR spacer2-CCRTM2-CCR endo2; (where: CCRAgB1 is a nucleic acid sequence encoding the antigen-binding domain of said first CCR; CCRspacer1 is a nucleic acid sequence encoding the spacer of said first CCR; CCRTM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR; CCR endo1 is a nucleic acid sequence encoding the endodomain of the first CCR; CCRAgB2 is a nucleic acid sequence encoding the antigen-binding domain of said second CCR; CCRspacer2 is a nucleic acid sequence encoding the spacer of said second CCR; CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; CCR endo2 is a nucleic acid sequence encoding the endodomain of the second CCR; Coexpr1 and coexpr2 are nucleic acid sequences that allow for coexpression of two adjacent sequences; CARAgB is a nucleic acid sequence encoding the antigen-binding domain of the CAR; CAR spacer is a nucleic acid sequence encoding the spacer of the CAR; CARTM is a nucleic acid sequence encoding the transmembrane domain of the CAR; CAR endo is a nucleic acid sequence encoding the endodomain of the CAR. 20. The nucleic acid construct according to item 19, comprising: (Item 21) 21. The nucleic acid construct of item 19 or 20, wherein coexpr encodes a sequence comprising a self-cleaving peptide. (Item 22) 22. The nucleic acid construct according to any of items 19 to 21, wherein to avoid homologous recombination, alternative codons are used in regions of the sequence that code for identical or similar amino acid sequences. (Item 23) A vector comprising the nucleic acid construct according to any one of items 19 to 22. (Item 24) 24. The retroviral or lentiviral vector or transposon of item 23. (Item 25) i) a vector comprising a nucleic acid sequence encoding a first CCR as defined in any of items 1 to 10; and ii) A vector comprising a nucleic acid sequence encoding a second CCR as defined in any of items 1 to 10. Including the kit. (Item 26) 26. The kit of item 25, also comprising a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor. (Item 27) i) a vector comprising a nucleic acid sequence encoding a CCR as defined in any of items 1 to 10; and ii) A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor. Including the kit. (Item 28) A method for producing the cell according to any one of Items 11 to 17, comprising the step of introducing into a cell the nucleic acid sequence according to Item 18; the nucleic acid construct according to any one of Items 19 to 22; the vector according to Item 23 or 24; or the vector kit according to any one of Items 25 to 27. (Item 29) 29. The method of claim 28, wherein the cell is derived from a sample isolated from a subject. (Item 30) 18. A pharmaceutical composition comprising a plurality of cells according to any one of items 11 to 17. (Item 31) 31. A method for treating and / or preventing a disease, comprising administering to a subject the pharmaceutical composition according to item 30. (Item 32) The process is as follows: (i) isolation of a cell-containing sample from a subject; (ii) transduction or transfection of said cells with a nucleic acid sequence according to item 18; a nucleic acid construct according to any of items 19 to 22; a vector according to item 23 or 24; or a kit of vectors according to any of items 25 to 27; and (iii) administering to the subject the cells from (ii). 32. The method according to claim 31, comprising: (Item 33) 33. The method of claim 32, wherein the sample is a T cell-containing sample. (Item 34) 34. The method according to item 32 or 33, wherein the disease is cancer. (Item 35) 31. The pharmaceutical composition according to item 30 for use in the treatment and / or prevention of a disease. (Item 36) 18. Use of a cell according to any of items 11 to 17 in the manufacture of a medicament for treating and / or preventing a disease. [Brief description of the drawings]

[0017] [Figure 1] Schematic diagram summarizing the structure of various cytokine receptors, the cell types that produce the cytokines, and the cell types that express the cytokine receptors.

[0018] [Diagram 2]Schematic diagram showing proposed chimeric cytokine receptors. (a) Cytokine receptors for cytokines IL2 and IL7 signal through a common gamma chain and a cytokine-specific alpha / beta chain. (b) One implementation of a chimeric cytokine receptor is to replace the ectodomains of the cytokine alpha / beta and gamma chains with different scFvs (or any other suitable binders) that recognize different epitopes of PSA. (c) An alternative approach is to replace the ectodomains of alpha / beta and gamma with the VH / VL of a PSA-specific antibody, where both VH and VL are involved in binding, such that they become one upon binding.

[0019] [Figure 3-1] Aggregation-based cytokine signaling enhancer. Schematic diagram showing a combination system of chimeric cytokine receptor and CAR. The cell contains two chimeric cytokine receptors that bind to different epitopes on the same soluble ligand. In the absence of soluble ligand (e.g., PSA) but in the presence of cell membrane antigen (e.g., PSMA), signaling occurs thought through the CAR. In the presence of soluble ligand, aggregation of the two chimeric cytokine receptors occurs, resulting in cytokine-based signal enhancement. [Figure 3-2] Aggregation-based cytokine signaling enhancer. Schematic diagram showing a combination system of chimeric cytokine receptor and CAR. The cell contains two chimeric cytokine receptors that bind to different epitopes on the same soluble ligand. In the absence of soluble ligand (e.g., PSA) but in the presence of cell membrane antigen (e.g., PSMA), signaling occurs thought through the CAR. In the presence of soluble ligand, aggregation of the two chimeric cytokine receptors occurs, resulting in cytokine-based signal enhancement.

[0020] [Figure 4]A theoretical construct map for the chimeric cytokine receptor / CAR combination system illustrated in FIG. 3 .

[0021] [Diagram 5] Schematic diagram illustrating an example of a structure for a chimeric transmembrane protein of the invention. The chimeric transmembrane protein comprises a dimerization domain and a cytokine receptor endodomain. The dimerization domain comprises antibody-type heavy and light chain constant regions, so the embodiment shown has a "Fab" type structure. Stable dimerization between these domains brings together the common gamma chain of the IL2 receptor with either the IL-2 receptor beta chain or the IL-7 receptor alpha chain, resulting in constitutive cytokine signaling.

[0022] [Figure 6] IL-2 signaling by chimeric transmembrane proteins. Two chimeric transmembrane proteins with the general structure shown in FIG. 5 were tested for their ability to induce IL-2 signaling. One chimeric transmembrane protein contained the IL2 receptor endodomain and the other contained the IL-7 receptor endodomain. IL-2 signaling was tested using the mouse cell line CTLL2, which depends on IL-2 signaling for growth. As a positive control, CTLL2 cells were cultured with 100 u / mL of mouse IL2. Cells expressing the chimeric transmembrane protein containing the IL2 receptor endodomain (Fab_IL2 endo) supported survival and growth of CTLL2 cells, whereas cells expressing the chimeric transmembrane protein containing the IL-7 receptor (Fab_IL7 endo) did not.

[0023] [Figure 7]Schematic diagram illustrating a panel of PSA chimeric cytokine receptors. A panel of chimeric cytokine receptors (CCRs) targeting PSA was developed using scFvs derived from two antibodies that bind different PSA epitopes: 5D5A5 and 5D3D11. Top left panel: CCR with an IL-2R endodomain with A5 on the chain with the IL2Rβ chain and D11 on the chain with the common γ chain; top right panel: CCR with an IL7R endodomain with A5 on the chain with the IL7Rα chain and D11 on the chain with the common γ chain; bottom left panel: CCR with an IL-2R endodomain with D11 on the chain with the IL2Rβ chain and A5 on the chain with the common γ chain; and bottom right panel: CCR with an IL-7R endodomain with D11 on the chain with the IL7Rα chain and A5 on the chain with the common γ chain. Negative controls were also made for each CCR, in which the IL2Rγ chain was replaced with a rigid linker.

[0024] [Figure 8-1] IL2 signaling from cells expressing PSA chimeric cytokine receptors-CTLL2 proliferation in the presence of PSA. CTLL2 cells were transduced with constructs expressing some of the PSA chimeric cytokine receptors depicted in FIG. 7. Cells were cultured in the presence or absence of IL2 (positive control) and in the presence or absence of 5 ng / mL or 5 μg / mL PSA. CTLL2 proliferation was assessed after 3 and 7 days. Anti-PSA chimeric cytokine receptors with IL2R endodomain supported proliferation of CTLL2 cells in the absence of IL2 and in the presence of PSA, whereas receptors with IL7R endodomain or any CCR containing a rigid linker instead of the common gamma chain did not. [Figure 8-2]IL2 signaling from cells expressing PSA chimeric cytokine receptors-CTLL2 proliferation in the presence of PSA. CTLL2 cells were transduced with constructs expressing some of the PSA chimeric cytokine receptors depicted in FIG. 7. Cells were cultured in the presence or absence of IL2 (positive control) and in the presence or absence of 5 ng / mL or 5 μg / mL PSA. CTLL2 proliferation was assessed after 3 and 7 days. Anti-PSA chimeric cytokine receptors with IL2R endodomain supported proliferation of CTLL2 cells in the absence of IL2 and in the presence of PSA, whereas receptors with IL7R endodomain or any CCR containing a rigid linker instead of the common gamma chain did not.

[0025] [Figure 9] IL2 signaling from cells expressing a PSA chimeric cytokine receptor in the presence of PSA-CTLL2 STAT5 phosphorylation. CTLL2 cells were left untransduced (WT); or transduced with a vector expressing a CCR for PSA (D11-CD8STK-IL2Rg_A5-hinge-IL2Rb) or an equivalent construct with a rigid linker instead of the common gamma chain (D11-CD8STK-RL_A5-hinge-IL2Rb). Cells were incubated with 500 μM pervanadate or 500 ng / mL PSA for 1 or 4 h. Phosphorylation of Y694 of STAT5 was then investigated using phosphoflow. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Summary of the Aspects of the Invention We have developed "chimeric cytokine receptors" (CCRs) in which the binding specificity of a non-cytokine binding molecule is grafted onto the endodomain of a cytokine receptor. Co-expression of such CCRs with chimeric antigen receptors (CARs) helps CAR T cells to engraft and expand in the harsh tumor microenvironment. The requirement for a ligand for the CCR as well as a ligand for the CAR to be present adds another layer of selectivity and helps prevent on-target off-tumor toxicity.

[0027] For example, we developed cells that co-express a CAR with a chimeric cytokine receptor that detects PSA and delivers IL2 / 15 or IL7 signals to the CAR T cells. In this way, the CAR T cells are stimulated to proliferate selectively only in the prostate cancer microenvironment, and in the absence of PSA (i.e., after the patient is in remission), cytokine stimulation is lost.

[0028] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: an exodomain that binds to a ligand selected from a tumor secreted factor, a chemokine, and a cell surface antigen; and Cytokine Receptor Endodomains The present invention provides a chimeric cytokine receptor (CCR) comprising:

[0029] In a first embodiment of the first aspect of the invention, the chimeric cytokine receptor comprises two polypeptides: (i) (a) a first antigen-binding domain that binds to a first epitope of the ligand; (b) The first chain of a cytokine receptor endodomain a first polypeptide comprising: (ii) (a) a second antigen-binding domain that binds to a second epitope of the ligand; (b) The second chain of a cytokine receptor endodomain. A second polypeptide comprising Figure 2b illustrates such an arrangement.

[0030] Each of the first and second antigen-binding domains may be, for example, a single chain variable fragment (scFv) or a single domain binder.

[0031] In a second embodiment of the first aspect of the invention, the chimeric cytokine receptor comprises two polypeptides: (i) (a) Heavy chain variable domain (VH) (b) The first chain of a cytokine receptor endodomain a first polypeptide comprising: (ii) (a) Light chain variable domain (VL) (b) The second chain of a cytokine receptor endodomain. A second polypeptide comprising Includes.

[0032] Figure 2c illustrates such an arrangement.

[0033] The first and second chains for a cytokine receptor endodomain may be different and may be selected from the α, β and γ chains of a type I cytokine receptor endodomain.

[0034] Alternatively, the first and second chains for a cytokine receptor endodomain may be the same and selected from the α, β and γ chains of a type I cytokine receptor endodomain.

[0035] For example, a cytokine receptor endodomain may be (i) IL-2 receptor β chain endodomain (ii) the α-chain endodomain of the IL-7 receptor; (iii) the α-chain endodomain of the IL-15 receptor; or (iv) Common γ chain receptor endodomain may include.

[0036] The cytokine receptor endodomain may comprise (i), (ii) or (iii); and (iv).

[0037] The ligand can be a tumor-secreted factor, for example, a tumor-secreted factor selected from prostate specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), and CA125.

[0038] The ligand can be a chemokine, for example, a chemokine selected from a chemokine selected from CXCL12, CCL2, CCL4, CCL5 and CCL22.

[0039] The ligand can be a cell surface molecule, such as a transmembrane protein. The ligand can be, for example, CD22.

[0040] In a second aspect, the present invention provides a cell comprising a chimeric cytokine receptor according to the first aspect of the invention.

[0041] The cell can contain a first chimeric cytokine receptor and a second chimeric cytokine receptor that bind to different epitopes on the same ligand.

[0042] The cell may comprise a first chimeric cytokine receptor comprising an α or β chain of a type I cytokine receptor endodomain, and a second chimeric cytokine receptor comprising a γ chain of a type I cytokine receptor endodomain, such that when the first chimeric cytokine receptor and the second cytokine receptor bind to a ligand, combined signal transduction occurs via the α / β and γ chains.

[0043] The cells may also contain a chimeric antigen receptor, for example a chimeric antigen receptor that binds to a tumor-associated cell surface antigen.

[0044] The chimeric antigen receptor may bind to a cell surface antigen associated with prostate cancer (e.g., prostate stem cell antigen (PSCA) or prostate specific membrane antigen (PSMA)).

[0045] When a CCR recognizes a cell surface antigen, the CCR and the CAR may recognize a cell surface antigen co-expressed on the same target (e.g., tumor) cell. For example, in the case of B cell malignancies, the CAR may recognize a cell surface antigen such as CD19, and the CCR may recognize a molecule (e.g., CD22) co-expressed on the target cell surface, thereby enhancing engraftment.

[0046] In a third aspect, the present invention provides a nucleic acid sequence encoding a chimeric cytokine receptor (CCR) according to the first aspect of the invention.

[0047] In a fourth aspect, the present invention provides a nucleic acid construct comprising a first nucleic acid sequence encoding a first CCR and a second nucleic acid sequence encoding a second CCR, the nucleic acid construct having the structure: AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 (where: AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR; spacer 1 is a nucleic acid sequence encoding the spacer of the first CCR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR; endo1 is a nucleic acid sequence encoding the endodomain of the first CCR; coexpr is a nucleic acid sequence that allows for coexpression of both CCRs, AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR; spacer2 is a nucleic acid sequence encoding the spacer of the second CCR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; Endo2 is a nucleic acid sequence encoding the endodomain of the second CCR. has. The nucleic acid construct may also encode a chimeric antigen receptor (CAR). In this embodiment, the nucleic acid construct has the structure: (i) CCRAgB1-CCR spacer1-CCRTM1-CCR end1-coexpr1-CCRAgB2-CCR spacer2-CCRTM2-CCR end2-coexpr2-CARAgB-CAR spacer-CARTM-CAR end; (ii) CCRAgB1-CCR spacer1-CCRTM1-CCR endo1-coexpr1-CARAgB-CAR spacer-CARTM-CAR endo-coexpr2-CCRAgB2-CCR spacer2-CCRTM2-CCR endo2; or (iii) CARAgB-CAR spacer-CARTM-CAR endo-coexpr1-CCRAgB1-CCR spacer1-CCRTM1-CCR endo1-coexpr2-CCRAgB2-CCR spacer2-CCRTM2-CCR endo2; (where: CCRAgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR; CCR spacer 1 is a nucleic acid sequence encoding the spacer of the first CCR; CCRTM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR; CCR endo1 is a nucleic acid sequence encoding the endodomain of the first CCR; CCRAgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR; CCR spacer2 is a nucleic acid sequence encoding the spacer of a second CCR; CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; CCR endo2 is a nucleic acid sequence encoding the endodomain of a second CCR; Coexpr1 and coexpr2 are nucleic acid sequences that allow for coexpression of two adjacent sequences; CARAgB is a nucleic acid sequence encoding the antigen-binding domain of the CAR; CAR spacer is a nucleic acid sequence encoding the spacer of the CAR; CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR; CAR endo is a nucleic acid sequence encoding the endodomain of CAR) may have:

[0048] Any or all of the sequences coexpr, coexpr1, coexpr2 may encode a sequence that includes a self-cleaving peptide.

[0049] To avoid homologous recombination, alternative codons may be used in the regions of the sequence that code for the same or similar amino acid sequences.

[0050] In a fifth aspect, the present invention provides a vector comprising a nucleic acid construct according to the fourth aspect of the invention.

[0051] The vector can be, for example, a retroviral or lentiviral vector or a transposon.

[0052] In a sixth aspect, the present invention provides a method for producing a composition comprising the steps of: i) a vector comprising a nucleic acid sequence encoding a first CCR according to the first aspect of the invention; and ii) A vector comprising a nucleic acid sequence encoding a second CCR according to the second aspect of the invention. A kit comprising:

[0053] The kit may also include a vector containing a nucleic acid sequence encoding the chimeric antigen receptor.

[0054] The above kit is i) a vector comprising a nucleic acid sequence encoding a CCR according to the first aspect of the invention; and ii) A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor. may include.

[0055] In a seventh aspect, the present invention provides a method for producing a cell according to the second aspect of the invention, the method comprising the step of introducing into a cell a nucleic acid sequence according to the third aspect of the invention; a nucleic acid construct according to the fourth aspect of the invention; a vector according to the fifth aspect of the invention; or a kit of vectors according to the sixth aspect of the invention.

[0056] The cells can be from a sample isolated from a subject.

[0057] In an eighth aspect, there is provided a pharmaceutical composition comprising a plurality of cells according to the second aspect of the invention.

[0058] In a ninth aspect, there is provided a method for treating and / or preventing a disease, the method comprising administering to a subject a pharmaceutical composition according to the eighth aspect of the invention.

[0059] The method comprises the steps of: (i) isolation of a cell-containing sample from a subject; (ii) transduction or transfection of a cell with a nucleic acid sequence according to the third aspect of the invention; a nucleic acid construct according to the fourth aspect of the invention; a vector according to the fifth aspect of the invention; or a kit of vectors according to the sixth aspect of the invention; and (iii) administering to the subject cells from (ii). may include.

[0060] The sample may be a T cell-containing sample.

[0061] The disease may be cancer.

[0062] There is also provided a pharmaceutical composition according to the eighth aspect of the invention for use in the treatment and / or prevention of disease.

[0063] There is also provided the use of a cell according to the second aspect of the invention in the manufacture of a medicament for treating and / or preventing a disease.

[0064] Further aspects of the present invention are summarized in the following numbered paragraphs:

[0065] 1. a dimerization domain; and Cytokine Receptor Endodomains A chimeric transmembrane protein comprising:

[0066] 2. The dimerization domain is a heavy chain constant domain (C H ) and the light chain constant domain (C L 2. The chimeric transmembrane protein of paragraph 1, comprising a dimerization portion of

[0067] 3. Two polypeptides: (i) (a) a first dimerization domain; and (b) The first chain of a cytokine receptor endodomain a first polypeptide comprising: (ii) (a) a second dimerization domain that dimerizes with the first dimerization domain; and (b) The second chain of a cytokine receptor endodomain. A second polypeptide comprising 4. The chimeric transmembrane protein of any of the preceding paragraphs, comprising:

[0068] 4. The chimeric transmembrane protein of paragraph 3, wherein the first and second dimerization domains dimerize spontaneously or in the presence of a chemical inducer of dimerization (CID).

[0069] 5. Two polypeptides: (i) (a) Heavy chain constant domain (CH) (b) The first chain of a cytokine receptor endodomain a first polypeptide comprising: (ii) (a) Light chain constant domain (CL) (b) The second chain of a cytokine receptor endodomain. A second polypeptide comprising 5. The chimeric transmembrane protein of paragraph 2, 3 or 4, comprising:

[0070] 6. A chimeric transmembrane protein according to paragraph 5, wherein the first and second chains for a cytokine receptor endodomain are different and are selected from the α, β and γ chains of a type I cytokine receptor endodomain.

[0071] 7. A chimeric transmembrane protein according to paragraph 5, wherein the first and second chains for a cytokine receptor endodomain are the same and are selected from the α, β and γ chains of a type I cytokine receptor endodomain.

[0072] 8. A cytokine receptor endodomain, (i) IL-2 receptor β chain endodomain (ii) the α-chain endodomain of the IL-7 receptor; or (iii) the α-chain endodomain of the IL-15 receptor; and / or (iv) Common γ chain receptor endodomain 4. The chimeric transmembrane protein of any of the preceding paragraphs, comprising:

[0073] 9. The chimeric transmembrane protein of paragraph 5, wherein the first polypeptide comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH); and the second polypeptide comprises a light chain variable domain (VL) and a light chain constant domain (CL).

[0074] 10. A chimeric transmembrane protein according to paragraph 9, comprising a Fab exodomain.

[0075] 11. A cell comprising a chimeric transmembrane protein according to any of the preceding paragraphs.

[0076] 12. The cell of paragraph 11, which also contains a chimeric antigen receptor.

[0077] 13. The cell of paragraph 12, wherein the chimeric antigen receptor binds to a tumor-associated cell surface antigen.

[0078] 14. A nucleic acid sequence encoding a chimeric transmembrane protein according to any one of paragraphs 1 to 10.

[0079] 15. A nucleic acid construct comprising a first nucleic acid sequence encoding a first polypeptide as defined in paragraph 3 and a second nucleic acid sequence encoding a second polypeptide as defined in paragraph 3, the nucleic acid construct having the structure: Dim1-TM1-End1-coexpr-Dim2-TM2-End2 (where: Dim1 is a nucleic acid sequence encoding the first dimerization domain; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence that allows for coexpression of both CCRs, Dim2 is a nucleic acid sequence encoding a second dimerization domain; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo2 is a nucleic acid sequence encoding the endodomain of the second polypeptide. has.

[0080] 16. The nucleic acid construct of paragraph 15, which also encodes a chimeric antigen receptor (CAR).

[0081] 17. A nucleic acid construct according to paragraph 15 or 16, wherein coexpr encodes a sequence comprising a self-cleaving peptide.

[0082] 18. A nucleic acid construct according to any of paragraphs 15 to 17, wherein alternative codons are used in regions of the sequence encoding identical or similar amino acid sequences, to avoid homologous recombination.

[0083] 19. A vector comprising a nucleic acid construct according to any one of paragraphs 15 to 18.

[0084] 20. A retroviral or lentiviral vector or transposon according to paragraph 19.

[0085] twenty one. i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined in paragraph 3; and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined in paragraph 3. Including the kit.

[0086] 22. The kit according to paragraph 21, also comprising a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

[0087] twenty three. i) a vector comprising a nucleic acid sequence encoding a chimeric transmembrane protein as defined in any of paragraphs 1 to 10; and ii) A kit comprising a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

[0088] 24. A method for producing a cell according to any one of paragraphs 11 to 13, the method comprising the step of introducing into a cell a nucleic acid sequence according to paragraph 14; a nucleic acid construct according to any one of paragraphs 15 to 18; a vector according to paragraph 19 or 20; or a kit of vectors according to any one of paragraphs 21 to 23.

[0089] 25. The method of paragraph 24, wherein the cells are derived from a sample isolated from a subject.

[0090] 26. A pharmaceutical composition comprising a plurality of cells according to any one of paragraphs 11 to 13.

[0091] 27. A method for treating and / or preventing a disease, the method comprising administering to a subject a pharmaceutical composition according to paragraph 26.

[0092] 28. The following process: (i) isolation of a cell-containing sample from a subject; (ii) transduction or transfection of a cell with a nucleic acid sequence according to paragraph 14; a nucleic acid construct according to any one of paragraphs 15 to 18; a vector according to paragraph 19 or 20; or a kit of vectors according to any one of paragraphs 21 to 23; and (iii) administering to the subject cells from (ii). 28. The method of claim 27, comprising:

[0093] 29. The method of paragraph 28, wherein the sample is a T cell-containing sample.

[0094] 30. The method of paragraph 28 or 29, wherein the disease is cancer.

[0095] 31. A pharmaceutical composition according to paragraph 26 for use in the treatment and / or prevention of a disease.

[0096] 32. Use of a cell according to any of paragraphs 11 to 13 in the manufacture of a medicament for treating and / or preventing a disease.

[0097] Detailed Description Chimeric cytokine receptors (CCRs) A chimeric cytokine receptor (CCR) is a molecule that contains a cytokine receptor endodomain and a heterologous ligand-binding exodomain. The heterologous exodomain binds to a ligand other than the cytokine for which the endodomain was derived. Thus, it is possible to alter the ligand specificity of a cytokine receptor by grafting in a heterologous binding specificity.

[0098] Chimeric cytokine receptors (i) the ligand-binding exodomain; (ii) an optional spacer; (iii) a transmembrane domain; and (iv) Cytokine receptor endodomains Includes.

[0099] Cytokine Receptors and Signal Transduction Many cellular functions are regulated by members of the cytokine receptor superfamily. Signaling by these receptors depends on their association with Janus kinases (JAKs), which couple ligand binding to tyrosine phosphorylation of signaling proteins recruited to the receptor complex. These include the signal transducers and activators of transcription (STATs), a family of transcription factors that contribute to the diversity of cytokine responses.

[0100] A chimeric cytokine receptor of the invention, upon binding to its ligand, may initiate one or more of the following intracellular signaling pathways: (i) JAK-STAT pathway (ii) the MAP kinase pathway; and (iii) The phosphoinositide 3-kinase (PI3K) pathway.

[0101] The JAK-STAT system consists of three major components: (1) receptors, (2) Janus kinases (JAKs) and (3) signal transducers and activators of transcription (STATs).

[0102] JAKs have tyrosine kinase activity and bind to cytokine receptors on the cell surface. Binding of a ligand to this receptor induces activation of JAKs. Upon increasing kinase activity, JAKs phosphorylate tyrosine residues on the receptor, creating sites for interaction with proteins containing SH2 domains that bind phosphotyrosines. STATs with SH2 domains that can bind to these phosphotyrosine residues are recruited to the receptor and are themselves tyrosine phosphorylated by JAKs. These phosphotyrosines then act as binding sites for the SH2 domains of other STATs, mediating their dimerization. Various STATs form hetero- or homodimers. Activated STAT dimers accumulate in the cell nucleus and activate the transcription of their target genes.

[0103] Cytokine Receptor Endodomains The chimeric cytokine receptors of the present invention comprise an endodomain that triggers "cytokine-type" cell signaling (alone or in the presence of another chimeric cytokine receptor) when the exodomain binds to its ligand.

[0104] The endodomain may be a cytokine receptor endodomain.

[0105] The endodomain may be derived from a type I cytokine receptor, which shares a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane.

[0106] The endodomain may be derived from a type II cytokine receptor, including those that bind type I and type II interferons, as well as those that bind members of the interleukin-10 family (interleukin-10, interleukin-20 and interleukin-22).

[0107] Type I cytokine receptors include (i) interleukin receptors (e.g., receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL13, IL-15, IL-21, IL-23 and IL-27); (ii) colony-stimulating factor receptors (e.g., receptors for erythropoietin, GM-CSF, and G-CSF); and (iii) hormone receptors / neuropeptide receptors (e.g., hormone receptors and prolactin receptors) Includes:

[0108] Members of the type I cytokine receptor family contain different chains, some of which are involved in ligand / cytokine interaction and others involved in signal transduction, for example, the IL-2 receptor contains an α chain, a β chain and a γ chain.

[0109] The common gamma chain of the IL-2 receptor (also known as CD132) is shared among the IL-2, IL-4, IL-7, IL-9, IL-13 and IL-15 receptors.

[0110] IL-2 IL-2 binds to the IL-2 receptor, which has three forms produced by different combinations of three different proteins often referred to as the α, β, and γ "chains"; these subunits are also part of the receptors for other cytokines. The β and γ chains of the IL-2R are members of the type I cytokine receptor family.

[0111] These three receptor chains are separately and differentially expressed on various cell types and can assemble in different combinations and orders to generate low-, intermediate- and high-affinity IL-2 receptors.

[0112] The α chain binds IL-2 with low affinity, whereas the combination of β and γ together forms a complex that binds IL-2 with intermediate affinity primarily on memory T cells and NK cells; all three receptor chains form a complex that binds IL-2 with high affinity (Kd approx. 10-11 M) on activated and regulatory T cells.

[0113] These three IL-2 receptor chains span the cell membrane and extend into the cell, delivering biochemical signals to the interior of the cell. The alpha chain is not involved in signal transduction, and the beta chain forms a complex with the tyrosine phosphatase JAK1. Similarly, the gamma chain forms a complex with another tyrosine kinase called JAK3. These enzymes are activated by IL-2 binding to the ectodomain of the IL-2R.

[0114] IL-2 signaling promotes the differentiation of early T cells into effector and memory T cells when they are also stimulated with antigen. Through their role in the generation of T cell immunological memory, which depends on the expansion in number and function of antigen-selected T cell clones, they also play a key role in long-term cell-mediated immunity.

[0115] A chimeric cytokine receptor of the present invention may comprise the IL-2 receptor beta chain and / or the IL-2 receptor (ie, common) gamma chain.

[0116] The amino acid sequences for the IL-2 β chain and common γ chain endodomains are shown as SEQ ID NOs:1 and 2. SEQ ID NO:1: Endodomain derived from the human common gamma chain: [ka] SEQ ID NO:2: Endodomain derived from human IL-2Rβ: [ka]

[0117] The term "derived from" means that the endodomain of the chimeric cytokine receptor of the present invention has a sequence identical to the wild-type sequence of the endogenous molecule, or a mutant form thereof that retains the ability to form a complex with JAK-1 or JAK-3 and to activate one of the signal transduction pathways mentioned above.

[0118] A "mutant" sequence has at least 80, 85, 90, 95, 98 or 99% sequence identity to a wild-type sequence (e.g., SEQ ID NO: 1 or 2), so long as the mutant sequence retains the function of the wild-type sequence, i.e., the ability to form a complex with JAK-1 or JAK-3, and, for example, the ability to activate the JAK-STAT signaling pathway.

[0119] The percentage identity between two polypeptide sequences can be readily determined by programs such as BLAST, which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0120] IL-7 The interleukin-7 receptor is composed of two chains: the interleukin-7 receptor-α chain (CD127) and the common gamma chain receptor (CD132). This common gamma chain receptor is shared with a variety of cytokines, including interleukin-2, -4, -9, and -15. The interleukin-7 receptor is expressed on a variety of cell types, including naive and memory T cells.

[0121] The interleukin-7 receptor plays a crucial role in lymphocyte development, particularly in V(D)J recombination. IL-7R also controls the accessibility of the genomic region containing the T cell receptor gamma gene through STAT5 and histone acetylation. Knockout studies in mice suggest that prevention of apoptosis is an essential function of this protein in T lymphocyte differentiation and activation.

[0122] A chimeric cytokine receptor of the present invention may comprise the α chain of the IL-7 receptor and / or the γ chain of the IL-7 receptor (ie, common), or a variant thereof.

[0123] The amino acid sequence for the endodomain of the α chain of IL-7 is shown as SEQ ID NO:3. SEQ ID NO:3 - Endodomain derived from human IL-7Rα: [ka] [ka]

[0124] IL-15 Interleukin 15 (IL-15) is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and several other cells) following viral infection. IL-15 induces cell proliferation in natural killer cells.

[0125] The interleukin-15 receptor consists of the interleukin-15 receptor alpha subunit and shares common beta and gamma subunits with the IL-2 receptor.

[0126] Spacer The chimeric cytokine receptors of the invention may comprise a spacer that connects the antigen-binding domain with the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to be oriented in various directions to allow antigen binding.

[0127] When the cell of the present invention comprises two or more chimeric cytokine receptors, the spacer can be the same or different.When the cell of the present invention comprises a chimeric cytokine receptor (CCR) and a chimeric antigen receptor (CAR), the spacer of the CCR and the CAR can be different, for example, have different lengths.The spacer of the CAR can be longer than the spacer of each CCR.

[0128] The spacer sequence may comprise, for example, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk. The linker may alternatively comprise an alternative linker sequence having similar length and / or domain spacing characteristics as an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk.

[0129] The human IgG1 spacer may be altered to remove the Fc binding motif.

[0130] Examples of amino acid sequences for these spacers are shown below: SEQ ID NO: 4 (Human IgG1 hinge-CH2CH3) [ka] SEQ ID NO:5 (human CD8 stalk): [ka] SEQ ID NO:6 (human IgG1 hinge): [ka]

[0131] Transmembrane domain The transmembrane domain is the sequence of the CCR that spans the membrane. The transmembrane domain may contain a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which provides good receptor stability.

[0132] Alternatively, the transmembrane domain may be derived from a cytokine receptor, for example the same cytokine from which the endodomain is derived.

[0133] The transmembrane domain can be derived, for example, from IL-2R, IL-7R or IL-15R. SEQ ID NO:7 - Transmembrane derived from human common gamma chain: [ka] SEQ ID NO:8 - Transmembrane derived from human IL-2Rβ: [ka] SEQ ID NO:9 - Transmembrane derived from human IL-7Rα: [ka] SEQ ID NO:10 - Transmembrane derived from human IL-15Rα: [ka]

[0134] Ligand-binding exodomain The ligand-binding domain comprises an antigen-binding domain, which binds to a target ligand for the CCR, i.e., a tumor-secreted factor or a chemokine or a cell surface antigen.

[0135] Numerous antigen-binding domains are known in the art, including those based on the antigen-binding site of antibodies, antibody mimetics, and T-cell receptors.For example, the antigen-binding domain may comprise a single-chain variable fragment (scFv) derived from a monoclonal antibody; a binding domain derived from the natural receptor of the target antigen; a peptide with sufficient affinity for the target ligand; a single domain binder (e.g., from camelids); an artificial single binder (e.g., Darpin); or a single chain derived from a T-cell receptor.

[0136] The term "ligand" is used synonymously with "antigen" to mean the entity that is specifically recognized and bound by the antigen-binding domain of a CCR.

[0137] When the ligand is a tumor-secreted factor, the antigen-binding domain may comprise an immunoglobulin-based antigen-binding site (e.g., an scFv or single domain binder).

[0138] Where the ligand is a chemokine, the antigen-binding domain may comprise the chemokine-binding portion of the natural receptor for that chemokine.

[0139] Ligand The CCRs of the present invention bind to a ligand.

[0140] The ligand can be a soluble ligand, such as a tumor-secreted factor or a chemokine.

[0141] Alternatively, the ligand may be a membrane-bound ligand (eg, a cell surface antigen).

[0142] The term "soluble ligand" is used to refer to a ligand or antigen that is not part of or attached to a cell and is free to move in the extracellular space, for example in the body fluids of a tissue of interest. Soluble ligands may be present in a cell-free state in the serum, plasma or other body fluids of an individual.

[0143] The soluble ligand may be associated with the presence or pathology of a particular disease, such as cancer.

[0144] Soluble ligands may be part of the cancer secretome, i.e., the collection of factors secreted by the tumor, and may originate from cancer stem cells, non-stem cells, or the surrounding stroma. Soluble ligands may be secreted or shed by tumor cells (see next section).

[0145] Soluble ligand can be characteristic of disease or diseased tissue.Soluble ligand can be found exclusively in diseased subjects, or can be found at higher levels in diseased subjects compared to healthy subjects; or in diseased tissue compared to healthy tissue.Soluble ligand can be expressed at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or 100,000-fold higher in diseased subjects compared to healthy subjects; or in diseased tissue compared to healthy tissue.

[0146] The terms "cell surface antigen" and "cell surface ligand" are used synonymously with "membrane-bound antigen" and "membrane-bound ligand," which refer to a ligand that is attached to or expressed on the cell surface. A cell surface ligand can be, for example, a transmembrane protein.

[0147] The cells on which the cell surface ligand is found may be target cells, such as cancer cells.

[0148] The cell surface ligand may be associated with the presence or pathology of a particular disease, such as cancer, or may be characteristic of the cell type of the target cell (e.g., B cell) without necessarily being associated with a pathological condition.

[0149] When a cell surface ligand is characteristic of a disease or diseased tissue, the cell surface ligand may be found exclusively on the relevant cells of a subject with a disease, or may be found at higher levels on the relevant cells of a subject with a disease compared to a healthy subject; or on the relevant cells of a diseased tissue compared to a healthy tissue. The cell surface ligand may be expressed at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold higher on the cells of a subject with a disease compared to a healthy subject; or on a diseased tissue compared to a healthy tissue.

[0150] Tumor-secreted factors The ligand recognized by the CCR can be a soluble ligand secreted or shed by the tumor.

[0151] This "tumor-secreted factor" can be, for example, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF) or cancer antigen-125 (CA-125).

[0152] The tumor-secreted factors may be soluble ligands that are not cytokines. Thus, the CCRs of the present invention graft binding specificity for a non-cytokine ligand onto the endodomain of a cytokine receptor.

[0153] Prostate-specific antigen (PSA) The soluble ligand can be prostate specific antigen (PSA).

[0154] Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), is a glycoprotein enzyme that in humans is encoded by the KLK3 gene. PSA is a member of the kallikrein-related peptidase family and is secreted by epithelial cells of the prostate gland.

[0155] PSA is present in small amounts in the serum of men with healthy prostates, but is elevated in individuals with prostate cancer and other prostate disorders.

[0156] PSA is a 237 residue glycoprotein that is activated by KLK2. Its physiological role is the liquefaction of the seminal clot components, resulting in the release of spermatozoa. In cancer, PSA may be involved in the process of neoplastic growth and metastasis.

[0157] PSA is a chymotrypsin-like serine protease with a typical His-Asp-Ser triad and a catalytic domain similar to that of other kallikrein-related peptidases. Crystal structures of PSA have been obtained i) in complex with the monoclonal antibody (mAb) 8G8F5, and ii) in a sandwich complex with two mAbs, 5D5A5 and 5D3D11 (Stura et al. (J. Mol. Biol. (2011) 414:530-544).

[0158] Various monoclonal antibodies are known, including clones 2G2-B2, 2D8-E8, IgG1 / K, as described in Bavat et al., Avicenna J. Med. Biotechnol. 2015, 7:2-7; and Leinonen (2004) 289:157-67.

[0159] A CCR of the invention can, for example, comprise the six CDRs or the VH and / or VL domains from a mAb that binds PSA (eg, 8G8F5, 5D5A5 or 5D3D11).

[0160] When a CCR contains two antigen-binding specificities that bind to different epitopes on PSA, one can be based, for example, on 5D3D11 and the other can be based, for example, on 5D5A5.

[0161] The amino acid sequences for VH and VL of 5D3D11 and 5D5A5 are shown below, with the complementarity determining regions (CDRs) highlighted in bold. [ka] [ka]

[0162] Where a cell comprises two CCRs, the antigen-binding domain of a first CCR may comprise the six CDRs derived from 5D5A5, and the antigen-binding domain of a second CCR may comprise the six CDRs derived from 5D3D11.

[0163] The antigen-binding domain of the first CCR may comprise the VH and / or VL domains from 5D5A5 or variants thereof; the antigen-binding domain of the second CCR may comprise the VH and / or VL domains from 5D3D11 or variants thereof. The variant VH and VL domains may have at least 80, 90, 95 or 99% identity with the sequences shown above, but retain PSA binding activity.

[0164] Cells expressing a CCR that binds PSA may be useful in the treatment of prostate cancer.

[0165] Carcinoembryonic antigen (CEA) The soluble ligand can be CEA.

[0166] Carcinoembryonic antigen (CEA) refers to a series of highly related glycoproteins involved in cell adhesion. CEA is normally produced in gastrointestinal tissues during fetal development, but its production stops before birth. Therefore, CEA is usually present at very low levels in the blood of healthy adults. However, its serum levels are elevated in some types of cancer, which means that it can be used as a tumor marker in clinical tests.

[0167] CEA is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein whose specialized sialofucosylated glycoforms serve as functional colon cancer L-selectin and E-selectin ligands that may be crucial for metastatic dissemination of colon cancer cells. Immunologically, they are characterized as members of the CD66 surface antigen cluster.

[0168] CEA and related genes constitute the CEA family, which belongs to the immunoglobulin superfamily. In humans, the carcinoembryonic antigen family consists of 29 genes, 18 of which are normally expressed. The following is a list of human genes encoding carcinoembryonic antigen-related cell adhesion proteins: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21.

[0169] Various antibodies targeting CEA are described in WO2011 / 034660.

[0170] Cells expressing a CCR to CEA may be useful, for example, in the treatment of colorectal cancer.

[0171] Vascular endothelial growth factor (VEGF) The soluble ligand can be VEGF.

[0172] Vascular endothelial growth factor (VEGF) is a signaling protein produced by cells that stimulates blood vessel development and angiogenesis. Vascular endothelial growth factor is part of a system that restores oxygen supply to tissues when blood circulation is inadequate. Serum concentrations of VEGF are high in bronchial asthma and diabetes mellitus. The normal function of VEGF is to create new blood vessels during embryonic development, to create new blood vessels after injury, to create muscle after exercise, and to create new vessels that bypass blocked vessels (collateral circulation).

[0173] When overexpressed, VEGF can contribute to disease: solid tumors cannot grow beyond a limited size without an adequate blood supply; cancers that express VEGF can grow and metastasize.

[0174] VEGFs are a subfamily of the platelet-derived growth factor family of cystine-knot growth factors. They are key signaling proteins involved in both vascular development (the de novo formation of the fetal circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).

[0175] The VEGF family includes five members in mammals: VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D.

[0176] Various antibodies against VEGF are known, such as bevacizumab (Avastin) and ranibizumab (Lucentis).

[0177] Cancer antigen 125 (CA-125) CA-125 is associated with ovarian cancer and is the most frequently used biomarker for its detection. Although CA-125 is best known as a marker for ovarian cancer, it may also be elevated in other cancers, including uterine, fallopian tube, lung, breast, and gastrointestinal cancers.

[0178] The sequence of human CA-125 (also known as mucin-16) is available under NCBI accession number 078966.

[0179] Several CA125-binding monoclonal antibodies are known, including OC125 and M11 (Nustad et al., 1996, Tumour Biol. 17:196-329). In this study, the specificity of 26 monoclonal antibodies against the CA125 antigen was examined. The CA125 antigen was found to have only two major antigenic domains, which classify the antibodies as OC125-like (group A) or M11-like (group B).

[0180] The chimeric cytokine receptors of the invention may comprise antigen-binding domains derived from such antibodies. Cells containing such CCRs may be useful, for example, in the treatment of ovarian cancer.

[0181] The tumor-secreted factor (or transmembrane protein, if membrane-bound) may be selected from the following non-exhaustive list:

[0182] Rearrangements and overexpression of the ALK gene leading to mutated forms of the ALK protein Alpha-fetoprotein (AFP) Beta-2-microglobulin (B2M) Beta-human chorionic gonadotropin (beta-hCG) BRAF V600 mutation resulting in mutated B-REF protein C-kit / CD117 CA15-3 / CA27.29 CA19-9 Calcitonin CD20 Chromogranin A (CgA) Cytokeratin fragment 21-1 EGFR gene mutation analysis Estrogen receptor (ER) / progesterone receptor (PR) Fibrin / fibrinogen HE4 HER2 / neu gene amplification or protein overexpression Immunoglobulins KRAS gene mutation analysis Lactate dehydrogenase Neuron-specific enolase (NSE) Nuclear matrix protein 22 Programmed Death-Ligand 1 (PD-L1) Thyroglobulin Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1)

[0183] Chemokines Chemokines are chemotactic cytokines. Cell migration is guided by gradients of chemokines embedded and immobilized in the extracellular matrix. Positively charged chemokines, such as CXCL12, bind to negatively charged ECM molecules. These gradients provide a route for homing of cancer and immune cells. The effect on T cells appears to be inhibitory to the homing of cytotoxic T cells, whereas regulatory T cells appear to be attracted.

[0184] Chemokines have a mass of approximately 8-10 kilodaltons and contain four cysteine ​​residues at conserved positions that are important for the formation of their three-dimensional shape.

[0185] Some chemokines are thought to be pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to sites of infection, whereas others are thought to be homeostatic and are involved in the control of cell migration in the normal processes of tissue maintenance or development.

[0186] Chemokines are classified into four major subfamilies: CXC, CC, CX3C and XC. All of these proteins exert their biological effects by interacting with G protein-coupled transmembrane receptors, called chemokine receptors, which are selectively found on the surface of target cells.

[0187] The main role of chemokines is to act as chemoattractants to guide cell migration. Cells attracted by chemokines follow the signal of increasing chemokine concentration toward the source of the chemokine. Some chemokines control cells of the immune system during processes of immune surveillance, such as directing lymphocytes to lymph nodes, so that they may screen for infiltrating pathogens by interacting with antigen-presenting cells present in these tissues. Other chemokines are inflammatory and are released by a wide variety of cells in response to bacterial infections, viruses, and other agents. Their release is often stimulated by proinflammatory cytokines such as interleukin-1. Inflammatory chemokines function primarily as chemoattractants for leukocytes, recruiting monocytes, neutrophils, and other blood-borne effector cells to sites of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate immune responses or promote wound healing. They are released by many different cell types and act to guide cells of both the innate and adaptive immune systems.

[0188] CC chemokines CC chemokine (or β-chemokine) proteins have two adjacent cysteines (amino acids) near the amino terminus. At least 27 distinct members of this subgroup have been described in mammals, called CC chemokine ligands (CCL)-1 through -28; CCL10 is identical to CCL9. Chemokines in this subfamily usually contain four cysteines (C4-CC chemokines), but a few CC chemokines have six cysteines (C6-CC chemokines). C6-CC chemokines include CCL1, CCL15, CCL21, CCL23, and CCL28. CC chemokines induce migration of monocytes and other cell types, such as NK cells and dendritic cells.

[0189] Examples of CC chemokines include monocyte chemoattractant protein-1 (MCP-1 or CCL2), which induces monocytes to leave the bloodstream and enter surrounding tissues to become tissue macrophages.

[0190] CCL5 (or RANTES) attracts cells such as T cells, eosinophils and basophils that express the receptor CCR5.

[0191] CXC chemokines The two N-terminal cysteines of CXC chemokines (or α-chemokines) are separated by one amino acid, represented in the name by "X". There are 17 different CXC chemokines described in mammals, which are subdivided into two categories: those that have a specific amino acid sequence (or motif) of glutamic acid-leucine-arginine (or ELR for short) immediately before the first cysteine ​​of the CXC motif (ELR positive) and those that do not have the ELR motif (ELR negative). ELR positive CXC chemokines specifically induce neutrophil migration and interact with the chemokine receptors CXCR1 and CXCR2.

[0192] C chemokine The third group of chemokines, known as the C chemokines (or gamma chemokines), differ from all other chemokines in that they have only two cysteines; one at the N-terminus and one downstream. Two chemokines have been described for this subgroup, termed XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β).

[0193] CX3C chemokine CX3C chemokines have three amino acids between two cysteines. The only CX3C chemokine discovered so far is called fractalkine (or CX3CL1). CX3C chemokines are secreted and bound to the surface of cells that express them, thereby acting as both chemoattractants and adhesion molecules.

[0194] Chemokine receptors are G protein-coupled receptors containing seven transmembrane domains found on the leukocyte surface. Approximately 19 different chemokine receptors have been characterized so far, which are divided into four families according to the type of chemokine they bind; CXCR, which binds CXC chemokines, CCR, which binds CC chemokines, CX3CR1, which binds only one CX3C chemokine (CX3CL1), and XCR1, which binds two XC chemokines (XCL1 and XCL2). Chemokine receptors share many structural features; they are similar in size (having about 350 amino acids), have a short acidic N-terminus, a seven-helical transmembrane domain with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus that contains serine and threonine residues that are important for the regulation of the receptor. The first two extracellular loops of chemokine receptors each have a conserved cysteine ​​residue that allows the formation of disulfide bridges between these loops. G proteins couple to the C-terminus of chemokine receptors to enable intracellular signal transduction following receptor activation, whereas the N-terminal domain of the chemokine receptor determines the specificity of ligand binding.

[0195] CXCL12 CXCL12 has strong chemotactic properties for lymphocytes. CXCL12 plays a key role in angiogenesis by recruiting endothelial progenitor cells (EPCs) from the bone marrow through a CXCR4-dependent mechanism. This function of CXCL12 makes it a crucial factor in carcinogenesis and neovascularization linked to tumor progression. CXCL12 also has a role in tumor metastasis, where cancer cells expressing the receptor CXCR4 are attracted to metastatic target tissues that release the ligand CXCL12.

[0196] The receptor for CXCL12 is CXCR4. A CCR of the invention may comprise a CXCL12-binding domain from CXCR4 linked to an endodomain derived from a cytokine receptor, such as the IL-2 receptor.

[0197] The paired expression of IL2 and CXCR4 can support the engraftment of therapeutic T cells for cancer treatment. In multiple myeloma, cells expressing such CCRs can recruit cells and change the bone marrow environment. Such cells also have applications in the treatment of solid cancers by modifying the microenvironment of solid tumors.

[0198] The amino acid sequence for CXCR4 is shown below as SEQ ID NO:17. [ka]

[0199] CXCR7 also binds to CXCL12.

[0200] CCL2 Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP1) and small inducible cytokine A2, recruits monocytes, memory T cells, and dendritic cells to sites of inflammation resulting from tissue injury or infection.

[0201] CCR2 and CCR4 are the two cell surface receptors that bind to CCL2.

[0202] CCR2 has the amino acid sequence shown as SEQ ID NO:18. [ka]

[0203] CCR4 has the amino acid sequence shown as SEQ ID NO:19. [ka]

[0204] A CCR of the present invention may comprise in its ligand binding domain the CCL2 binding site of CCR2 or CCR4.

[0205] cell surface antigen The ligand can be a cell surface antigen, such as a transmembrane protein.

[0206] The cell surface antigen can be CD22.

[0207] CD22, or cluster of differentiation 22, is a molecule that belongs to the SIGLEC family of lectins. CD22 is found on the surface of mature B cells and, to a lesser extent, on some immature B cells. Generally speaking, CD22 is a regulatory molecule that prevents overactivation of the immune system and the development of autoimmune diseases.

[0208] CD22 is a carbohydrate-binding transmembrane protein that specifically binds sialic acid and contains an immunoglobulin (Ig) domain at its N-terminus. The presence of the Ig domain makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0209] High expression of CD22 is found in non-Hodgkin's lymphoma and other lymphomas. Various monoclonal antibodies that target CD22 are known, including epratuzumab, inotuzumab ozogamicin, m971 and m972.

[0210] Chimeric antigen receptors (CARs) The cells of the present invention may also comprise one or more chimeric antigen receptors. The CAR may be specific for a tumor-associated antigen.

[0211] Classical CARs are chimeric type I transmembrane proteins that link an extracellular antigen recognition domain (binder) to an intracellular signaling domain (endodomain). Binders are typically single-chain variable fragments (scFVs) derived from monoclonal antibodies (mAbs), but can be based on other formats, including antibody-like or ligand-based antigen binding sites. The transmembrane domain anchors the protein to the cell membrane and links the spacer to the endodomain.

[0212] Early CAR designs had endodomains derived from the intracellular portion of either FcεR1 or the γ-chain of CD3ζ. Thus, these first generation receptors transmitted immunological signal 1, which was sufficient to induce T cell killing of allogeneic target cells, but did not sufficiently activate T cells to cause proliferation and survival. To overcome this limitation, composite endodomains were constructed: fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ results in second generation receptors that can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal to induce T cell proliferation, namely immunological signal 2. Some receptors have also been described that contain endodomains from the TNF receptor family, such as the closely related OX40 and 41BB, which transmit survival signals. Even more potent third generation CARs have now been described, with endodomains that can transmit activation, proliferation and survival signals.

[0213] CAR-encoding nucleic acid can be transferred into T cells, for example, by using retroviral vector.In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer.When CAR binds to target antigen, this leads to the transmission of activation signal to the T cells in which it is expressed.That is, CAR induces the specificity and cytotoxicity of T cells toward cells expressing target antigen.

[0214] The cells of the invention may contain one or more CARs.

[0215] The CAR(s) may comprise an antigen-binding domain, a spacer domain, a transmembrane domain, and an endodomain. The endodomain may comprise or be associated with a domain that transmits an activation signal of a T cell.

[0216] Antigen-binding domain CAR antigen-binding domain The antigen binding domain is the part of the CAR that recognizes the antigen.

[0217] Numerous antigen-binding domains are known in the art, including those based on the antigen-binding site of antibodies, antibody mimetics, and T-cell receptors.For example, the antigen-binding domain may comprise a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder (e.g., from a camelid); an artificial single binder (e.g., Darpin); or a single chain derived from a T-cell receptor.

[0218] The term "ligand" is used synonymously with "antigen" to mean the entity that is specifically recognized and bound by the antigen-binding domain of a CAR.

[0219] cell surface antigen CARs can recognize cell surface antigens, i.e., entities such as transmembrane proteins expressed on the surface of target cells, such as tumor cells.

[0220] The CAR can specifically bind to a tumor-associated cell surface antigen.

[0221] Various tumor-associated antigens (TAA) are known, some of which are shown in Table 1. The antigen-binding domain used in the present invention may be a domain capable of binding to the TAA shown therein. [Table 1]

[0222] Where the CAR recognizes a B cell lymphoma or leukemia antigen (e.g., CD19, CD20, CD52, CD160, or CD5), the CCR may recognize another B cell antigen, such as CD22.

[0223] Prostate cancer associated antigens The CAR can specifically bind to a cell surface antigen associated with prostate cancer (e.g., prostate stem cell antigen (PSCA) or prostate specific membrane antigen (PSMA)).

[0224] PSCA is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein that is upregulated in many prostate cancers and is also detected in bladder and pancreatic cancers.

[0225] Various anti-PSCA antibodies are known, for example, 7F5 (Morgenroth et al. (Prostate (2007) 67:1121-1131); 1G8 (Hillerdal et al. (2014) BMC Cancer 14:30); and Ha1-4.117 (Abate-Daga et al. (2014) 25:1003-1012).

[0226] The CCR-expressing cells of the invention may also express an anti-PSCA CAR, which may include an antigen-binding domain based on one of these antibodies.

[0227] PSMA is a membrane-located zinc metalloenzyme. It is highly expressed in the human prostate, 100-fold higher than expression in most other tissues. In cancer, PSMA is upregulated and is called the second most commonly upregulated gene in prostate cancer, 8-12-fold higher than in noncancerous prostate. In addition to expression in the human prostate and prostate cancer, PSMA has also been found to be highly expressed in the neovasculature of tumors, but not in the normal vasculature of all types of solid tumors (e.g., kidney, breast, colon, etc.).

[0228] Various anti-PSMA antibodies are known, such as 7E11, J591, J415 and Hybritech PEQ226.5 and PM2J004.5, each of which binds to a different epitope on PSMA (Chang et al. (1999) Cancer Res 15:3192-8).

[0229] The CCR-expressing cells of the invention may also express an anti-PSMA CAR, which may comprise an antigen-binding domain based on one of these antibodies.

[0230] For example, the CCR may comprise a J591-based scFv having the sequence shown as SEQ ID NO:20. [ka]

[0231] CAR transmembrane domain The transmembrane domain is the sequence of CAR that spans the membrane. The CAR transmembrane domain may contain a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which provides good receptor stability.

[0232] CAR signal peptide The CARs and CCRs described herein can contain a signal peptide so that, when expressed in a cell, such as a T cell, they direct the nascent protein to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.

[0233] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that have a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps enforce the proper topology of the polypeptide during translocation. At the end of the signal peptide, there is typically a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave during or after translocation is complete to generate a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0234] The signal peptide may be located at the amino terminus of the molecule.

[0235] The signal peptide can comprise the sequence set forth in SEQ ID NO: 21, 22 or 23, or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions), provided that the signal peptide still functions to drive cell surface expression of the CAR. [ka]

[0236] The signal peptide of SEQ ID NO:21 is small and highly efficient, derived from the TCR β chain, and is predicted to provide for efficient removal by signal peptidases, resulting in approximately 95% cleavage after the terminal glycine. [ka]

[0237] The signal peptide of SEQ ID NO:22 is derived from IgG1. [ka]

[0238] The signal peptide of SEQ ID NO:23 is derived from CD8a.

[0239] CAR endodomain The endodomain is the part of a classical CAR that is located on the intracellular side of the membrane.

[0240] The endodomain is the signaling part of classical CARs. After antigen recognition by the antigen-binding domain, individual CAR molecules cluster, native CD45 and CD148, and are excluded from the synapse, transmitting the signal to the cell.

[0241] The CAR endodomain may be or may include an intracellular signaling domain. In an alternative embodiment, the CAR endodomain may interact with an intracellular signaling molecule present in the cytoplasm, resulting in signal transduction.

[0242] The intracellular signaling domain or separate intracellular signaling molecule may be or may include a T cell signaling domain.

[0243] The most commonly used signaling component is the endodomain component of the CD3-zeta endodomain, which contains three ITAMs. It transmits activation signals to T cells after antigen is bound. CD3-zeta may not provide a sufficiently competent activation signal, so additional costimulatory signaling may be required. For example, chimeric CD28 and OX40 may be used in conjunction with CD3-zeta to transmit proliferation / survival signals, or all three may be used together.

[0244] The CAR may comprise only the CD3-zeta endodomain, may comprise the CD3-zeta endodomain together with the CD28 or OX40 endodomain, or may comprise the CD28 endodomain as well as the OX40 and CD3-zeta endodomain.

[0245] The CAR endodomain may comprise one or more of the following: ICOS endodomain, CD27 endodomain, BTLA endodomain, CD30 endodomain, GITR endodomain and HVEM endodomain.

[0246] The endodomain may comprise a sequence as set forth in SEQ ID NOs: 24-32 or a variant thereof having at least 80% sequence identity. [ka] [ka] [ka]

[0247] The variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 24-32, provided that the sequence provides an effective intracellular signaling domain.

[0248] nucleic acid The invention also provides nucleic acids encoding the CCRs of the invention.

[0249] The nucleic acid has the structure: AgB-spacer-TM-end (where: AgB1 is a nucleic acid sequence encoding the antigen-binding domain of CCR; spacer 1 is a nucleic acid sequence encoding the spacer of CCR; TM1 is a nucleic acid sequence encoding the transmembrane domain of CCR; Endo1 is a nucleic acid sequence encoding the endodomain of CCR. may have:

[0250] nucleic acid construct The present invention further provides a nucleic acid construct comprising a first nucleic acid sequence encoding a first CAR as defined in relation to the first aspect of the invention; and a second nucleic acid sequence encoding a second CAR as defined in relation to the first aspect of the invention.

[0251] The nucleic acid construct has the following structure: AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2 (where: AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR; spacer 1 is a nucleic acid sequence encoding the spacer of the first CCR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR; endo1 is a nucleic acid sequence encoding the endodomain of the first CCR; coexpr is a nucleic acid sequence that allows for coexpression of both CCRs, AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR; spacer2 is a nucleic acid sequence encoding the spacer of the second CCR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; Endo2 is a nucleic acid sequence encoding the endodomain of the second CCR. may have:

[0252] When the nucleic acid construct is expressed in a cell, such as a T cell, it encodes a polypeptide that is cleaved at the cleavage site such that the first and second CCRs are co-expressed at the cell surface.

[0253] The first and second CCRs may bind to different epitopes on the same antigen.

[0254] The first and second CCRs may have complementary endodomains, for example an endodomain derived from the α or β chain of a cytokine receptor and an endodomain derived from the γ chain of the same cytokine receptor.

[0255] The present invention also provides nucleic acid constructs encoding the CCRs and CARs of the present invention. Such constructs have the structure: CCRAgB-CCR spacer-CCRTM-CCR end-coexpr-CARAgB-CAR spacer-CARTM-CAR end or CARAgB-CARspacer-CARTM-CARend-coexpr-C CRAgB-CCR spacer-CCRTM-CCR end (where: CCRAgB is a nucleic acid sequence encoding the antigen-binding domain of CCR; CCR spacer is a nucleic acid sequence encoding the spacer of a CCR; CCRTM is a nucleic acid sequence encoding the transmembrane domain of CCR; CCR endo is a nucleic acid sequence encoding the endodomain of CCR; coexpr is a nucleic acid sequence allowing for coexpression of both the CCR and the CAR, CARAgB is a nucleic acid sequence encoding the antigen-binding domain of the CAR; CAR spacer is a nucleic acid sequence encoding the spacer of the CAR; CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR; CAR endo is a nucleic acid sequence encoding the endodomain of CAR) may have:

[0256] The present invention also provides a nucleic acid construct encoding the first and second CCRs and CAR of the present invention. The first and second CCRs may bind to distinct epitopes on the same antigen. Such a construct may have the structure: (i) CCRAgB1-CCR spacer1-CCRTM1-CCR end1-coexpr1-CCRAgB2-CCR spacer2-CCRTM2-CCR end2-coexpr2-CARAgB-CAR spacer-CARTM-CAR end; (ii) CCRAgB1-CCR spacer1-CCRTM1-CCR endo1-coexpr1-CARAgB-CAR spacer-CARTM-CAR endo-coexpr2-CCRAgB2-CCR spacer2-CCRTM2-CCR endo2; or (iii) CARAgB-CAR spacer-CARTM-CAR endo-coexpr1-CCRAgB1-CCR spacer1-CCRTM1-CCR endo1-coexpr2-CCRAgB2-CCR spacer2-CCRTM2-CCR endo2; (where: CCRAgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR; CCR spacer 1 is a nucleic acid sequence encoding the spacer of the first CCR; CCRTM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR; CCR endo1 is a nucleic acid sequence encoding the endodomain of the first CCR; CCRAgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR; CCR spacer2 is a nucleic acid sequence encoding the spacer of a second CCR; CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; CCR endo2 is a nucleic acid sequence encoding the endodomain of a second CCR; Coexpr1 and coexpr2 are nucleic acid sequences that allow for coexpression of two adjacent sequences; CARAgB is a nucleic acid sequence encoding the antigen-binding domain of the CAR; CAR spacer is a nucleic acid sequence encoding the spacer of the CAR; CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR; CAR endo is a nucleic acid sequence encoding the endodomain of CAR) may have:

[0257] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0258] It is understood by those skilled in the art that, as a result of the degeneracy of the genetic code, many different polynucleotides and nucleic acids can code for the same polypeptide. Furthermore, it should be understood that those skilled in the art may use routine techniques to make substitutions of nucleotides that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polynucleotides described herein are to be expressed.

[0259] Nucleic acids according to the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that contain synthetic or modified nucleotides within them. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the purposes of use as described herein, the polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or extend the lifespan of the polynucleotide of interest.

[0260] The terms "variant", "homolog" or "derivative" in reference to a nucleotide sequence include any substitution, alteration, modification, substitution, deletion or addition of one (or more) nucleic acid(s) from or to that sequence.

[0261] In the above structure, "coexpr" is a nucleic acid sequence that allows for the co-expression of both the first and second CARs. It may be a sequence that codes for a cleavage site, such that the nucleic acid construct generates and contains two or more CCRs linked by the cleavage site, or one CCR and one CAR. The cleavage site may be self-cleaving, such that once the polypeptide is generated, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

[0262] The cleavage site may be any sequence that allows the first and second CCR or one CCR and one CAR to become split.

[0263] The term "cleavage" is used herein for convenience, however, the cleavage site may separate the peptide into individual entities by mechanisms other than classical cleavage. For example, in the case of the foot and mouth disease virus (FMDV) 2A autocleaving peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolysis by host cell proteinases, autoproteolysis or translational effects (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). When the cleavage site is located between nucleic acid sequences encoding proteins, the exact mechanism of such "cleavage" is not important for the purposes of the present invention, as long as it allows the expression of those proteins as separate entities.

[0264] The cleavage site may be a furin cleavage site.

[0265] Furin is an enzyme that belongs to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process latent precursor proteins to their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type 1 matrix metalloprotease, pro-nerve growth factor beta subunit, and von Willebrand factor. Furin cleaves proteins immediately downstream of a basic amino acid target sequence (canonically Arg-X-(Arg / Lys)-Arg') and is concentrated in the Golgi apparatus.

[0266] The cleavage site may be the tobacco etch virus (TEV) cleavage site.

[0267] TEV protease is a highly sequence-specific cysteine ​​protease and a chymotrypsin-like protease. Because TEV protease is highly specific for its target cleavage site, it is often used for controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (where "\" represents the peptide bond to be cleaved). Mammalian cells, such as human cells, do not express TEV protease. Therefore, in embodiments in which the present nucleic acid construct contains a TEV cleavage site and is expressed in a mammalian cell, an exogenous TEV protease must also be expressed in the mammalian cell.

[0268] The cleavage site may encode a self-cleaving peptide.

[0269] A "self-cleaving peptide" refers to a peptide which functions such that when the polypeptide constitutes the protein and the self-cleaving peptide is produced, the polypeptide is immediately "cleaved" or separated into distinct, discontinuous first and second polypeptides without the need for any external cleavage activity.

[0270] The self-cleaving peptide can be a 2A self-cleaving peptide from an aphthovirus or cardiovirus. The initial 2A / 2B cleavage of aphthoviruses and cardioviruses is mediated by 2A "cleaving" at its own C-terminus. In apthoviruses (e.g., foot and mouth disease virus (FMDV) and equine rhinitis A virus), the 2A region is a short section of about 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), becomes an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al. (2001) as above).

[0271] "2A-like" sequences are found in repeat sequences in picornaviruses other than aphthoviruses or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses and Trypanosoma spp., as well as bacterial sequences (Donnelly et al. (2001) as above). The cleavage site may be one of these 2A-like sequences, e.g.: [ka] [ka] may include.

[0272] The cleavage site may comprise a 2A-like sequence shown as SEQ ID NO: 38 (RAEGRGSLLTCGDVEENPGP).

[0273] The present invention also provides a kit comprising one or more nucleic acid sequences encoding the first and second CCRs according to the first aspect of the invention, or one or more CCRs and one or more CARs according to the invention.

[0274] SEQ ID NOs: 45 and 46 provide the complete amino acid sequence of the anti-PSMA CAR and anti-PSA CCR fusion. Subheadings are provided to indicate respective portions of the sequence, although in reality the various elements are connected to provide one continuous sequence.

[0275] The nucleic acid construct of the present invention may encode a fusion protein as shown in SEQ ID NO:45 or 46. SEQ ID NO:45 - Exemplary construct having IL-2R beta chain Signal sequence derived from human CD8a: [ka] scFv aPSMA (J591 H / L) [ka] Linker SDPA Human IgG1Fc spacer (HCH2CH3pvaa): [ka] Transmembrane domain derived from human CD28: [ka] Endodomain derived from TCRz: [ka] 2A peptide derived from the Thosea asigna virus capsid protein: [ka] Signal sequence derived from mouse kappa VIII: [ka] scFv aPSA (5D5A5 H / L): [ka] Linker: [ka] Human CD8aSTK spacer: [ka] Transmembrane domain derived from human common gamma chain: [ka] Endodomain derived from the human common gamma chain: [ka] 2A peptide from Equine rhinitis A virus polyprotein: [ka] Signal sequence derived from mouse kappa VIII: [ka] scFv aPSA (5D3D11 H / L): [ka] Linker: [ka] Human CD28STK spacer: [ka] Transmembrane derived from human IL-2Rβ: [ka] Endodomain derived from human IL-2Rβ: [ka] SEQ ID NO:46 - Exemplary construct having IL-7R alpha chain Signal sequence derived from human CD8a: [ka] scFv aPSMA(J591 H / L) [ka] Linker SDPA Human IgG1Fc spacer (HCH2CH3pvaa): [ka] Transmembrane domain derived from human CD28: [ka] Endodomain derived from TCRz: [ka] 2A peptide derived from the Thosea asigna virus capsid protein: [ka] Signal sequence derived from mouse kappa VIII: [ka] scFv aPSA (5D5A5 H / L): [ka] Linker: [ka] Human CD8aSTK spacer: [ka] Transmembrane domain derived from human common gamma chain: [ka] Endodomain derived from the human common gamma chain: [ka] 2A peptide from Equine rhinitis A virus polyprotein: [ka] Signal sequence derived from mouse kappa VIII: [ka] scFv aPSA (5D3D11 H / L): [ka] Linker: [ka] Human CD28STK spacer: [ka] Transmembrane derived from human IL-7Rα: [ka] Endodomain derived from human IL-7Rα: [ka] [ka]

[0276] vector The present invention also provides a vector or a kit of vectors comprising one or more nucleic acid sequences encoding one or more CCRs and optionally one or more CARs according to the first aspect of the invention. Such vectors may be used to introduce the nucleic acid sequences into a host cell so as to express the CCRs according to the first aspect of the invention.

[0277] The vector can be, for example, a plasmid or a viral vector (eg, a retroviral or lentiviral vector) or a transposon-based vector or a synthetic mRNA.

[0278] The vector may be capable of transfecting or transforming T cells or NK cells.

[0279] cell The present invention provides cells comprising one or more CCRs of the invention and optionally one of more CARs.

[0280] The cell may comprise a nucleic acid or vector of the invention.

[0281] The cell may be a cytolytic immune cell, such as a T cell or a NK cell.

[0282] T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of the T cell receptor (TCR) on the cell surface. There are different types of T cells, as outlined below.

[0283] T helper cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or THF, which promote different types of immune responses by secreting various cytokines.

[0284] Cytolytic T cells (TC cells or CTLs) destroy virus-infected and tumor cells and also play a role in transplant rejection. CTLs express CD8 on their surface. These cells recognize targets by binding to MHC class I-associated antigens, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0285] Memory T cells are a subset of antigen-specific T cells that persist long after the resolution of an infection. They rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen, providing the immune system with "memory" against past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0286] Regulatory T cells (Treg cells), previously known as suppressor T cells, are crucial for maintaining immune tolerance: their main role is to suppress T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

[0287] Two major classes of CD4+ Treg cells have been described - naturally occurring Treg cells and adaptive Treg cells.

[0288] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+Treg cells) arise in the thymus and are associated with interactions between both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated with TSLP and developing T cells. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, leading to the fatal autoimmune disease IPEX.

[0289] Adaptive Treg cells (also known as Tr1 or Th3 cells) can arise during a normal immune response.

[0290] The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide a rapid response to endogenous signals from virus-infected cells in an MHC-independent manner.

[0291] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third type of cell differentiated from a common lymphoid precursor that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus, from where they then enter the circulatory system.

[0292] The CCR-expressing cells of the present invention may be any of the cell types listed above.

[0293] The T or NK cells according to the first aspect of the invention may be generated ex vivo from the patient's own peripheral blood (first party), or in the context of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0294] Alternatively, T or NK cells according to the first aspect of the invention may be obtained from ex vivo differentiation of inducible or embryonic precursor cells into T or NK cells. Alternatively, immortalized T cell lines may be used which retain lytic function and can act as therapeutic agents.

[0295] In all of these embodiments, CCR-expressing cells are generated by introducing DNA or RNA encoding the or each CCR by one of a number of means, including transduction with a viral vector, transfection with DNA or RNA.

[0296] The cells of the present invention may be ex vivo T or NK cells from a subject. The T or NK cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The T or NK cells may be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody, before being transduced with a nucleic acid encoding a molecule that provides a CCR according to the first aspect of the present invention.

[0297] The T or NK cells of the present invention are (i) isolation of a T or NK cell-containing sample from a subject or other source listed above; and (ii) transduction or transfection of T or NK cells with one or more nucleic acid sequences encoding a CCR. It can be made by:

[0298] Those T or NK cells can then be purified, e.g., selected, based on expression of the antigen-binding domain of the antigen-binding polypeptide.

[0299] Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the present invention.

[0300] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, diluent or excipient.The pharmaceutical composition may optionally comprise one or more additional pharma- ceutical active polypeptides and / or compounds.Such a formulation may be, for example, in a form suitable for intravenous infusion.

[0301] Treatment The invention provides methods for treating and / or preventing disease, the methods comprising administering to a subject a cell of the invention (e.g., a cell of the invention in a pharmaceutical composition as described above).

[0302] Methods for treating diseases relate to therapeutic uses of the cells of the invention, where the cells can be administered to a subject having a disease or condition to alleviate, reduce or ameliorate at least one symptom associated with an existing disease and / or to slow, reduce or prevent the progression of the disease.

[0303] The method for preventing disease relates to the prophylactic use of the cells of the present invention.Herein, such cells can be administered to a subject who is not yet afflicted with disease and / or does not show any symptoms of disease, to prevent the disease or to attenuate the cause of the disease, or to reduce or prevent the occurrence of at least one symptom associated with the disease.The subject can be predisposed to the disease or can be considered to be at risk of developing the disease.

[0304] The above method is (i) isolating a T or NK cell-containing sample; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the invention; (iii) administering cells from (ii) to a subject. may include.

[0305] The T or NK cell-containing sample may be isolated from the subject or from other sources, such as those described above. T or NK cells may be isolated from the subject's own peripheral blood (first party), or from hematopoietic stem cell transplantation settings from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0306] The present invention provides a CCR-expressing cell of the present invention for use in the treatment and / or prevention of disease.

[0307] The present invention also relates to the use of a CCR-expressing cell of the invention in the manufacture of a medicament for treating and / or preventing a disease.

[0308] The disease treated and / or prevented by the methods of the present invention may be a cancerous disease (e.g., bladder cancer, breast cancer, colon cancer, uterine cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer and thyroid cancer).

[0309] When the ligand recognized by the CCR is PSA, the cancer may be prostate cancer.

[0310] The cells of the present invention may be capable of killing target cells, such as cancer cells. Target cells may be characterized by the presence of tumor-secreted or chemokine ligands in the vicinity of the target cells. Target cells may be characterized by the presence of soluble ligands, along with the expression of tumor-associated antigens (TAA) on the target cell surface.

[0311] The cells and pharmaceutical compositions of the invention may be for use in the treatment and / or prevention of the diseases described above.

[0312] The cells and pharmaceutical compositions of the invention may be for use in any of the methods described above.

[0313] Chimeric transmembrane proteins The present invention also provides a chimeric transmembrane protein comprising: a dimerization domain; and a cytokine receptor endodomain.

[0314] Dimerization may occur spontaneously, in which case the chimeric transmembrane protein is constitutively active, or it may occur only in the presence of a chemical inducer of dimerization (CID), in which case the transmembrane protein triggers cytokine-type signaling only in the presence of the CID.

[0315] Suitable dimerization domains and CIDs are described in WO2015 / 150771, the contents of which are incorporated herein by reference.

[0316] For example, one dimerization domain may comprise the rapamycin binding domain of FK binding protein 12 (FKBP12) and the other may comprise the FKBP12-rapamycin binding (FRB) domain of mTOR; the CID may be rapamycin or a derivative thereof.

[0317] One dimerization domain may comprise the FK506 (tacrolimus) binding domain of FK binding protein 12 (FKBP12) and the other dimerization domain may comprise the cyclosporine binding domain of cyclophilin A; the CID may be an FK506 / cyclosporine fusion or a derivative thereof.

[0318] One dimerization domain may comprise an estrogen binding domain (EBD) and the other dimerization domain may comprise a streptavidin binding domain; the CID may be an estrone / biotin fusion protein or a derivative thereof.

[0319] One dimerization domain may comprise a glucocorticoid binding domain (GBD) and the other dimerization domain may comprise a dihydrofolate reductase (DHFR) binding domain; the CID may be a dexamethasone / methotrexate fusion protein or a derivative thereof.

[0320] One dimerization domain may comprise an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and the other dimerization domain may comprise a dihydrofolate reductase (DHFR) binding domain; the CID may be an O6-benzylguanine derivative / methotrexate fusion protein or a derivative thereof.

[0321] One dimerization domain may comprise a retinoic acid receptor domain and the other dimerization domain may comprise an ecodysone receptor domain; the CID may be RSL1 or a derivative thereof.

[0322] When the dimerization domain spontaneously heterodimerizes, it can be based on the dimerization domain of an antibody. In particular, it can include the dimerization portion of the heavy chain constant domain (CH) and the light chain constant domain (CL). The "dimerization portion" of the constant domain is the part of the sequence that forms interchain disulfide bonds.

[0323] A chimeric cytokine receptor may, for example, comprise the Fab portion of an antibody as an exodomain, as diagrammed in FIG.

[0324] A chimeric transmembrane protein comprises two polypeptides: (i) (a) a first dimerization domain; and (b) The first chain of a cytokine receptor endodomain a first polypeptide comprising: (ii) (a) a second dimerization domain that dimerizes with the first dimerization domain; and (b) The second chain of a cytokine receptor endodomain. A second polypeptide comprising may include.

[0325] The above sections defining the cytokine receptor endodomain of a chimeric cytokine receptor also apply to the chimeric transmembrane proteins of the present invention.

[0326] The sections above relating to nucleic acids, vectors, kits, cells, pharmaceutical compositions and methods also apply to the chimeric transmembrane proteins of the invention.

[0327] The present invention is further illustrated by examples, which are meant to serve to aid those of ordinary skill in the art in practicing the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0328] Example 1 - In vitro testing T cells are transduced with a PSMA-specific CAR or a construct that co-expresses a PSMA-specific CAR with a PSA-specific CCR. T cells are co-cultured with PSMA-expressing target cells that do or do not secrete PSA. This co-culture is performed in the presence or absence of exogenous IL2. This co-culture is performed at different effector-to-target ratios. This co-culture is repeated serially with T cells that are repeatedly challenged with target cells. T cell proliferation and target cell killing are measured. In this way, the contribution of the CCR to T cell proliferation and survival can be measured. Furthermore, the contribution capacity of serially re-challenging repeatedly.

[0329] Example 2 - In vivo testing A human prostate cancer cell line that expresses PSMA, secretes PSA, and expresses firefly luciferase is implanted into NSG mice. T cells are transduced with a PSMA-specific CAR or a construct that co-expresses a PSMA-specific CAR with a PSA-specific CCR. The mice are administered T cells. Tumor burden can be measured continuously using bioluminescence imaging to evaluate the response to CAR T cells. Mice within each cohort can be sacrificed at various time points, and tumor burden can be measured directly by macroscopic measurement and immunohistochemistry. In addition, T cell engraftment / expansion within tumor beds or lymphoid tissues (e.g., lymph nodes, spleen, and bone marrow) is measured by flow cytometry of the tissues.

[0330] Example 3 - Generation and testing of constitutively active cytokine signaling molecules Constitutively active cytokine signaling chimeric transmembrane proteins were created by linking a cytokine receptor endodomain to a "Fab" type exodomain (Figure 5). This structure uses the dimerization domains from the natural dimerization components of antibodies, namely the heavy and light chain constant regions. The chimeric transmembrane proteins have two chains; a first polypeptide containing the antibody light chain kappa chain and the IL2 receptor common gamma chain as endodomains; and a second polypeptide containing an endodomain containing either the antibody heavy chain CH1 and the IL2 receptor beta chain (resulting in a constitutively active IL2-signaling molecule); or the IL7 receptor (resulting in a constitutively active IL7-signaling molecule). The constitutively active cytokine signaling chimeric transmembrane proteins tested in this study included the heavy and light chain variable regions of scFv. These domains are not necessary for dimerization to occur. The signal is independent of antigen binding and the structure can be equally "headless" (as shown in Figure 5) or can include another entity such as a tag protein.

[0331] The nucleic acid sequences encoding these two polypeptides were cloned in frame, separated by a sequence encoding the 2A peptide.

[0332] CTLL-2 (ATCC (registered trademark) TIB-214 TMCTLL-2 cells are murine cytotoxic T lymphocyte cells that depend on IL-2 for growth. In the absence of IL-2, the cells undergo apoptosis. CTLL-2 cells were transduced with a vector expressing a chimeric protein containing the IL2 receptor endodomain (Fab_IL2endo) or a vector expressing a chimeric protein containing the IL7 receptor endodomain (Fab_IL7endo), or left untransduced (WT). As a positive control, all three types of cells were co-cultured with 100 U / ml of murine IL2. Cell proliferation was assessed after 3 and 7 days of culture, and the results are shown in Figure 6.

[0333] Untransduced CTLL2 cells proliferated in the presence of 100 U / mL mouse IL2 together with CTLL2 cells transduced with either construct (Fab_IL2endo or Fab_IL7endo) (Figure 6, left panel). However, in the absence of exogenously added IL2, only cells transduced with the construct carrying the IL2R endodomain (Fab_IL2endo) survived and proliferated. This indicates that the chimeric transmembrane receptor provides the necessary IL2 signal to CTLL2 cells.

[0334] Example 4 - Creation and testing of chimeric cytokine receptors for PSA A panel of chimeric cytokine receptors targeting PSA was developed using scFv derived from two antibodies that bind different PSA epitopes: 5D5A5 and 5D3D11. The crystal structure of PSA was obtained in a sandwich complex with these two (Stura et al. (2011) as above).

[0335] A schematic illustrating some of the panels of the CCR is shown in FIG.

[0336] The panel includes the following structures: A5-CD8stk-IL2Rg_D11-hinge-IL2Rb: CCR with IL-2R endodomain with A5 on the chain with the common γ chain and D11 on the chain with the IL2R β chain; D11-CD8stk-IL2Rg_A5-hinge-IL2Rb: CCR with IL-2R endodomain with D11 on the chain with the common γ chain and A5 on the chain with the IL2R β chain; D11-CD8stk-RL_A5-hinge-IL2Rb: a negative control construct equivalent to D11-CD8stk-IL2Rg_A5-hinge-IL2Rb, but in which the IL2R gamma chain is replaced by a rigid linker; D11-CD8stk-IL2Rg_A5-hinge-IL7Ra: CCR with an IL-7R endodomain with D11 on the chain with the common gamma chain and A5 on the chain with the IL7R alpha chain; and D11-CD8stk-RL_A5-hinge-IL7Ra: A negative control construct equivalent to D11-CD8stk-IL2Rg_A5-hinge-IL7Ra, but in which the IL2R gamma chain is replaced by a rigid linker. was included.

[0337] CTLL2 cells were transduced with vectors expressing these constructs. Cells were cultured in the presence or absence of IL2 (the presence of IL2 served as a positive control) and in the presence or absence of 5 ng / mL or 5 μg / mL PSA. Proliferation of CTLL2 cells was assessed after 3 and 7 days, and the results are shown in FIG. 8.

[0338] CTLL2 cells expressing a CCR with an IL7 endodomain did not support survival and proliferation of CTLL2 cells (Figure 8, last two panels). The presence of mouse IL-2 in these cells supported the growth and proliferation of CTLL2 cells on day 3, but by day 7, the majority of the cells had undergone apoptosis.

[0339] An anti-PSA chimeric cytokine receptor bearing the IL2R endodomain supported proliferation of CTLL2 cells in the absence of IL2 and in the presence of both 5 ng / ml and 5 μg / ml of PSA (Figure 8, first panel), with 5 μg / ml resulting in higher survival and proliferation, especially at day 7.

[0340] Both anti-PSA chimeric cytokine receptors with IL2R endodomains, i.e., A5-CD8stk-IL2Rg_D11-hinge-IL2Rb and D11-CD8stk-IL2Rg_A5-hinge-IL2Rb, suggest that the relative positioning of the two PSA binding domains: 5D5A5 and 5D3D11, is not important for function.

[0341] Replacement of the common gamma chain with a rigid linker abolished the ability of the CCR to support the survival and proliferation of CTLL2 cells (FIG. 8, third panel).

[0342] As an alternative readout for IL2 signaling, phosphorylation of Y694 in STAT5 was investigated using phosphoflow.

[0343] CTLL2 cells were either not transduced (WT); transduced with a PSA CCR construct carrying the IL2R endodomain (D11-CD8STK-IL2Rg_A5-hinge-IL2Rb); or with an equivalent negative control construct in which the IL2R gamma chain was replaced with a rigid linker (D11-CD8STK-RL_A5-hinge-IL2Rb). The cells were incubated overnight in the absence of exogenously added IL-2. The next day, cells were incubated with 500 μM pervanadate (a positive control that inhibits phosphatases and leads to STAT5 phosphorylation) or 500 ng / mL PSA for 1 or 4 h. After incubation, cells were fixed, permeabilized, and analyzed by flow cytometry.

[0344] The results are shown in Figure 9. In cells expressing the PSA CCR, the presence of PSA increased STAT5 phosphorylation over time (Figure 9, middle panel). No such increase in phosphorylation was seen in untransduced CTLL2 cells or in CTLL2 cells transduced with an equivalent construct in which the IL2Rγ chain was replaced with a tight linker (Figure 9, right panel).

[0345] These results are consistent with the CTLL2 survival / proliferation data shown in Figure 8 and demonstrate that cytokine signaling can be induced in T cells by using a chimeric cytokine receptor for a soluble ligand (here PSA).

[0346] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. Two polypeptides: (i) (a) Heavy chain constant domain (CH) (b) the first chain of the GM-CSF receptor endodomain a first polypeptide comprising: (ii) (a) a light chain constant domain (CL), which spontaneously dimerizes with the heavy chain constant domain; (b) the second chain of the GM-CSF receptor endodomain A second polypeptide comprising A chimeric transmembrane protein comprising: wherein spontaneous dimerization of the heavy chain constant domain and the light chain constant domain brings together a first chain of the GM-CSF receptor endodomain with a second chain of the GM-CSF receptor endodomain to provide constitutive signaling.

2. The chimeric transmembrane protein of claim 1, wherein the first polypeptide comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH), and the second polypeptide comprises a light chain variable domain (VL) and a light chain constant domain (CL).

3. The chimeric transmembrane protein of claim 2 , comprising a Fab exodomain.

4. A cell comprising the chimeric transmembrane protein of any one of claims 1 to 3.

5. The cell of claim 4, which also contains a chimeric antigen receptor.

6. The cell of claim 5 , wherein the chimeric antigen receptor binds to a tumor-associated cell surface antigen.

7. A nucleic acid encoding the chimeric transmembrane protein according to any one of claims 1 to 3.

8. (a) Heavy chain constant domain (CH) (b) the first chain of the GM-CSF receptor endodomain a first nucleic acid sequence encoding a first polypeptide comprising: (a) a light chain constant domain (CL), which spontaneously dimerizes with the heavy chain constant domain; (b) the second chain of the GM-CSF receptor endodomain wherein the nucleic acid construct comprises a second nucleic acid sequence encoding a second polypeptide comprising the structure: CH-TM1-endo1-coexpr-CL-TM2-endo2 (where: CH is a nucleic acid sequence encoding the heavy chain constant domain of the first polypeptide; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence enabling co-expression of both the first and second polypeptides, CL is a nucleic acid sequence encoding the light chain constant domain of the second polypeptide; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo2 is a nucleic acid sequence encoding the endodomain of the second polypeptide. A nucleic acid construct having the following structure: wherein spontaneous dimerization of the heavy chain constant domain and the light chain constant domain brings together a first chain of the GM-CSF receptor endodomain with a second chain of the GM-CSF receptor endodomain to provide constitutive signaling.

9. 9. The nucleic acid construct of claim 8, which also encodes a chimeric antigen receptor (CAR).

10. 10. The nucleic acid construct of claim 8 or 9, wherein coexpr encodes a sequence comprising a self-cleaving peptide.

11. A nucleic acid construct according to any of claims 8 to 10, wherein alternative codons are used in regions of the sequence encoding identical or similar amino acid sequences to avoid homologous recombination.

12. A vector comprising the nucleic acid construct according to any one of claims 8 to 11.

13. The vector of claim 12, wherein the vector is a retroviral vector, a lentiviral vector, or a transposon.

14. i) (a) Heavy chain constant domain (CH) (b) the first chain of the GM-CSF receptor endodomain a vector comprising a nucleic acid sequence encoding a first polypeptide comprising: ii) (a) a light chain constant domain (CL), which spontaneously dimerizes with the heavy chain constant domain; (b) the second chain of the GM-CSF receptor endodomain A vector comprising a nucleic acid sequence encoding a second polypeptide comprising A kit comprising: wherein spontaneous dimerization of the heavy chain constant domain and the light chain constant domain brings together a first chain of the GM-CSF receptor endodomain with a second chain of the GM-CSF receptor endodomain to provide constitutive signaling.

15. 15. The kit of claim 14, also comprising a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

16. i) a vector comprising a nucleic acid sequence encoding a chimeric transmembrane protein according to any one of claims 1 to 3; and ii) A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor. Including the kit.

17. A method for producing a cell according to any one of claims 4 to 6, comprising the step of introducing into a cell a nucleic acid according to claim 7; a nucleic acid construct according to any one of claims 8 to 11; a vector according to claim 12 or 13; or a kit of vectors according to any one of claims 14 to 16, wherein the cell is derived from a sample isolated from a subject.

18. A pharmaceutical composition comprising a plurality of cells according to any one of claims 4 to 6.

19. 19. The pharmaceutical composition according to claim 18 for treating and / or preventing a disease.

20. The following steps: (i) the cell is transduced or transfected with a nucleic acid according to claim 7; a nucleic acid construct according to any one of claims 8 to 11; a vector according to claim 12 or 13; or a kit of vectors according to any one of claims 14 to 16, (ii) administering to the subject the cells from (i).

20. The pharmaceutical composition according to claim 19, characterized in that

21. 21. The pharmaceutical composition of claim 20, wherein the sample is a T cell-containing sample.

22. The pharmaceutical composition of claim 20 or 21, wherein the disease is cancer.

23. 19. A pharmaceutical composition according to claim 18 for use in the treatment and / or prevention of a disease.

24. Use of a cell according to any one of claims 4 to 6 in the manufacture of a medicament for treating and / or preventing a disease.

25. A composition comprising the cells according to any one of claims 4 to 6 for treating and / or preventing a disease.

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