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 the Invention The present invention relates to chimeric cytokine receptors (CCRs), and to cells that express such chimeric cytokine receptors and optionally chimeric antigen receptors on their cell surface.

Background Art

[0002] Background of the Invention Chimeric Antigen Receptor (CAR) 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] A chimeric antigen receptor is a protein that links the specificity of a monoclonal antibody (mAb) to the effector functions of T cells. The normal form of a chimeric antigen receptor is in the form of a type I transmembrane domain protein having a transmembrane domain all linked to an antigen recognition amino-terminal, a spacer, and a complex 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 signal transduction endodomain via a spacer and a transmembrane domain. The above molecules result in activation of T cells in response to recognition by the scFv of its target. When T cells express such a CAR, the T cells recognize and kill target cells that express the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently being tested in clinical trials for the treatment of various cancers.

Summary of the Invention

Means for Solving the Problems

[0005] CAR-based approach for treating prostate cancer Prostate cancer is the second most common cancer among men worldwide and the sixth leading cause of cancer-related death. Worldwide, there are approximately 1,100,000 new cases and 300,000 deaths each year, constituting 4 percent of all cancer deaths. One in six men is estimated to be diagnosed with this disease during his lifetime.

[0006] Initial treatment for prostate cancer can consist of surgery, radiation therapy, hormonal therapy, or any combination thereof. Hormonal therapy consists of reducing the level of testosterone, a male hormone that promotes uncontrolled cell growth. Usually, chemotherapy is reserved for advanced-stage cancer.

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

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

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

[0010] This may be because in vivo, CAR T cells struggle to overcome the harsh cancer microenvironment. 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 cytokine administration 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 oncogenesis (Nagarkatti et al. (1994) PNAS 91:7638-7642; Hassuneh et al. (1997) Blood 89:610-620).

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

[0013] On-target off-tumour toxicity Because cancer can lose specificity, it is relatively rare to effectively explain cancer by the presence of a single antigen.

[0014] Most cancers cannot be distinguished from normal tissues based on a single antigen. Therefore, a significant amount of "on-target off-tumor" toxicity occurs, whereby normal tissues are damaged by the treatment. For example, targeting CD20 to treat B-cell lymphoma with rituximab causes depletion of the entire normal B-cell compartment, targeting CD52 to treat chronic lymphocytic leukemia causes depletion of the entire lymphoid compartment, and targeting CD33 to treat acute myeloid leukemia causes damage to the entire bone marrow compartment, etc.

[0015] The predicted problems of "on-target off-tumor" toxicity are supported by clinical trials. For example, the approach of targeting ERBB2 killed patients with colon cancer that had metastasized to the lungs 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" toxicity is reduced or eliminated. The present invention provides, for example, the following items. (Item 1) An ectodomain that binds to a ligand selected from tumor-secreted factors, chemokines, and cell surface antigens; and A cytokine receptor endodomain A chimeric cytokine receptor (CCR) comprising the same. (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 the same; and (ii) (a) A second antigen-binding domain that binds to a second epitope of the ligand (b) The second chain of the cytokine receptor endodomain A second polypeptide comprising The chimeric cytokine receptor according to item 1, comprising (Item 3) The chimeric cytokine receptor according to item 2, wherein each of the first and second antigen-binding domains is a single-chain variable fragment (scFv). (Item 4) The chimeric cytokine receptor according to item 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) The first chain of the cytokine receptor endodomain A first polypeptide comprising; and (ii) (a) Light chain variable domain (V L ) (b) The second chain of the cytokine receptor endodomain A second polypeptide comprising The chimeric cytokine receptor according to item 1, comprising (Item 6) The chimeric cytokine receptor according to item 5, wherein the first and second chains for the cytokine receptor endodomain are different and are selected from the α chain, β chain, and γ chain of the type I cytokine receptor endodomain. (Item 7) The chimeric cytokine receptor according to item 5, wherein the first and second chains for the cytokine receptor endodomain are the same and are selected from the α chain, β chain, and γ chain of the type I cytokine receptor endodomain. (Item 8) The cytokine receptor endodomain is (i) The β chain endodomain of the IL-2 receptor (ii) The α chain endodomain of the IL-7 receptor; or (iii) The α-chain end domain of the IL-15 receptor; and / or (iv) The common γ-chain receptor end domain The chimeric cytokine receptor according to any one of the preceding items, comprising (Item 9) The chimeric cytokine receptor according to any one of the preceding items, wherein the ligand is a tumor-secreted factor selected from prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), and CA125. (Item 10) 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 the chimeric cytokine receptor according to any one of the preceding items. (Item 12) The cell according to 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) When the first chimeric cytokine receptor and the second cytokine receptor bind to the ligand, the first chimeric cytokine receptor comprises the α-chain or β-chain of the type I cytokine receptor end domain, and the second chimeric cytokine receptor comprises the γ-chain of the type I cytokine receptor end domain, such that combined signal transduction occurs via the α / β-chain and γ-chain of the type I cytokine receptor end domain. The cell according to Item 12. (Item 14) The cell according to any one of Items 11 to 13, further comprising a chimeric antigen receptor. (Item 15) The cell according to Item 14, wherein the chimeric antigen receptor binds to a tumor-associated cell surface antigen. (Item 16) The cell according to Item 15, wherein the chimeric antigen receptor binds to a cell surface antigen associated with prostate cancer. (Item 17) The cell according to 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 a 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-Spacer 1-TM1-End 1-coexpr-AbB2-Spacer 2-TM2-End 2 (wherein 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; End 1 is a nucleic acid sequence encoding the end domain of the first CCR; coexpr is a nucleic acid sequence enabling co-expression of both CCRs, AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR; Spacer 2 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; End 2 is a nucleic acid sequence encoding the end domain of the second CCR) having the nucleic acid construct. (Item 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 Spacer 1-CCRTM1-CCR End 1-coexpr1-CCRAgB2-CCR Spacer 2-CCRTM2-CCR End 2-coexpr2-CARAgB-CAR Spacer-CARTM-CAR End; (ii) CCRAgB1 - CCR Spacer 1 - CCRTM1 - CCR End 1 - coexpr1 - CARAgB - CAR Spacer - CARTM - CAR End - coexpr2 - CCRAgB2 - CCR Spacer 2 - CCRTM2 - CCR End 2; or (iii) CARAgB - CAR Spacer - CARTM - CAR End - coexpr1 - CCRAgB1 - CCR Spacer 1 - CCRTM1 - CCR End 1 - coexpr2 - CCRAgB2 - CCR Spacer 2 - CCRTM2 - CCR End 2; (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 End 1 is a nucleic acid sequence encoding the end - domain of the first CCR; CCRAgB2 is a nucleic acid sequence encoding the antigen - binding domain of the second CCR; CCR Spacer 2 is a nucleic acid sequence encoding the spacer of the second CCR; CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; CCR End 2 is a nucleic acid sequence encoding the end - domain of the second CCR; Coexpr1 and coexpr2 are nucleic acid sequences that enable co - expression 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 End is a nucleic acid sequence encoding the end - domain of the CAR) The nucleic acid construct according to item 19, having (Item 21) The nucleic acid construct according to item 19 or 20, wherein coexpr encodes an array containing a self-cleaving peptide. (Item 22) The nucleic acid construct according to any one of items 19 to 21, wherein alternative codons are used in regions of sequences encoding the same or similar amino acid sequences to avoid homologous recombination. (Item 23) A vector comprising the nucleic acid construct according to any one of items 19 to 22. (Item 24) The retroviral vector or lentiviral vector or transposon according to item 23. (Item 25) i) A vector comprising a nucleic acid sequence encoding a first CCR as defined in any one of items 1 to 10; and ii) A vector comprising a nucleic acid sequence encoding a second CCR as defined in any one of items 1 to 10 A kit comprising. (Item 26) The kit according to item 25, further 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 one of items 1 to 10; and ii) A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor A kit comprising. (Item 28) A method for producing the cell according to any one of items 11 to 17, comprising the step of introducing into the cell a nucleic acid sequence according to item 18; a nucleic acid construct according to any one of items 19 to 22; a vector according to item 23 or 24; or a kit of vectors according to any one of items 25 to 27. (Item 29) The method according to item 28, wherein the cell is derived from a sample isolated from a subject. (Item 30) A pharmaceutical composition comprising a plurality of cells according to any one of items 11 to 17. (Item 31) A method for treating and / or preventing a disease, comprising the step of administering to a subject the pharmaceutical composition according to item 30. (Item 32) The following steps: (i) Isolation of a cell-containing sample from a subject; (ii) Transduction or transfection of the cells with 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 kit of vectors according to any one of items 25 to 27; and (iii) Administering the cells from (ii) to the subject The method according to item 31, comprising the above steps. (Item 33) The method according to item 32, wherein the sample is a T cell-containing sample. (Item 34) The method according to item 32 or 33, wherein the disease is cancer. (Item 35) The pharmaceutical composition according to item 30 for use in the treatment and / or prevention of a disease. (Item 36) Use of the cells according to any one of items 11 to 17 in the manufacture of a medicament for treating and / or preventing a disease. Brief Description of the Drawings

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Figure 9

Modes for Carrying Out the Invention

[0026] Summary of Aspects of the Invention The inventors have developed a "chimeric cytokine receptor" (CCR) in which the binding specificity of a non-cytokine binding molecule is transplanted into the endodomain of a cytokine receptor. Co-expression of such a CCR and a chimeric antigen receptor (CAR) helps CAR T cells to engraft and expand in a harsh tumor microenvironment. The requirements for the ligands for the CCRs that should be present as well as the ligands for the CARs add another layer of selectivity and serve to prevent on-target off-tumor toxicity.

[0027] For example, the inventors have developed cells that co-express a chimeric cytokine receptor that detects PSA and transmits an IL2 / 15 or IL7 signal to CAR T cells. Thus, CAR T cells are stimulated to selectively proliferate only in the prostate cancer microenvironment, and in the absence of PSA (i.e., after the patient has achieved remission), cytokine stimulation is lost.

[0028] In a first aspect, the invention provides an ectodomain that binds to a ligand selected from tumour secreted factors, chemokines and cell surface antigens; and a cytokine receptor endodomain 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) a first chain of the cytokine receptor endodomain a first polypeptide comprising; and (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. It includes. Figure 2b illustrates such an arrangement.

[0030] Each of the first and second antigen-binding domains can 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 present invention, the chimeric cytokine receptor comprises two polypeptides: (i) (a) Heavy chain variable domain (VH) (b) The first chain of the cytokine receptor endodomain A first polypeptide comprising; and (ii) (a) Light chain variable domain (VL) (b) The second chain of the cytokine receptor endodomain A second polypeptide comprising It includes.

[0032] Figure 2c illustrates such an arrangement.

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

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

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

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

[0037] The ligand can be a tumor-secreted factor, such as 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, such as a chemokine selected from chemokines 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 the chimeric cytokine receptor described in the first aspect of the present invention.

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

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

[0043] The cell can also include a chimeric antigen receptor, such as a chimeric antigen receptor that binds to a tumor-associated cell surface antigen.

[0044] The chimeric antigen receptor can 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 the CCR recognizes a cell surface antigen, the CCR and the CAR can recognize a cell surface antigen co-expressed on the same target (e.g., tumor) cells. For example, in the case of B cell malignancies, the CAR can recognize a cell surface antigen such as CD19, and the CCR can enhance engraftment by recognizing a molecule (e.g., CD22) co-expressed on the target cell surface.

[0046] In a third aspect, the present invention provides a nucleic acid sequence encoding the chimeric cytokine receptor (CCR) described in the first aspect of the present 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 - End1 - coexpr - AbB2 - Spacer2 - TM2 - End2 (where AgB1 is a nucleic acid sequence encoding the antigen - binding domain of the first CCR; Spacer1 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; End1 is a nucleic acid sequence encoding the end domain of the first CCR; coexpr is a nucleic acid sequence enabling co - expression 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; End2 is a nucleic acid sequence encoding the end domain of the second CCR). having. This 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 End1 - coexpr1 - CARAgB - CAR Spacer - CARTM - CAR End - coexpr2 - CCRAgB2 - CCR Spacer2 - CCRTM2 - CCR End2; or (iii) CARAgB - CAR Spacer - CARTM - CAR End - coexpr1 - CCRAgB1 - CCR Spacer1 - CCRTM1 - CCR End1 - coexpr2 - CCRAgB2 - CCR Spacer2 - CCRTM2 - CCR End2; (where CCRAgB1 is a nucleic acid sequence encoding the antigen - binding domain of the first CCR; CCR Spacer1 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 End1 is a nucleic acid sequence encoding the end domain 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 the second CCR; CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; CCR End2 is a nucleic acid sequence encoding the end domain of the second CCR; Coexpr1 and coexpr2 are nucleic acid sequences enabling co - expression 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; The CAR end is a nucleic acid sequence encoding the end domain of the CAR) may have.

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

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

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

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

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

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

[0054] The above kit i) a vector comprising a nucleic acid sequence encoding the CCR according to the first aspect of the present 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 the cell according to the second aspect of the present invention, the method comprising introducing into a cell a kit of a nucleic acid sequence according to the third aspect of the present invention; a nucleic acid construct according to the fourth aspect of the present invention; a vector according to the fifth aspect of the present invention; or a vector according to the sixth aspect of the present invention.

[0056] The cell can be derived 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 present 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 the pharmaceutical composition according to the eighth aspect of the present invention.

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

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

[0061] The disease can be cancer.

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

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

[0064] A further aspect of the invention is summarized in the following numbered paragraphs:

[0065] 1. A dimerization domain; and a cytokine receptor endodomain comprising chimeric transmembrane protein.

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

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

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

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

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

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

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

[0073] 9. The chimeric transmembrane protein according to 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. The chimeric transmembrane protein according to paragraph 9, comprising a Fab ectodomain.

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

[0076] 12. The cell according to paragraph 11, further comprising a chimeric antigen receptor.

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

[0078] 14. A nucleic acid sequence encoding the 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 a first dimerization domain; TM1 is a nucleic acid sequence encoding a transmembrane domain of the first polypeptide; End1 is a nucleic acid sequence encoding an end domain of the first polypeptide; coexpr is a nucleic acid sequence enabling co-expression of both CCRs, Dim2 is a nucleic acid sequence encoding a second dimerization domain; TM2 is a nucleic acid sequence encoding a transmembrane domain of the second polypeptide; End2 is a nucleic acid sequence encoding an end domain of the second polypeptide). having.

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

[0081] 17. The 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 one of paragraphs 15 to 17, wherein alternative codons are used in regions of the sequence encoding the same or a similar amino acid sequence in order 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 vector or a lentiviral vector or a transposon according to paragraph 19.

[0085] 21. 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 A kit comprising the same.

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

[0087] 23. i) A vector comprising a nucleic acid sequence encoding a chimeric transmembrane protein as defined in any one 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 the 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 according to paragraph 24, wherein the cell is 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 the pharmaceutical composition according to paragraph 26.

[0092] 28. The following steps: (i) Isolation of a cell-containing sample from a subject; (ii) Transduction or transfection of the cells 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 the cells from (ii) to the subject The method according to claim 27, comprising.

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

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

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

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

[0097] Detailed description Chimeric cytokine receptor (CCR) A chimeric cytokine receptor (CCR) is a molecule that includes a cytokine receptor endodomain and a heterologous ligand-binding ectodomain. The heterologous ectodomain binds to ligands other than cytokines that are selective for the cytokine receptor from which the endodomain is derived. Thus, it is possible to alter the ligand specificity of a cytokine receptor by transplanting heterologous binding specificity.

[0098] A chimeric cytokine receptor comprises (i) a ligand-binding ectodomain; (ii) an optional spacer; (iii) a transmembrane domain; and (iv) a cytokine receptor endodomain and includes.

[0099] Cytokine Receptors and Signal Transduction The functions of many cells are controlled by members of the cytokine receptor superfamily. Signal transduction by these receptors depends on the association of these receptors with Janus kinases (JAKs). Janus kinases link ligand binding to tyrosine phosphorylation of signal transduction proteins recruited to the receptor complex. These include signal transducer and activator of transcription (STAT), a family of transcription factors that contribute to the diversity of cytokine responses.

[0100] When the chimeric cytokine receptor of the present invention binds to its ligand, one or more of the following intracellular signal transduction pathways can be induced: (i) the JAK-STAT pathway (ii) the MAP kinase pathway; and (iii) the phosphoinositide 3-kinase (PI3K) pathway.

[0101] The JAK-STAT system consists of three main components: (1) a receptor, (2) a Janus kinase (JAK), and (3) a signal transducer and activator of transcription (STAT).

[0102] JAK has tyrosine kinase activity and binds to cytokine receptors on the cell surface. Binding of a ligand to this receptor induces activation of JAK. When JAK's kinase activity is enhanced, it phosphorylates tyrosine residues on the receptor, creating a site for interaction with proteins containing SH2 domains that bind to phosphotyrosine. STATs, which have SH2 domains capable of binding to these phosphotyrosine residues, are recruited to the receptor and are themselves phosphorylated on tyrosine by JAK. These phosphotyrosines then act as binding sites for the SH2 domains of other STATs, mediating their dimerization. Various STATs form hetero- or homo-dimers. The activated STAT dimers accumulate in the cell nucleus and activate transcription of their target genes.

[0103] Cytokine receptor endodomain The chimeric cytokine receptor of the present invention includes an endodomain that, when the exodomain binds to its ligand, elicits "cytokine-type" cell signaling (either alone or in the presence of another chimeric cytokine receptor).

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

[0105] The endodomain can be derived from a type I cytokine receptor. Type I cytokine receptors share a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane.

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

[0107] Type I cytokine receptors include (i) Interleukin receptors (for example, 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 (for example, receptors for erythropoietin, GM-CSF and G-CSF); and (iii) Hormone receptors / neuropeptide receptors (for example, hormone receptors and prolactin receptors) are included.

[0108] Members of the type I cytokine receptor family contain different chains, some of which are involved in ligand / cytokine interaction and others 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 receptor, the IL-4 receptor, the IL-7 receptor, the IL-9 receptor, the IL-13 receptor and the IL-15 receptor.

[0110] IL-2 IL-2 binds to IL-2 receptors that have three forms generated by different combinations of three different proteins often referred to as α, β and γ "chains"; these subunits are also part of 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 expressed separately and differently on various cell types and can assemble in different combinations and orders to generate low-affinity, medium-affinity and high-affinity IL-2 receptors.

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

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

[0114] The signal transduction of IL-2 promotes the differentiation of naive T cells into effector T cells and memory T cells when the naive T cells are also stimulated by an antigen. Through their role in the generation of T cell immune memory, which depends on the expansion of the number and function of the T cell clones selected by the antigen, those T cell clones also play a key role in long-term cellular immunity.

[0115] The chimeric cytokine receptor of the present invention may include the β chain of the IL-2 receptor and / or the γ chain of the IL-2 receptor (i.e., common).

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

Chemical formula

Chemical formula

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

[0118] As 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 signal transduction pathway, the "mutant" sequence has at least 80, 85, 90, 95, 98 or 99% sequence identity with the wild-type sequence (e.g., SEQ ID NO: 1 or 2).

[0119] The percentage identity between two polypeptide sequences can be readily measured 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 γ-chain receptor (CD132). This common γ-chain receptor is shared by various cytokines including interleukin-2, -4, -9 and -15. The interleukin-7 receptor is expressed on various 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 genomic regions containing the T cell receptor gamma gene by STAT5 and histone acetylation. Knockout studies in mice have suggested that the prevention of apoptosis is an essential function of this protein in T lymphocyte differentiation and activation.

[0122] The chimeric cytokine receptor of the present invention may include the α chain of the IL-7 receptor and / or the γ chain of the IL-7 receptor (i.e., 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:

Chemical formula

Chemical formula

[0124] IL-15 Interleukin 15 (IL-15) is a cytokine that has structural similarity to IL-2. Similar to IL-2, IL-15 binds to a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132), and signals through that complex. IL-15 is secreted by monocytes (and some other cells) after viral infection. IL-15 induces the cell proliferation of natural killer cells.

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

[0126] Spacer The chimeric cytokine receptor of the present invention may include a spacer that connects the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to face in various directions that enable antigen binding.

[0127] If the cells of the present invention contain two or more chimeric cytokine receptors, the spacers may be the same or different. If the cells of the present invention contain a chimeric cytokine receptor (CCR) and a chimeric antigen receptor (CAR), the spacers of the CCR and the CAR may be different, for example, they may have different lengths. The spacer of the CAR may be longer than the spacer of each CCR.

[0128] The spacer sequence may include, for example, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk. The linker may alternatively include an alternative linker sequence having a length and / or domain spacing property similar to that of the IgG1 Fc region, the IgG1 hinge, or the CD8 stalk.

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

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

Chemical formula

Chemical formula

Chemical formula

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

[0132] Alternatively, the transmembrane domain can be derived from the same cytokine as the cytokine from which the endodomain is derived, such as a cytokine receptor.

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

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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

[0135] Numerous antigen-binding domains are known in the art and include those based on the antigen-binding sites of antibodies, antibody mimetics and T cell receptors. For example, the antigen-binding domain can be 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 having sufficient affinity for the target ligand; a single-domain binder (e.g., from camelids); an artificial binder single (e.g., Darpin); or can include a single-chain derived from a T cell receptor.

[0136] The term "ligand" is used as a synonym for "antigen" meaning an entity that is specifically recognized and bound by the antigen-binding domain of the CCR.

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

[0138] When 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 CCR of the present invention binds to a ligand.

[0140] That ligand may be a soluble ligand (e.g., a tumor-secreted factor or chemokine).

[0141] Alternatively, that ligand may be a membrane-bound ligand (e.g., a cell surface antigen).

[0142] The term "soluble ligand" is used to denote a ligand or antigen that is not part of a cell or attached to a cell and moves freely in the extracellular space, e.g., in the body fluids of the tissue of interest. Soluble ligands may exist cell-free in an individual's serum, plasma or other body fluids.

[0143] Soluble ligands may be associated with the presence or pathology of certain diseases such as cancer.

[0144] Soluble ligands may be part of the cancer secretome, i.e., a collection of factors secreted by tumors, and may be derived 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 ligands can be characteristic of a disease or diseased tissue. Soluble ligands can be found exclusively in a subject having the disease, or in a subject having the disease as compared to a healthy subject; or at a higher level in diseased tissue as compared to healthy tissue. Soluble ligands can be expressed at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or 100,000-fold higher in a subject having the disease as compared to a healthy subject; or in diseased tissue as compared to healthy tissue.

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

[0147] The cells in which cell surface ligands are found can be target cells such as cancer cells.

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

[0149] When a cell surface ligand is characteristic of a disease or diseased tissue, that cell surface ligand can be found exclusively on the relevant cells of a subject having the disease, or on the relevant cells of a subject having the disease as compared to a healthy subject; or at a higher level on the relevant cells of diseased tissue as compared to healthy tissue. Cell surface ligands can 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 having the disease as compared to a healthy subject; or in diseased tissue as compared to healthy tissue.

[0150] Tumor Secretion Factor The ligand recognized by CCR can be a soluble ligand secreted by or shed from 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 factor can be a soluble ligand that is not a cytokine. Thus, the CCR of the present invention has transplanted the 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 encoded by the KLK3 gene in humans. PSA is a member of the kallikrein-related peptidase family and is secreted by epithelial cells of the prostate.

[0155] PSA is present in small amounts in the serum of men with a healthy prostate but is increased 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 clotting components of semen, leading to the release of sperm. In cancer, PSA can be involved in the processes of neoplastic growth and metastasis.

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

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

[0159] The CCR of the present invention may include, for example, six CDRs or VH and / or VL domains derived from an mAb that binds to PSA (e.g., 8G8F5, 5D5A5 or 5D3D11).

[0160] If the CCR includes two antigen-binding specificities that bind to different epitopes on PSA, one may be obtained, for example, based on 5D3D11 and the other may be obtained, for example, based on 5D5A5.

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

Chemical formula

Chemical formula

[0162] If the cell contains two CCRs, the antigen-binding domain of the first CCR may include six CDRs derived from 5D5A5, and the antigen-binding domain of the second CCR may include six CDRs derived from 5D3D11.

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

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

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

[0166] Carcinoembryonic antigen (CEA) refers to a closely related series of glycoproteins involved in cell adhesion. CEA is normally produced in gastrointestinal tissues during fetal development, but its production ceases before birth. Thus, CEA is usually present in very low levels in the blood of healthy adults. However, its serum levels increase 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, and its specialized sialofucosylated glycoform may function as a functional colon cancer L-selectin and E-selectin ligand that is extremely important for the metastatic seeding of colon cancer cells. Immunologically, they are characterized as members of the CD66 surface antigen classification.

[0168] CEA and related genes constitute the CEA family belonging 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 CCR for 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 angiogenesis and neovascularization. Vascular endothelial growth factor is part of a system that restores oxygen supply to tissues when blood circulation is insufficient. The serum concentration of VEGF is high in bronchial asthma and type 2 diabetes. The normal functions of VEGF are to create new blood vessels during embryonic development, create new blood vessels after injury, create muscles after exercise, and create new blood vessels (collateral blood vessels) that bypass blocked vessels.

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

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

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

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

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

[0178] The sequence of human CA-125 (also known as mucin-16) is available from 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 specificities of 26 monoclonal antibodies against the CA125 antigen were investigated. The CA125 antigen was found to have only two major antigenic domains, and these antigenic domains classify the antibodies as OC125-like (group A) or M11-like (group B).

[0180] The chimeric cytokine receptor of the present invention may include an antigen-binding domain derived from such an antibody. 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] Rearrangement and overexpression of the ALK gene resulting in a mutant form of the ALK protein Alpha-fetoprotein (AFP) Beta-2-microglobulin (B2M) Beta-human chorionic gonadotropin (beta-hCG) BRAF V600 mutation resulting in a mutated B-RAF 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 Amplification of the HER2 / neu gene or overexpression of the protein Immunoglobulin 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] Chemokine Chemokines are chemotactic cytokines. Cell migration is guided by the gradient 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 path for the homing of cancer cells and immune cells. The effect on T cells is considered inhibitory to the homing of cytotoxic T cells, while regulatory T cells are considered attracted.

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

[0185] Some chemokines are thought to be pro-inflammatory and can be induced during the immune response to recruit cells of the immune system to the site of infection, while others are thought to be constitutive and are involved in the control of cell migration in 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 the increasing chemokine concentration towards the source of chemokines. Some chemokines control immune system cells during processes such as immune surveillance mechanisms that direct lymphocytes towards lymph nodes, so these chemokines can screen for pathogen invasion by interacting with antigen-presenting cells present in these tissues. Other chemokines are inflammatory and are released from a variety of cells in response to bacterial infections, viruses and other agents. Their release is often stimulated by pro-inflammatory cytokines such as interleukin-1. Inflammatory chemokines mainly function as chemoattractants for white blood cells and recruit monocytes, neutrophils, and other effector cells derived from the blood to the site of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate an immune response 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 chemokine CC chemokine (or β-chemokine) proteins have two adjacent cysteines (amino acids) near the amino terminus. There are at least 27 different members of this subgroup reported for mammals, called CCL-1 to -28; CCL10 is the same as CCL9. Chemokines of this subfamily usually contain four cysteines (C4-CC chemokines), although a few CC chemokines have six cysteines (C6-CC chemokines). Examples of C6-CC chemokines include CCL1, CCL15, CCL21, CCL23 and CCL28. CC chemokines induce the migration of monocytes and other cell types (e.g., 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 the surrounding tissue 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 by "X" in this name. There are 17 different CXC chemokines reported in mammals, and they are subdivided into two categories: those with a specific amino acid sequence (or motif) of glutamic acid-leucine-arginine (or abbreviated as ELR) immediately before the first cysteine of the CXC motif (ELR-positive), and those without the ELR motif (ELR-negative). ELR-positive CXC chemokines specifically induce neutrophil migration and interact with chemokine receptors CXCR1 and CXCR2.

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

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

[0194] Chemokine receptors are G protein-coupled receptors that contain seven transmembrane domains found on the surface of white blood cells. Approximately 19 different chemokine receptors have been characterized to date, and they are divided into four families according to the type of chemokine to which they bind; CXCR that binds to CXC chemokines, CCR that binds to CC chemokines, CX3CR1 that binds to the only CX3C chemokine (CX3CL1), and XCR1 that binds to 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, seven helix transmembrane domains with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus containing serine and threonine residues important for receptor regulation. The first two extracellular loops of chemokine receptors each have conserved cysteine residues that allow the formation of disulfide bridges between these loops. The G protein is conjugated with the C-terminus of the chemokine receptor to enable intracellular signal transduction after receptor activation, whereas the N-terminal domain of the chemokine receptor determines the specificity of ligand binding.

[0195] CXCL12 CXCL12 has strong chemotaxis for lymphocytes. CXCL12 plays an important 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 very important factor in tumorigenesis and angiogenesis associated with tumor progression. CXCL12 also has a role in tumor metastasis, and cancer cells expressing the receptor CXCR4 are attracted to the metastatic target tissue that releases the ligand CXCL12.

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

[0197] Co-expression of IL2 and CXCR4 can support the engraftment of therapeutic T cells for cancer treatment. In multiple myeloma, cells expressing such CCR can mobilize cells and change the bone marrow environment. Such cells also have uses 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.

Chemical formula

[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 caused by tissue damage or infection.

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

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

Chemical formula

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

Chemical formula

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

[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, that is, surface antigen classification 22, is a molecule belonging 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 sugar-binding transmembrane protein that specifically binds to sialic acid and has an immunoglobulin (Ig) domain located at the N-terminus. Due to the presence of the Ig domain, CD22 is a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

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

[0210] Chimeric antigen receptor (CAR) The cells of the present invention may also contain one or more chimeric antigen receptors. The CAR can 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). The binder is typically a single-chain variable fragment (scFV) derived from a monoclonal antibody (mAb), but can also be based on other formats including antibody-like antigen-binding sites or ligand-based antigen-binding sites. The transmembrane domain anchors the protein in the cell membrane and attaches a spacer to the endodomain.

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

[0213] The CAR-encoding nucleic acid can be transferred into T cells, for example, using a retroviral vector. Thus, large numbers of antigen-specific T cells can be generated for adoptive cell transfer. When the CAR binds to the target antigen, this results in the transmission of activation signals to the T cells in which it is expressed. That is, the CAR induces the specificity and cytotoxicity of T cells towards cells expressing the target antigen.

[0214] The cells of the present invention may comprise 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 associate with a domain that transmits an activation signal for T cells.

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

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

[0218] The term "ligand" is used as a synonym for "antigen" meaning an entity specifically recognized and bound by the antigen-binding domain of the CAR.

[0219] Cell surface antigen The CAR may recognize a cell surface antigen, i.e., an entity such as a transmembrane protein expressed on the surface of target cells such as tumor cells.

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

[0221] Various tumor-associated antigens (TAAs) 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 TAAs shown therein.

Table 1

[0222] When 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-related antigen The CAR may 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. PSCA is upregulated in many prostate cancers and is also detected in bladder and pancreatic cancers.

[0225] Various anti-PSCA antibodies are known, e.g., 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 present invention may also express an anti-PSCA CAR that may contain an antigen-binding domain based on one of these antibodies.

[0227] PSMA is a zinc metalloenzyme present in the membrane. PSMA is strongly expressed in the human prostate, which is 100 times higher than its expression in most other tissues. In cancer, PSMA expression is upregulated and it is called the second most upregulated gene in prostate cancer, which is 8 - 12 times higher than in non-cancerous prostate. In addition to its expression in the human prostate and prostate cancer, PSMA has also been found to be highly expressed in the tumor neovasculature, but not highly expressed 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, and each of these binds to a different epitope of PSMA (Chang et al. (1999) Cancer Res 15:3192 - 8).

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

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

Chemical formula

[0231] CAR transmembrane domain The transmembrane domain is the sequence of the 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 may contain a signal peptide, such that when they are expressed in cells such as T cells, the nascent protein is directed 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 tend to form a single alpha-helix. The signal peptide may begin with a short stretch of positively charged amino acids, which helps to reinforce 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 signal peptidase. Signal peptidase may cleave during or after translocation to generate a free signal peptide and a mature protein. The free signal peptide is then digested by specific proteases.

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

[0235] The signal peptide may include the sequence shown in SEQ ID NO: 21, 22 or 23, or variants thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions), provided that the signal peptide still functions to cause cell surface expression of the CAR.

Chemical formula

[0236] The signal peptide of SEQ ID NO: 21 is small and highly efficient and is derived from the TCRβ chain. It is predicted to result in efficient removal by signal peptidase, as it causes approximately 95% cleavage after the terminal glycine.

Chemical formula

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

Chem.

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

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

[0240] The end domain is the signaling portion of a classical CAR. After antigen recognition by the antigen-binding domain, individual CAR molecule clusters, native CD45 and CD148 are excluded from the synapse and the signal is transmitted to the cell.

[0241] The CAR end domain can be or can include an intracellular signaling domain. In alternative embodiments, the end domain of the present CAR can interact with intracellular signaling molecules present in the cytoplasm and can effect signaling.

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

[0243] The most commonly used signaling component is the end domain component of the CD3-zeta end domain that contains three ITAMs. This transmits an activation signal to the T cell after antigen binding. Since CD3-zeta may not provide a fully competent activation signal, additional co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 can be used in combination with CD3-zeta to transmit a proliferation / survival signal, or all three can be used together.

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

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

[0246] The end domain may comprise the sequence shown in SEQ ID NOs: 24 to 32 or a variant thereof having at least 80% sequence identity.

Chemical formula

Chemical formula

Chemical formula

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

[0248] Nucleic acid The present invention also provides a nucleic acid encoding the CCR of the present 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; Spacer1 is a nucleic acid sequence encoding the spacer of CCR; TM1 is a nucleic acid sequence encoding the transmembrane domain of CCR; End 1 is a nucleic acid sequence encoding the end domain 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 present invention; and a second nucleic acid sequence encoding a second CAR as defined in relation to the first aspect of the present invention.

[0251] The nucleic acid construct has the following structure: AgB1 - Spacer 1 - TM1 - End 1 - coexpr - AgB2 - Spacer 2 - TM2 - End 2 (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; End 1 is a nucleic acid sequence encoding the end domain of the first CCR; coexpr is a nucleic acid sequence enabling co - expression of both CCRs, AgB2 is a nucleic acid sequence encoding the antigen - binding domain of the second CCR; Spacer 2 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; End 2 is a nucleic acid sequence encoding the end domain of the second CCR) may have.

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

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

[0254] The first and second CCRs can have complementary end domains, for example, an end domain derived from the α or β chain of a cytokine receptor and an end domain 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-CAR spacer-CARTM-CAR end-coexpr-CCRAgB-CCR spacer-CCRTM-CCR end (where CCRAgB is a nucleic acid sequence encoding the antigen-binding domain of the CCR; CCR spacer is a nucleic acid sequence encoding the spacer of the CCR; CCRTM is a nucleic acid sequence encoding the transmembrane domain of the CCR; CCR end is a nucleic acid sequence encoding the end domain of the CCR; coexpr is a nucleic acid sequence that enables co-expression 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 the CAR; CAR end is a nucleic acid sequence encoding the end domain of the CAR) and can have.

[0256] The present invention also provides nucleic acid constructs encoding the first and second CCRs and CARs of the present invention. The first and second CCRs can bind to distinct epitopes on the same antigen. Such constructs 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 End1 - coexpr1 - CARAgB - CAR Spacer - CARTM - CAR End - coexpr2 - CCRAgB2 - CCR Spacer2 - CCRTM2 - CCR End2; or (iii) CARAgB - CAR Spacer - CARTM - CAR End - coexpr1 - CCRAgB1 - CCR Spacer1 - CCRTM1 - CCR End1 - coexpr2 - CCRAgB2 - CCR Spacer2 - CCRTM2 - CCR End2; (where CCRAgB1 is a nucleic acid sequence encoding the antigen - binding domain of the first CCR; CCR Spacer1 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 End1 is a nucleic acid sequence encoding the end domain 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 the second CCR; CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR; CCR End2 is a nucleic acid sequence encoding the end domain of the second CCR; Coexpr1 and coexpr2 are nucleic acid sequences that enable co - expression 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; The CAR end is a nucleic acid sequence encoding the end domain of the CAR) may have.

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

[0258] As a result of the degeneracy of the genetic code, it is understood by those skilled in the art that numerous different polynucleotides and nucleic acids can encode the same polypeptide. Further, in order to reflect the codon usage frequency of any particular host organism in which the polynucleotides described herein are to be expressed, it should be understood that those skilled in the art may make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein using routine techniques.

[0259] The nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides containing synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and 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, those 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" with respect to a nucleotide sequence include any substitution, change, modification, replacement, deletion or addition of one (or more than one) nucleic acid from or to that sequence.

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

[0262] The cleavage site can be any sequence that enables the first and second CCRs or one CCR and one CAR to be cleaved.

[0263] The term "cleavage" is used herein for convenience, but the cleavage site can separate the above peptides into individual entities by a mechanism other than classical cleavage. For example, in the case of the foot-and-mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolysis by host cell proteases, 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 those proteins are expressed as separate entities.

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

[0265] Furin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process latent precursor proteins into 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, the beta subunit of pro-nerve growth factor, and von Willebrand factor. Furin cleaves proteins immediately downstream of a basic amino acid target sequence (typically, Arg-X-(Arg / Lys)-Arg’) and is concentrated in the Golgi apparatus.

[0266] The cleavage site can be a cleavage site of tobacco etch virus (TEV).

[0267] TEV protease is a highly sequence-specific cysteine protease and a chymotrypsin-like protease. Because TEV protease is very specific for its target cleavage site, it is often used for the 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. Thus, in embodiments where the present nucleic acid construct contains a TEV cleavage site and is expressed in mammalian cells, exogenous TEV protease must also be expressed in the mammalian cells.

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

[0269] The term "self-cleaving peptide" refers to a peptide in which the polypeptide constitutes the protein and, when the self-cleaving peptide is generated, the polypeptide functions to be "cleaved" immediately without requiring any external cleavage activity or to be separated into different discontinuous first and second polypeptides.

[0270] The self-cleaving peptide can be a 2A self-cleaving peptide derived from aphthovirus or cardiovirus. The first 2A / 2B cleavage of aphthovirus and cardiovirus is mediated by 2A that "cleaves" at its own C-terminus. In aphthoviruses (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, which, together with the N-terminal residue (conserved proline residue) of protein 2B, becomes an autonomous element capable of mediating "cleavage" at its own C-terminus (Donelly et al. (2001) as described above).

[0271] "2A-like" sequences are found in picornaviruses other than aphthovirus or cardiovirus, "picornavirus-like" insect viruses, rotavirus C and Trypanosoma spp in repetitive sequences and bacterial sequences (Donelly et al. (2001) as described above). The cleavage site can include one of these 2A-like sequences, for example:

Chemical formula

Chemical formula

[0272] The cleavage site can include a 2A-like sequence represented as SEQ ID NO: 38 (RAEGRGSLLTCGDVEENPGP).

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

[0274] SEQ ID NOs: 45 and 46 provide the complete amino acid sequences of a fusion of an anti-PSMA CAR and an anti-PSA CCR. The headings are given to display a part of each of those sequences, but in reality, various elements are connected to result in 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. Exemplary construct with SEQ ID NO: 45 - IL-2R beta chain Signal sequence derived from human CD8a:

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[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 according to the first aspect of the present invention and optionally one or more CARs. Such vectors can be used to introduce their nucleic acid sequences into host cells so as to express the CCRs according to the first aspect of the present invention.

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

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

[0279] Cell The present invention provides a cell comprising one or more CCRs of the present invention and optionally one or more (one of more) CARs.

[0280] The above-mentioned cell may contain the nucleic acid or vector of the present invention.

[0281] The above-mentioned cell can be a cytolytic immune cell such as a T cell or an NK cell.

[0282] T cells or T lymphocytes are a type of lymphocyte that play 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 their cell surface. There are various types of T cells as outlined below.

[0283] Helper T helper cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the 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 TFH, which promote various types of immune responses by secreting various cytokines.

[0284] Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize targets by binding to antigens associated with MHC class I, 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 anergic states and prevent 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. When re-exposed to their homologous antigens, they rapidly expand into a large number of effector T cells, providing an immune system with "memory" of 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), formerly known as suppressor T cells, are very important for maintaining immune tolerance. Their main role is to halt T cell-mediated immunity towards the end of an immune response and suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

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

[0288] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are associated with the interaction between developing T cells and both myeloid (CD11c+) dendritic cells and plasmacytoid (CD123+) dendritic cells activated by TSLP. 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 cells or Th3 cells) can arise during a normal immune response.

[0290] The cell can 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 (which belong to the group of natural lymphocytes) are defined as large granular lymphocytes (LGL) and constitute a third type of cell differentiated from a common lymphoid progenitor cell that gives rise to B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus and then enter the circulatory system from there.

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

[0293] The T or NK cells described in the first aspect of the present invention can be produced ex vivo from the patient's own peripheral blood (Group 1), or in the context of a hematopoietic stem cell transplant from a donor peripheral blood (Group 2), or from a peripheral blood from an unrelated donor (Group 3).

[0294] Alternatively, the T or NK cells according to the first aspect of the present invention can be obtained from the ex vivo differentiation of inducible precursor cells or embryonic precursor cells into T or NK cells. Alternatively, an immortalized T cell line that retains the lytic function and can act as a therapeutic agent can be used.

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

[0296] The cells of the present invention can be ex vivo T or NK cells derived from a subject. The T or NK cells can be derived from a peripheral blood mononuclear cell (PBMC) sample. The T or NK cells can be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody, prior to transduction with a nucleic acid encoding a molecule that provides the 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 sample containing T or NK cells from a subject or other origin listed above; and (ii) transduction or transfection of the T or NK cells with one or more nucleic acid sequences encoding the CCR and can be produced thereby.

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

[0299] Pharmaceutical composition 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 pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active polypeptides and / or compounds. Such a formulation may be in a form suitable for, for example, intravenous infusion.

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

[0302] The method for treating a disease relates to the therapeutic use of the cells of the present invention. As used herein, those cells can be administered to a subject having the disease or condition to alleviate, reduce or improve at least one symptom associated with the existing disease and / or to slow down, reduce or prevent the progression of the disease.

[0303] The method for preventing a disease relates to the prophylactic use of the cells of the present invention. As used herein, such cells can be administered to a subject who has not yet contracted the disease and / or does not exhibit any symptoms of the disease to prevent the disease or weaken its cause, or to reduce or prevent the occurrence of at least one symptom associated with the disease. The subject may have a predisposition to the disease or may be considered at risk of developing the disease.

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

[0305] A T- or NK-cell-containing sample can be isolated from a subject or other origin, such as an origin as described above. T- or NK-cells can be isolated from the subject's own peripheral blood (first party), or in the context of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

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

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

[0308] Diseases that can be treated and / or prevented by the method of the present invention can be cancerous diseases (e.g., bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer).

[0309] If the ligand recognized by CCR is PSA, the cancer can be prostate cancer.

[0310] The cells of the present invention may be able to kill target cells such as cancer cells. The target cells may be characterized by the presence of a tumor-secreted ligand or chemokine ligand in the vicinity of the target cells. The target cells may be characterized by the presence of a soluble ligand together with the expression of a tumor-associated antigen (TAA) on the surface of the target cells.

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

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

[0313] Chimeric transmembrane protein 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. Alternatively, dimerization may occur only in the presence of a dimerization chemical inducer (CID), in which case the transmembrane protein elicits cytokine-type signaling only in the presence of CID.

[0315] Suitable dimerization domains and CIDs are described in WO2015 / 150771, the content of which is 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 include an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain, and the other dimerization domain may include 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 include a retinoic acid receptor domain, and the other dimerization domain may include an ecdysone receptor domain; the CID may be RSL1 or a derivative thereof.

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

[0323] The chimeric cytokine receptor may include, as an exodomain, the Fab portion of an antibody, as schematically illustrated in FIG. 5, for example.

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

[0325] The above terms defining the cytokine receptor endodomain of the chimeric cytokine receptor also apply to the chimeric transmembrane protein of the present invention.

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

[0327] The present invention is further illustrated by examples, which are meant to assist those skilled 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 test T cells are transduced with a PSMA-specific CAR or a construct co-expressing a PSMA-specific CAR and a PSA-specific CCR. The T cells are co-cultured with PSMA-expressing target cells that secrete 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 continuously repeated using T cells repeatedly challenged with target cells. The proliferation of T cells and the killing of target cells are measured. In this way, the contribution of CCR to the proliferation and survival of T cells can be measured. Furthermore, the contribution ability of the continuous ability to be repeatedly re-challenged.

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

[0330] Example 3 - Preparation and testing of constitutively active cytokine signaling molecules Constitutively active cytokine signaling chimeric transmembrane proteins were prepared by linking cytokine receptor endodomains to "Fab"-type exodomains (Figure 5). This structure uses the natural dimerization components of antibodies, namely, dimerization domains derived from the heavy chain constant region and the light chain constant region. The chimeric transmembrane protein has two chains; a first polypeptide containing the antibody light chain κ chain and the IL2 receptor common γ chain as the endodomain; and a second polypeptide having an endodomain containing either the antibody heavy chain CH1 and the IL2 receptor β 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 contained the heavy chain variable region and the light chain variable region of the scFv. These domains are not required for dimerization to occur. Their signal is independent of antigen binding, and their structure can be equally "headless" (as shown in Figure 5) or can contain another entity such as a tag protein.

[0331] The nucleic acid sequences encoding these two polypeptides were cloned in-frame with the sequence encoding the 2A peptide interrupted in between.

[0332] CTLL-2 (ATCC® TIB-214 TMis a mouse cytotoxic T lymphocyte cell that depends on IL-2 for growth. In the absence of IL-2, those cells undergo apoptosis. CTLL-2 cells were either transduced with a vector expressing a chimeric protein containing the IL2 receptor endodomain (Fab_IL2 end) or a vector expressing a chimeric protein containing the IL7 receptor endodomain (Fab_IL7 end), or left untransduced (WT). As a positive control, all three types of cells were co-cultured with 100 U / ml of mouse IL2. Cell proliferation was evaluated 3 and 7 days after culturing, and the results are shown in Figure 6.

[0333] Untransduced CTLL2 cells proliferated in the presence of 100 U / mL of mouse IL2 together with CTLL2 cells transduced with either construct (Fab_IL2 end or Fab_IL7 end) (Figure 6, left panel). However, in the absence of exogenously added IL2, only cells transduced with the construct having the IL2R endodomain (Fab_IL2 end) survived and proliferated. This indicates that the chimeric transmembrane receptor provides the necessary IL2 signal to CTLL2 cells.

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

[0335] A schematic diagram illustrating some of the panel of CCRs is shown in Figure 7.

[0336] That panel included the following constructs: A5-CD8stk-IL2Rg_D11-hinge-IL2Rb: A CCR having an IL-2R endodomain with D11 on a chain having A5 and an IL2Rβ chain on a chain having a common γ chain; CCR having an IL-2R end domain with D11 and an A5 on the chain having an IL2Rβ chain and a common γ chain, and an A5 on the chain having D11; D11-CD8stk-RL_A5-Hinge-IL2Rb: A negative control construct equivalent to D11-CD8stk-IL2Rg_A5-Hinge-IL2Rb, but with the IL2Rγ chain replaced by a rigid linker; CCR having an IL-7R end domain with D11 and an A5 on the chain having an IL7Rα chain and a common γ chain; and D11-CD8stk-RL_A5-Hinge-IL7Ra: A negative control construct equivalent to D11-CD8stk-IL2Rg_A5-Hinge-IL7Ra, but with the IL2Rγ chain replaced by a rigid linker were included.

[0337] CTLL2 cells were transfected with vectors expressing these constructs. The cells were cultured in the presence or absence of IL2 (the presence of IL2 serves as a positive control) and in the presence or absence of 5 ng / mL or 5 μg / mL of PSA. The growth of CTLL2 cells was evaluated after 3 and 7 days, and the results are shown in Figure 8.

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

[0339] The anti-PSA chimeric cytokine receptor having an IL2R end domain supported the 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), and 5 μg / ml resulted in higher survival and proliferation, particularly on day 7.

[0340] Both anti-PSA chimeric cytokine receptors having the IL2R end domain, namely, 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] By replacing the common γ chain with a rigid linker, the ability of CCR to support the survival and proliferation of CTLL2 cells was lost (Figure 8, third panel).

[0342] As another readout for IL2 signaling, phosphorylation of Y694 of STAT5 was investigated using phospho-flow.

[0343] Either not transduced into CTLL2 cells (WT); transduced with a PSA CCR construct having the IL2R end domain (D11-CD8STK-IL2Rg_A5-hinge-IL2Rb); or transduced with an equivalent negative control construct (D11-CD8STK-RL_A5-hinge-IL2Rb) in which the IL2Rγ chain was replaced with a rigid linker. Those cells were incubated overnight in the absence of exogenously added IL-2. The next day, the cells were incubated for 1 or 4 hours with 500 μM pervanadic acid (a positive control that inhibits phosphatases and results in phosphorylation of STAT5) or 500 ng / mL PSA. After incubation, the cells were fixed, permeabilized, and analyzed by flow cytometry.

[0344] The results are shown in Fig. 9. In cells expressing PSA CCR, the presence of PSA increased the phosphorylation of STAT5 over time (Fig. 9, middle panel). Such an increase in phosphorylation was not seen in untransduced CTLL2 cells or CTLL2 cells transduced with an equivalent construct in which the IL2Rγ chain was replaced with a rigid linker (Fig. 9, right panel).

[0345] These results are consistent with the CTLL2 survival / proliferation data shown in Fig. 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 of the publications cited in the above specification are hereby incorporated by reference into this specification. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. While the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the methods described for carrying out the invention that are obvious to experts in molecular biology or related fields are also intended to be included within the scope of the following claims.

Claims

[Claim 1] The method or chimeric cytokine receptor (CCR) described in the specification.

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