Chimeric cytokine receptors including the shortened forms IL2Rβ and IL2Rγ

Abstract

The present invention provides a chimeric cytokine receptor comprising a cytokine receptor endodomain comprising a first chain and a second chain, wherein both the first and second chains of the cytokine receptor endodomain are shortened. The present invention also provides cells comprising such chimeric cytokine receptors, optionally combined with chimeric antigen receptors (CARs), and their use in the treatment of diseases such as autoimmune diseases and cancer.

Description

Technical Field

[0001] The present invention relates to chimeric cytokine receptors (CCRs). In particular, the present invention relates to CCRs in which both chains of the cytokine receptor endodomain are shortened.

Background Art

[0002] Chimeric antigen receptor (CAR) A number of immunotherapeutic agents, including therapeutic monoclonal antibodies (mAbs), bispecific T cell engagers, and chimeric antigen receptors (CARs), have been described for use in cancer treatment.

[0003] A chimeric antigen receptor is a protein that transfers the specificity of a monoclonal antibody (mAb) to the effector function of a T cell. Their normal form is that of a type I transmembrane domain protein having a transmembrane domain linked to an antigen recognition amino terminus, 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 transmembrane domain. Such molecules result in the activation of T cells in response to recognition by the scFv of their target. When a T cell expresses such a CAR, the T cell recognizes and kills 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 in clinical trials for the treatment of various cancers.

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

[0006] Initial treatment for prostate cancer may consist of surgery, radiation therapy, or hormone therapy, or any combination thereof. Hormone therapy involves lowering levels of testosterone, a male hormone that promotes uncontrolled cell growth. Chemotherapy is usually reserved for advanced-stage cancer.

[0007] If prostate cancer continues to grow despite the reduction in testosterone levels due to hormone therapy, treatment options are limited. Typically, cancer vaccine cyproisel-T (Provenge®), a dendritic cell-based therapeutic cancer vaccine designed to induce a targeted immune response against prostatic acid phosphatase (PAP) antigen, is added in sequence to hormone therapy. This may include radiopharmaceuticals (e.g., radium-223 chloride), second-line hormone therapy (e.g., abiraterone or enzalutamide), and / or chemotherapy (docetaxel and cabazitaxel). While each of these treatments can slow cancer growth and alleviate symptoms caused by the disease over several months, the disease eventually becomes resistant to them.

[0008] In preclinical studies, 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 therapies.

[0009] Mice treated with T cells genetically modified with PSCA CAR showed delayed tumor growth. However, although the cells exhibited high in vitro cytotoxicity, in vivo, while tumor growth was delayed, mice with tumors were not cured.

[0010] This may be because, in vivo, CAR T cells struggle to overcome the harsh tumor microenvironment. In particular, CAR T cells may not engraft and proliferate 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, and constitutive production of cytokines can lead to uncontrolled proliferation and transformation.

[0012] Therefore, there is a need for alternative CAR T cell approaches that promote T cell engraftment and proliferation to counteract the effects of the harsh tumor microenvironment.

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

[0014] Most cancers cannot be distinguished from normal tissue based on a single antigen. Consequently, significant "on-target-off-tumor" toxicity occurs, thereby damaging normal tissue with the therapy. For example, targeting CD20 to treat B-cell lymphoma with rituximab can deplete the entire normal B-cell compartment, targeting CD52 to treat chronic lymphocytic leukemia can deplete the entire lymphatic compartment, and targeting CD33 to treat acute myeloid leukemia can damage the entire myeloid compartment.

[0015] The anticipated problem of "on-target off-tumor" toxicity is supported by clinical trials. For example, approaches targeting ERBB2 have resulted in death in patients whose colon cancer has metastasized to the lungs and liver. ERBB2 is overexpressed in some patients with colon cancer, but it is also expressed in several normal tissues, including the heart and normal vascular structures.

[0016] Therefore, there is a need for improved cancer treatment approaches that reduce or eliminate such "on-target off-tumor" toxicity.

[0017] Patent Document 1 describes two types of chimeric cytokine receptors (CCRs). The first type of CCR transfers the binding specificity of a non-cytokine-binding molecule to the endodomain of the cytokine receptor. In the presence of a ligand for the CCR, cytokine signaling is transmitted to CCR-expressing cells. The second type of CCR includes a dimerization domain and a cytokine receptor endodomain. Dimerization may occur spontaneously, in which case the CCR is constitutively active. Alternatively, dimerization may occur only in the presence of a chemical inducer of dimerization (CID), in which case the transmembrane protein induces cytokine-type signaling only in the presence of the CID.

[0018] Such co-expression of CCRs and chimeric antigen receptors (CARs) helps CAR T cells engraft and proliferate in the harsh tumor microenvironment.

[0019] In vivo proliferation of CCR-transduced cells occurs when the proliferation rate is faster than the rate of cell death. When the proliferation rate equals the rate of cell death, the cells reach a homogenized (steady) state. If the rate of death is faster than the rate of proliferation, the cells will not persist. In some cases, hyperphysiological activation of the CCR may be required to ensure that the cells persist in vivo. In other cases, reduced proliferation may be required to adapt to the rate of cell death in order to maintain cellular homogeny. In other cases, some cells are hypersensitive to CCR signaling, and excessive activation of the CCR may alter cellular function or differentiation, requiring a reduction in CCR signaling.

[0020] Shortening of cytokine receptor endodomain Patent Document 2 describes a chimeric cytokine receptor having a series of C-terminal truncations on one of the chains of the cytokine receptor endodomain. It has been found that the first deletion improves cell proliferation, and subsequent longer deletions reduce cytokine signaling in an analog-like manner. Therefore, by selecting an appropriate truncation, it is possible to select the desired level of cytokine signaling.

Prior Art Documents

Patent Documents

[0021]

Patent Document 1

Patent Document 2

Summary of the Invention

[0022] Summary of Aspects of the Invention The inventors have found that when the endodomains of both chains of the chimeric cytokine receptor endodomain are truncated, an additive effect is achieved, and the combination of both the truncated IL2 receptor β chain and the truncated common γ chain exhibits improved signal transduction.

[0023] Thus, in a first aspect, the present invention provides two polypeptides: (i) (a) a first dimerization domain, and (b) a truncated IL2 receptor β chain endodomain comprising the sequence shown as SEQ ID NO: 2 having a C-terminal truncation of about 10 amino acids to about 70 amino acids, a first polypeptide comprising; (ii) (a) a second dimerization domain that dimerizes with the first dimerization domain, and (b) a truncated common γ chain endodomain comprising the sequence shown as SEQ ID NO: 1 having a C-terminal truncation of about 10 amino acids to about 30 amino acids, a second polypeptide comprising; This provides a chimeric cytokine receptor, including the following:

[0024] The truncated IL2 receptor β-chain endodomain of the first polypeptide may have the sequence shown as Sequence ID No. 58.

[0025] The common gamma chain end domain of the abbreviated form of the second polypeptide may have the sequence shown as Sequence ID No. 62.

[0026] The first and second dimerizing domains can dimerize spontaneously or in the presence of a dimerizing chemical inducer (CID) or a protein ligand.

[0027] When the first dimerization domain and the second dimerization domain spontaneously dimerize, the first dimerization domain may contain a heavy chain constant domain (CH), and the second dimerization domain may contain a light chain constant domain (CL), or The first dimerization domain may include a light chain constant domain (CL), and the second dimerization domain may include a heavy chain constant domain (CH).

[0028] When the first and second dimerization domains dimerize in the presence of a protein ligand, the chimeric cytokine receptor is composed of two polypeptides: (i) (a) A first antigen-binding domain that binds to the first epitope of the ligand, and (b) A shortened IL2 receptor β-chain endodomain containing the sequence shown as Sequence ID No. 2, having a C-terminal shortening of approximately 10 to 70 amino acids, A first polypeptide containing, (ii) (a) A second antigen-binding domain that binds to a second epitope of the ligand, and (b) A shortened common gamma chain end domain containing the sequence shown as Sequence ID No. 1, having a C-terminal shortening of approximately 10 to 30 amino acids, A second polypeptide containing, It may include.

[0029] The first antigen-binding domain and the second antigen-binding domain may each be, for example, a single-stranded variable fragment (scFv) or a single-domain binder (dAb).

[0030] Alternatively, chimeric cytokine receptors consist of two polypeptides: (i) (a) Heavy chain variable domain (VH), and (b) A shortened IL2 receptor β-chain endodomain containing the sequence shown as Sequence ID No. 2, having a C-terminal shortening of approximately 10 to 70 amino acids, A first polypeptide containing, (ii) (a) Light chain variable domain (VL), and (b) A shortened common gamma chain end domain containing the sequence shown as Sequence ID No. 1, having a C-terminal shortening of approximately 10 to 30 amino acids, A second polypeptide containing, It may include, VH and VL associate to form a ligand-binding site.

[0031] The ligand may be a tumor secretion factor selected from prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and vascular endothelial growth factor (VEGF) and CA125.

[0032] The ligand may be a chemokine selected from CXCL12, CCL2, CCL4, CCL5, and CCL22.

[0033] In a second embodiment, cells comprising the chimeric cytokine receptor described in the first embodiment of the present invention are provided.

[0034] Cells may also contain chimeric antigen receptors.

[0035] In a third embodiment, a nucleic acid construct encoding a chimeric cytokine receptor according to the first aspect of the present invention is provided, comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide.

[0036] Nucleic acid constructs can also encode chimeric antigen receptors (CARs).

[0037] Nucleic acid constructs may include MND promoters.

[0038] In a fourth embodiment, a vector comprising a nucleic acid construct according to a third aspect of the present invention is provided.

[0039] The vector may be a lentiviral vector. The vector may also be a lentiviral vector containing an MND promoter.

[0040] In the fifth embodiment, i) A vector comprising a nucleic acid sequence encoding a first polypeptide as described in a first aspect of the present invention, ii) A vector comprising a nucleic acid sequence encoding the second polypeptide described in the first aspect of the present invention, A kit is provided that includes the following.

[0041] The kit may also include a vector containing a nucleic acid sequence encoding a chimeric antigen receptor.

[0042] In a sixth aspect, a method for producing cells according to a second aspect of the present invention is provided, comprising the step of introducing a nucleic acid construct according to a third aspect of the present invention, a vector according to a fourth aspect of the present invention, or a kit of a vector according to a fifth aspect of the present invention into cells ex vivo.

[0043] The cells may originate from a sample isolated from the subject.

[0044] In a seventh embodiment, a pharmaceutical composition comprising a plurality of cells according to a second aspect of the present invention is provided.

[0045] In an eighth embodiment, a method for treating a disease is provided, comprising the step of administering a pharmaceutical composition according to the seventh embodiment of the present invention to a subject.

[0046] The method involves the following steps: (i) Isolation step of a cell-containing sample derived from the subject, (ii) A cell transfection or cell transfection step using a nucleic acid construct according to the third aspect of the present invention, a vector according to the fourth aspect of the present invention, or a vector according to the fifth aspect of the present invention, (iii) The step of administering cells from (ii) to the subject, It may include.

[0047] The sample may contain T cells.

[0048] The disease may be, for example, cancer, an infectious disease, or an autoimmune disease.

[0049] In a ninth embodiment, a pharmaceutical composition according to the seventh embodiment of the present invention is provided for use in the treatment of a disease.

[0050] In a tenth embodiment, the use of cells according to a second embodiment of the present invention in the manufacture of a pharmaceutical product for treating a disease is provided.

[0051] Further aspects of the present invention Further aspects of the present invention are shown in the following numbered paragraphs:

[0052] 1. A vector containing a nucleic acid sequence encoding a chimeric cytokine receptor (CCR) under the control of the MND promoter.

[0053] 2. The above CCR consists of two polypeptides: (i) (a) an external domain containing the first dimerization domain, and (b) The first chain of the cytokine receptor endodomain, A first polypeptide containing, (ii) (a) an external domain including the first dimerization domain and a second dimerization domain that spontaneously dimerizes, (b) The second chain of the cytokine receptor endodomain, A second polypeptide containing, Includes, The vector according to paragraph 1, wherein the vector comprises a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide.

[0054] 3. The vector according to paragraph 2, wherein one of the first dimerization domain and the second dimerization domain contains a heavy chain constant domain (CH) and the other contains a light chain constant domain (CL).

[0055] 4. The vector described in paragraph 2 or 3, wherein the cytokine receptor endodomain is a type 1 cytokine receptor endodomain.

[0056] 5. The vector described in paragraph 4, wherein the cytokine receptor endodomain is an IL2 receptor endodomain, an IL7 receptor endodomain, or a GM-CSF receptor endodomain.

[0057] 6. The vector described in paragraph 2 or 3, wherein the cytokine receptor endodomain is the IL18 receptor endodomain.

[0058] 7. A vector, including a nucleic acid sequence encoding a chimeric antigen receptor (CAR), as described in any one of the preceding paragraphs.

[0059] 8. A lentiviral vector, as described in any one of the preceding paragraphs.

[0060] 9. Cells transfected or transfected with the vector described in any one of the preceding paragraphs.

[0061] 10. A method for producing cells as described in paragraph 9, comprising the step of transfecting or transfecting cells with a vector described in any one of paragraphs 1 to 8.

[0062] 11. A pharmaceutical composition comprising multiple cells as described in paragraph 9.

[0063] 12. A method for treating a disease, comprising the step of administering a pharmaceutical composition described in paragraph 11 to a subject. [Brief explanation of the drawing]

[0064] [Figure 1] This is a schematic diagram summarizing the structures of various cytokine receptors, the cell types that produce cytokines, and the cell types that express cytokine receptors. [Figure 2] This is a schematic diagram showing the proposed chimeric cytokine receptor. (a) The cytokine receptors for cytokines IL2 and IL7 signal via a common γ chain and cytokine-specific α / β chains. (b) One embodiment of the chimeric cytokine receptor is to replace the external domains of the cytokine α / β and γ chains with different scFv (or any other suitable binder) that recognize different epitopes of PSA. (c) An alternative approach is to replace the external domains of α / β and γ with VH / VL of a PSA-specific antibody, where both VH and VL are involved in binding, and as a result they become one through binding. [Figure 3] This figure illustrates a cytokine signaling enhancer based on aggregation. It is a schematic diagram showing a combination system of chimeric cytokine receptors and CARs. This cell contains two chimeric cytokine receptors that bind to different epitopes on the same soluble ligand. In the absence of the soluble ligand (e.g., PSA) but in the presence of a cell membrane antigen (e.g., PSMA), signaling occurs via CARs. In the presence of the soluble ligand, aggregation of these two chimeric cytokine receptors occurs, resulting in cytokine-based signal enhancement. [Figure 4]This figure shows a map of the theoretical constructs of the chimeric cytokine receptor / CAR combination system shown in Figure 3. [Figure 5] This is a schematic diagram of the CCR. Each polypeptide in the CCR contains a dimerization domain and a cytokine receptor endodomain. Since the dimerization domain includes an antibody-type heavy chain constant region and a light chain constant region, the shown embodiment has a "Fab" type structure. Sustained dimerization between these domains leads to constitutive cytokine signaling by integrating the IL-2 receptor common γ chain with either the IL-2 receptor β chain or the IL-7 receptor α chain. [Figure 6] This figure shows IL-2 signaling by CCRs. CCRs with the general structure shown in Figure 5 were tested for their ability to induce IL-2 signaling. One CCR had an IL-2 receptor endodomain, and the other had an IL-7 receptor endodomain. IL-2 signaling was tested using the mouse cell line CTLL2, which is dependent on IL-2 signaling for growth. As a positive control, CTLL2 cells were cultured with 100 U / mL of mouse IL-2. Cells expressing a CCR containing an IL-2 receptor endodomain (Fab_IL2 end) supported the survival and growth of CTLL2 cells, whereas cells expressing a CCR containing an IL-7 receptor (Fab_IL7 end) did not. [Figure 7]This is a schematic diagram showing a panel of PSA chimeric cytokine receptors. A panel of PSA-targeting chimeric cytokine receptors (CCRs) was developed using scFv derived from two antibodies that bind to different PSA epitopes: 5D5A5 and 5D3D11. The upper left panel shows a CCR with an IL-2R end-domain having A5 on the chain with the IL2Rβ chain and D11 on the chain with the common γ chain; the upper right panel shows a CCR with an IL7R end-domain having A5 on the chain with the IL7Rα chain and D11 on the chain with the common γ chain; the lower left panel shows a CCR with an IL-2R end-domain having D11 on the chain with the IL2Rβ chain and A5 on the chain with the common γ chain; and the lower right panel shows a CCR with an IL-7R end-domain having D11 on the chain with the IL7Rα chain and A5 on the chain with the common γ chain. Negative controls were also prepared for each CCR, in which the IL2Rγ chain was replaced with a rigid linker. [Figure 8] This figure shows IL2 signaling-CTLL2 proliferation from cells expressing PSA chimeric cytokine receptors in the presence of PSA. CTLL2 cells were transduced with constructs expressing several of the PSA chimeric cytokine receptors shown in Figure 7. The cells were cultured in or without IL2 (positive control), and in or without 5 ng / mL or 5 μg / mL of PSA. CTLL2 proliferation was evaluated at 3 and 7 days. Anti-PSA chimeric cytokine receptors with an IL2R endodomain supported the proliferation of CTLL2 cells in the absence of IL2 and in the presence of PSA, but receptors with an IL7R endodomain, or CCRs containing a rigid linker instead of a common gamma chain, did not. [Figure 9]This figure shows IL2 signaling from cells expressing the PSA chimeric cytokine receptor in the presence of PSA, followed by CTLL2 STAT5 phosphorylation. CTLL2 cells were either left untransduced (WT) or transduced with a vector expressing a CCR for PSA (D11-CD8STK-IL2Rg_A5-hinge-IL2Rb) or an equivalent construct with a rigid linker instead of a common gamma chain (D11-CD8STK-RL_A5-hinge-IL2Rb). The cells were incubated with either 500 μM pervanadate or 500 ng / mL PSA for 1 or 4 hours. Phosphorylation of STAT5 at Y694 was then investigated using phosphoflow. [Figure 10] This figure shows the proliferation signal mediated by IL2Rβ chain shortening. a) Diagram of different IL2Rβ chain shortenings. Each shortening was paired with the full-length IL2R common γ chain. b) Transduced T cells were cultured for 4 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable transduced cells was assessed by flow cytometry. These values ​​were normalized to the value obtained on day 0 and plotted on the y-axis as a multiple change from day 0. The boxes represent the median values ​​for four different donors. [Figure 11] This figure shows the general structure of receptors derived from the type I cytokine receptor family. The extracellular cytokine receptor module contains four conserved cysteine ​​residues involved in disulfide bonding. The WSXWS (Tre, Ser, any amino acid, Tre, Ser) motif, essential for receptor processing, ligand binding, and receptor activation, is also located in the extracellular domain. In the intracellular portion, two short domains, referred to as box 1 and box 2, are important for JAK binding. Tyrosine residues are present in the intracellular portion and are phosphorylated upon receptor activation. [Figure 12]This figure shows the proliferation signal mediated by IL2Rγ chain shortening. a) Diagram of different IL2Rγ chain shortenings. Each shortening was paired with a full-length IL2Rβ chain. b) Transduced T cells were cultured for 4 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable transduced cells was assessed by flow cytometry. These values ​​were normalized to the value obtained on day 0 and plotted on the y-axis as a multiple change from day 0. The boxes represent the median values ​​for four different donors. [Figure 13] This figure compares proliferation signals mediated by truncated IL2R endodomains alone or in combination. T cells were transduced using vectors expressing either the untruncated IL2R CCR (full-length IL2 CCR), IL2 CCR with full-length IL2Rβ chain and truncated IL2Rγ chain (IL2Rγ aa284~349), IL2 CCR with truncated IL2Rβ chain and full-length IL2Rγ chain (IL2Rβ aa266~511), or IL2 CCR with truncated IL2Rβ chain and truncated IL2Rγ chain (IL2Rγ aa284~349 / IL2Rβ aa266~511). Transduced T cells were cultured for 7 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable transduced cells was evaluated by flow cytometry. These values ​​were normalized to the value obtained on day 0 and plotted on the y-axis as a multiple change from day 0. The boxes represent the median values ​​for four separate donors. [Modes for carrying out the invention]

[0065] Detailed explanation Chimeric cytokine receptor (CCR) Chimeric cytokine receptors are described in Patent Document 1.

[0066] A chimeric cytokine receptor (CCR) is a molecule that contains either a cytokine receptor endodomain and either a heterologous ligand-binding exodomain or a dimerization domain that combines two chains of the cytokine receptor endodomain. This latter type of CCR will be described in more detail below.

[0067] In ligand-binding cytokine receptors (CCRs), heterologous exodomains bind to ligands other than cytokines, which are selective for the cytokine receptor from which the endodomain originates. In this way, by modifying the ligand specificity of the cytokine receptor, it is possible to alter its ligand specificity.

[0068] Ligand-bound chimeric cytokine receptors are (i) Ligand-binding exodomain and (ii) Optional spacers, (iii) Transmembrane domain and (iv) Cytokine receptor endodomain and Includes.

[0069] Alternatively, the CCR may include a dimerization domain and a cytokine receptor endodomain.

[0070] This type of chimeric cytokine receptor is (i) Dimerized exodomain and (ii) Optional spacers, (iii) Transmembrane domain and (iv) Cytokine receptor endodomain and Includes.

[0071] Dimerization can occur spontaneously, in which case the CCR is constitutively active. Alternatively, dimerization may occur only in the presence of a dimerizing chemical inducer (CID), in which case the transmembrane protein triggers cytokine-type signaling only in the presence of the CID.

[0072] Suitable dimerization domains and CIDs are described in International Publication No. 2015 / 150771 (which is incorporated herein by reference).

[0073] For example, one dimerized domain may contain the rapamycin-binding domain of FK-binding protein 12 (FKBP12), and the other may contain the FKBP12-rapamycin-binding (FRB) domain of mTOR, and CID may be rapamycin or a derivative thereof.

[0074] One dimerization domain may contain the FK506 (tacrolimus) binding domain of FK-binding protein 12 (FKBP12), and the other dimerization domain may contain the cyclosporine binding domain of cyclophylline A, and CID may be an FK506 / cyclosporine fusion or a derivative thereof.

[0075] One dimerization domain may contain an estrogen-binding domain (EBD), and the other dimerization domain may contain a streptavidin-binding domain, and CID may be an estrone / biotin fusion protein or a derivative thereof.

[0076] One dimerized domain may contain a glucocorticoid-binding domain (GBD), and the other dimerized domain may contain a dihydrofolate reductase (DHFR)-binding domain, and CID may be a dexamethasone / methotrexate fusion protein or a derivative thereof.

[0077] One dimerization domain may contain an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain, and the other dimerization domain may contain a dihydrofolate reductase (DHFR) binding domain, and CID may be an O6-benzylguanine derivative / methotrexate fusion protein or a derivative thereof.

[0078] One dimerized domain may contain a retinoic acid receptor domain, and the other dimerized domain may contain an ecdysone receptor domain, and CID may be RSL1 or a derivative thereof.

[0079] If a dimerizing domain spontaneously undergoes heterodimerization, it may be based on the antibody's dimerizing domain. In particular, it may include dimerized portions of the heavy chain constant domain (CH) and the light chain constant domain (CL). The "dimerized portion" of the constant domain is a part of the sequence that forms an interchain disulfide bond.

[0080] Chimeric cytokine receptors may contain the Fab portion of an antibody as an exodomain, for example, as schematically shown in Figure 5.

[0081] Chimeric cytokine receptors consist of two polypeptides: (i) (a) the first dimerization domain, and (b) The first chain of the cytokine receptor endodomain, A first polypeptide containing, (ii) (a) A first dimerization domain and a second dimerization domain that undergoes dimerization, and (b) Second chain of the cytokine receptor endodomain, A second polypeptide containing, Includes.

[0082] Cytokine receptors and signal transduction The function of many cells is regulated by members of the cytokine receptor superfamily. Signaling by these receptors depends on association with Janus kinase (JAK), which links ligand binding to tyrosine phosphorylation of signaling proteins recruited to the receptor complex. These include signal transducers and activators of transcription (STAT), a family of transcription factors that contribute to the diversity of cytokine responses.

[0083] When chimeric cytokine receptors bind to their ligands or dimerize, they can trigger one or more of the following intracellular signaling pathways: (i) JAK-STAT routes (ii) MAP kinase pathway, and (iii) The phosphoinositide 3-kinase (PI3K) pathway.

[0084] The JAK-STAT system consists of three main components: (1) a receptor, (2) Janus kinase (JAK), and (3) a signaling transcription factor (STAT).

[0085] JAK possesses tyrosine kinase activity and binds to cytokine receptors on the cell surface. Binding of ligands to these receptors induces JAK activation. Increased kinase activity phosphorylates tyrosine residues on the receptors, creating sites for interaction with proteins containing phosphotyrosine-binding SH2 domains. STATs with SH2 domains capable of binding to these phosphotyrosine residues are recruited to the receptors and are themselves tyrosine-phosphorylated by JAK. These phosphotyrosines then act as binding sites for the SH2 domains of other STATs, mediating their dimerization. Various STATs form heterodimers or homodimers. Activated STAT dimers accumulate in the cell nucleus and activate the transcription of their target genes.

[0086] Cytokine receptor endodomain CCRs contain an endodomain that induces "cytokine-type" cellular signaling (either alone or in the presence of another chimeric cytokine receptor).

[0087] The endodomain may be a cytokine receptor endodomain.

[0088] The endodomain may originate from type I cytokine receptors. Type I cytokine receptors share a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane.

[0089] The endodomain may be derived from type II cytokine receptors. 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).

[0090] Type I cytokine receptors include: (i) Interleukin receptors (e.g., receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, and IL-27); (ii) Colony-stimulating factor receptors (e.g., receptors for erythropoietin, GM-CSF, and G-CSF); and, (iii) Hormone receptors / neuropeptide receptors (e.g., hormone receptors and prolactin receptors); It includes.

[0091] Members of the type I cytokine receptor family contain different chains, some of which are involved in ligand / cytokine interactions, while others are involved in signal transduction. For example, the IL-2 receptor contains α, β, and γ chains.

[0092] The common gamma chain of the IL-2 receptor (also known as CD132) is shared among the IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15 receptors.

[0093] IL-2 IL-2 binds to the IL-2 receptor, which has three forms produced by different combinations of three different proteins, often referred to as α, β, and γ "chains"; these subunits are also part of the receptors for other cytokines. The β and γ chains of IL-2R are members of the type I cytokine receptor family.

[0094] These three receptor chains can be expressed separately and differently on various cell types and, by assembling in different combinations and orders, can generate low-affinity, medium-affinity, and high-affinity IL-2 receptors.

[0095] The α chain binds to IL-2 with low affinity, while the β and γ combination forms a complex that binds to IL-2 with moderate affinity, mainly on memory T cells and NK cells. All three receptor chains bind with high affinity (Kd approximately 10) on activated T cells and regulatory T cells. -11 M) forms a complex that binds to IL-2.

[0096] These three IL-2 receptor chains span the cell membrane and extend into the cell, thereby transmitting biochemical signals into the intracellular space. The α chain is not involved in signal transduction, while the β chain forms a complex with the tyrosine phosphatase JAK1. Similarly, the γ chain forms a complex with another tyrosine kinase called JAK3. These enzymes are activated by IL-2 binding to the external domain of IL-2R.

[0097] IL-2 signaling promotes the differentiation of early T cells into effector T cells and memory T cells when they are also stimulated by an antigen. Through their role in the development of T cell immunological memory, which depends on the number and functional expansion of T cell clones selected by the antigen, they also play an important role in long-term cellular immunity.

[0098] The chimeric cytokine receptor may include the β chain of the IL-2 receptor and / or the γ chain of the IL-2 receptor (i.e., common).

[0099] The amino acid sequences for the β-chain and common γ-chain endodomains of IL-2 are shown as Sequence ID No. 1 and Sequence ID No. 2. Sequence ID 1: Endodomain derived from the common human gamma chain: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET Sequence ID 2: Endodomain derived from human IL-2Rβ: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0100] The term "derived from ~" means that the endodomain of the chimeric cytokine receptor has the same sequence as the wild-type sequence of the endogenous molecule, or a variant of that molecule that retains the ability to form a complex with JAK-1 or JAK-3 and to activate one of the signaling pathways described above.

[0101] A "mutant" sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the wild-type sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), wherein the mutant sequence is limited to retaining the function of the wild-type sequence, namely the ability to form a complex with JAK-1 or JAK-3, and, for example, the ability to activate the JAK-STAT signaling pathway.

[0102] The percentage identity between two polypeptide sequences can be easily determined by programs such as BLAST, which are freely available at http: / / blast.ncbi.nlm.nih.gov.

[0103] IL-7 The interleukin-7 receptor consists of two chains: the interleukin-7 receptor-α chain (CD127) and the common gamma chain receptor (CD132). The common gamma chain receptor is shared with various cytokines, including interleukin-2, interleukin-4, interleukin-9, and interleukin-15. The interleukin-7 receptor is expressed on various cell types, including naive T cells and memory T cells.

[0104] The interleukin-7 receptor plays a crucial role in lymphocyte development, particularly in V(D)J recombination. IL-7R also regulates accessibility to genomic regions, including the T cell receptor γ gene, through STAT5 and histone acetylation. Knockout studies in mice suggest that inhibiting apoptosis is an essential function of this protein in T lymphocyte differentiation and activation.

[0105] Chimeric cytokine receptors may include the α chain of the IL-7 receptor and / or the γ chain of the IL-7 receptor (i.e., common), or variants thereof.

[0106] The amino acid sequence for the endodomain of the α-chain of IL-7 is shown as Sequence ID No. 3. Endodomain derived from Sequence ID No. 3 - Human IL-7Rα: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSSNQEEAYVTMSSFYQNQ

[0107] IL-15 Interleukin-15 (IL-15) is a cytokine that is structurally similar to IL-2. Like IL-2, IL-15 binds to a complex composed of the IL-2 / IL-15 receptor β chain (CD122) and common γ chain (γ-C, CD132), and signals are transmitted through this complex. IL-15 is secreted by mononuclear phagocytes (and some other cells) after viral infection. IL-15 induces the proliferation of natural killer cells.

[0108] The interleukin-15 receptor consists of the interleukin-15 receptor α subunit and shares β and γ subunits with the IL-2 receptor.

[0109] The amino acid sequence for the endodomain of IL-15Rα is shown as Sequence ID No. 60.

[0110] Endodomain derived from Sequence ID No. 60 - Human IL-15Rα: SRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

[0111] IL-18 IL-18 belongs to the IL-1 superfamily and is primarily produced by macrophages, but is also produced by other cell types and has multifaceted functions in cellular stimulation. IL-18 is a pro-inflammatory cytokine that promotes type 1 responses. Together with IL-12, it induces cellular immunity after infection with microbial products such as lipopolysaccharide (LPS). In combination with IL-12, IL-18 acts on CD4, CD8 T cells, and NK cells to induce the production of IFNγ, a type II interferon that plays a crucial role in the activation of macrophages or other cells. This combination of IL-18 and IL-12 has been shown to inhibit IL-4-dependent IgE and IgG1 production and enhance IgG2a production in B cells. In vivo, IL-18 does not induce IFNγ production in the absence of IL-12 or IL-15, but it has been reported to play an important role in the differentiation of naive T cells into Th2 cells and stimulate mast cells and basophils to produce chemical mediators such as IL-4, IL-13, and histamine.

[0112] The IL-18 receptor consists of the interleukin-18 receptor 1 (IL18R1), an inductive component that binds to mature IL-18 with low affinity, and the constitutively expressed co-receptor interleukin-18 receptor accessory protein (IL18RAP). IL-18 binds to the ligand receptor IL18R1, inducing the recruitment of IL18RAP to form a high-affinity complex, which then signals via the toll / interleukin-1 receptor (TIR) ​​domain. This signaling domain recruits the MyD88 adapter protein, which activates the inflammatory program and the NF-κB pathway.

[0113] The amino acid sequence for human IL18 receptor 1 (also known as IL18 receptor α-chain) is available from Uniprot accession number Q13478 and is shown below as Sequence ID No. 67. In this sequence, residues 22-329 represent the extracellular domain, residues 330-350 represent the transmembrane domain, and residues 351-541 represent the cytoplasmic domain. Sequence ID 67-Human IL18R1 MNCRELPLTLWVLISVSTAESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKF FQITCENSYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIM TPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKADMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLE KHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES

[0114] The amino acid sequence for the human IL18 receptor accessory protein (also known as IL18RAP or IL18 receptor β-chain) is available from Uniprot accession number O95256 and is shown below as Sequence ID No. 68. In this sequence, residues 20-356 represent the extracellular domain, residues 357-377 represent the transmembrane domain, and residues 378-599 represent the cytoplasmic domain. Sequence ID 68 - Human IL18RAP MLCLGWIFLWLVAGERIKGFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKP QTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKLKPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWE VSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYS LCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW

[0115] GMCSF Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts, and functions as a cytokine.

[0116] The granulocyte-macrophage colony-stimulating factor receptor is a heterodimer composed of at least two different subunits (an α chain and a β chain, which is also present in the receptors for IL-3 and IL-5).

[0117] The amino acid sequence for the human GMCSF receptor α chain is available from Uniprot accession number P15509 and is shown below as Sequence ID No. 69. In this sequence, residues 23-320 represent the extracellular domain, residues 321-346 represent the transmembrane domain, and residues 347-400 represent the cytoplasmic domain. GMCSF receptor α chain (SEQ ID NO: 69) MLLLVTSLLLCELPHPAFLLIPEKSDLRTVAPASSLNVRFDSRTMNLSWDCQENTTFSKCFLTDKKNRVVEPRLSNNECSCTFREICLHEGVTFEVHVNTSQRGFQQKLLYPNSGREGTAAQNFSCFIYNADLMNCTWARGPTAPRDVQYFLYIRNSKRRREIRCPYYIQDSGTHVGCHLLDNLSGLTSRNYFLVNGTSRE IGIQFFDSLLDTKKIERFNPPSNVTVRCNTTHCLVRWKQPRTYQKLSYLDFQYQLDVHRKNTQPGTENLLINVSGDLENRYNFPSSEPRAKHSVKIRAAD VRILNWSSWSEAIEFGSDDGNLGSVYIYVLLIVGTLVCGIVLGLFFKRFLRIQRLFPPVPQIKDKLNDNHEVEDEIIWEEFTPEEGKGYREEVLTVKEIT

[0118] The amino acid sequence for the common human β-chain is available from Uniprot accession number P32927 and is shown below as Sequence ID No. 70. In this sequence, residues 17-443 represent the extracellular domain, residues 444-460 represent the transmembrane domain, and residues 461-897 represent the cytoplasmic domain. Human common β-chain (SEQ ID NO: 70) MVLAQGLLSMALLALCWERSLAGAEETIPLQTLRCYNDYTSHITCRWADTQDAQRLVNVTLIRRVNEDLLEPVSCDLSDDMPWSACPHPRCVPRRCVIPCQSFVVTDVDYFS FQPDRPLGTRLTVTLTQHVQPPEPRDLQISTDQDHFLLTWSVALGSPQSHWLSPGDLEFEVVYKRLQDSWEDAAILLSNTSQATLGPEHLMPSSTYVARVRTRLAPGSRLSG RPSKWSPEVCWDSQPGDEAQPQNLECFFDGAAVLSCSWEVRKEVASSVSFGLFYKPSPDAGEEECSPVLREGLGSLHTRHHCQIPVPDPATHGQYIVSVQPRRAEKHIKSSV NIQMAPPSLNVTKDGDSYSLRWETMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPSTRYWARVRVRTSRTGYNGIWSEWSEARSWDTESVLPMWVLALI VIFLTIAVLLALRFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASD LPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGG GPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSPRNNP VPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC

[0119] Shortened cytokine receptor endodomain In the chimeric cytokine receptor of the present invention, both cytokine receptor endodomain chains are shortened. By shortening the C-terminus of one or both of the cytokine receptor endodomain chains, it is possible to regulate the activity of the CCR.

[0120] Figure 11 shows a schematic diagram illustrating the general structure of the cytokine receptor endodomain. The endodomain contains elements known as box 1 and box 2, which are important for JAK binding. A series of tyrosine residues are present in the intracellular portion and are phosphorylated upon receptor activation.

[0121] The sequence of the endodomain derived from human IL-2Rβ is shown above as Sequence ID No. 2. The Box 1 motif is derived from amino acids 278-286 in the full-length sequence and has the sequence KCNTPDPS (Sequence ID No. 47). The Box 2 motif is derived from amino acids 323-333 in the full-length sequence and has the sequence SPLEVLERDKV (Sequence ID No. 48). IL2BR endodomain showing box 1 and box 2 and tyrosine residues (SEQ ID NO: 2) TIFF2026510989000001.tif43170

[0122] If the CCR contains a receptor endodomain derived from the type I cytokine receptor family, it may contain an endodomain that is shortened at the C-terminus but retains the box 1 and box 2 motifs.

[0123] The end-domain derived from human IL-2Rβ is 286 amino acids long, as shown in SEQ ID NO: 2 and Figure 10a. The truncated form of IL-2Rβ may lack up to 218 amino acids from the C-terminus. This means that the box 1 and box 2 motifs remain intact in the first 68 amino acids of the sequence. The truncated form of IL-2Rβ may have C-terminal shortenings of up to 200 amino acids, up to 180 amino acids, up to 160 amino acids, up to 140 amino acids, up to 120 amino acids, up to 100 amino acids, up to 80 amino acids, up to 60 amino acids, up to 40 amino acids, or up to 20 amino acids.

[0124] Truncations of IL-2Rβ can range from 10-200 amino acids, 20-180 amino acids, 40-180 amino acids, 60-160 amino acids, 80-140 amino acids, or 100-120 amino acids. As shown in Figure 10b, truncations of 40 amino acids (i.e., IL2Rβ aa266-511) to 180 amino acids (i.e., IL2Rβ aa266-371) result in a gradual decrease in cytokine signaling activity, making it possible to "tune down" cytokine signaling by selecting deletions within this range.

[0125] The truncated form of IL-2Rβ may have one of the sequences shown as sequence numbers 49 to 59. The truncated form of IL-2Rβ may have a sequence "between" two of the truncated sequences shown as sequence numbers 49 to 59. For example, the sequence "between" IL2Rβ aa266~411 (sequence number 53) and IL2Rβ aa266~431 (sequence number 54) could be aa266~412, aa266~413, aa266~429, aa266~430, etc. IL2Rβ aa266~331 (SEQ ID NO: 49): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKV IL2Rβ aa266~351 (SEQ ID NO: 50): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSS IL2Rβ aa266~371 (Sequence ID 51): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLP IL2Rβ aa266~391 (SEQ ID NO: 52): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEED IL2Rβ aa266~411 (SEQ ID NO: 53): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPL IL2Rβ aa266~431 (SEQ ID NO: 54): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFS IL2Rβ aa266~451 (SEQ ID NO: 55): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAG IL2Rβ aa266~471 (Sequence ID 56): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQP IL2Rβ aa266~491 (SEQ ID NO: 57): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEAC QVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELV IL2Rβ aa266~511 (Sequence ID 58): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYS EEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSF IL2Rβ aa266~531 (Sequence ID 59): NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAG APTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPL

[0126] As shown in Figure 11 and the annotated version of Sequence ID No. 2 above, IL-2Rβ has six tyrosine residues in its endodomain. These tyrosine residues are phosphorylated upon receptor activation. Truncate forms of IL-2Rβ may lack one or more tyrosine residues in their endodomain compared to the wild-type sequence. Truncate forms of the IL-2Rβ endodomain may lack one, two, three, four, five, or all six tyrosine residues compared to the wild-type sequence.

[0127] The endodomain derived from the common human gamma chain has the sequence shown above as Sequence ID No. 1, which has 86 amino acids. Shortened versions of this sequence may have C-terminal shortenings of up to 60, 50, 40, 30, 20, or 10 amino acids.

[0128] The truncated form of the human common gamma chain may have one of the sequences shown as SEQ ID NOs. 61 to 66. The truncated form of the human common gamma chain may have a sequence "between" two of the truncated sequences shown as SEQ ID NOs. 61 to 66, for example, the sequence "between" IL2Rγ aa284~359 (SEQ ID NOs. 61) and IL2Rγ aa284~349 (SEQ ID NOs. 62) could be aa284~358, aa284~357, aa284~350, aa284~349, etc. IL2Rγ aa284~359 (Sequence ID 61): ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWA IL2Rγ aa284~349 (Sequence ID 62): ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGAS IL2Rγ aa284~339 (SEQ ID NO: 63): ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKG IL2Rγ aa284~329 (Sequence ID 64): ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERL IL2Rγ aa284~309 (SEQ ID NO: 65): ERTMPRIPTLKNLEDLVTEYHGNFSA IL2Rγ aa284~289 (Sequence ID 66): ERTMPR

[0129] The common gamma chain has four tyrosine residues in its endodomain. The truncated form of the common gamma chain endodomain may lack one or more tyrosine residues compared to the wild-type sequence. The truncated form of the common gamma chain endodomain may lack one, two, three, or all four tyrosine residues compared to the wild-type sequence.

[0130] The endodomain derived from human IL-7Rα has the sequence shown above as Sequence ID No. 3, which has 195 amino acids. Shortened versions of this sequence may have C-terminal shortenings of up to 120, 100, 80, 60, 40, or 20 amino acids.

[0131] Human IL-7Rα has three tyrosine residues in its endodomain. The truncated form of the human IL-7Rα endodomain may lack one or more tyrosine residues compared to the wild-type sequence. The truncated form of the human IL-7Rα endodomain may lack one, two, or all three tyrosine residues compared to the wild-type sequence.

[0132] The endodomain of IL-15Rα, shown above as SEQ ID NO: 60, has 38 amino acids. Shortened versions of this sequence may have C-terminal shortenings of, for example, up to 20, up to 15, up to 10, or up to 5 amino acids.

[0133] For any given cytokine receptor end-domain, the truncated form may have up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10% C-terminal deletions of amino acids compared to the wild-type end-domain sequence. The deletions may be 10%–60%, 20%–50%, or 30%–40%.

[0134] In chimeric cytokine receptors, one or more chains may have a truncated sequence. For example, in a CCR having an IL-2 receptor endodomain containing the IL-2 receptor β chain and / or the IL-2 receptor (i.e., common) γ chain, the IL-2 receptor β chain and / or common γ chain may be truncated.

[0135] Methods for regulating CCR activity The activity of chimeric cytokine receptors (CCRs) can be regulated by shortening one or more chains in the cytokine receptor endodomain. CCR activity, and therefore cytokine signaling, can increase or decrease in response to this shortening.

[0136] As shown in Example 3, by preparing a construct with a deletion panel and investigating its effect on cytokine signaling by observing parameters such as cell proliferation, it is possible to confirm the effect of shortening the cytokine receptor endodomain in CCR.

[0137] Furthermore, it is possible to adjust cytokine signaling to a desired level by selecting a shortening or combination of shortenings of the cytokine receptor endodomain that results in a desired level of activity for cytokine signaling mediated by CCR.

[0138] The activity of chimeric cytokine receptors (CCRs) can be reduced by shortening one or more chains in the cytokine receptor endodomain.

[0139] For example, the activity of CCRs containing the IL-2 receptor β-end domain can be reduced by shortening the IL-2Rβ at the C-terminus by 40 to 180 amino acids.

[0140] Spacer Chimeric cytokine receptors may include spacers that connect the antigen-binding domain or dimerization domain to the transmembrane domain and spatially separate the antigen-binding domain or dimerization domain from the endodomain. Flexible spacers allow the antigen-binding domain to be oriented in various directions that enable antigen binding.

[0141] When cells express chimeric cytokine receptors (CCRs) and chimeric antigen receptors (CARs), the spacers of the CCRs and CARs may be the same or different, for example, having different lengths. The spacer of the CAR may be longer than the spacer of the CCR.

[0142] The spacer sequence may include, for example, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk. Alternatively, the linker may include an alternative linker sequence having similar length and / or domain spacing characteristics to the IgG1 Fc region, IgG1 hinge, or CD8 stalk.

[0143] The human IgG1 spacer may be modified to remove the Fc-binding motif.

[0144] Examples of amino acid sequences for these spacers are shown below: Sequence ID 4 (Human IgG1 hinge-CH2CH3) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD Sequence ID 5 (Human CD8 Stork): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI Sequence ID 6 (Human IgG1 Hinge): AEPKSPDKTHTCPPCPKDPK

[0145] transmembrane domain The transmembrane domain is a sequence of CCRs that spans the membrane. It may contain a hydrophobic α-helix. The transmembrane domain may originate from CD28, which contributes to good receptor stability.

[0146] Alternatively, the transmembrane domain may originate from the same cytokine as the cytokine receptor, for example, the endodomain.

[0147] The transmembrane domain may originate from, for example, IL-2R, IL-7R, or IL-15R. Sequence ID 7 - Transmembrane transfer derived from the human common gamma chain: VVISVGSMGLIISLLCVYFWL Transmembrane transmembrane derived from SEQ ID NO: 8-Human IL-2Rβ: IPWLGHLLVGLSGAFGFIILVYLLI Transmembrane transmembrane derived from Sequence ID No. 9 - human IL-7Rα: PILLTISILSFFSVALLVILACVLW Transmembrane transmembrane derived from Sequence ID No. 10 - human IL-15Rα: AISTSTVLLCGLSAVSLLACYL

[0148] Ligand-binding exodomain The ligand-binding domain includes an antigen-binding domain. The antigen-binding domain binds to target ligands for CCRs, i.e., tumor secretion factors, chemokines, or cell surface antigens.

[0149] Numerous antigen-binding domains are known in the art, including those based on antigen-binding sites of antibodies, antibody mimetics, and T cell receptors. For example, antigen-binding domains may include single-strand variable fragments (scFv) derived from monoclonal antibodies; binding domains derived from native receptors for target antigens; peptides with sufficient affinity for target ligands; single-domain binders (e.g., from camelids); artificial single binders (as Darpin); or single-stranded domains derived from T cell receptors.

[0150] The term "ligand" is used synonymously with "antigen," meaning an entity that is specifically recognized and bound to the antigen-binding domain of the CCR.

[0151] If the ligand is a tumor secretion factor, the antigen-binding domain may include an immunoglobulin-based antigen-binding site (e.g., scFv or a single-domain binder).

[0152] If the ligand is a chemokine, the antigen-binding domain may include the chemokine-binding portion of the innate receptor for the chemokine.

[0153] Ligand The CCR of the present invention can bind to a ligand.

[0154] The ligand may be a soluble ligand such as a tumor secretion factor or a chemokine.

[0155] Alternatively, the ligand may be a membrane-bound ligand such as a cell surface antigen.

[0156] The term "soluble ligand" is used to refer to a ligand or antigen that is not part of a cell or attached to a cell, but is freely mobile in the extracellular space, for example, in the fluids of the tissue of interest. Soluble ligands may exist in a cell-free state in the serum, plasma, or other fluids of an individual.

[0157] Soluble ligands may be associated with the presence or pathology of certain diseases, such as cancer.

[0158] Soluble ligands may be part of the cancer secretome, i.e., a collection of factors secreted by tumors, and may originate from cancer stem cells, non-stem cells, or the surrounding stroma. Soluble ligands may be secreted or shed by tumor cells (see the next section).

[0159] Soluble ligands may be characteristic of disease or affected tissue. They may be exclusively found, or at higher levels, in diseased subjects compared to healthy subjects, or in affected tissue compared to healthy tissue. Soluble ligands may be expressed at levels at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, or 100000-fold higher in diseased subjects compared to healthy subjects, or in affected tissue compared to healthy tissue.

[0160] The terms "cell surface antigen" and "cell surface ligand" are used synonymously with "membrane-bound antigen" and "membrane-bound ligand," which refer to ligands that are attached to or expressed on the cell surface. A cell surface ligand may, for example, be a transmembrane protein.

[0161] Cells exhibiting cell surface ligands may be target cells, such as cancer cells.

[0162] Cell surface ligands may be associated with the presence or pathology of specific diseases, such as cancer. Alternatively, cell surface ligands may be characteristic of the cell type of target cells (e.g., B cells) without necessarily being associated with disease status.

[0163] If a cell surface ligand is characteristic of a disease or affected tissue, it may be found exclusively or at higher levels on the relevant cells of a diseased subject compared to a healthy subject, or on the relevant cells of an affected tissue compared to a healthy tissue. The cell surface ligand may be expressed at levels at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, or 100000-fold higher on the cells of a diseased subject compared to a healthy subject, or on the cells of an affected tissue compared to a healthy tissue.

[0164] Tumor secreted factors The ligands recognized by CCR may be soluble ligands secreted by or shed from the tumor.

[0165] These "tumor-secreting factors" may be, for example, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), or cancer antigen-125 (CA-125).

[0166] Tumor secretory factors can be soluble ligands that are not cytokines. Therefore, CCRs transfer binding specificity for non-cytokine ligands onto the endodomain of cytokine receptors.

[0167] Prostate-specific antigen (PSA) The soluble ligand may be a prostate-specific antigen (PSA).

[0168] Prostate-specific antigen (PSA), also known as γ-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 prostate epithelial cells.

[0169] PSA is present in small amounts in the serum of men with a healthy prostate, but it is elevated in individuals with prostate cancer and other prostate disorders.

[0170] PSA is a 237-residue glycoprotein activated by KLK2. Its physiological role is the liquefaction of the coagulant components of semen, leading to sperm release. In cancer, PSA may be involved in the processes of neoplasm growth and metastasis.

[0171] PSA is a chymotrypsin-like serine protease possessing a typical His-Asp-Ser triple structure and a catalytic domain similar to that of other kallikrein-related peptidases. The crystal structure of PSA was obtained i) as a complex with the monoclonal antibody (mAb) 8G8F5, and ii) as a sandwich complex with two mAbs, 5D5A5 and 5D3D11 (Stura et al. (J. Mol. Biol. (2011) 414:530-544)).

[0172] Various monoclonal antibodies are known, including clones 2G2-B2, 2D8-E8, and IgG1 / K, described in Bavat et al. Avicenna J. Med. Biotechnol. 2015, 7:2-7 and Leinonen (2004) 289:157-67.

[0173] The CCR may include, for example, six CDR or VH and / or VL domains (or more) derived from a PSA-conjugated mAb (e.g., 8G8F5, 5D5A5, or 5D3D11).

[0174] If the CCR includes two antigen-binding specificities that bind to different epitopes on the PSA, one may be obtained based on, for example, 5D3D11, and the other may be obtained based on, for example, 5D5A5.

[0175] The amino acid sequences for VH and VL of 5D3D11 and 5D5A5 are shown below. The complementarity-determining region (CDR) is highlighted in bold. 5D3D11 VH (Sequence ID 11) TIFF2026510989000002.tif211705D3D11 VL (Sequence ID 12) TIFF2026510989000003.tif141705D5A5 VH (Sequence ID 13) TIFF2026510989000004.tif211705D5A5 VL(Sequence ID 14) ScFv (Sequence ID 15) based on TIFF2026510989000005.tif141705D5A5 QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGGSGG GGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKLEIK ScFv (sequence number 16) based on 5D3D11 QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGGSGG GGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGTKVEIK

[0176] If a cell contains two CCRs, the antigen-binding domain of the first CCR may contain six CDRs derived from 5D5A5, and the antigen-binding domain of the second CCR may contain six CDRs derived from 5D3D11.

[0177] The antigen-binding domain of the first CCR may contain VH and / or VL domains (or more) derived from 5D5A5 or variants thereof, and the antigen-binding domain of the second CCR may contain VH and / or VL domains (or more) derived from 5D3D11 or variants thereof. The variant VH and VL domains may have at least 80%, 90%, 95%, or 99% identity with the sequences shown above, but retain PSA-binding activity.

[0178] Cells expressing PSA-binding CCR may be useful in the treatment of prostate cancer.

[0179] Carcinoembryonic antigen (CEA) The soluble ligand could be CEA.

[0180] Carcinoembryonic antigens (CEAs) refer to a series of highly related glycoproteins involved in cell adhesion. Normally, CEAs are produced in gastrointestinal tissue during fetal development, but production ceases before birth. Therefore, CEAs are usually present in very low levels in the blood of healthy adults. However, serum levels are elevated in some types of cancer, meaning they can be used as tumor markers in clinical testing.

[0181] CEAs are glycosylphosphatidylinositol (GPI) cell surface anchored glycoproteins, and their specialized sialofucosylated glycoforms act as functional colon cancer L-selectin and E-selectin ligands, which may be crucial for metastatic dissemination of colon cancer cells. Immunologically, they are characterized as members of the CD66 surface antigen classification.

[0182] CEA and related genes constitute the CEA family, which belongs to the immunoglobulin superfamily. In humans, the carcinoembryonic antigen family consists of 29 genes, 18 of which are normally expressed. The following is a list of human genes encoding carcinoembryonic antigen-associated cell adhesion proteins: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21.

[0183] Various antibodies targeting CEA are described in International Publication No. 2011 / 034660.

[0184] Cells expressing CCR against CEA may be useful, for example, in the treatment of colorectal cancer.

[0185] Vascular endothelial growth factor (VEGF) The soluble ligand could be VEGF.

[0186] Vascular endothelial growth factor (VEGF) is a signaling protein produced by cells that stimulates vascular formation and angiogenesis. It is part of a system that restores oxygen supply to tissues when blood circulation is insufficient. Serum levels of VEGF are high in bronchial asthma and diabetes mellitus. The normal functions of VEGF include creating new blood vessels during embryonic development, creating new blood vessels after injury, creating muscle after exercise, and creating new blood vessels (collateral circulation) that bypass blocked blood vessels.

[0187] VEGF, when overexpressed, can contribute to disease. Solid tumors cannot grow beyond a certain size without an adequate blood supply; cancers that can express VEGF are capable of growing and metastasizing.

[0188] VEGFs are a subfamily of platelet-derived growth factors within the cystine-knot growth factor family. They are important signaling proteins involved in both vascularization (de novo formation of the fetal circulatory system) and angiogenesis (growth of blood vessels from existing vascular structures).

[0189] The VEGF family in mammals includes five members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D.

[0190] Various antibodies against VEGF are known (e.g., bevacizumab (Avastin) and ranibizumab (Lucentis)).

[0191] Cancer antigen 125 (CA-125) CA-125 is associated with ovarian cancer and is the most frequently used biomarker for detecting ovarian cancer. While CA-125 is best known as a marker for ovarian cancer, it may also be elevated in other cancers, including endometrial cancer, fallopian tube cancer, lung cancer, breast cancer, and gastrointestinal cancer.

[0192] The sequence for human CA-125 (also known as mucin-16) is available from NCBI, accession number 078966.

[0193] Numerous CA125-conjugating monoclonal antibodies are known, including OC125 and M11 (Nustad et al. 1996, Tumour Biol. 17:196-329). This study investigated the specificity of 26 monoclonal antibodies against the CA125 antigen. It was found that the CA125 antigen possesses only two major antigenic domains, which classify the antibodies as either OC125-like (Group A) or M11-like (Group B).

[0194] Chimeric cytokine receptors may contain antigen-binding domains derived from such antibodies. Cells containing such CCRs may be useful, for example, in the treatment of ovarian cancer.

[0195] Tumor secretory factors (or, in the case of membrane-bound forms, transmembrane proteins) may be selected from the following non-exclusive list:

[0196] Rearrangement and overexpression of the ALK gene resulting in ALK protein mutations Alpha-fetoprotein (AFP) β-2-microglobulin (B2M) β-Human chorionic gonadotropin (β-hCG) BRAF V600 mutation resulting in a mutated B-REF protein C-kit / CD117 CA15-3 / CA27.29 CA19-9 Calcitonin CD20 Chromogranin A (CgA) Cytokeratin Fragment 21-1 EGFR gene mutation analysis Estrogen receptor (ER) / Progesterone receptor (PR) Fibrin / Fibrinogen HE4 Amplification or overexpression of the HER2 / neu gene Immunoglobulins KRAS gene mutation analysis Lactate dehydrogenase Neuron-specific enolase (NSE) Nuclear matrix protein 22 Programmed dead ligand 1 (PD-L1) thyroglobulin Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1)

[0197] Chemokines Chemokines are chemotactic cytokines. Cell migration is guided by gradients of chemokines embedded and immobilized in the extracellular matrix. Positively charged chemokines, such as CXCL12, bind to negatively charged ECM molecules. These gradients provide pathways for homing cancer cells and immune cells. Their effects on T cells appear to be inhibitory to homing of cytotoxic T cells, while regulatory T cells appear to be attracted.

[0198] Chemokines have a mass of approximately 8 to 10 kilodaltons and contain four cysteine ​​residues in conserved positions that are important for the formation of their three-dimensional shape.

[0199] Some chemokines are thought to be pro-inflammatory, induced during immune responses to recruit immune system cells to the site of infection, while others are thought to be homeostatic, involved in maintaining tissues or regulating cell migration in normal developmental processes.

[0200] 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-bound transmembrane receptors called chemokine receptors, which are selectively found on the surface of their target cells.

[0201] The primary role of chemokines is to act as chemical attractants to guide cell migration. Cells attracted by chemokines follow a signal of increasing chemokine concentration toward the source of the chemokine. Some chemokines regulate cells of the immune system during processes of immune surveillance, such as directing lymphocytes to lymph nodes, and they can screen for pathogen infiltration by interacting with antigen-presenting cells present in these tissues. Other chemokines are inflammatory and are released from a wide 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 primarily function as chemical attractants to leukocytes, recruiting monocytes, neutrophils, and other effector cells from the blood to the site of infection or tissue damage. Certain inflammatory chemokines activate cells to trigger an immune response or promote wound healing. They are released by many different cell types and work to guide cells in both the innate and adaptive immune systems.

[0202] CC Chemokine CC chemokines (or β-chemokines) are proteins that have two adjacent cysteine ​​amino acids near their amino terminus. There are at least 27 different members of this subgroup reported in mammals, called CC chemokine ligands (CCL)-1 to -28, where CCL10 is the same as CCL9. Chemokines of this subfamily typically contain four cysteine ​​(C4-CC chemokines), but a few CC chemokines have six cysteine ​​(C6-CC chemokines). Examples of C6-CC chemokines include CCL1, CCL15, CCL21, CCL23, and CCL28. CC chemokines induce migration of monocytes and other cell types (e.g., NK cells and dendritic cells).

[0203] An example of a CC chemokine is monocyte chemoattractant protein-1 (MCP-1 or CCL2), which induces monocytes to leave the bloodstream, enter surrounding tissues, and become tissue macrophages.

[0204] CCL5 (or RANTES) attracts cells such as T cells, eosinophils, and basophils that express the receptor CCR5.

[0205] CXC Chemokine The two N-terminal cysteines of a CXC chemokine (or α-chemokine) are separated by a single amino acid, represented by "X" in the name. There are 17 distinct CXC chemokines described in mammals, which are subdivided into two categories: those that have a specific amino acid sequence (or motif) of glutamate-leucine-arginine (or ELR) immediately before the first cysteine ​​of the CXC motif (ELR-positive) and those that do not have an ELR motif (ELR-negative). ELR-positive CXC chemokines specifically induce neutrophil migration and interact with chemokine receptors CXCR1 and CXCR2.

[0206] C-chemokine The third group of chemokines, known as C-chemokines (or γ-chemokines), differs from all other chemokines in that they contain only two cysteine ​​molecules, one N-terminal cysteine, and one downstream cysteine. Two chemokines have been described from this subgroup, called XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β).

[0207] CX3C Chemokine CX3C chemokines have three amino acids between two cysteine ​​molecules. The only CX3C chemokine discovered to date is called fractalkine (or CX3CL1). Upon secretion, it is immediately tethered to the surface of the cell expressing it, thereby acting as both a chemoattractant and an adhesion molecule.

[0208] Chemokine receptors are G protein-coupled receptors containing seven transmembrane domains and are found on the surface of leukocytes. Approximately 19 different chemokine receptors have been characterized to date, and they are divided into four families depending on the type of chemokine they bind to: CXCR, which binds to CXC chemokines; CCR, which binds to CC chemokines; CX3CR1, which binds to the sole CX3C chemokine (CX3CL1); and XCR1, which binds to two XC chemokines (XCL1 and XCL2). They share many structural features; they are similar in size (having approximately 350 amino acids), have a short acidic N-terminus, seven helical 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 a conserved cysteine ​​residue that allows for the formation of disulfide crosslinks between these loops. The G protein, coupled to the C-terminus of the chemokine receptor, enables intracellular signal transduction after receptor activation, while the N-terminal domain of the chemokine receptor determines ligand binding specificity.

[0209] CXCL12 CXCL12 exhibits strong chemotaxis towards lymphocytes. CXCL12 plays a crucial role in angiogenesis by recruiting endothelial progenitor cells (EPCs) from the bone marrow via a CXCR4-dependent mechanism. This function of CXCL12 is a critical factor in neovascularization, which is linked to carcinogenesis and tumor progression. CXCL12 also plays a role in tumor metastasis, where cancer cells expressing the receptor CXCR4 are attracted to metastatic target tissues that release the ligand CXCL12.

[0210] The receptor for CXCL12 is CXCR4. CCR may contain a CXCL12-binding domain derived from CXCR4, which is linked to an endodomain derived from a cytokine receptor such as the IL-2 receptor.

[0211] IL2-CXCR4 coupled expression may support the engraftment of therapeutic T cells for cancer therapy. In multiple myeloma, cells expressing such CCRs can be recruited and alter the bone marrow environment. Such cells also have applications in the treatment of solid tumors by modifying the solid tumor microenvironment.

[0212] The amino acid sequence for CXCR4 is shown below as Sequence ID No. 17. Sequence ID 17 1 msiplpllqi ytsdnyteem gsgdydsmke pcfreenanf nkiflptiys iifltgivgn 61 glvilvmgyq kklrsmtdky rlhlsvadll fvitlpfwav davanwyfgn flckavhviy 121 tvnlyssvli lafisldryl aivhatnsqr prkllaekvv yvgvwipall ltipdfifan 181 vseaddryic drfypndlwv vvfqfqhimv glilpgivil scyciiiskl shskghqkrk 241 alkttvilil affacwlpyy igisidsfil leiikqgcef entvhkwisi tealaffhcc 301 lnpilyaflg akfktsaqha ltsvsrgssl kilskgkrgg hssvsteses ssfhss

[0213] CXCR7 also binds to CXCL12.

[0214] CCL2 Chemokine (CC motif) ligand 2 (CCL2) is also known as monocyte chemotactic protein 1 (MCP1) and small inducible cytokine A2. CCL2 recruits monocytes, memory T cells, and dendritic cells to inflammatory sites resulting from tissue damage or infection.

[0215] CCR2 and CCR4 are two cell surface receptors that bind to CCL2.

[0216] CCR2 has an amino acid sequence shown as SEQ ID NO: 18. SEQ ID NO: 18 1 mlstsrsrfi rntnesgeev ttffdydyga pchkfdvkqi gaqllpplys lvfifgfvgn 61 mlvvlilinc kklkcltdiy llnlaisdll flitlplwah saanewvfgn amcklftgly 121 higyfggiff iilltidryl aivhavfalk artvtfgvvt svitwlvavf asvpgiiftk 181 cqkedsvyvc gpyfprgwnn fhtimrnilg lvlpllimvi cysgilktll rcrnekkrhr 241 avrviftimi vyflfwtpyn ivillntfqe ffglsncest sqldqatqvt etlgmthcci 301 npiiyafvge kfrslfhial gcriaplqkp vcggpgvrpg knvkvttqgl ldgrgkgksi 361 grapeaslqd kega

[0217] CCR4 has an amino acid sequence shown as SEQ ID NO: 19. SEQ ID NO: 19 1 mnptdiadtt ldesiysnyy lyesipkpct kegikafgel flpplyslvf vfgllgnsvv 61 vlvlfkykrl rsmtdvylln laisdllfvf slpfwgyyaa dqwvfglglc kmiswmylvg 121 fysgiffvml msidrylaiv havfslrart ltygvitsla twsvavfasl pgflfstcyt 181 ernhtycktk yslnsttwkv lssleinilg lviplgimlf cysmiirtlq hcknekknka 241 vkmifavvvl flgfwtpyni vlfletlvel evlqdctfer yldyaiqate tlafvhccln 301 piiyfflgek frkyilqlfk tcrglfvlcq ycgllqiysa dtpsssytqs tmdhdlhdal

[0218] CCR may contain a CCL2 binding site of CCR2 or CCR4 in its ligand-binding domain.

[0219] cell surface antigen Ligands can be cell surface antigens such as transmembrane proteins.

[0220] The cell surface antigen may be CD22.

[0221] CD22, or surface antigen classification-22, is a molecule belonging to the SIGLEC family of lectins. It 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.

[0222] CD22 is a sugar-binding transmembrane protein that specifically binds to sialic acid and has an immunoglobulin (Ig) domain at its N-terminus. The presence of the Ig domain makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0223] Increased CD22 expression is observed in non-Hodgkin lymphoma and other lymphomas. Various monoclonal antibodies targeting CD22 are known, including epratuzumab, inotuzumab ozogamicin, m971, and m972.

[0224] Chimeric antigen receptor (CAR) The cells of the present invention may also include one or more chimeric antigen receptors. The CAR(s) may be specific to tumor-associated antigens.

[0225] Classical carriers (CARs) are chimeric type I transmembrane proteins that connect an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). The binder is typically a single-strand 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 to the cell membrane and connects the spacer to the endodomain.

[0226] Early CAR designs had endodomains derived from either the intracellular portion of the γ chain of FcεR1 or CD3ζ. Therefore, these first-generation receptors transmitted immunological signals 1, which were sufficient to induce T cell killing of allogeneic target cells, but they could not sufficiently activate T cells to proliferate and survive. To overcome this limitation, complex endodomains were constructed: fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ resulted in second-generation receptors capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28, which delivers the most potent costimulatory signal, i.e., immunological signal 2, that induces T cell proliferation. Several receptors have also been described that contain TNF receptor family endodomains, such as the closely related OX40 and 41BB, which transmit survival signals. Even more potent third-generation CARs, possessing endodomains capable of transmitting activation, proliferation, and survival signals, are now being described.

[0227] CARs targeting pathogenic autoreactive B cells have also been developed, which contain an autoantigen (or its autoantibody binding portion) as the target domain, rather than antibody-derived fragments such as scFv.

[0228] CAR-encoding nucleic acids can be introduced into T cells, for example, using a retroviral vector. In this way, a large number 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 an activation signal to the T cell in which it is expressed. Thus, the CAR induces the specificity and cytotoxicity of T cells towards cells expressing the target antigen.

[0229] The cells of the present invention may comprise one or more CARs.

[0230] 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 a T cell activation signal.

[0231] CAR antigen-binding domain The antigen-binding domain is part of the CAR that recognizes the antigen.

[0232] A number of antigen-binding domains are known in the art and include antibodies, antibody mimetics, and those based on the 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 single binder (such as a Darpin); or a single-chain derived from a T cell receptor.

[0233] The term "ligand" is used synonymously with "antigen" to mean an entity specifically recognized and bound by the antigen-binding domain of the CAR.

[0234] Cell surface antigen The CAR can 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.

[0235] CARs can specifically bind to tumor-associated cell surface antigens.

[0236] Various tumor-associated antigens (TAAs) are known, some of which are shown in Table 1. The antigen-binding domain of a CAR or CCR may be a domain capable of binding to the TAA shown therein.

[0237] [Table 1]

[0238] If a CAR recognizes a B-cell lymphoma or leukemia antigen (e.g., CD19, CD20, CD52, CD160, or CD5), a CCR may recognize another B-cell antigen, such as CD22.

[0239] Prostate cancer-related antigens CARs can specifically bind to cell surface antigens associated with prostate cancer (e.g., prostate stem cell antigen (PSCA) or prostate-specific membrane antigen (PSMA)).

[0240] PSCA is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein. It is upregulated in many prostate cancers and is also detected in bladder and pancreatic cancers.

[0241] Various anti-PSCA antibodies are known, such as 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).

[0242] CCR-expressing cells may also express anti-PSCA CARs, which may contain an antigen-binding domain based on one of these antibodies.

[0243] PSMA is a membrane-based zinc metalloenzyme. PSMA is strongly expressed in the human prostate, 100 times more so than in most other tissues. In cancer, its expression is upregulated, and it is called the second most upregulated gene in prostate cancer, 8 to 12 times more so than in non-cancerous prostate. In addition to its expression in the human prostate and prostate cancer, PSMA is also highly expressed in the neovascular structures of tumors, but it has been found not to be highly expressed in the normal vascular structures of all types of solid tumors (e.g., kidney, breast, colon, etc.).

[0244] Various anti-PSMA antibodies are known, such as 7E11, J591, J415, and Hybritech PEQ226.5 and PM2J004.5, each of which binds to a different epitope of PSMA (Chang et al. (1999) Cancer Res 15:3192-8).

[0245] CCR-expressing cells may also express anti-PSMA CARs, which may contain an antigen-binding domain based on one of these antibodies.

[0246] For example, a CAR may contain an scFv based on J591 having the sequence shown as sequence number 20. Sequence ID 20 (J591 scFv) EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGG SGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR

[0247] CAR transmembrane domain The transmembrane domain is a sequence of CARs that spans the membrane. It may contain a hydrophobic α-helix. The CAR transmembrane domain may be derived from CD28, which contributes to good receptor stability.

[0248] CAR signal peptide The CARs and CCRs described herein may include signal peptides, which, when expressed in cells such as T cells, direct the nascent proteins to the endoplasmic reticulum and subsequently to the cell surface where they are expressed.

[0249] The core of a signal peptide may contain a long stretch of hydrophobic amino acids that tend to form a single α-helix. A signal peptide may begin with a short, positively charged stretch of amino acids, which helps to enhance the proper topology of the polypeptide during rearrangement. Typically, the terminal end of a signal peptide contains a stretch of amino acids that are recognized and cleaved by a signal peptidase. The signal peptidase may cleave during or after rearrangement to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0250] Signal peptides can be located at the amino terminus of a molecule.

[0251] The signal peptide may include the sequence shown as SEQ ID NOs: 21, 22, or 23, or variants thereof having 5, 4, 3, 2, or 1 amino acid mutation (insertion, substitution, or addition), provided that the signal peptide still functions to induce cell surface expression of CAR and / or CCR.

[0252] Sequence ID 21:MGTSLLCWMALCLLGADHADG The signal peptide of SEQ ID NO: 21 is small, highly efficient, and derived from the TCRβ chain. Its approximately 95% cleavage after the terminal glycine suggests efficient removal by signal peptidases.

[0253] Sequence ID 22: MSLPVTALLLPLALLLHAARP The signal peptide in SEQ ID NO: 22 is derived from IgG1.

[0254] Sequence ID 23: MAVPTQVLGLLLLWLTDARC The signal peptide in SEQ ID NO: 23 is derived from CD8a.

[0255] CAR end domain Endodomains are part of the classical CAR located on the intracellular side of the membrane.

[0256] The endodomain is the signaling region of classical CARs. Following antigen recognition by the antigen-binding domain, individual CAR molecular clusters, native CD45 and CD148, are excluded from the synapse, and the signal is transmitted to the cell.

[0257] The CAR endodomain may be or may contain an intracellular signaling domain. In an alternative embodiment, the endodomain of the CAR may interact with intracellular signaling molecules present in the cytoplasm to induce signaling.

[0258] An intracellular signaling domain or a separate intracellular signaling molecule may be or may contain a T cell signaling domain.

[0259] The most commonly used signaling domain component is the CD3-ζ endodomain containing three ITAMs. This transmits an activation signal to T cells after antigen binding. CD3-ζ may not provide a sufficiently qualified activation signal, and additional co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 can be used with CD3-ζ to transmit proliferation / survival signals, or all three can be used together.

[0260] CAR may include the CD3-ζ end domain alone, the CD3-ζ end domain and either the CD28 or OX40 end domain, or the CD28 end domain and both the OX40 and CD3-ζ end domains.

[0261] A CAR end domain may include 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.

[0262] The end domain may contain the sequences shown as SEQ ID NOs. 24 to 32, or variants thereof having at least 80% sequence identity. Sequence ID 24-CD3ζ Endodomain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence IDs 25-CD28 and CD3ζ end-domain SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence IDs 26-CD28, OX40, and CD3ζ end-domain SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 27 - ICOS end domain CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL Sequence ID 28-CD27 End Domain QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP Sequence ID 29 - BTLA end-domain RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS Sequence ID 30-CD30 end domain HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK Sequence ID 31 - GITR end-domain QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV Column number 32 - HVEM end domain CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH

[0263] The mutant sequences may have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with sequence numbers 24 to 32, provided that the sequences provide an effective intracellular signaling domain.

[0264] nucleic acid The present invention also provides nucleic acids that encode CCR or a part thereof.

[0265] Nucleic acids have a structure: AgB-Spacer-TM-End (In the formula, AgB1 is a nucleic acid sequence that encodes the antigen-binding domain of the first or second polypeptide of CCR. Spacer 1 is a nucleic acid sequence that encodes a spacer for the first polypeptide or the second polypeptide. TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first polypeptide or the second polypeptide. End 1 may have a nucleic acid sequence that encodes the end domain of the first polypeptide or the second polypeptide.

[0266] Alternatively, nucleic acids have a structure: Dim-Spacer-TM-End (In the formula, Dim is a nucleic acid sequence that encodes the dimerization domain of the first or second polypeptide of the CCR. Spacer 1 is a nucleic acid sequence that encodes a spacer for the first polypeptide or the second polypeptide. TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first polypeptide or the second polypeptide. End 1 may have a nucleic acid sequence that encodes the end domain of the first polypeptide or the second polypeptide.

[0267] nucleic acid construct The present invention further provides nucleic acid constructs that encode CCR.

[0268] A nucleic acid construct encoding a chimeric cytokine receptor may comprise a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the nucleic acid construct has the following structure: Dim1-TM1-end1-coexpr-Dim2-TM2-end2 (In the formula, Dim1 is a nucleic acid sequence that encodes the first dimerization domain, TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first polypeptide. End 1 is a nucleic acid sequence that encodes the end domain of the first polypeptide. coexpr is a nucleic acid sequence that enables the co-expression of both CCRs. Dim2 is a nucleic acid sequence that encodes the second dimerization domain. TM2 is a nucleic acid sequence that encodes the transmembrane domain of the second polypeptide. End 2 has a nucleic acid sequence that encodes the end domain of the second polypeptide.

[0269] A nucleic acid construct encoding a chimeric cytokine receptor may comprise a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the nucleic acid construct has the following structure: AgB1-Spacer 1-TM1-End 1-coexpr-AbB2-Spacer 2-TM2-End 2 (In the formula, AgB1 is a nucleic acid sequence that encodes the antigen-binding domain of the first polypeptide. Spacer 1 is a nucleic acid sequence that encodes a spacer for the first polypeptide. TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first polypeptide. End 1 is a nucleic acid sequence that encodes the end domain of the first polypeptide. coexpr is a nucleic acid sequence that enables the co-expression of both polypeptides. AgB2 is a nucleic acid sequence that encodes the antigen-binding domain of the second polypeptide. Spacer 2 is a nucleic acid sequence that encodes a spacer for the second polypeptide. TM2 is a nucleic acid sequence that encodes the transmembrane domain of the second polypeptide. End 2 has a nucleic acid sequence that encodes the end domain of the second polypeptide.

[0270] A nucleic acid construct encoding a chimeric cytokine receptor may comprise a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the nucleic acid construct has the following structure: VH-Spacer 1-TM1-End 1-coexpr-VL-Spacer 2-TM2-End 2 (In the formula, VH is a nucleic acid sequence that encodes the VH domain of the first polypeptide. Spacer 1 is a nucleic acid sequence that encodes a spacer for the first polypeptide. TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first polypeptide. End 1 is a nucleic acid sequence that encodes the end domain of the first polypeptide. coexpr is a nucleic acid sequence that enables the co-expression of both polypeptides. VL is a nucleic acid sequence that encodes the VL domain of the second polypeptide. Spacer 2 is a nucleic acid sequence that encodes a spacer for the second polypeptide. TM2 is a nucleic acid sequence that encodes the transmembrane domain of the second polypeptide. End 2 has a nucleic acid sequence that encodes the end domain of the second polypeptide.

[0271] When a nucleic acid construct is expressed in cells such as T cells, it encodes a polypeptide that is cleaved at a cleavage site so that a first polypeptide and a second polypeptide are co-expressed on the cell surface.

[0272] When CCR binds to a ligand, the first and second polypeptides can bind to different epitopes on the same antigen.

[0273] The first polypeptide and the second polypeptide have complementary endodomains, for example, one derived from the α-chain or β-chain of the cytokine receptor, and the other derived from the γ-chain of the same cytokine receptor.

[0274] The present invention also provides nucleic acid constructs encoding CCR and CAR.

[0275] As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are intended to be synonymous with each other.

[0276] As a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that numerous different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, it should be understood that, in order to reflect the codon usage frequency of any particular host organism in which the polynucleotide is to be expressed, those skilled in the art may use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein.

[0277] Nucleic acids may include DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides containing synthetic or modified nucleotides. Numerous different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate skeletons, 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, polynucleotides may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or extend the lifespan of the polynucleotide of interest.

[0278] The terms “mutant,” “homolog,” or “derivative” in reference to a nucleotide sequence include any substitution, mutation, modification, replacement, deletion, or addition of one (or more) nucleic acids to or from the sequence.

[0279] In the structure described above, "coexpr" is a nucleic acid sequence that enables the co-expression of both the first and second polypeptides. It may be a sequence encoding a cleavage site, and as a result, the nucleic acid construct contains two or more CCR-forming polypeptides linked by the cleavage site(s), or a CCR and a CAR. The cleavage site(s) may be self-cleaving, and as a result, once the polypeptides are formed, they are immediately cleaved into individual peptides without the need for any external cleavage activity.

[0280] The cleavage site may be any sequence that allows for the separation of the first polypeptide and the second polypeptide, or the CCR and CAR.

[0281] The term "cleavage" is used herein for convenience, but cleavage sites can separate peptides into individual entities by mechanisms other than classical cleavage. For example, in the case of the foot-and-mouth disease virus (FMDV) 2A autocleavable peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolysis by host cell proteinases, autoprotein degradation, or translational effects (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important to the purposes of this invention, as long as the cleavage sites are located between protein-coding nucleic acid sequences and result in the expression of the protein as a distinct entity.

[0282] The cleavage site may be a furin cleavage site.

[0283] 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 substrates for furin include proparathyroid hormone, transforming growth factor β1 precursor, proalbumin, pro-β-secretase, membrane type I matrix metalloproteinase, pro-nerve growth factor β-subunit, and von Willebrand factor. Furin cleaves proteins immediately downstream of the basic amino acid target sequence (typically Arg-X-(Arg / Lys)-Arg') and is enriched in the Golgi apparatus.

[0284] The cleavage site could be the cleavage site of the tobacco etch virus (TEV).

[0285] TEV proteases are highly sequence-specific cysteine ​​proteases and chymotrypsin-like proteases. They are highly specific to their target cleavage site and are therefore frequently used for controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (where "\" represents the peptide bond to be cleaved). Mammalian cells, such as human cells, do not express TEV proteases. Therefore, in embodiments in which this nucleic acid construct contains a TEV cleavage site and is expressed in mammalian cells, exogenous TEV proteases must also be expressed in mammalian cells.

[0286] The cleavage site may encode a self-cleaving peptide.

[0287] A "self-cleaving peptide" refers to a peptide that constitutes a protein and, once a self-cleaving peptide is generated, functions to be immediately "cleaved" or separated into different, distinct first and second polypeptides, without requiring any external cleavage activity.

[0288] Self-cleaving peptides can be 2A self-cleaving peptides derived from aphthoviruses or cardioviruses. The initial 2A / 2B cleavage of aphthoviruses and cardioviruses is mediated by 2A, which "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 (a conserved proline residue) of protein 2B, represents an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al. (2001) above).

[0289] "2A-like" sequences are found in picornaviruses other than aftviruses or cardioviruses, "picornavirus-like" insect viruses, rotavirus type C, and in repeat sequences within Trypanosoma species and bacterial sequences (Donnelly et al. (2001) above). The cleavage site may contain one of these 2A-like sequences, as shown below. YHADYYKQRLIHDVEMNPGP(Sequence ID 33) HYAGYFADLLIHDIETNPGP(Sequence ID 34) QCTNYALLKLAGDVESNPGP(Sequence ID 35) ATNFSLLKQAGDVEENPGP(Sequence ID 36) AARQMLLLLSGDVETNPGP(Sequence ID 37) RAEGRGSLLTCGDVEENPGP(Sequence ID 38) TRAEIEDELIRAGIESNPGP (Sequence ID 39) TRAEIEDELIRADIESNPGP (Sequence ID 40) AKFQIDKILISGDVELNPGP(Sequence ID 41) SSIIRTKMLVSGDVEENPGP(Sequence ID 42) CDAQRQKLLLSGDIEQNPGP(Sequence ID 43) YPIDFGGFLVKADSEFNPGP (Sequence ID 44)

[0290] The cleavage site may contain a 2A-like sequence, indicated by sequence number 38 (RAEGRGSLLTCGDVEENPGP).

[0291] The present invention also provides a kit comprising a first polypeptide and a second polypeptide of a CCR according to the present invention, or one or more nucleic acid sequences encoding one or more CCRs and one or more CARs.

[0292] Sequence IDs 45 and 46 show the complete amino acid sequences of the fusion between anti-PSMA CAR and anti-PSA CCR. Subheadings are shown to label each part of the sequence, but in reality, various elements are joined together to produce a single continuous sequence. Exemplary constructs containing the IL-2Rβ chain (SEQ ID NO: 45) Signal sequence derived from human CD8a: MSLPVTALLLPLALLLHAA scFv aPSMA(J591 H / L) EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGG SGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR Linker SDPA Human IgG1Fc spacer (HCH2CH3pvaa): EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Transmembrane transmembrane derived from human CD28: FWVLVVVGGVLACYSLLVTVAFIIFWV End domains derived from TCRz: RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A peptide derived from the Thosea asigna viral capsid protein: RAEGRGSLLTCGDVEENPGP Signal sequence derived from mouse κVIII: METDTLILWVLLLLVPGSTG scFv aPSA(5D5A5 H / L): QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGGSGG GGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKLEIK Linker: SDPA Human CD8a STK spacer: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI Transmembrane transfer derived from the common human gamma chain: VVISVGSMGLIISLLCVYFWL Endodomain derived from the common human gamma chain: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET 2A peptide derived from equine rhinitis A virus polyprotein: QCTNYALLKLAGDVESNPGP Signal sequence derived from mouse κVIII: METDTLILWVLLLLVPGSTG scFv aPSA(5D3D11 H / L): QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGGSGG GGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGTKVEIK Linker: SDPA Human CD28STK Spacer: KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Transmembrane transmembrane derived from human IL-2Rβ: IPWLGHLLVGLSGAFGFIILVYLLI Endodomain derived from human IL-2Rβ: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV Exemplary constructs containing the sequence number 46-IL-7Rα chain Signal sequence derived from human CD8a: MSLPVTALLLPLALLLHAA scFv aPSMA(J591 H / L) EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGG SGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR Linker SDPA Human IgG1Fc spacer (HCH2CH3pvaa): EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Transmembrane transmembrane derived from human CD28: FWVLVVVGGVLACYSLLVTVAFIIFWV End domains derived from TCRz: RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2A peptide derived from Zosea acident virus capsid protein: RAEGRGSLLTCGDVEENPGP Signal sequence derived from mouse κVIII: METDTLILWVLLLLVPGSTG scFv aPSA(5D5A5 H / L): QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGGSGG GGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKLEIK Linker: SDPA Human CD8a STK spacer: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI Transmembrane transfer derived from the common human gamma chain: VVISVGSMGLIISLLCVYFWL Endodomain derived from the common human gamma chain: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET 2A peptide derived from equine rhinitis A virus polyprotein: QCTNYALLKLAGDVESNPGP Signal sequence derived from mouse κVIII: METDTLILWVLLLLVPGSTG scFv aPSA(5D3D11 H / L): QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGGSGG GGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGTKVEIK Linker: SDPA Human CD28STK Spacer: KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Transmembrane transmembrane derived from human IL-7Rα: PILLTISILSFFSVALLVILACVLW Endodomain derived from human IL-7Rα: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSSNQEEAYVTMSSFYQNQ

[0293] vector The present invention also provides vectors, or kits of vectors, comprising one or more nucleic acid sequences encoding a CCR and one or more CARs optionally. Such vectors can be used to introduce nucleic acid sequences(s) into host cells to express a CCR and optionally CARs.

[0294] The vector may be, for example, a plasmid or viral vector (e.g., a retroviral vector or a lentiviral vector), a transposon-based vector, or synthetic mRNA.

[0295] The vector can be used to transfect or transduce cytolytic immune cells such as T cells or NK cells.

[0296] Lentiviral vectors may be selected from the group consisting of, for example, human immunodeficiency virus (HIV), Visna-Maedi virus (VMV), canine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0297] The vector, especially if it is a lentiviral vector, may contain a myeloproliferative sarcoma virus enhancer, a negative control region deletion, and a dl587rev primer-binding site substitution (MND) promoter. MND is a synthetic promoter containing two elements: the U3 region of a modified Moloney mouse leukemia retrovirus with long-terminal repeats and an enhancer derived from myeloproliferative sarcoma virus. It is a constitutive promoter that is highly expressed in the hematopoietic system.

[0298] The MND promoter contains the nucleotide sequence shown as SEQ ID NO: 71. MND promoter (SEQ ID NO: 71) tttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctaggatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagca gttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaa ggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagccca

[0299] cell The present invention provides cells comprising one or more CCRs and optionally one or more CARs.

[0300] The cells may contain the nucleic acid or vector of the present invention.

[0301] The cells may be cytolytic immune cells such as T cells or NK cells.

[0302] T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cellular immunity. They can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of T cell receptors (TCRs) on their cell surface. There are various types of T cells, as outlined below.

[0303] Helper T cells (TH cells) assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and 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.

[0304] 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 their 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 an anergic state, preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0305] Memory T cells are a subset of antigen-specific T cells that persist for a long period after the resolution of an infection. Upon re-exposure to their allogeneic antigen, they rapidly proliferate into a large number of effector T cells, thus providing an immune system with a "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.

[0306] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary role is to halt T-cell immunity towards the end of the immune response and suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0307] The two main classes of CD4+ Treg cells—naturally occurring Treg cells and adaptive Treg cells—are described.

[0308] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+Treg cells) originate in the thymus and are associated with interactions 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 disrupt the development of regulatory T cells, potentially leading to the fatal autoimmune disease IPEX.

[0309] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0310] The cells may be natural killer cells (i.e., NK cells). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0311] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute a third type of cell differentiated from common lymphoid progenitor cells that give 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 before entering circulation.

[0312] The CCR-expressing cells of the present invention may be any of the cell types described above and can be produced ex vivo from the patient's own peripheral blood (first party), from the status of hematopoietic stem cell transplantation derived from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0313] Alternatively, CCR-expressing cells may be induced from the ex vivo differentiation of T cell or NK cell-inducible progenitor cells or embryonic progenitor cells. Or, immortalized T cell lines that retain lytic function and can act as therapeutic agents may be used.

[0314] In all of these embodiments, CCR-expressing cells are generated by introducing CCR or the DNA or RNA encoding each CCR by one of many means, including transduction with a viral vector or transfection with DNA or RNA.

[0315] The cells of the present invention may be ex vivo cells derived from a subject. The cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The cells may be activated and / or proliferated by treatment with an anti-CD3 monoclonal antibody, for example, before being transduced with a nucleic acid encoding a molecule that provides CCR.

[0316] The cells of the present invention are (i) Isolation of the subject or cell-containing sample from other sources listed above, (ii) Transduction or transfection of cells with one or more nucleic acid sequences encoding CCR, It can be produced by [method].

[0317] Next, cells can be purified and selected, for example, based on the expression of the antigen-binding domain of the antigen-binding polypeptide.

[0318] Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the present invention.

[0319] The pharmaceutical composition may additionally contain pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical composition may optionally contain one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be, for example, in a form suitable for intravenous infusion.

[0320] Treatment method The present invention provides a method for treating and / or preventing a disease or disorder, comprising the step of administering the cells of the present invention (for example, in a pharmaceutical composition as described above) to a subject.

[0321] The method for treating diseases relates to the therapeutic use of cells of the present invention. In this specification, cells may be administered to a subject having a pre-existing disease or condition to alleviate, reduce, or improve at least one symptom associated with the disease, and / or to slow, reduce, or halt the progression of the disease.

[0322] Methods for preventing disease relate to the prophylactic use of cells of the present invention. In this specification, such cells may be administered to subjects who are not yet infected with a disease and / or who do not exhibit any symptoms of the disease, in order to prevent or reduce the cause of the disease, or to reduce or prevent the occurrence of at least one symptom associated with the disease. Subjects may be predisposed to the disease or may be considered to be at risk of developing the disease.

[0323] When the cells of the present invention express a CAR for a target antigen, a method is provided for depleting target antigen-expressing cells from a subject, comprising the step of administering the cells of the present invention to the subject.

[0324] This method, (i) A step of isolating the cell-containing sample, (ii) A step of transfecting or transfecting such cells with a nucleic acid sequence or vector provided by the present invention, (iii) The step of administering cells from (ii) to the subject, It may include.

[0325] Cell-containing samples may be isolated from the subject or from other sources, such as those described above. Cells may be isolated from the subject's own peripheral blood (first source), from a hematopoietic stem cell transplantation using donor peripheral blood (second source), or from peripheral blood from an unrelated donor (third source).

[0326] The present invention provides CCR-expressing cells used for the treatment and / or prevention of diseases or disorders.

[0327] The present invention also relates to the use of CCR-expressing cells in the manufacture of pharmaceuticals for treating and / or preventing diseases or disorders.

[0328] The disease or disorder may be, for example, a cancerous disease, an infectious disease, an allergy, or an autoimmune disease.

[0329] The diseases treated and / or prevented by the method of the present invention may include cancerous diseases such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell cancer), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0330] If the ligand recognized by CCR is PSA, the cancer may be prostate cancer.

[0331] The cells of the present invention may be able to kill target cells such as cancer cells. Target cells may be characterized by the presence of tumor-secreted ligands or chemokine ligands in the vicinity of the target cells. Target cells may be characterized by the expression of tumor-associated antigens (TAAs) on the surface of the target cells, as well as the presence of soluble ligands.

[0332] The diseases treated and / or prevented by the method of the present invention may include autoimmune diseases such as Addison's disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes mellitus, mucosal or mucocutaneous pemphigus vulgaris, or membranous nephropathy.

[0333] The disease or disorder may be characterized by an inappropriate or undesirable immune response, such as graft rejection, GvHD, or hemophilia.

[0334] The cells and pharmaceutical compositions of the present invention may be used for the treatment and / or prevention of the diseases described above.

[0335] When a disease or disorder is characterized by the presence of autoantibodies and / or activation of autoreactive B cells (e.g., systemic lupus erythematosus (SLE)), the cells of the present invention can target B cell antigens such as CD19.

[0336] The cells and pharmaceutical compositions of the present invention may be used in any of the methods described above.

[0337] The present invention is further illustrated by examples, which are intended to assist those skilled in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. [Examples]

[0338] Example 1 - Preparation and testing of constitutively active cytokine signaling molecules A constitutively active cytokine signaling CCR was constructed by ligating the cytokine receptor endodomain to a "Fab" type exodomain (Figure 5). This structure utilizes the natural dimerization components of the antibody, namely the dimerization domains derived from the heavy chain constant region and the light chain constant region. The CCR has two chains: a first polypeptide containing the antibody light κ chain and the IL2 receptor common γ chain as the endodomain, and a second polypeptide 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) as the endodomain. The constitutively active cytokine signaling CCR proteins tested in this study contained the heavy chain variable region and the light chain variable region of scFv. These domains are not required for dimerization to occur. The signal is independent of antigen binding, and the structure can similarly be "headless" (as shown in Figure 5) or may contain another entity such as a protein tag.

[0339] The nucleic acid sequences encoding these two polypeptides were cloned in frames separated by the sequence encoding the 2A peptide.

[0340] CTLL-2 (ATCC™ TIB-214™) is a mouse cytotoxic T lymphocyte cell that depends on IL-2 for growth. In the absence of IL-2, this cell undergoes apoptosis. CTLL-2 cells were transduced using a vector expressing a chimeric protein containing an IL2 receptor endodomain (Fab_IL2 endo) or a vector expressing a chimeric protein containing an IL7 receptor endodomain (Fab_IL7 endo), or left untransduced (WT). As a positive control, all three cell types were co-cultured with 100 U / ml of mouse IL-2. Cell proliferation was evaluated at 3 and 7 days after culture, and the results are shown in Figure 6.

[0341] CTLL2 cells that were not transduced proliferated in the presence of 100 U / mL of mouse IL2, along 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 containing the IL2R end domain (Fab_IL2 end) survived and proliferated. This indicates that CCR provides the necessary IL2 signal to CTLL2 cells.

[0342] Example 2 - Preparation and testing of chimeric cytokine receptors for PSA A panel of chimeric cytokine receptors targeting PSA was developed using scFv derived from two antibodies that bind to different PSA epitopes: 5D5A5 and 5D3D11. The crystal structure of PSA was obtained as a sandwich complex with these two antibodies (as shown above in Stura et al. (2011)).

[0343] A schematic diagram showing some of the CCR panels is shown in Figure 7.

[0344] The panel includes the following structures: A5-CD8stk-IL2Rg_D11-hinge-IL2Rb: A CCR having an IL-2R endodomain with A5 on a common γ chain and D11 on a chain with an IL2Rβ chain; D11-CD8stk-IL2Rg_A5-hinge-IL2Rb: A CCR having an IL-2R endodomain with D11 on a common γ chain and A5 on a chain with an IL2Rβ chain; 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; D11-CD8stk-IL2Rg_A5-hinge-IL7Ra: A CCR having an IL-7R endodomain with D11 on a chain having a common γ chain and A5 on a chain having an IL7Rα 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; It included.

[0345] CTLL2 cells were transduced with vectors expressing these constructs. The cells were cultured in the presence or absence of IL2 (the presence of IL2 acted as a positive control), and in the presence or absence of 5 ng / mL or 5 μg / mL of PSA. The proliferation of CTLL2 cells was evaluated at 3 and 7 days, and the results are shown in Figure 8.

[0346] CTLL2 cells expressing CCR with an IL7 endodomain did not support the survival and proliferation of CTLL2 cells (Figure 8, last two panels). The presence of mouse IL-2 in these cells supported the growth and proliferation of CTLL2 cells on day 3, but by day 7, the majority of the cells underwent apoptosis.

[0347] Anti-PSA chimeric cytokine receptors possessing an IL2R endodomain supported the proliferation of CTLL2 cells in the absence of IL2 and in the presence of PSA at both 5 ng / ml and 5 μg / ml (Figure 8, first panel), with higher survival and proliferation at 5 μg / ml, particularly on day 7.

[0348] Both anti-PSA chimeric cytokine receptors possessing an IL2R endodomain, namely A5-CD8stk-IL2Rg_D11-hinge-IL2Rb and D11-CD8stk-IL2Rg_A5-hinge-IL2Rb, demonstrate that the relative positioning of the two PSA-binding domains: 5D5A5 and 5D3D11, is not important for function.

[0349] By replacing the common gamma chain with a rigid linker, the CCR lost its ability to support the survival and proliferation of CTLL2 cells (Figure 8, third panel).

[0350] As another read for IL2 signaling, phosphorylation of Y694 in STAT5 was investigated using phosphoflow.

[0351] CTLL2 cells were either untransduced (WT), transduced with a PSA CCR construct containing the IL2R endodomain (D11-CD8STK-IL2Rg_A5-hinge-IL2Rb), or transduced with an equivalent negative control construct in which the IL2Rγ chain was replaced with a rigid linker (D11-CD8STK-RL_A5-hinge-IL2Rb). Cells were incubated overnight in the absence of exogenously added IL-2. The following day, cells were incubated for 1 or 4 hours with 500 μM pervanadate (a positive control that inhibits phosphatase and leads to STAT5 phosphorylation) or 500 ng / mL PSA. After incubation, cells were fixed, permeabilized, and analyzed by flow cytometry.

[0352] The results are shown in Figure 9. In cells expressing PSA CCR, the presence of PSA increased STAT5 phosphorylation over time (Figure 9, center panel). No such increase in phosphorylation was observed in CTLL2 cells that were not transduced, or in CTLL2 cells transduced with an equivalent construct in which the IL2Rγ chain was replaced with a rigid linker (Figure 9, right panel).

[0353] These results are consistent with the CTLL2 survival / proliferation data shown in Figure 8, demonstrating that cytokine signaling can be induced in T cells using a chimeric cytokine receptor for a soluble ligand (in this case, PSA).

[0354] Example 3 - Production and testing of a chimeric cytokine receptor having a shortened IL-2 receptor β-chain endodomain. General structure: RQR8-2A-CL-SP1-TM1-IL2Rγ-2Aw-SP2-CH-TM2-IL2Rβ (In the formula, RQR8 is a marker gene described in International Publication No. 2013 / 153391. 2A and 2Aw are self-cleaving peptides, and the sequence encoding 2Aw is codon-wobbled to prevent homologous recombination. CL is a light κ chain, SP1 and SP2 are spacers. TM1 and TM2 are transmembrane domains. IL2Rγ is an end-domain derived from the common IL2R γ chain. CH is in the heavy chain steady region, IL2Rβ is the truncated or full-length IL-2 receptor β-chain endodomain. A structure with the following characteristics was constructed.

[0355] The construct was created using a series of shortened IL-2 receptor β-chain endodomains shown in Figure 10a and the key shown in Figure 10b.

[0356] T cells were transduced using vectors expressing each construct and cultured for 4 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable transduced cells was evaluated by flow cytometry. The results are shown in Figure 10b. Shortening the IL-2 receptor β-chain endodomain by 20 or 40 amino acids (i.e., from amino acids 266-551 to 266-531 and 266-511, respectively) increased proliferation, with the highest level of proliferation observed at IL2Rβ aa266-511. Further shortening of the IL-2 receptor β-chain endodomain resulted in a stepwise decrease in proliferation: aa266-471 > aa266-451 > aa266-411 > aa266-391 > aa266-371, reaching a plateau at which further deletions did not significantly affect the proliferation level. Therefore, shortening one or both cytokine receptor endodomains can reduce or enhance the activity of the chimeric cytokine receptor. Furthermore, by selecting endodomain shortenings that result in a desired level of activity, it is possible to "adjust" the CCR to produce a desired level of cytokine (in this case, IL-2).

[0357] Example 4 - Preparation and testing of chimeric cytokine receptors having a shortened common γ-chain endodomain. General structure: RQR8-2A-CL-SP1-TM1-IL2Rγ-2Aw-SP2-CH-TM2-IL2Rβ (In the formula, RQR8 is a marker gene described in International Publication No. 2013 / 153391. 2A and 2Aw are self-cleaving peptides, and the sequence encoding 2Aw is treated with codon fluctuations to prevent homologous recombination. CL is a light κ chain, SP1 and SP2 are spacers. TM1 and TM2 are transmembrane domains. IL2Rγ is a shortened or full-length end domain derived from the common IL2R γ chain. CH is in the heavy chain steady region, IL2Rβ is the IL-2 receptor β-chain endodomain. A structure with the following characteristics was constructed.

[0358] The construct was created using a series of truncated IL-2 receptor β-chain endodomains shown in Figure 12a and the key shown in Figure 12b.

[0359] T cells were transduced using vectors expressing each construct and cultured for 4 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable transduced cells was evaluated by flow cytometry. The results are shown in Figure 12b. Shortening the IL-2 receptor common gamma chain end domain by 20 amino acids (i.e., from amino acids 284-369 to amino acids 284-349) increased proliferation, with the highest level of proliferation observed with IL2Rβ aa266-511. Further shortening of the IL-2 receptor common gamma chain end domain decreased proliferation.

[0360] Example 5 - Investigation of the effect of combining shortened endodomains in IL2R CCR General structure: RQR8-2A-CL-SP1-TM1-IL2Rγ-2Aw-SP2-CH-TM2-IL2Rβ (In the formula, RQR8 is a marker gene described in International Publication No. 2013 / 153391. 2A and 2Aw are self-cleaving peptides, and the sequence encoding 2Aw is treated with codon fluctuations to prevent homologous recombination. CL is a light κ chain, SP1 and SP2 are spacers. TM1 and TM2 are transmembrane domains. IL2Rγ is either a full-length endodomain derived from the common IL2R γ chain or a shortened form having amino acids 284-349. CH is in the heavy chain steady region, IL2Rβ is either the full-length IL-2 receptor β-chain endodomain or a truncated form containing 266-511 amino acids. A structure with the following characteristics was constructed.

[0361] T cells were transduced with vectors expressing either the unshortened IL2R CCR (full-length IL2 CCR), the IL2 CCR with the full-length IL2Rβ chain and the shortened IL2Rγ chain (IL2Rγ aa284~349), the IL2 CCR with the shortened IL2Rβ chain and the full-length IL2Rγ chain (IL2Rβ aa266~511), or the IL2 CCR with the shortened IL2Rβ chain and the shortened IL2Rγ chain (IL2Rγ aa284~349 / IL2Rβ aa266~511), and cultured for 4 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable transduced cells was evaluated by flow cytometry. The results are shown in Figure 13. As previously observed, shortening the IL-2 receptor β-chain endodomain (by 40 amino acids) or the IL2R common γ-chain (by 20 amino acids) increased proliferation. However, the greatest increase in proliferation was observed in constructs containing both truncated IL2Rβ and IL2Rγ chains.

[0362] All publications cited in the above specification constitute part of this specification by reference. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and systems described in the present invention without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it should be understood that the invention as described in the claims should not be excessively limited to such specific embodiments. In fact, various modifications of the form described for the implementation of the invention that are apparent to those skilled in the art in molecular biology or related fields are intended to be within the scope of the appended claims.

Claims

1. Two polypeptides: (i) (a) the first dimerization domain, and (b) A shortened IL2 receptor β-chain endodomain containing the sequence shown as SEQ ID NO: 2, having a C-terminal shortening of approximately 10 to 70 amino acids, A first polypeptide containing, (ii) (a) the first dimerization domain and the second dimerization domain that dimerizes, (b) A shortened common γ chain end domain containing the sequence shown as Sequence ID No. 1, having a C-terminal shortening of approximately 10 to 30 amino acids, A second polypeptide containing, Chimeric cytokine receptors, including those mentioned above.

2. The chimeric cytokine receptor according to claim 1, wherein the IL2 receptor β-chain end domain of the abbreviated form of the first polypeptide has the sequence shown as Sequence ID No.

58.

3. The chimeric cytokine receptor according to claim 1 or 2, wherein the common γ-chain end domain of the shortened form of the second polypeptide has the sequence shown as Sequence ID No.

62.

4. The chimeric cytokine receptor according to any one of claims 1 to 3, wherein the first dimerizing domain and the second dimerizing domain spontaneously dimerize.

5. The chimeric cytokine receptor according to any one of claims 1 to 4, wherein the first dimerizing domain and the second dimerizing domain dimerize in the presence of a dimerizing chemical inducer (CID) or a ligand.

6. The first dimerization domain includes a heavy chain steady domain (CH), and the second dimerization domain includes a light chain steady domain (CL), or The chimeric cytokine receptor according to claim 4, wherein the first dimerization domain comprises a light chain constant domain (CL), and the second dimerization domain comprises a heavy chain constant domain (CH).

7. Two polypeptides: (i) (a) A first antigen-binding domain that binds to the first epitope of the ligand, and (b) A shortened IL2 receptor β-chain endodomain containing the sequence shown as SEQ ID NO: 2, having a C-terminal shortening of approximately 10 to 70 amino acids, A first polypeptide containing, (ii) (a) A second antigen-binding domain that binds to a second epitope of the ligand, and (b) A shortened common γ chain end domain containing the sequence shown as Sequence ID No. 1, having a C-terminal shortening of approximately 10 to 30 amino acids, A second polypeptide containing, The chimeric cytokine receptor according to claim 5, comprising:

8. The chimeric cytokine receptor according to claim 7, wherein each of the first antigen-binding domain and the second antigen-binding domain is a single-stranded variable fragment (scFv).

9. The chimeric cytokine receptor according to claim 7, wherein each of the first antigen-binding domain and the second antigen-binding domain is a single-domain binder (dAb).

10. Two polypeptides: (i) (a) Heavy chain variable domain (VH), and (b) A shortened IL2 receptor β-chain endodomain containing the sequence shown as SEQ ID NO: 2, having a C-terminal shortening of approximately 10 to 70 amino acids, A first polypeptide containing, (ii) (a) Light chain variable domain (VL), and (b) A shortened common γ chain end domain containing the sequence shown as Sequence ID No. 1, having a C-terminal shortening of approximately 10 to 30 amino acids, A second polypeptide containing, Includes, The chimeric cytokine receptor according to claim 5, wherein the VH and VL associate to form a ligand-binding site.

11. The chimeric cytokine receptor according to any one of claims 7 to 10, wherein the ligand is a tumor secretion factor selected from prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), and CA125.

12. The chimeric cytokine receptor according to any one of claims 7 to 10, wherein the ligand is a chemokine selected from CXCL12, CCL2, CCL4, CCL5, and CCL22.

13. A cell comprising a chimeric cytokine receptor according to any one of claims 1 to 12.

14. The cell according to claim 13, which also includes a chimeric antigen receptor.

15. A nucleic acid construct encoding a chimeric cytokine receptor according to any one of claims 1 to 13, comprising a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide.

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

17. A nucleic acid construct according to claim 15 or 16, comprising an MND promoter.

18. A vector comprising a nucleic acid construct according to any one of claims 15 to 17.

19. The vector according to claim 18, which is a lentiviral vector.

20. i) A vector comprising a nucleic acid sequence encoding the first polypeptide according to any one of claims 1 to 13, ii) A vector comprising a nucleic acid sequence encoding a second polypeptide according to any one of claims 1 to 13, A kit that includes this.

21. The kit according to claim 20, further comprising a vector containing a nucleic acid sequence encoding a chimeric antigen receptor.

22. A method for producing cells according to claim 13 or 14, comprising the step of introducing a nucleic acid construct according to any one of claims 15 to 17, a vector according to claim 18 or 19, or a kit of a vector according to claim 20 or 21 into cells using ex vivo.

23. The method according to claim 22, wherein the cells are derived from a sample isolated from a subject.

24. A pharmaceutical composition comprising a plurality of cells as described in claim 13 or 14.

25. A method for treating a disease, comprising the step of administering the pharmaceutical composition described in claim 24 to a subject.

26. The following steps: (i) Isolation step of a cell-containing sample derived from the subject, (ii) A step of transfecting or transfecting the cells using a nucleic acid construct according to any one of claims 15 to 17, a vector according to claim 18 or 19, or a kit of a vector according to claim 20 or 21, (iii) The step of administering the cells from (ii) to the subject, The method according to claim 25, including the method described in claim 25.

27. The method according to claim 26, wherein the sample is a T cell-containing sample.

28. The method according to claim 26 or 27, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

29. A pharmaceutical composition according to claim 24, used for the treatment of a disease.

30. Use of cells according to claim 13 or 14 in the manufacture of a pharmaceutical product for treating a disease.

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