Chimeric cytokine receptors

Abstract

The present invention provides a chimeric cytokine receptor (CCR) comprising two polypeptides: (i) (a) an extracellular domain comprising a first dimerization domain; (b) a first polypeptide comprising an interleukin-18 receptor 1 (IL18R1) intracellular domain; and (ii) (a) an extracellular domain comprising a second dimerization domain; (b) a second polypeptide comprising an interleukin-18 receptor accessory protein (IL18RAP) intracellular domain. The first and second polypeptides spontaneously dimerize to cause IL18-type signal transduction that occurs through the IL18R1 and IL18RAP intracellular domains. The present invention also provides a cell comprising such a CCR and, optionally, a chimeric antigen receptor (CAR), as well as the use of such a cell in, for example, the treatment of cancer or autoimmune disease.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a chimeric cytokine receptor having an IL-18 receptor intracellular domain. [Background technology]

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

[0003] Chimeric antigen receptors are proteins that transfer the specificity of monoclonal antibodies (mAbs) to the effector functions of T cells. Their usual form is that of a type I transmembrane domain protein with an antigen-recognizing amino terminus, a spacer, and a transmembrane domain, all connected to a compound intracellular domain that transmits T cell survival and activation signals.

[0004] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody that recognizes a target antigen fused to a signaling intracellular domain via a spacer and a transmembrane domain. Such molecules result in the activation of T cells in response to recognition by the scFv of its target. When T cells express such CARs, they recognize and kill target cells that express the target antigen. Several CARs have been raised against tumor-associated antigens, and adoptive transfer methods using T cells expressing such CARs are currently in clinical trials for the treatment of various cancers.

[0005] To date, most of the success of CAR T cells has been in hematological malignancies. CARs targeted to the B cell antigen CD19 were first used successfully to treat chronic lymphoblastic leukemia (CLL). In August 2017, the FDA approved the use of CART19 (Kymriah) to treat pediatric relapsed or refractory acute lymphoblastic leukemia (ALL), and in October of the same year, another CD19-targeting CAR (Yescarta) was approved by the FDA for adult relapsed or refractory large B-cell lymphoma. In addition, the European Medicines Agency (EMA) also approved the use of both these drugs in June of 2018. However, despite extensive studies, CAR T cell therapy for solid tumors has been largely unsuccessful.

[0006] One of the key hurdles that needs to be overcome for successful cell-based immunotherapeutic treatment of solid tumors is the tumor microenvironment (TME), which has been widely characterized as hostile to T cells: CAR T cells may not be able to engraft and expand within the TME. The persistence and activity of CAR T cells can be enhanced by administration of cytokines or by CAR T cells that constitutively produce cytokines. However, these approaches have limitations: systemic administration of cytokines can be toxic; constitutive production of cytokines can lead to uncontrolled proliferation and transformation (Nagarkatti et al (1994) PNAS 91:7638-7642; Hassuneh et al (1997) Blood 89:610-620). Chimeric cytokine receptors (CCRs) WO2017 / 029512 describes constitutively active "chimeric cytokine receptors" (CCRs) with a Fab-like extracellular domain and the intracellular domain of the IL2 or IL7 receptor. Co-expression of the IL7 CCR with a chimeric antigen receptor (CAR) was shown to support the engraftment and expansion of CAR T cells in vivo (Achkova et al Society for the Immunotherapy of Cancer (SITC) meeting 2019 Abstract P146). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2017 / 029512 [Non-patent literature]

[0008] [Non-Patent Document 1] Nagarkatti et al (1994) PNAS 91:7638-7642 [Non-Patent Document 2] Hassuneh et al (1997) Blood 89:610-620 [Non-Patent Document 3] Achkova et al Society for the Immunotherapy of Cancer (SITC) meeting 2019 Abstract P146 [Brief description of the drawings]

[0009] [Figure 1-1]Schematic diagram illustrating IL-18 chimeric cytokine receptors. A. Tethered IL18-CCR: truncated IL18 linked to either IL18R1 or IL18RAP using a flexible linker. B. Ligand-inducible IL18-CCR: ligand (e.g., PSA) specific scFv is linked to IL18R1 or IL18RAP intracellular domain via a spacer and transmembrane domain. C. TGFβ-IL18 switch receptor: IL18R1 or IL18RAP intracellular domain is fused to either TGFβR1 or TGFβR2 extracellular domain. D. TGFβ-IL18 CAR enhancer: TGFβ binds to activated IL18 receptor and enhances first generation CAR. E. Constitutively active IL18-CCR: heavy chain constant domain (CH) and light chain constant domain (CL) derived from antibody linked to IL18R1 or IL18RAP intracellular domain. [Figure 1-2] Same as above. [Diagram 2] (FIG. 2A) Schematic illustrating the vectors used for in vitro studies. (FIG. 2B) Graphs showing target cell killing, IFNγ and IL-2 release after co-culture of transduced T cells with target cells. [Diagram 3] Schematic diagram illustrating the experimental outline of the CT26-GD2+ immunocompetent animal model for in vivo studies. [Figure 4] Graph showing tumor volume in CT26-GD2+ immune competent mouse model following treatment with cells expressing only the GD2 CAR or co-expressing the GD2 CAR with IL2, IL7 or IL18 CCR. [Diagram 5] Graph showing T cells in peripheral blood of CT26-GD2+ immune competent model mice following treatment with cells expressing only the GD2 CAR or co-expressing the GD2 CAR with IL2, IL7 or IL18 CCR. [Figure 6]Kaplan Meyer survival curves of CT26-GD2+ immunocompetent model mice following treatment with cells expressing only the GD2 CAR or co-expressing the GD2 CAR with IL2, IL7 or IL18 CCR. [Figure 7] Plot showing the results of principal component analysis (PCA) calculated from the analysis of an 800-gene CAR T cell panel for activated and non-activated CAR-T cells expressing either IL7- or IL18-CCR. [Figure 8-1] Volcano plot showing gene expression in activated and non-activated CAR-T cells expressing IL18-CCR. IL7 CCR was used as the baseline for calculation of differential gene expression. [Figure 8-2] Volcano plot showing gene expression in activated and non-activated CAR-T cells expressing IL18-CCR. IL7 CCR was used as the baseline for calculation of differential gene expression. [Figure 9] Graph showing CBA assay derived from co-culture of CAR-T cells expressing either IL7- or IL18-CCR with SupT1 GD2+ target cells at an E:T ratio of 1:4. [Figure 10] Schematic of an IL-18 CCR made using coiled-coil domains derived from acid / basic leucine zippers. One polypeptide of the CCR contains a first pair of alpha-helical coiled coils (coiled-coil 1, e.g., acid zipper) and the other contains a second pair (coiled-coil 2, e.g., basic zipper). These domains spontaneously dimerize, bringing together the IL18R1 and IL18RAP intracellular domains and conferring constitutive IL-18 signaling. [Figure 11]Schematic of an IL-18 CCR made using coiled-coil domains derived from the SNAP-25 / SNARE heterotetrameric complex. In this system, one polypeptide of the CCR contains the A chain of the SNAP-25 / SNARE heterotetrameric complex; one polypeptide contains the B chain of the SNAP-25 / SNARE heterotetrameric complex; one polypeptide contains the C chain of the SNAP-25 / SNARE heterotetrameric complex; and one polypeptide contains the D chain of the SNAP-25 / SNARE heterotetrameric complex. These domains spontaneously heterodimerize, bringing together two copies of the IL18R1 and IL18RAP intracellular domains and conferring constitutive IL-18 signaling. Summary of the Invention [Means for solving the problem]

[0010] Summary of aspects of the invention The inventors surprisingly found that cells co-expressing a CAR and a CCR with an IL-18 receptor intracellular domain exhibit improved CAR-T cell persistence and anti-tumor activity in vivo in immune competent animal models than comparable cells co-expressing a CAR and an IL-7 or IL-2 CCR.

[0011] Thus, in a first aspect, the present invention provides a method for the preparation of a polypeptide comprising: (i)(a) an extracellular domain comprising a first dimerization domain; (b) Interleukin-18 receptor 1 (IL18R1) intracellular domain a first polypeptide comprising: (ii) (a) an extracellular domain that comprises a first dimerization domain and a second dimerization domain that spontaneously dimerizes; (b) Interleukin-18 receptor accessory protein (IL18RAP) intracellular domain A second polypeptide comprising The present invention provides a chimeric cytokine receptor (CCR) comprising:

[0012] The dimerization domain may be based on an antibody dimerization domain, in which one of the first and second dimerization domains may comprise a heavy chain constant domain (CH) and the other may comprise a light chain constant domain (CL).

[0013] Alternatively, the first and second dimerization domains may comprise coiled-coil domains.

[0014] For example, the first and second dimerization domains may comprise leucine zipper domains, such as Fos / Jun domains or docking domain (DDD1) / anchor domain (AD1) leucine zipper domains.

[0015] The CCR may be a tetramer. In this embodiment, the CCR comprises four polypeptides: (i)(a) an extracellular domain comprising a first tetramerization domain; (b) Interleukin-18 receptor 1 (IL18R1) intracellular domain a first polypeptide comprising: (ii)(a) an extracellular domain that includes a second tetramerization domain; (b) Interleukin-18 receptor accessory protein (IL18RAP) intracellular domain a second polypeptide comprising: (i)(a) an extracellular domain containing a third tetramerization domain; (b) Interleukin-18 receptor 1 (IL18R1) intracellular domain a third polypeptide comprising: (ii)(a) an extracellular domain containing a fourth tetramerization domain; (b) Interleukin-18 receptor accessory protein (IL18RAP) intracellular domain A fourth polypeptide comprising Includes.

[0016] In this embodiment, the tetramerization domain may include, for example, strands A, B, C and D of the SNAP-25 / SNARE heterotetrameric complex.

[0017] In a second aspect, the present invention provides a cell comprising a chimeric cytokine receptor according to the first aspect of the invention.

[0018] The cells may also comprise a chimeric antigen receptor.

[0019] In a third aspect, there is provided a nucleic acid construct encoding a chimeric cytokine receptor (CCR) according to the first aspect of the invention, the nucleic acid construct comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide.

[0020] To produce a tetrameric CCR, the nucleic acid construct may comprise a first nucleic acid sequence encoding a first polypeptide; a second nucleic acid sequence encoding a second polypeptide; a third nucleic acid sequence encoding a third polypeptide; and a fourth nucleic acid sequence encoding a fourth polypeptide.

[0021] The nucleic acid construct may also include a nucleic acid sequence encoding a chimeric antigen receptor.

[0022] In a fourth aspect, there is provided a vector comprising a nucleic acid construct according to the third aspect of the invention.

[0023] In a fifth aspect, i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined above; and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined above. A kit is provided comprising:

[0024] To produce tetrameric CCR, the kit contains: i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined above; ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined above; iii) a vector comprising a nucleic acid sequence encoding a third polypeptide as defined above; and iv) a vector comprising a nucleic acid sequence encoding a fourth polypeptide as defined above. may include:

[0025] In a sixth aspect, there is provided a method for producing a cell according to the second aspect of the invention, the method comprising introducing into a cell ex vivo a nucleic acid construct according to the third aspect of the invention; a vector according to the fourth aspect of the invention; or a kit of vectors according to the fifth aspect of the invention.

[0026] In a seventh aspect, there is provided a pharmaceutical composition comprising a plurality of cells according to the second aspect of the invention.

[0027] In an eighth aspect, there is provided a pharmaceutical composition according to the seventh aspect of the invention for use in the treatment of cancer or an autoimmune disease.

[0028] In a ninth aspect, there is provided a method for treating cancer or an autoimmune disease comprising administering to a subject a pharmaceutical composition according to the seventh aspect of the invention.

[0029] In an eleventh aspect, there is provided the use of a plurality of cells according to the second aspect of the invention for use in the manufacture of a pharmaceutical composition for the treatment of cancer or an autoimmune disease.

[0030] Further aspects of the invention Further aspects of the present invention are presented in the following numbered paragraphs: 1. Two polypeptides: (i)(a) an extracellular domain comprising a first dimerization domain; (b) Interleukin-18 receptor 1 (IL18R1) intracellular domain a first polypeptide comprising: (ii)(a) an extracellular domain that includes a second dimerization domain; (b) Interleukin-18 receptor accessory protein (IL18RAP) intracellular domain A second polypeptide comprising A chimeric cytokine receptor (CCR). 2. The CCR of paragraph 1, wherein the first and second dimerization domains spontaneously dimerize. 3. The CCR described in paragraph 2, wherein one of the first and second dimerization domains comprises a heavy chain constant domain (CH) and the other comprises a light chain constant domain (CL). 4. The CCR described in paragraph 1, wherein the first and second dimerization domains dimerize in the presence of a ligand. 5. The CCR according to paragraph 4, wherein the first and second dimerization domains are single chain variable fragments (scFv) or single domain binders (dAbs) that bind independently to a ligand. 6. The CCR according to paragraph 4, wherein one of the first and second dimerization domains comprises a heavy chain variable domain (VH) and the other comprises a light chain variable domain (VL) and together binds to a ligand. 7. The CCR described in paragraph 4, wherein one of the first and second dimerization domains comprises the extracellular region of transforming growth factor beta receptor I (TBRI) and the other comprises the extracellular region of transforming growth factor beta receptor II (TBRII), and the ligand is transforming growth factor beta (TGFβ). 8. The first polypeptide comprises: (i) an antigen-binding domain, (ii) a linker, (iii) the extracellular domain of the first transforming growth factor beta receptor (TBR); (iv) a first transmembrane domain, (v) interleukin-18 receptor 1 (IL18R1) intracellular domain, and (vi) CD3z intracellular domain Including, The second polypeptide is (i) the extracellular domain of a second TBR; (ii) a second transmembrane domain, and (iii) Interleukin-18 receptor accessory protein (IL18RAP) intracellular domain Including, or the first polypeptide is (i) an antigen-binding domain, (ii) a linker, (iii) the extracellular domain of the first transforming growth factor beta receptor (TBR); (iv) a first transmembrane domain, and (v) the IL18RAP intracellular domain, and (vi) CD3z intracellular domain Including, The second polypeptide is (i) the extracellular domain of a second TBR; (ii) a second transmembrane domain, and (iii) IL18R1 intracellular domain The CCR set out in paragraph 8, including: 9. A cell comprising a chimeric cytokine receptor according to any of the preceding paragraphs. 10. The cell of paragraph 6, which also contains a chimeric antigen receptor. 11. A nucleic acid construct encoding a chimeric cytokine receptor (CCR) described in any of paragraphs 1 to 8, wherein the nucleic acid construct comprises a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide. 12. A vector comprising the nucleic acid construct according to paragraph 11. 13.i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined in any of paragraphs 1 to 8; and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined in any of paragraphs 1 to 8. Kit including: 14. A method for producing a cell according to paragraph 9 or 10, comprising the step of introducing into a cell ex vivo a nucleic acid construct according to paragraph 11; a vector according to paragraph 12; or a kit of vectors according to paragraph 13. 15. A pharmaceutical composition comprising a plurality of cells according to paragraph 11 or 12. 16. The pharmaceutical composition according to paragraph 15 for use in the treatment of cancer or an autoimmune disease. 17. A method for treating cancer or an autoimmune disease, comprising administering to a subject the pharmaceutical composition described in paragraph 15. 18. Use of a plurality of cells according to paragraphs 9 or 10 for use in the manufacture of a pharmaceutical composition for the treatment of cancer or an autoimmune disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description Chimeric cytokine receptors (CCRs) A chimeric cytokine receptor (CCR) is a molecule that contains a cytokine receptor intracellular domain and an extracellular domain. The extracellular domain may be derived from a protein other than a cytokine receptor. The extracellular domain may be derived from a protein other than the IL-18 receptor.

[0032] The extracellular domain may render the chimeric cytokine receptor constitutively active. For example, a CCR may contain an extracellular domain that spontaneously dimerizes.

[0033] In the arrangement shown diagrammatically in Figure IE, one polypeptide of the CCR contains a heavy chain constant domain (CH) and the other contains a light chain constant domain (CL), which spontaneously dimerize, bringing together the IL18R1 and IL18RAP intracellular domains and conferring constitutive IL-18 signaling.

[0034] Alternatively, a constitutively active CCR of the invention may comprise a coiled-coil domain that confers spontaneous dimerization or multimerization (eg tetramerization).

[0035] In the arrangement shown diagrammatically in Figure 10, one polypeptide of the CCR contains a first pair of alpha-helical coiled coils (coiled-coil 1, e.g., acid zipper) and the other contains a second pair (coiled-coil 2, e.g., basic zipper). These domains spontaneously dimerize, bringing together the IL18R1 and IL18RAP intracellular domains and conferring constitutive IL-18 signaling.

[0036] In the arrangement shown diagrammatically in Figure 11, one polypeptide of the CCR comprises the A chain of the SNAP-25 / SNARE heterotetrameric complex; one polypeptide comprises the B chain of the SNAP-25 / SNARE heterotetrameric complex; one polypeptide comprises the C chain of the SNAP-25 / SNARE heterotetrameric complex; and one polypeptide comprises the D chain of the SNAP-25 / SNARE heterotetrameric complex. These domains spontaneously heterodimerize, bringing together two copies of the IL18R1 and IL18RAP intracellular domains and conferring constitutive IL-18 signaling.

[0037] In the configuration shown diagrammatically in Figure 1A, the receptor comprises IL-18Rα and β chains, one of which also contains an IL-18 molecule tethered to a polypeptide by a linker. The tethered IL-18, due to its permanent physical proximity, binds and dimerizes with the IL18R1 and IL18RAP chains, conferring constitutive IL-18 signaling.

[0038] Alternatively, the extracellular domain may render the chimeric cytokine receptor active in the presence of a chemical inducer of dimerization (CID) or ligand.

[0039] In the arrangement shown diagrammatically in Figure 1B, the first and second polypeptides of the CCR each comprise a binding domain, such as an scFv or dAb, that binds to a ligand. In the presence of a ligand, both extracellular domains bind, bringing the intracellular domains together and conferring ligand-dependent IL-18 signaling.

[0040] The ligand may be, for example, prostate specific antigen (PSA), carcinoembryonic antigen (CEA) and factors secreted by tumors, such as vascular endothelial growth factor (VEGF) or CA125.

[0041] In the arrangement shown in FIG. 1C, the first and second polypeptides of the CCR each comprise an extracellular domain derived from a TGFβ receptor. For example, one polypeptide may comprise an extracellular domain derived from a TGFβ receptor I, and the other polypeptide may comprise an extracellular domain derived from a TGFβ receptor II. In the presence of TGFβ, the two TGFβ receptor intracellular domains combine to trigger IL-18 type signal transduction.

[0042] The arrangement shown in FIG. 1D is also an IL-18 type signaling, which is also induced by the presence of TGFβ. In this arrangement, one polypeptide contains an ITAM-containing intracellular domain and an antigen-binding domain. This polypeptide acts like a CAR in that it induces cell activation in the presence of a target antigen. This polypeptide also contains one of the IL-18 receptor intracellular domains and a TGFβ receptor extracellular domain. The other polypeptide in the CCR contains a second TGFβ receptor extracellular domain and another IL-18 receptor intracellular domain chain. In the presence of TGFβ, the two TGFβ receptor intracellular domains are integrated to cause IL-18 type signaling. In the presence of both TGFβ and a target antigen, the cell receives both an activation signal and an IL-18 type growth signal.

[0043] IL18 receptor intracellular domain IL-18 belongs to the IL-1 superfamily and is produced primarily by macrophages, but also by other cell types, with pleiotropic functions in cell stimulation. IL-18 is a proinflammatory cytokine that facilitates type 1 responses. Together with IL-12, it induces cell-mediated immunity after infection with microbial products such as lipopolysaccharide (LPS). IL-18, together with IL12, acts on CD4, CD8 T cells and NK cells to induce the production of IFNγ, a type II interferon that plays a key 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, in the absence of IL-12 or IL-15, IL-18 does not induce IFNγ production, but it plays an important role in the differentiation of naive T cells into Th2 cells and stimulates mast cells and basophils to produce chemical mediators such as IL-4, IL-13, and histamine.

[0044] The IL-18 receptor consists of an inducible component, interleukin-18 receptor 1 (IL18R1), which binds mature IL-18 with low affinity, and a constitutively expressed co-receptor, interleukin-18 receptor accessory protein (IL18RAP). IL-18 binds to the ligand receptor IL18R1 and induces the recruitment of IL18RAP to form a high-affinity complex that signals through the toll / interleukin-1 receptor (TIR) ​​domain. This signaling domain recruits the MyD88 adaptor protein, which activates proinflammatory programs and the NF-κB pathway.

[0045] The amino acid sequence of human IL18 receptor 1 (also known as the IL18 receptor alpha chain) is available from Uniprot accession number Q13478 and is shown below as SEQ ID NO: 1. 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. [ka]

[0046] For embodiments of the invention in which a single polypeptide comprises the IL18R1 extracellular and intracellular domains, such as the embodiment illustrated in FIG. 1A, the polypeptide may comprise residues 22-541 of SEQ ID NO:1.

[0047] For embodiments of the invention in which a single polypeptide comprises an IL18R1 intracellular domain and a heterologous extracellular domain, the polypeptide may comprise residues 330-541 of SEQ ID NO:1 (if the polypeptide comprises the IL18R1 transmembrane domain - SEQ ID NOs:8 and 9 below) or residues 351-541 (if the polypeptide has a heterologous transmembrane domain).

[0048] The CCRs of the present invention may comprise variants of part or all of the sequence shown as SEQ ID NO:1, which may have, for example, 70, 80, 90, 95 or 99% identity to the sequence shown as SEQ ID NO:1 (or an identified portion thereof), provided that a polypeptide comprising the variant sequence retains the ability to induce an IL-18 signal in a cell when dimerized with a polypeptide comprising the IL18RAP intracellular domain.

[0049] The amino acid sequence of human IL18 receptor accessory protein (IL18RAP, also known as the IL18 receptor β chain) is available from Uniprot accession number O95256 and is shown below as SEQ ID NO: 2. 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. [ka]

[0050] For embodiments of the invention in which a single polypeptide comprises the IL18RAP extracellular and intracellular domains, such as the embodiment illustrated in FIG. 1A, the polypeptide may comprise residues 20-599 of SEQ ID NO:2.

[0051] For embodiments of the invention in which a single polypeptide comprises an IL18RAP intracellular domain and a heterologous extracellular domain, such as the embodiment illustrated in Figures 1B-E, the polypeptide may comprise residues 357-599 of SEQ ID NO:2 (if the polypeptide comprises the IL18RAP transmembrane domain - see SEQ ID NOs:12 and 13 below) or residues 378-599 (if the polypeptide has a heterologous transmembrane domain).

[0052] The CCRs of the present invention may comprise variants of part or all of the sequence shown as SEQ ID NO:2, which may have, for example, 70, 80, 90, 95 or 99% identity to the sequence shown as SEQ ID NO:2 (or an identified portion thereof), provided that a polypeptide comprising the variant sequence retains the ability to induce an IL-18 signal in a cell when dimerized with a polypeptide comprising the IL18R1 intracellular domain.

[0053] IL18 intracellular domain truncation It is known that truncations of some type I cytokine receptor intracellular domains can increase proliferation when the CCR is expressed in T cells relative to the wild-type version. WO2021 / 023987 describes a study in which the IL-2 receptor beta chain of a constitutively active CCR was given a consecutive 20-mer deletion. The CCR was expressed in T cells and the absolute number of live, transduced cells was assessed by flow cytometry. Truncation of the IL-2 receptor beta chain intracellular domain 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 for IL2R beta aa266-511. Further truncations of the IL-2 receptor beta chain intracellular domain resulted in a stepwise decrease in proliferation in the order aa266-471>aa266-451>aa266-411>aa266-391>aa266-371, at which point it reached a plateau with further deletions having no significant effect on proliferation levels. Thus, the activity of the chimeric cytokine receptor can be decreased by truncation of one or both cytokine receptor intracellular domains. Also, the CCR can be "tuned" to have a desired level of cytokine production by selecting a truncation of the intracellular domain that confers a desired level of activity.

[0054] Truncated IL18R1 or IL18RAP may have a truncation of 10 to 180 amino acids from the C-terminus, for example, 20 to 170 amino acids, 40 to 160, 60 to 150, 80 to 130 or 100 to 120 amino acids. Truncation may include removal of 1 to 40 amino acids from the C-terminus, for example, 5 to 35, 10 to 30, or 15 to 25 amino acids from the C-terminus.

[0055] Table 1 shows truncations of IL18R1 and IL18RAP intracellular domain sequences. A CCR of the invention may comprise one of the truncated forms of IL18R1 and / or IL18RAP shown in Table 1. The truncated forms of IL18R1 and / or IL18RAP may have one of the sequences shown as SEQ ID NOs: 36-53, and 62. The truncated forms of IL18R1 and / or IL18RAP may have a sequence "between" two truncated sequences shown in Table 1, for example, the sequence "between" IL18R1 truncation 10 (SEQ ID NO: 44), which is missing the last 11 amino acids of the intracellular domain sequence, and IL18R1 truncation 9 (SEQ ID NO: 43), which is missing the last 31 amino acids of the intracellular domain sequence, is a truncation of the last 30, 29, 28...etc...14, 13 and 12 amino acids of the IL18R1 intracellular domain sequence.

[0056] The truncation may be such that a CCR made with a truncated IL18R1 or IL18RAP intracellular domain, when expressed in a T cell, increases proliferation of the T cell compared to a comparable cell expressing an equivalent CCR having a wild-type (i.e., non-truncated) intracellular domain sequence. [Table 1-1] [Table 1-2]

[0057] Extracellular domain dimerization The chimeric cytokine receptors of the present invention comprise first and second polypeptides that dimerize to bring together the IL18R1 and IL18RAP intracellular domains.

[0058] Dimerization may occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active, or it may occur only in the presence of a chemical inducer of dimerization (CID) or ligand, in which case the transmembrane protein will trigger cytokine-type signaling only in the presence of the CID or ligand.

[0059] Constitutively active systems When the dimerization domain spontaneously heterodimerizes, it may be based on the dimerization domain of an antibody. In particular, it may comprise the dimerization portion of a heavy chain constant domain (CH) and a light chain constant domain (CL). The "dimerization portion" of the constant domain is the portion of the sequence that forms interchain disulfide bonds.

[0060] A suitable arrangement of such a CCR with a "Fab" type extracellular domain is shown diagrammatically in FIG. 1E.

[0061] The light chain constant domain may comprise the sequence shown as SEQ ID NO:3 and the heavy chain constant domain may comprise the sequence shown as SEQ ID NO:4. [ka]

[0062] An exemplary sequence of a "Fab-type" CCR shown in FIG. 1E is shown below, with the individual components identified.

[0063] “Fab type” CCR [ka] [ka]

[0064] Another type of CCR that spontaneously dimerizes (or multimerizes) is one that contains a coiled-coil domain.

[0065] A coiled coil is a structural motif in which 2-7 alpha helices are wound together like strands of rope. Coiled coils usually contain a repeating pattern of hydrophobic (h) and charged (c) amino acid residues, called heptad repeats, hxxhcxc. Packing at the coiled-coil interface is exceptionally tight, showing nearly perfect van der Waals contacts between the side chains. Many endogenous proteins contain coiled-coil domains, such as leucine zippers.

[0066] Leucine zippers are supersecondary structures that function as dimerization domains. Their presence generates adhesive forces in parallel alpha helices. A single leucine zipper consists of multiple leucine residues spaced approximately 7 residues apart, forming an amphipathic alpha helix with a hydrophobic region running along one side. This hydrophobic region provides an area for dimerization, allowing the motif to "zip" together. Leucine zippers are typically 20-40 amino acids long, e.g., approximately 30 amino acids. Such leucine zippers are found in transcription factors such as c-fos and c-jun, which form the adaptor protein-1 transcription complex. Jun-Fos leucine zippers have previously been used to dimerize scFv antibodies, interferon gamma receptor β subunits, and receptors for the cytokines granulocyte-macrophage colony-stimulating factor and growth hormone.

[0067] The first and / or second heterodimerization domain may comprise the sequence set forth as SEQ ID NO: 54 or 55. The first heterodimerization domain may comprise the sequence set forth as SEQ ID NO: 54 and the second heterodimerization domain may comprise the sequence set forth as SEQ ID NO: 55, or vice versa. SEQ ID NO:54: QLEKELQALEKENAQLEWELQALEKELAQ SEQ ID NO:55: QLEKKLQALKKKNAQLKWKLQALKKKLAQ

[0068] In certain embodiments, the first and second heterodimerization domains may be acidic (e.g., SEQ ID NO: 54) or basic (e.g., SEQ ID NO: 55) leucine zippers. In particular, if the first heterodimerization domain is an acidic leucine zipper, the second heterodimerization domain is a basic leucine zipper, and vice versa.

[0069] The first and second heterodimerization domains may be a dimerization and docking domain (DDD1) and an anchoring domain (AD1).

[0070] DDD1 is a short alpha-helical structure derived from protein kinase A (PKA). AD1 is a short alpha-helical structure derived from A-kinase anchor protein (AKAP).

[0071] The DDD1 domain may comprise the sequence shown as SEQ ID NO:56. SEQ ID NO:56: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

[0072] The AD1 domain may comprise the sequence shown as SEQ ID NO:57. SEQ ID NO:57: VQIEYLAKQIVDNAIQQA

[0073] The coiled-coil domain may form dimers or multimers, particularly multimers that are of order 2 (e.g., 2, 4, 6 or 8 chains). The CCR may form a tetramer, for example, by using a coiled-coil domain derived from the SNAP-25 / SNARE heterotetrameric complex. This complex contains four chains: chain A, chain B, chain C and chain D, as shown below. SNAP-25 and SNAREs: Parallel Heterotetramers [ka]

[0074] As shown diagrammatically in FIG. 11, by linking the tetramerization domains of two of these chains containing the IL18RAP intracellular domain and two of these chains containing the IL18BR1 intracellular domain, two copies of the IL18RAP intracellular domain are combined with two copies of the IL18BR1 intracellular domain to confer constitutive IL-8 signaling.

[0075] Another constitutively active CCR is one in which the first or second polypeptide contains an essential IL-18 molecule. Such an arrangement is shown diagrammatically in FIG. 1A, where the receptor contains IL-18Rα and β chains, one of the chains also contains an IL-18 molecule tethered to the polypeptide by a linker. The tethered IL-18, due to its permanent physical proximity, binds and dimerizes with the IL18R1 and IL18RAP chains, conferring constitutive IL-18 signaling.

[0076] The sequence of human IL-18 is available from Uniprot accession number Q14116 and is shown below as SEQ ID NO: 14. The CCRs of the invention may comprise a truncated form of IL-18 lacking the signal sequence. Such a truncated sequence is shown below as SEQ ID NO: 15. [ka]

[0077] The IL-18 molecule, or a truncated IL-18 molecule, can be tethered to a first or second polypeptide. It can be N-terminal to the IL18R1 extracellular domain or the IL18RAP extracellular domain. It can be tethered by a linker, such as a serine-glycine linker. The linker can be, for example, 10-40 amino acids in length, for example, 20-30 or 24-28 amino acids in length.

[0078] Two exemplary sequences for creating the CCR shown in Figure 1A are shown below, with the individual components identified: in the first sequence, a truncated IL-18 molecule is linked to the N-terminus of IL18R1, and in the second sequence, a truncated IL-18 molecule is linked to the N-terminus of IL18RAP.

[0079] Tethered IL-18 - Version 1 [ka]

[0080] Tethered IL-18 - Version 2 [ka] [ka]

[0081] To generate the type of CCR illustrated in Figure 1A, it is only necessary to recombinantly express a polypeptide chain containing a linked IL-18 molecule. This polypeptide will pair with an endogenous chain of the complementary part of the IL18 receptor to produce a CCR. Thus, the above version 1, in which IL-18 is tethered to IL18R1 via a linker, will pair with endogenous IL18RAP; and the above version 2, in which IL-18 is tethered to IL18RAP via a linker, will pair with endogenous IL18R1.

[0082] CID / ligand activation system As an alternative to a constitutively active system, dimerization may occur only in the presence of a chemical inducer of dimerization (CID) or ligand, in which case the transmembrane protein triggers cytokine-type signaling only in the presence of the CID or ligand.

[0083] Suitable dimerization domains and CIDs are described in WO2015 / 150771, the contents of which are incorporated herein by reference.

[0084] For example, one dimerization domain may comprise the rapamycin binding domain of FK binding protein 12 (FKBP12) and the other may comprise the FKBP12-rapamycin binding (FRB) domain of mTOR; the CID may be rapamycin or a derivative thereof.

[0085] One dimerization domain may comprise the FK506 (tacrolimus) binding domain of FK binding protein 12 (FKBP12) and the other dimerization domain may comprise the cyclosporine binding domain of cyclophilin A; the CID may be an FK506 / cyclosporine fusion or a derivative thereof.

[0086] One dimerization domain may comprise an estrogen binding domain (EBD) and the other dimerization domain may comprise a streptavidin binding domain; the CID may be an estrone / biotin fusion protein or derivative thereof.

[0087] One dimerization domain may comprise a glucocorticoid binding domain (GBD) and the other dimerization domain may comprise a dihydrofolate reductase (DHFR) binding domain; the CID may be a dexamethasone / methotrexate fusion protein or a derivative thereof.

[0088] One dimerization domain may comprise an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and the other dimerization domain may comprise a dihydrofolate reductase (DHFR) binding domain; the CID may be an O6-benzylguanine derivative / methotrexate fusion protein or derivative thereof.

[0089] One dimerization domain may comprise a retinoic acid receptor domain and the other dimerization domain may comprise an ecodysone receptor domain; the CID may be RSL1 or a derivative thereof.

[0090] Rather than being activated by chemical inducers of dimerization administered exogenously to the patient, CCRs may dimerize in the presence of naturally occurring ligands, such as factors secreted by tumors or TGFβ.

[0091] In the arrangement shown diagrammatically in Figure IB, the first and second polypeptides of the CCR each contain a binding domain, such as an svFv or dAb, that binds to a ligand. In the presence of a ligand, both extracellular domains bind, bringing the intracellular domains together and conferring ligand-dependent IL-18 signaling.

[0092] The ligand may be, for example, a factor secreted by a tumor or a chemokine or a cell surface antigen.

[0093] Several ligand-binding domains are known in the art, including those based on the antigen-binding site of antibodies, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise a single-chain variable fragment (scFv) derived from a monoclonal antibody; a binding domain derived from a natural receptor for the target antigen; a peptide with sufficient affinity for the target ligand; a single domain binder such as camelids; a single artificial binder as a darpin; or a single chain derived from a T-cell receptor.

[0094] The term "ligand" is used synonymously with "antigen" to mean the entity that is specifically recognized and bound by the antigen-binding domain of a CCR.

[0095] The ligand may be prostate specific antigen (PSA), carcinoembryonic antigen (CEA) and factors secreted by tumors such as vascular endothelial growth factor (VEGF) or CA125.

[0096] Two exemplary sequences for making a CCR that senses PSA as shown in Figure 1B are shown below, and individual components are identified. The PSA binding domain of CCR is 5D3D11 and 5D5A5, which are described in WO2017 / 029512. In the first version, 5D3D11 scFv is linked to IL18R1 intracellular domain via a spacer and a transmembrane domain; 5D5A5 scFv is linked to IL18RAP intracellular domain via a spacer and a transmembrane domain. In the second version, 5D5A5 scFv is linked to IL18R1 intracellular domain via a spacer and a transmembrane domain; 5D3D11 scFv is linked to IL18RAP intracellular domain via a spacer and a transmembrane domain.

[0097] PSA Sensing - Version 1 [ka] [ka] [ka]

[0098] PSA Sensing - Version 2 [ka] [ka]

[0099] The CCR of the present invention can be induced by the presence of TGFβ in the extracellular environment.For example, in the configuration shown in FIG. 1C, the first and second polypeptides of the CCR each comprise an extracellular domain derived from a TGFβ receptor.For example, one polypeptide may comprise an extracellular domain derived from a TGFβ receptor I, and the other polypeptide may comprise an extracellular domain derived from a TGFβ receptor II.In the presence of TGFβ, the two TGFβ receptor intracellular domains are integrated to cause IL-18 type signal transduction.

[0100] Two exemplary sequences for creating a TGFβ-induced CCR as shown in FIG. 1C are shown below, with the individual components identified. In the first version, the TGFβR2 extracellular domain is linked to the IL18R1 intracellular domain via a spacer and a transmembrane domain; the TGFβR1 extracellular domain is linked to the IL18RAP intracellular domain via a spacer and a transmembrane domain. In the second version, the TGFβR1 extracellular domain is linked to the IL18RAP intracellular domain via a spacer and a transmembrane domain; the TGFβR2 extracellular domain is linked to the IL18R1 intracellular domain via a spacer and a transmembrane domain.

[0101] TGF beta-IL18 switch receptor: version 1 [ka] [ka]

[0102] TGF beta-IL18 switch receptor-version 2 [ka]

[0103] The configuration shown in FIG. 1D is also induced by the presence of TGFβ. In this configuration, one polypeptide contains an ITAM-containing intracellular domain and an antigen-binding domain. This polypeptide acts like a CAR in that it induces cell activation in the presence of a target antigen. This polypeptide also contains one of the IL-18 receptor intracellular domains and a TGFβ receptor extracellular domain. The other polypeptide in the CCR contains a second TGFβ receptor extracellular domain and another IL-18 receptor intracellular domain chain. In the presence of TGFβ, the two TGFβ receptor intracellular domains are integrated to cause IL-18-type signal transduction. In the presence of both TGFβ and a target antigen, the cell receives both an activation signal and an IL-18-type growth signal.

[0104] Two exemplary sequences for creating such a TGFbeta-IL18 CAR enhancer are shown below, with the individual components identified. This system includes an anti-EGFRvIII domain antibody (dAb) as the antigen-binding domain, and therefore EGFRvIII can be used as a model target antigen. Equivalent systems can be created that target other target antigens by swapping in the dAb or scFv for other binding specificities. In the first version of the sequence shown below, the anti-EGFRvIII dAb and TGFβR2 extracellular domain are linked to the IL18R1 and CD3z intracellular domains via a spacer and a transmembrane domain; the TGFβR1 extracellular domain is linked to the IL18RAP intracellular domain via a spacer and a transmembrane domain. In the second version, the anti-EGFRvIII dAb and the TGFβR1 extracellular domain are linked to the IL18RAP and CD3z intracellular domain via a spacer and a transmembrane domain; the TGFβR2 extracellular domain is linked to the IL18R1 intracellular domain via a spacer and a transmembrane domain.

[0105] TGF beta-IL18 CAR enhancer-version 1 [ka] [ka]

[0106] TGF beta-IL18 CAR enhancer-version 2 [ka] [ka]

[0107] Spacer The chimeric cytokine receptors of the invention, particularly the ligand-activated version shown diagrammatically in Figure IB, may include a spacer to connect the ligand-binding domain with the transmembrane domain and to spatially separate the ligand-binding domain from the intracellular domain. The flexible spacer allows the ligand-binding domain to be oriented in different directions to allow antigen binding.

[0108] When the cell of the present invention comprises two or more chimeric cytokine receptors, the spacer can be the same or different.When the cell of the present invention comprises a chimeric cytokine receptor (CCR) and a chimeric antigen receptor (CAR), the spacer of CCR and CAR can be different, for example, have different length.The spacer of CAR can be longer than the spacer of CCR or each CCR.

[0109] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk. Alternatively, the linker may comprise an alternative linker sequence having similar length and / or domain spacing characteristics as an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk.

[0110] The human IgG1 spacer can be altered to remove the Fc binding motif.

[0111] The spacer can be modified to remove the cysteine ​​residue so that it is monomeric. The sequence of the monomeric CD8 stalk is shown above as SEQ ID NO:24.

[0112] Transmembrane domain The transmembrane domain is the sequence of the CCR that spans the membrane. It may contain a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, conferring good receptor stability.

[0113] The constitutively active CCRs shown generally in Figures 1A and 1E and the ligand-activated CCRs shown generally in Figure 1B may comprise the IL18R1 and IL18RAP transmembrane domains shown above as SEQ ID NOs: 8 and 12, respectively. Alternatively, the TM domain of one or both chains may be exchanged for a different TM domain sequence.

[0114] The TGFβ-activated CCR system shown generally in Figures 1C and 1D may comprise the TGFβ1 and TGFβ2 transmembrane domains shown above as SEQ ID NOs: 32 and 30, respectively. Alternatively, the TM domain of one or both chains may be replaced with a different TM domain sequence.

[0115] Ligand The first and second polypeptides of the CCR type shown diagrammatically in FIG. 1B specifically bind to a ligand.

[0116] The ligand may be a soluble ligand, such as a factor secreted by a tumor or a chemokine.

[0117] Alternatively, the ligand may be a membrane-bound ligand, such as a cell surface antigen.

[0118] The term "soluble ligand" is used to indicate a ligand or antigen that is not part of or bound to a cell, but is free to move in the extracellular space, for example in the body fluids of a tissue of interest. Soluble ligands may be present in a cell-free state in the serum, plasma or other body fluids of an individual.

[0119] The soluble ligand may be associated with the presence or pathology of a particular disease, such as cancer.

[0120] Soluble ligands may be part of the cancer secretome, i.e., the collection of factors secreted by the tumor, derived from cancer stem cells, non-stem cells or the surrounding stroma. Soluble ligands may be secreted or excreted by tumor cells (see next subsection).

[0121] Soluble ligands can be characteristic of disease or diseased tissue. They can be found exclusively or at higher levels in diseased subjects versus healthy subjects, or in diseased tissue versus healthy tissue. Soluble ligands can be expressed at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold higher in diseased subjects versus healthy subjects, or in diseased tissue versus healthy tissue.

[0122] The terms "cell surface antigen" and "cell surface ligand" are used interchangeably with "membrane-bound antigen" and "membrane-bound ligand" and refer to a ligand that is bound or expressed on the surface of a cell. A cell surface ligand may be, for example, a transmembrane protein.

[0123] The cells on which the cell surface ligand is found may be target cells, such as cancer cells.

[0124] The cell surface ligand may be associated with the presence or pathology of a particular disease, such as cancer, or may be characteristic of the cell type of the target cell (e.g., a B cell) without necessarily being associated with a disease state.

[0125] If a cell surface ligand is characteristic of a disease or diseased tissue, it may be found exclusively or at higher levels on relevant cells in diseased subjects versus healthy subjects, or in diseased tissue versus healthy tissue. The cell surface ligand may be expressed at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold higher on cells of diseased subjects versus healthy subjects, or in diseased tissue versus healthy tissue.

[0126] Factors secreted by tumors The ligand recognized by the CCR may be a soluble ligand secreted or shed by the tumor.

[0127] This "tumor-secreted factor" may be, for example, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF) or cancer antigen-125 (CA-125).

[0128] Tumor secreted factors may be soluble ligands that are not cytokines. Thus, the CCRs of the invention graft binding specificity for a non-cytokine ligand onto the intracellular domain of a cytokine receptor.

[0129] Prostate-specific antigen (PSA) The soluble ligand may be prostate specific antigen (PSA).

[0130] Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), is a glycoprotein enzyme encoded in humans by the KLK3 gene. PSA is a member of the kallikrein-related peptidase family and is secreted by epithelial cells of the prostate gland.

[0131] PSA is present in small amounts in the serum of men with healthy prostates, but is elevated in individuals with prostate cancer and other prostate disorders.

[0132] PSA is a 237-residue glycoprotein that is activated by KLK2. Its physiological role is the liquefaction of the seminal clot components leading to the release of spermatozoa. In cancer, PSA may be involved in the processes of neoplastic growth and metastasis.

[0133] PSA is a chymotrypsin-like serine protease with a typical His-Asp-Ser triad and a catalytic domain similar to those of other kallikrein-related peptidases. Crystal structures of PSA were obtained i) in complex with monoclonal antibody (mAb) 8G8F5 and ii) in sandwich complex with mAbs 5D5A5 and 5D3D11 (Stura et al (J. Mol. Biol. (2011) 414:530-544).

[0134] Various monoclonal antibodies are known, including clones 2G2-B2, 2D8-E8, IgG1 / K described in Bavat et al Avicenna J. Med. Biotechnol. 2015, 7:2-7 and Leinonen (2004) 289:157-67.

[0135] A CCR of the invention may comprise, for example, the six CDRs or the VH and / or VL domains from a PSA-binding mAb such as 8G8F5, 5D5A5 or 5D3D11.

[0136] When a CCR comprises two antigen-binding specificities that bind to different epitopes on PSA, one may be based, for example, on 5D3D11 and one may be based, for example, on 5D5A5.

[0137] The amino acid sequences of the VH and VL of 5D3D11 and 5D5A5 are shown above as SEQ ID NOs:21, 23, 26 and 27.

[0138] Carcinoembryonic antigen (CEA) The soluble ligand may be CEA.

[0139] Carcinoembryonic antigen (CEA) describes a set of highly related glycoproteins involved in cell adhesion. CEA is normally produced in the gastrointestinal tissues during fetal development, but production stops before birth. Therefore, CEA is normally present only at very low levels in the blood of healthy adults. However, its serum levels are elevated in some types of cancer, which means that it can be used as a tumor marker in clinical trials.

[0140] CEA are glycosylphosphatidylinositol (GPI) cell surface anchored glycoproteins whose special sialofucosylated glycoforms act as functional colon cancer L-selectin and E-selectin ligands and may be important for metastatic dissemination of colon cancer cells. Immunologically, they have been characterized as members of the cluster of differentiation antigens CD66.

[0141] CEA and related genes constitute the CEA family, which belongs to the immunoglobulin superfamily. In humans, the carcinoembryonic antigen family consists of 29 genes, 18 of which are normally expressed. The following is a list of human genes encoding carcinoembryonic antigen-related cell adhesion proteins: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21.

[0142] Various antibodies targeting CEA are described in WO2011 / 034660.

[0143] Cells expressing a CCR to CEA may be useful, for example, in the treatment of colorectal cancer.

[0144] Vascular endothelial growth factor (VEGF) The soluble ligand may be VEGF.

[0145] Vascular endothelial growth factor (VEGF) is a signaling protein produced by cells that stimulates blood vessel formation and angiogenesis. It is part of a system that restores oxygen supply to tissues when blood circulation is insufficient. Serum concentrations of VEGF are high in bronchial asthma and diabetes. The normal function of VEGF is to create new blood vessels during embryonic development, after injury, in muscles after exercise, and to bypass blocked blood vessels (collateral circulation).

[0146] When VEGF is overexpressed, it can contribute to disease: solid tumors cannot grow beyond a limited size without an adequate blood supply; tumors that can express VEGF can grow and metastasize.

[0147] VEGFs are a subfamily of the platelet-derived growth factor family of cysteine-knot growth factors. They are key signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from preexisting vasculature).

[0148] The VEGF family includes five members in mammals: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D.

[0149] Various antibodies against VEGF are known, such as bevacizumab (Avastin) and ranibizumab (Lucentis).

[0150] Cancer antigen 125 (CA-125) CA-125 is associated with ovarian cancer and is the most frequently used biomarker for its detection. Although CA-125 is best known as a marker for ovarian cancer, it can also be elevated in other cancers, including endometrial, fallopian tube, lung, breast, and gastrointestinal cancers.

[0151] The sequence of human CA-125 (also known as mucin-16) is available from NCBI accession number 078966.

[0152] Several CA125-binding monoclonal antibodies are known, including OC125 and M11 (Nustad et al 1996, Tumour Biol. 17:196-329). In this study, the specificity of 26 monoclonal antibodies against the CA125 antigen was investigated. The CA125 antigen was found to carry only two major antigenic domains, classifying the antibodies as OC125-like (group A) or M11-like (group B).

[0153] The chimeric cytokine receptors of the invention may comprise antigen-binding domains derived from such antibodies. Cells containing such CCRs may be useful, for example, in the treatment of ovarian cancer.

[0154] The tumor secreted factor (or transmembrane protein, if membrane bound) may be selected from the following non-exhaustive list:

[0155] Mutant ALK protein resulting in ALK gene rearrangements and overexpression Alpha-fetoprotein (AFP) Beta-2-microglobulin (B2M) Beta-human chorionic gonadotropin (beta-hCG) Mutant B-REF proteins conferring BRAF V600 mutations C-kit / CD117 CA15-3 / CA27.29 CA19-9 Calcitonin CD20 Chromogranin A (CgA) Cytokeratin fragment 21-1 EGFR gene mutation analysis Estrogen receptor (ER) / progesterone receptor (PR) Fibrin / fibrinogen HE4 HER2 / neu gene amplification or protein overexpression Immunoglobulins KRAS gene mutation analysis Lactate dehydrogenase Neuron-specific enolase (NSE) Nuclear matrix protein 22 Programmed cell death ligand 1 (PD-L1) Thyroglobulin Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1)

[0156] Chemokines Chemokines are chemotactic cytokines. Cell migration is guided by chemokine gradients embedded and immobilized in the extracellular matrix. Positively charged chemokines, such as CXCL12, bind to negatively charged ECM molecules. These gradients provide a pathway for cancer and immune cell homing. The effect on T cells is thought to be inhibitory to the homing of cytotoxic T cells, whereas regulatory T cells are thought to be attracted.

[0157] Chemokines have a mass of approximately 8-10 kilodaltons and contain four cysteine ​​residues at conserved positions that are key to forming their three-dimensional shape.

[0158] Some chemokines are thought to be pro-inflammatory and can be induced during immune responses to recruit cells of the immune system to sites of infection, whereas others are thought to be homeostatic and are involved in controlling cell migration during the normal processes of tissue maintenance or development.

[0159] Chemokines have been classified into four main subfamilies: CXC, CC, CX3C and XC. All these proteins exert their biological effects by interacting with G protein-coupled transmembrane receptors, called chemokine receptors, which are selectively found on the surface of their target cells.

[0160] The main role of chemokines is to act as chemoattractants that guide the migration of cells. Cells attracted by chemokines follow the signal of increasing chemokine concentration toward the source of the chemokine. Some chemokines control cells of the immune system during the process of immune surveillance, such as directing lymphocytes to lymph nodes so that they can screen for invading pathogens by interacting with antigen-presenting cells present in these tissues. Other chemokines are inflammatory and are released from various cells in response to bacterial infections, viruses and other agents. Their release is often stimulated by inflammatory cytokines such as interleukin-1. Inflammatory chemokines function primarily as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate immune responses or promote wound healing. They are released by many different cell types and act to guide cells of both the innate and adaptive immune systems.

[0161] CC chemokines CC chemokine (or β-chemokine) proteins have two adjacent cysteines (amino acids) near their amino termini. There have been at least 27 different members of this subgroup reported for mammals, called CC chemokine ligand (CCL)-1 to -28; CCL10 is identical to CCL9. Chemokines in this subfamily usually contain four cysteines (C4-CC chemokines), but a few CC chemokines have six cysteines (C6-CC chemokines). C6-CC chemokines include CCL1, CCL15, CCL21, CCL23 and CCL28. CC chemokines induce migration of monocytes as well as other cell types such as NK cells and dendritic cells.

[0162] Examples of CC chemokines include monocyte chemoattractant protein-1 (MCP-1 or CCL2), which induces monocytes to leave the bloodstream, enter surrounding tissues, and become tissue macrophages.

[0163] CCL5 (or RANTES) attracts cells such as T cells, eosinophils and basophils that express the receptor CCR5.

[0164] CXC chemokines The two N-terminal cysteines of CXC chemokines (or α-chemokines) are separated by one amino acid, represented in the name with an "X". There are 17 different CXC chemokines described in mammals, which are subdivided into two categories: those that have a specific amino acid sequence (or motif) of glutamic acid-leucine-arginine (or ELR for short) immediately before the first cysteine ​​of the CXC motif (ELR positive), and those that do not have the ELR motif (ELR negative). ELR positive CXC chemokines specifically induce the migration of neutrophils and interact with the chemokine receptors CXCR1 and CXCR2.

[0165] C chemokine The third group of chemokines is known as the C chemokines (or gamma chemokines) and differs from all other chemokines in that it has only two cysteines; one N-terminal cysteine ​​and one downstream. Two chemokines have been described for this subgroup, called XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β).

[0166] CX3C chemokine CX3C chemokines have three amino acids between two cysteines. The only CX3C chemokine discovered so far is called fractalkine (or CX3CL1). It is secreted and anchored to the surface of any cell that expresses it, thereby acting both as a chemoattractant and as an adhesion molecule.

[0167] Chemokine receptors are G protein-coupled receptors that contain seven transmembrane domains found on the surface of leukocytes. To date, approximately 19 different chemokine receptors have been characterized and are divided into four families depending on the type of chemokine they bind; CXCR, which binds CXC chemokines, CCR, which binds CC chemokines, CX3CR1, which binds only one CX3C chemokine (CX3CL1), and XCR1, which binds two XC chemokines (XCL1 and XCL2). They share many structural features; they are similar in size (with 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 that contains serine and threonine residues that are important for receptor regulation. The first two extracellular loops of chemokine receptors each have a conserved cysteine ​​residue that allows the formation of disulfide bridges between these loops. G proteins couple to the C-terminus of chemokine receptors to enable intracellular signaling following receptor activation, whereas the N-terminal domain of the chemokine receptor determines ligand binding specificity.

[0168] CXCL12 CXCL12 is strongly chemotactic for lymphocytes. CXCL12 plays a key role in angiogenesis by recruiting endothelial progenitor cells (EPCs) from the bone marrow via a CXCR4-dependent mechanism. It is this function of CXCL12 that makes it a critical factor in neovascularization associated with carcinogenesis and tumor progression. CXCL12 also has a role in tumor metastasis, where cancer cells expressing the receptor CXCR4 are attracted to metastatic target tissues that release the ligand CXCL12.

[0169] The receptor for CXCL12 is CXCR4. A CCR of the invention may comprise a CXCL12-binding domain derived from CXCR4 linked to an intracellular domain derived from a cytokine receptor, such as the IL-2 receptor.

[0170] CXCR4-coupled expression of IL2 supports the engraftment of therapeutic T cells for cancer therapy. In multiple myeloma, cells expressing such CCRs can migrate cells and change the bone marrow environment. Such cells are also useful in the treatment of solid tumors by modifying the solid tumor microenvironment.

[0171] CXCR7 also binds CXCL12.

[0172] CCL2 Chemokine (CC motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP1) and small molecule-inducible cytokine A2, recruits monocytes, memory T cells, and dendritic cells to sites of inflammation created by either tissue injury or infection.

[0173] CCR2 and CCR4 are the two cell surface receptors that bind to CCL2.

[0174] A CCR of the present invention may comprise the CCL2 binding site of CCR2 or CCR4 in its ligand binding domain.

[0175] cell surface antigen The ligand may be a cell surface antigen, such as a transmembrane protein.

[0176] The cell surface antigen may be CD22.

[0177] CD22, or Cluster of Differentiation-22, is a molecule that belongs 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.

[0178] CD22 is a carbohydrate-binding transmembrane protein that specifically binds sialic acid and contains an immunoglobulin (Ig) domain at its N-terminus. The presence of the Ig domain makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0179] Increased expression of CD22 is found in non-Hodgkin's lymphoma and other lymphomas. A variety of monoclonal antibodies that target CD22 are known, including epratuzumab, inotuzumab ozogamicin, m971 and m972.

[0180] Chimeric antigen receptors (CARs) The cells of the present invention may also comprise one or more chimeric antigen receptors. The CAR may be specific for tumor-associated antigens. Alternatively, the CAR may comprise an autoantigen, or a part thereof, in its extracellular domain, such that it binds to the autoantibody produced by pathogenic B cells.

[0181] Classical CARs are chimeric type I transmembrane proteins that connect an extracellular antigen recognition domain (binder) to an intracellular signaling domain (intracellular domain). The binder is typically a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be antibody-like or based on other formats that contain a ligand-based antigen binding site. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the intracellular domain.

[0182] Early CAR designs had intracellular domains derived from the intracellular portion of the γ-chain of either FcεR1 or CD3ζ. As a result, these first generation receptors delivered immunological signal 1, which was sufficient to induce T cell killing of cognate target cells, but failed to fully activate the T cell to proliferate and survive. To overcome this limitation, composite intracellular domains were constructed: fusions of the intracellular portion of a T cell costimulatory molecule with that of CD3ζ result in second generation receptors that can deliver simultaneous activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal, i.e., immunological signal 2, which induces T cell proliferation. Some receptors have also been described that contain TNF receptor family intracellular domains, such as the closely related OX40 and 41BB, which deliver survival signals. Even more potent third generation CARs are now described, with intracellular domains that can deliver activation, proliferation and survival signals.

[0183] The nucleic acid encoding CAR can be transferred to 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 CAR binds to target antigen, this results in the transmission of activation signal to the T cell in which it is expressed.Thus, CAR directs the specificity and cytotoxicity of T cells to cells that express target antigen.

[0184] The cells of the invention may comprise one or more CARs.

[0185] A CAR may comprise an antigen-binding domain, a spacer domain, a transmembrane domain and an intracellular domain. The intracellular domain may comprise or be associated with a domain that transmits a T cell activation signal.

[0186] CAR antigen-binding domain The antigen-binding domain is the portion of the CAR that recognizes the antigen.

[0187] Several antigen-binding domains are known in the art, including those based on the antigen-binding site of antibodies, antibody mimetics, and T-cell receptors. For example, an antigen-binding domain may comprise a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as camelids; a single artificial binder such as a darpin; or a single chain derived from a T-cell receptor.

[0188] cell surface antigen A CAR may recognize a cell surface antigen, i.e., an entity such as a transmembrane protein expressed on the surface of a target cell, such as a tumor cell.

[0189] CARs are capable of specifically binding to tumor-associated cell surface antigens.

[0190] Various tumor-associated antigens (TAA) are known, some of which are shown in Table 2. The antigen-binding domain used in the present invention may be a domain capable of binding to a TAA as shown therein. [Table 2]

[0191] Where the CAR recognizes a B cell lymphoma or leukemia antigen (such as CD19, CD20, CD52, CD160 or CD5), the CCR may recognize another B cell antigen, such as CD22.

[0192] Prostate cancer associated antigens The CAR may specifically bind to a cell surface antigen associated with prostate cancer, such as prostate stem cell antigen (PSCA) or prostate specific membrane antigen (PSMA).

[0193] PSCA is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein that is upregulated in the majority of prostate cancers and is also detected in bladder and pancreatic cancers.

[0194] 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).

[0195] The CCR-expressing cells of the invention may also express an anti-PSCA CAR, which may comprise an antigen-binding domain based on one of these antibodies.

[0196] PSMA is a membrane-located zinc metalloenzyme. It is strongly expressed in the human prostate, 100-fold higher than expression in many other tissues. In cancer, its expression is upregulated, and it has been called the second most upregulated gene in prostate cancer, increasing 8-12-fold compared to non-cancerous prostate. In addition to its expression in the human prostate and prostate cancer, PSMA has also been found to be highly expressed in tumor neovasculature, but not in normal vasculature, in all types of solid tumors, such as kidney, breast, and colon.

[0197] Various anti-PSMA antibodies are known, such as 7E11, J591, J415, and Hybritech PEQ226.5 and PM2J004.5, each binding to a different epitope on PSMA (Chang et al (1999) Cancer Res 15:3192-8).

[0198] The CCR-expressing cells of the invention may also express an anti-PSMA CAR, which may comprise an antigen-binding domain based on one of these antibodies.

[0199] For example, the CCR may comprise a J591-based scFv having the sequence shown as SEQ ID NO:35. [ka]

[0200] Autoantibody Receptors Autoimmune disease is a condition resulting from an abnormal immune response against a functioning body part. Autoimmunity is the presence of an autoreactive immune response (e.g., autoantibodies, autoreactive T cells) with or without resulting damage or pathology. Autoantibodies are the hallmark of many autoimmune disorders.

[0201] CAR-based approaches can be used to treat autoimmune diseases and other disorders associated with abnormal or undesirable immune responses.CAR extracellular domains can bind to one or more autoantibodies to identify and kill pathogenic B cells associated with autoimmune diseases.Extracellular domains can include autoantibody binding entities, such as anti-idiotypic antibodies, or they can simply include autoantigens themselves, or parts thereof.

[0202] Autoimmune disorders include celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis (MS), alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mucous membrane pemphigus vulgaris (mPV), mucocutaneous pemphigus vulgaris (mcPV), membranous nephropathy (MN) and myasthenia gravis (MG).

[0203] The extracellular domain of the CAR may contain all or part of an autoantigen, such as the cell adhesion proteins desmoglein 3 (DSG3) or DSG1; muscle-specific receptor tyrosine kinase (MuSK); acetylcholine receptor (AChR); phospholipase A2 receptor (PLA2R) or aquaporin-4 (AQP4).

[0204] Other undesirable immune responses include allergies, host-versus-graft responses, and immune responses to therapy, such as hemophilia A patients who develop antibodies to factor VIII. In this latter example, the use of a CAR that contains part or all of FVIII as its extracellular domain can destroy antibodies and B cells that neutralize the FVIII clotting factor.

[0205] CAR transmembrane domain The transmembrane domain is the sequence of the CAR that spans the membrane. It may contain a hydrophobic alpha helix. The CAR transmembrane domain may be derived from CD28, conferring good receptor stability.

[0206] CAR signal peptide The CARs and CCRs described herein may contain a signal peptide such that, when expressed in a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface, where it is expressed.

[0207] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that have a tendency to form a single alpha helix. The signal peptide may begin with a short positively charged stretch of amino acids that help enforce the proper topology of the polypeptide during translocation. At the end of the signal peptide, there is typically a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave either during or after the completion of translocation to generate a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0208] The signal peptide may be at the amino terminus of the molecule.

[0209] The signal peptide may comprise the sequence shown as SEQ ID NO:5 or 11 above.

[0210] CAR intracellular domain The intracellular domain is the signaling portion of the CAR. It may be part of or associated with the intracellular domain of the CAR. After antigen recognition, the receptor cluster, native CD45 and CD148, are excluded from the synapse and the signal is transmitted to the cell. The most commonly used intracellular domain component is that of CD3-zeta, which contains three ITAMs. It transmits activation signals to T cells after antigen binding. CD3-zeta cannot provide a fully competent activation signal and additional costimulatory signaling may be required. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.). For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0211] The intracellular domain is (i) an ITAM-containing intracellular domain, such as the intracellular domain derived from CD3 zeta; and / or (ii) a costimulatory domain, such as the intracellular domain from CD28 or ICOS; and / or (iii) a domain that transmits a survival signal, for example, an intracellular domain of the TNF receptor family such as OX-40, 4-1BB, CD27, or GITR may include:

[0212] Some systems have been described in which antigen recognition moiety is on a separate molecule from signal transduction moiety, such as those described in WO2015 / 150771; WO2016 / 124930 and WO2016 / 030691.The CAR of the present invention can therefore comprise an antigen binding moiety that comprises an antigen binding domain and a transmembrane domain, which can interact with separate intracellular signal transduction components that comprise signal transduction domains.The vector of the present invention can express such a CAR signal transduction system that comprises an antigen binding moiety and an intracellular signal transduction component.

[0213] Nucleic acid sequences / constructs The present invention also provides nucleic acid sequences and nucleic acid constructs encoding the CCRs of the present invention.

[0214] The present invention provides a nucleic acid sequence for producing a chimeric cytokine receptor as shown diagrammatically in Figure 1A. As explained above, it is only necessary to encode one of the two chains of this CCR by recombinant means, since it will associate with the complementary endogenous chain in the cell. The nucleic acid sequence encodes a polypeptide comprising interleukin-18 receptor 1 (IL18R1) or interleukin-18 receptor accessory protein (IL18RAP) tethered to an IL-18 molecule, and has the following general structure: IL18-L-IL18R1ecto-TM-IL18R1endo or IL18-L-IL18RAPecto-TM-IL18RAPendo (where: IL18 is a nucleic acid sequence encoding an IL18 molecule with a truncated or non-functional signal peptide, L is a nucleic acid sequence encoding a flexible linker, IL18R1ecto is a nucleic acid sequence encoding the interleukin-18 receptor 1 (IL18R1) extracellular domain; TM is a nucleic acid sequence encoding a transmembrane domain, IL18R1endo is a nucleic acid sequence encoding the IL18R1 intracellular domain, IL18RAPecto is a nucleic acid sequence encoding the extracellular domain of the interleukin-18 receptor accessory protein (IL18RAP); IL18RAPendo is a nucleic acid sequence encoding the intracellular domain of IL18RAP. may have the following structure:

[0215] A nucleic acid construct of the invention may comprise a first nucleic acid sequence encoding a first polypeptide, and a second nucleic acid sequence encoding a second polypeptide.

[0216] The nucleic acid construct has the following structure: ecto1-spacer1-TM1-IL18R1endo-coexpr-ecto2-spacer2-TM2-IL18RAPendo, or ecto1-spacer1-TM1-IL18RAPendo-coexpr-ecto2-spacer2-TM2-IL18R1endo (where: ecto1 is a nucleic acid sequence encoding the extracellular domain of the first polypeptide; spacer1 is a nucleic acid sequence encoding the spacer of the first polypeptide; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; IL18R1endo is a nucleic acid sequence encoding the interleukin-18 receptor 1 (IL18R1) intracellular domain; coexpr is a nucleic acid sequence allowing for the simultaneous expression of both polypeptides as separate entities, ecto2 is a nucleic acid sequence encoding the extracellular domain of the second polypeptide; spacer2 is a nucleic acid sequence encoding the spacer of the second polypeptide; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide, IL18RAPendo is a nucleic acid sequence encoding the intracellular domain of the interleukin-18 receptor accessory protein (IL18RAP) may have the following structure:

[0217] The extracellular domains of the first and second polypeptides may be capable of spontaneously dimerizing to produce a constitutively active CCR. Endo1 and endo2 may encode two domains capable of forming a coiled-coil structure, such as, for example, a heavy chain constant domain (HC) and a light chain constant domain (LC) in any order; or a leucine zipper.

[0218] When the nucleic acid construct is expressed in a cell, such as a T cell, it encodes a polypeptide that is cleaved at the cleavage site such that the first and second polypeptides are co-expressed at the cell surface.

[0219] The present invention also provides a nucleic acid construct encoding the first and second polypeptides of the present invention and a CAR. Such a construct has the structure: (i) CCRecto1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CCRecto2-CCRspacer2-CCRTM2-CCRendo2-coexpr2-CARAgB-CARspacer-CARTM-CARendo; (ii) CCRecto1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CARAgB-CARspacer-CARTM-CARendo-coexpr2-CCRecto2-CCRspacer2-CCRTM2-CCRendo2; or (iii) CARAgB-CAR spacer-CARTM-CARendo-coexpr1-CCRecto1-CCR spacer1-CCRTM1-CCRendo1-coexpr2-CCRecto2-CCR spacer2-CCRTM2-CCRendo2 (where: CCRecto1 is a nucleic acid sequence encoding the extracellular domain of the first polypeptide of CCR; CCR spacer 1 is a nucleic acid sequence encoding the spacer of the first polypeptide of CCR, CCRTM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide of CCR; CCRendo1 is a nucleic acid sequence encoding the intracellular domain of the first polypeptide of CCR; CCRecto2 is a nucleic acid sequence encoding the extracellular domain of the second polypeptide of CCR; CCR spacer2 is a nucleic acid sequence encoding the spacer of the second polypeptide of CCR, CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide of CCR, CCRendo2 is a nucleic acid sequence encoding the intracellular domain of the second polypeptide of CCR, coexpr1 and coexpr2 are nucleic acid sequences, which may be the same or different, that allow for the simultaneous expression of the two flanking sequences, CARAgB is a nucleic acid sequence encoding the antigen-binding domain of the CAR, CAR spacer is a nucleic acid sequence encoding the spacer of the CAR, CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR, CARendo is a nucleic acid sequence encoding the intracellular domain of CAR) may have the following structure:

[0220] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0221] Those skilled in the art will understand that several different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it should be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0222] Nucleic acids according to the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that contain synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the purposes of the uses described herein, polynucleotides can be modified by any method available in the art. Such modifications can be carried out to enhance the in vivo activity or life span of the polynucleotide of interest.

[0223] The terms "variant," "homolog" or "derivative" in reference to a nucleotide sequence include any substitution, variation, modification, replacement, deletion or addition of one or more nucleic acid(s) from or to the sequence.

[0224] In the above structure, "coexpr" is a nucleic acid sequence that allows simultaneous expression of both the first and second CAR. It may be a sequence that codes for a cleavage site, such that the nucleic acid construct comprises two or more CCRs, or one CCR and a CAR, linked by a cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

[0225] The cleavage site may be the first and second polypeptides of the CCR; or any sequence that allows the CCR and the CAR to be separated.

[0226] The term "cleavage" is used herein for convenience, however, the cleavage site may separate the peptide into individual entities by mechanisms other than classical cleavage. For example, for the foot and mouth disease virus (FMDV) 2A autocleaving peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolysis by host cell proteinases, autoproteolysis or translational effects (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041). When the cleavage site is located between nucleic acid sequences encoding proteins, the exact mechanism of such "cleavage" is not important for the purposes of the present invention, as long as it allows the proteins to be expressed as separate entities.

[0227] The cleavage site may be a furin cleavage site.

[0228] Furin is an enzyme that belongs to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process latent precursor proteins to their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane-type matrix metalloproteinase 1, pro-nerve growth factor beta subunit, and von Willebrand factor. Furin cleaves proteins immediately downstream of a basic amino acid target sequence (canonically Arg-X-(Arg / Lys)-Arg') and is abundant in the Golgi apparatus.

[0229] The cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.

[0230] TEV protease is a highly sequence-specific cysteine ​​protease that is a chymotrypsin-like protease. It is very specific for its target cleavage site and is therefore frequently used for controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S, where "\" indicates the peptide bond to be cleaved. Mammalian cells, such as human cells, do not express TEV protease. Thus, in embodiments in which the nucleic acid construct of the invention contains a TEV cleavage site and is expressed in a mammalian cell, an exogenous TEV protease must also be expressed in the mammalian cell.

[0231] The cleavage site may encode a self-cleaving peptide.

[0232] "Autocleaving peptide" refers to a peptide that functions such that when proteins and polypeptides that include the autocleaving peptide are produced, they are immediately "cleaved" or separated into distinct and separate first and second polypeptides without the need for any external cleavage activity.

[0233] The autocleaving peptide may be a 2A autocleaving peptide derived from an aphthovirus or a cardiovirus. Primary 2A / 2B cleavage of aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In aphthoviruses such as foot and mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), represents an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al (2001) supra).

[0234] "2A-like" sequences have been found in picornaviruses other than aphthoviruses or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses, and repetitive sequences within trypanosoma species and bacterial sequences (Donnelly et al (2001) supra). A nucleic acid construct of the invention may encode one or these 2A-like cleavage sequences. A suitable sequence is given above (SEQ ID NO: 10).

[0235] vector The invention also provides a vector, or a kit of vectors, comprising one or more nucleic acid sequences encoding a CCR according to the invention and, optionally, one or more CARs. Such vectors can be used to introduce the nucleic acid sequences into a host cell such that it expresses a CCR according to the invention.

[0236] The vector may be, for example, a plasmid or a viral vector, such as a retroviral or lentiviral vector, or a transposon-based vector or a synthetic mRNA.

[0237] The vector may be capable of transfecting or transducing a cell, such as a T cell or a NK cell.

[0238] cell The invention provides cells comprising a CCR of the invention and optionally one or more CARs.

[0239] The cell may comprise a nucleic acid or vector of the invention.

[0240] The cell may be a cytolytic immune cell, such as a T cell or a NK cell.

[0241] T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of the T cell receptor (TCR) on the cell surface. There are various types of T cells, as summarized below.

[0242] T helper cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are 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 secrete different cytokines to facilitate different types of immune responses.

[0243] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected and tumor cells and are also involved in graft 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. IL-10, secreted by regulatory T cells, can inactivate CD8+ cells, via adenosine and other molecules, into an anergic state that prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0244] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has cleared. They rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with a "memory" for 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 may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0245] Regulatory T cells (Treg cells), previously known as suppressor T cells, are important for the maintenance of immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

[0246] Two main classes of CD4+ Treg cells have been described - naturally occurring Treg cells and adaptive Treg cells.

[0247] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+Treg cells) arise in the thymus and are associated with the interaction between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated by TSLP.Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3.Mutations in the FOXP3 gene can prevent regulatory T cell development and cause the fatal autoimmune disease IPEX.

[0248] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0249] The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0250] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third type of cell differentiated from a common lymphoid precursor cell that gives rise to B and T lymphocytes. NK cells are known to enter the circulation after differentiation and maturation in the bone marrow, lymph nodes, spleen, tonsils and thymus.

[0251] The CCR-expressing cells of the invention may be any of the cell types described above.

[0252] T or NK cells according to the first aspect of the invention can be generated ex vivo either from the patient's own peripheral blood (first party), or in the setting of a hematopoietic stem cell transplant from the peripheral blood of a donor (second party), or from peripheral blood from an unrelated donor (third party).

[0253] Alternatively, T or NK cells according to the first aspect of the invention may be derived from ex vivo differentiation of inducible or embryonic precursor cells into T or NK cells. Alternatively, immortalized T cell lines can be used which retain their lytic function and can act as therapeutic agents.

[0254] In all of these embodiments, CCR-expressing cells are generated by introducing the CCR or DNA or RNA encoding the respective CCR by one of a number of means, including transduction with a viral vector, transfection with DNA or RNA.

[0255] The cells of the invention may be ex vivo T or NK cells derived from a subject. The T or NK cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The T or NK cells may be activated and / or expanded, for example by treatment with an anti-CD3 monoclonal antibody, prior to transduction with a nucleic acid encoding a molecule that provides a CCR according to the first aspect of the invention.

[0256] The T or NK cells of the present invention (i) isolation of a T or NK cell-containing sample from a subject or other source listed above; and (ii) Transduction or transfection of T or NK cells with one or more nucleic acid sequences encoding a CCR. It can be prepared by:

[0257] T or NK cells can then be purified, e.g., selected, based on expression of the antigen-binding domain of the antigen-binding polypeptide.

[0258] Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the present invention.

[0259] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, diluent or excipient.The pharmaceutical composition may optionally comprise one or more additional pharma- ceutically active polypeptides and / or compounds.Such a preparation may be, for example, in a form suitable for intravenous infusion.

[0260] Treatment The present invention provides a method for treating and / or preventing a disease, comprising the step of administering a cell of the present invention (eg, in a pharmaceutical composition described above) to a subject.

[0261] The method for treating disease relates to the therapeutic use of the cells of the present invention. The cells can be administered to a subject having an existing disease or condition to alleviate, reduce or ameliorate at least one symptom associated with the disease, and / or to slow down, reduce or block the progression of the disease.

[0262] The method for preventing disease relates to the prophylactic use of the cells of the present invention.Such cells may be administered to a subject who has not yet suffered from disease and / or does not show any symptoms of disease, in order to prevent or attenuate the cause of disease, or to reduce or prevent the occurrence of at least one symptom associated with disease.The subject may have a predisposition to disease, or may be considered to be at risk of developing disease.

[0263] The method is: (i) isolating a T or NK cell-containing sample; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii) administering cells derived from (ii) to a subject. may include:

[0264] A T or NK cell-containing sample can be isolated from a subject or from other sources, for example, as described above. T or NK cells can be isolated from the subject's own peripheral blood (first party), or in the setting of a hematopoietic stem cell transplant from the peripheral blood of a donor (second party), or from peripheral blood from an unrelated donor (third party).

[0265] The present invention provides a CCR-expressing cell of the invention for use in the treatment and / or prevention of disease.

[0266] The present invention also relates to the use of a CCR-expressing cell of the invention in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0267] The disease treated and / or prevented by the methods of the present invention may be a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

[0268] In particular, the cancer may be a solid cancer. The cancer may result in so-called "cold tumours", i.e., tumours that are unlikely to induce a strong immune response. Cold tumours tend to be surrounded by cells that can attack tumour cells and inhibit T cells from killing them. Cold tumours have low immune infiltration and usually do not respond to immunotherapy. Examples of cold tumours include colon, prostate, ovarian and pancreatic cancers, neuroblastoma and glioblastoma.

[0269] The cancer may be one that contains relatively low levels of neoantigens. Neoantigens are tumor-specific antigens that are generated by nonsynonymous mutations that occur during cell transformation. Because they are foreign to the host genome, they do not undergo central tolerance and can be highly immunogenic. Examples of cancers that contain low levels of neoantigens include prostate, pancreatic and ovarian cancers, neuroblastoma, renal carcinoma and Merkel cell carcinoma.

[0270] If the CCR is activated by the ligand PSA, the cancer may be prostate cancer.

[0271] The cells of the present invention may be capable of killing target cells, such as cancer cells. The target cells may be characterized by the presence of a tumor-secreted or chemokine ligand in the vicinity of the target cells. The target cells may be characterized by the presence of a soluble ligand along with the expression of a tumor-associated antigen (TAA) on the target cell surface.

[0272] The disease treated and / or prevented by the methods of the present invention may be an autoimmune disease, allergy, or other condition associated with an inappropriate or unwanted immune response.

[0273] Autoimmune disorders include celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis (MS), alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mucous membrane pemphigus vulgaris (mPV), mucocutaneous pemphigus vulgaris (mcPV), membranous nephropathy (MN) and myasthenia gravis (MG).

[0274] The cells of the present invention may be cells that produce autoreactive or other undesirable antibodies (such as antibodies against a graft or treatment). The cells of the present invention may be capable of killing B cells that produce autoantibodies. Target cells may be characterized by the expression of and / or the ability to secrete undesirable antibodies, such as autoantibodies.

[0275] The cells and pharmaceutical compositions of the invention may be for use in the treatment and / or prevention of the above mentioned diseases.

[0276] The cells and pharmaceutical compositions of the invention may be for use in any of the methods described above.

[0277] The present invention will now be further described by way of examples, which are meant to serve to assist those of ordinary skill in the art in the practice of the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0278] Example 1 Investigating the effect of co-expression of CAR with IL-18 CCR in vitro

[0279] A constitutively active IL-18 signaling chimeric cytokine receptor was produced by linking the IL-18 receptor intracellular domain to a "Fab" type extracellular domain (see the embodiment shown diagrammatically in FIG. 1E). This structure uses the natural dimerization components of antibodies, i.e., the dimerization domains derived from the heavy and light chain constant regions. The chimeric cytokine receptor has two chains; a first polypeptide comprising an antibody kappa light chain (shown as SEQ ID NO:3) and an IL18R1 TM domain (SEQ ID NO:8) and an intracellular domain (SEQ ID NO:9); and a second polypeptide comprising an antibody heavy chain CH1 (SEQ ID NO:4) and an IL18RAP TM domain (SEQ ID NO:12) and an intracellular domain (SEQ ID NO:13).

[0280] The nucleic acid sequences encoding these two polypeptides, separated by the 2A peptide coding sequence, were cloned in frame.

[0281] The vectors were constructed as illustrated diagrammatically in Figure 2A. Both vectors expressed a) the selection suicide gene RQR8 described in WO2013 / 153391; and b) a second generation CAR with a GD2 binding domain and a 41BB-CD3z intracellular domain described in WO2015 / 132604. One vector also expressed the IL-18 CCR.

[0282] T cells were transduced with these constructs and then cocultured with either wild-type SupT1 cells (which do not express GD2) or SupT1 target cells transduced to express GD2. Briefly, 12.5 × 10 4 5 x 10 transduced CAR T cells 4 1000 IL-18 CCR were co-cultured with 1000 IL-18 CCR target cells to obtain an E:T ratio of 1:4. Target cell killing was analyzed by FACS and IL2 and IFNγ release into the supernatant was analyzed by ELISA. The results are shown in Figure 2B. Co-expression of IL-18 CCR maintained target cell killing but improved cytokine release compared to cells expressing CAR alone.

[0283] Example 2 Comparison of the effects of co-expression of IL2, IL7 or IL18 CCRs in vivo in a CT26-GD2+ colon cancer animal model In vivo experiments were performed to evaluate the engraftment and antitumor activity of murine T cells co-expressing CAR and CCR in an established colon cancer immunocompetent model in Balb / c mice.

[0284] The experimental design is shown diagrammatically in Figure 3. Briefly, 6- to 8-week-old Balb / c female mice were inoculated with 1 × 10 6 CT26 colon cancer cells were subcutaneously injected. After 9 days of tumor engraftment, mice were sublethally irradiated with 5 Gy of total body irradiation (TBI). Mouse CAR-T cells were generated by transducing cells with either a single vector expressing the GD2 CAR (GD2 CAR); or a bicistronic vector co-expressing the GD2 CAR and one of the IL2-CCR, IL-7 CCR, or IL-18 CCR. One day after TBI, 1 × 10 6 Transduced CAR T cells were administered by intravenous injection. Tumor growth was monitored twice weekly by caliper. Peripheral blood was collected every 7 days and analyzed for the presence of transduced CAR T cells by markers CD3 and Thy1.1. The CD3 marker was used to identify lymphocyte populations within total CD45+ immune cells. THY1.1 was used to identify transduced CD3 T cells within the total T cell population. When tumors were 1 cm 3 Mice were euthanized when they reached their maximum length or width or when they suddenly lost more than 20% of their body weight. Spleens and tumors were collected at the time of euthanasia for CAR T cell tracking.

[0285] The results are shown in Figure 4 (tumor volume) and Figure 5 (peripheral blood T cell counts).

[0286] CT26 GD2 +Tumors were not recruited by cells expressing the GD2 CAR alone or cells co-expressing the GD2 CAR with IL2-CCR or IL7-CCR. In contrast, CAR T cells transduced with a bicistronic vector expressing the IL18-CCR showed potent antitumor activity, prolonging survival of Balb / c mice with established tumor burdens, with tumors remaining undetectable by day 50 in four of eight mice.

[0287] Enhanced tumor control by IL18-CCR GD2 CAR T cells was also accompanied by higher CAR T cell engraftment: IL18 CCR CAR T cells demonstrated the highest degree of engraftment, peaking at day 10 post-infusion, and sustained high peripheral persistence of the transduced CAR T cells until the end of the experiment.

[0288] Kaplan Meyer survival curves (Figure 6) showed the extent of improved survival in animals receiving CAR-T cells expressing IL2 or IL7 CCRs, which was greatly increased in animals receiving CAR-T cells expressing IL18 CCRs: 50% of mice in the IL18 CCR transduced cohort survived to day 50 after CAR T cell injection.

[0289] Example 3 Investigating differences in gene expression in activated and non-activated CAR-T cells expressing IL7 or IL-18 CCRs The expression of an 800-gene panel was analyzed for activated and non-activated CAR-T cells expressing IL7- or IL-18 CCRs. The results of the principal component analysis (PCA) are shown in Figure 7. Clear differences were observed between CAR-T cells expressing IL7- or IL-18 CCRs in both the non-activated and activated states.

[0290] To explore this further, expression changes for individual genes were analyzed for IL18 CCR expressing cells in both activated and non-activated states, using IL7 CCR as the baseline for differential gene expression calculations.

[0291] The results are shown in Figure 8. Expression of some genes, notably those encoding the chemokines CCL22, CCL17, CXCL8 and interleukin IL1A, is significantly upregulated in CAR-T cells expressing the IL-18 CCR compared to CAR-T cells expressing the IL-7 CCR.

[0292] This observation suggests that the mechanisms by which IL-7 and IL-18 CCR support CAR-T function may be fundamentally different. Without wishing to be bound by theory, we predict that IL-18 CCR functions by interacting with the host immune system, rather than having a direct effect on CAR-T cells themselves. This is consistent with the fact that IL-18, as a cytokine, does not directly induce T cell proliferation, unlike IL-7. The differential expression of chemokines such as CCL22, CXCL8 and CCL17 suggests that IL-18 CCR functions primarily by recruiting cells of the host immune system to tumor sites.

[0293] This mechanism is believed to support the observed results in the CT-26-GD2+ mouse model. This immunocompetent animal model can be considered as a cold tumor, i.e., one that is likely to be immune-compromised. Cold tumors tend to be surrounded by cells that can suppress T cells, and they typically do not respond to immunotherapy. Immunocompetent animal models also have low levels of neoantigens. Using this model, CAR-T cells expressing IL-18 CCR were significantly better at controlling tumors than CAR T cells expressing IL-7 CCR. Recruitment of host immune cells to the tumor site by CAR-T cells expressing IL-18 CCR may be more important in addressing this colon cancer tumor model than boosting the activity of the CAR-T cells themselves.

[0294] In contrast, hot tumours, which are more likely to elicit strong immune responses, or tumours in which there are higher levels of neoantigens capable of eliciting neoantigen-induced tumour killing activity, are more likely to be boosted by the proliferative effects of, for example, IL2- or IL7-CCR.

[0295] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art of molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. Two polypeptides: (i) (a) an extracellular domain comprising a first dimerization domain; (b) Interleukin-18 receptor 1 (IL18R1) intracellular domain a first polypeptide comprising: (ii) (a) an extracellular domain that comprises a second dimerization domain that spontaneously dimerizes with the first dimerization domain; (b) Interleukin-18 receptor accessory protein (IL18RAP) intracellular domain A second polypeptide comprising Including, One of the first and second dimerization domains comprises a heavy chain constant domain (CH) and the other comprises a light chain constant domain (CL); Chimeric cytokine receptors (CCRs).

2. A cell comprising the chimeric cytokine receptor of claim 1.

3. The cell of claim 2 , which also contains a chimeric antigen receptor.

4. The nucleic acid construct encoding a chimeric cytokine receptor (CCR) of claim 1, wherein the nucleic acid construct comprises a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide.

5. 5. The nucleic acid construct of claim 4, further comprising a nucleic acid sequence encoding a chimeric antigen receptor.

6. A vector comprising the nucleic acid construct of claim 4.

7. i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined in claim 1; and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined in claim 1. Kit including:

8. A method for producing a cell according to claim 2 or 3, comprising the step of introducing a nucleic acid construct according to claim 4 or 5; a vector according to claim 6; or a kit of vectors according to claim 7 into a cell ex vivo.

9. A pharmaceutical composition comprising a plurality of cells according to claim 2 or 3.

10. 10. The pharmaceutical composition of claim 9 for use in the treatment of cancer or an autoimmune disease.

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