RU2826122C1 - Chimeric cytokine receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to chimeric cytokine receptors (CCR), and can be used in medicine for treating cancer. Disclosed is a nucleic acid construct for producing a cell which expresses a chimeric transmembrane protein, wherein said nucleic acid construct encodes a chimeric transmembrane protein and comprises a first nucleic acid sequence encoding a first polypeptide comprising a heavy chain (CH) constant domain dimerization domain and a first endodomain chain of a type I cytokine receptor; and a second nucleic acid sequence coding a second polypeptide comprising a light chain constant domain (CL) dimerisation domain which spontaneously heterodimerizes with said heavy chain constant domain (CH) dimerisation domain, and a second chain of endodomain of the type I cytokine receptor.

EFFECT: said chimeric transmembrane protein provides a constitutively active cytokine signal in said cell.

17 cl, 9 dwg, 1 tbl, 4 ex

Description

FIELD OF INVENTION

The present invention relates to a chimeric cytokine receptor (CCR) and a cell that expresses such a chimeric cytokine receptor, and, additionally, a chimeric antigen receptor on the cell surface.

LEVEL OF TECHNOLOGY

Chimeric antigen receptors (CARs)

A number of immunotherapeutic agents have been described for use in the treatment of cancer, including therapeutic monoclonal antibodies (mAbs), T-cell-attracting bispecific activators, and chimeric antigen receptors (CARs).

Chimeric antigen receptors are proteins that graft the specificity of a monoclonal antibody (mAb) to the effector function of a T cell. Their usual form is a type 1 transmembrane domain protein with an antigen-recognizing amino terminus, a spacer, and a transmembrane domain, all linked to a composite endodomain that transmits T cell survival and activation signals.

The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies that recognize a target antigen, linked via a spacer and a transmembrane domain to a signaling endodomain. Such molecules lead to T cell activation in response to the scFv's recognition of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for the treatment of various cancers.

CAR-based approaches to prostate cancer treatment

Prostate cancer is the second most common cancer in men worldwide and the sixth leading cause of cancer death. Globally, there are approximately 1,100,000 new cases and 300,000 deaths each year, accounting for 4% of all cancer deaths. It is estimated that 1 in every 6 men will be diagnosed with the disease in their lifetime.

Initial treatment for prostate cancer may include surgery, radiation, or hormone therapy, or any combination of each. Hormone therapy works by lowering levels of testosterone, the male hormone that fuels uncontrolled cell growth. Chemotherapy is generally reserved for advanced cancers.

When prostate cancer continues to grow despite testosterone suppression with hormonal therapy, treatment options are limited. Typically, the sipuleucel-T (Provenge®) cancer vaccine, a therapeutic cancer vaccine designed to induce an immune response directed by dendritic cells against the prostate-associated acid phosphatase (PAP) antigen, a radiopharmaceutical (such as radium-223 chloride), secondary hormonal therapy (such as abiraterone or enzalutamide), and/or chemotherapy (docetaxel and cabazitaxel) are added sequentially to hormonal therapy. Although each of these treatments may slow the growth of the cancer for several months and reduce the symptoms caused by the disease, the disease eventually becomes resistant to them.

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

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

This may be due to the fact that CAR T cells in vivo tend to overcome the microenvironment of malignant carcinoma. In particular, CAR T cells may be unable to enter and spread within the tumor bed of prostate cancer.

The viability and activity of CAR T cells can be enhanced by administration of cytokines or CAR T cells that produce cytokines constitutively. 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).

Therefore, there is a need for alternative CAR T cell approaches that promote T cell engraftment and expansion to counter the complex effects of the harmful tumor microenvironment.

Non-tumor target toxicity

It is relatively rare for the presence of a single antigen to be effective in describing cancer, which may lead to a lack of specificity.

Most cancers cannot be distinguished from normal tissues on the basis of a single antigen. Consequently, significant “off-target” toxicity occurs when normal tissues are damaged by therapy. For example, targeting CD20 for the treatment of B-cell lymphoma with rituximab depletes the entire pool of normal B cells, targeting CD52 for chronic lymphocytic leukemia depletes the entire lymphatic pool, targeting CD33 for acute myeloid leukemia damages the entire myeloid pool, and so on.

The predicted problem of “off-target” toxicity has been confirmed in clinical trials. For example, an approach targeting ERBB2 caused the death of a patient with colon cancer metastatic to the lungs and liver. ERBB2 is overexpressed in colon cancer in some patients, but it is also expressed in several healthy tissues, including the heart and healthy vasculature.

There is therefore a need for improved approaches to cancer therapy that reduce or eliminate such off-target toxicity.

DESCRIPTION OF FIGURES

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

Figure 2 : Schematic diagram showing the putative chimeric cytokine receptor

(a) The IL2 and IL7 cytokine receptors signal through the common gamma chain and the cytokine-specific alpha/beta chain.

(b) One embodiment of a chimeric cytokine receptor is to replace the ectodomain of the cytokine alpha/beta and gamma chain with different scFvs (or any other suitable binder) that recognize different PSA epitopes.

(c) An alternative approach is to replace the alpha/beta and gamma VH/VL ectodomains of the PSA-specific antibody, where VH and VL participate in binding, so that binding brings them together.

Figure 3 : Aggregation-based cytokine signaling enhancer.

Schematic diagram showing a chimeric cytokine receptor and CAR combining system. The cell contains two chimeric cytokine receptors that bind different epitopes to the same soluble ligand. In the absence of soluble ligand (e.g. PSA) but in the presence of a cell surface antigen (e.g. PSMA), signaling occurs through the CAR. In the presence of soluble ligand, aggregation of the two chimeric cytokine receptors occurs, resulting in amplification of the cytokine-based signal.

Figure 4 : Theoretical design map for the chimeric cytokine receptor/CAR combination system shown in Fig. 3.

Figure 5 : Schematic diagram illustrating an example of a structure for a chimeric transmembrane protein of the present invention. The chimeric transmembrane protein comprises a dimerization domain and a cytokine receptor endodomain. The embodiment shown has a "Fab" type structure since the dimerization domain contains the constant regions of the heavy and light chain of an antibody. Constant dimerization between these domains combines the common γ-chain of the IL2 receptor with either the β-chain of the IL-2 receptor or the α-chain of the IL-7 receptor, resulting in constitutive cytokine signaling.

Figure 6 : IL-2 signaling by a chimeric transmembrane protein.

Two chimeric transmembrane proteins sharing the general structure shown in Fig. 5 were tested for their ability to induce IL-2 signaling. One chimeric transmembrane protein contained the IL2 receptor endodomain and the other contained the IL-7 receptor endodomain. IL-2 signaling was tested using the murine CTLL2 cell line, whose growth is dependent on IL-2 signaling. As a positive control, CTLL2 cells were cultured with 100 U/ml murine IL2. Cells expressing the chimeric transmembrane protein containing the IL2 receptor endodomain (Fab_IL2endo) supported the survival and growth of CTLL2 cells, whereas cells expressing the chimeric transmembrane protein containing the IL-7 receptor (Fab_IL7endo) did not.

Figure 7 : Schematic diagram showing the PSA group of chimeric cytokine receptors

A panel of chimeric cytokine receptors (CCRs) targeting PSA was developed using scFvs derived from two antibodies that bind to different PSA epitopes: 5D5A5 and 5D3D11.

Upper left panel: CCR with the IL-2R endodomain, sharing A5 on the chain with the IL2R β-chain and D11 on the chain with the common γ-chain;

Upper right panel: CCR with the IL7R endodomain, sharing A5 on the IL7R α-chain chain and D11 on the γ-chain chain;

Lower left panel: CCR with the IL-2R endodomain, sharing D11 on the chain with the IL2R β-chain and A5 on the chain with the common γ-chain; and

Lower right panel: CCR with the IL-7R endodomain sharing D11 on the IL7R α-chain chain and A5 on the γ-chain chain.

A negative control was also created for each CCR in which the IL2Rγ chain was replaced with a rigid linker.

Figure 8 : IL2 signaling from PSA chimeric cytokine receptor-expressing cells in the presence of PSA - CTLL2 proliferation.

CTLL2 cells were transduced with constructs expressing some of the PSA receptors shown in Fig. 7. Cells were cultured in the absence and presence of IL2 (positive control) and in the absence and presence of 5 ng/ml or 5 μg/ml PSA. CTLL2 proliferation was assessed at 3 and 7 days.

An anti-PSA chimeric cytokine receptor with the IL2 endodomain supported CTLL2 cell proliferation in the absence of IL2 and in the presence of PSA, but not the IL7R endodomain-bearing receptor or any of the CCRs containing a rigid linker in place of the common γ chain.

Figure 9 : IL2 signaling from cells expressing the PSA chimeric cytokine receptor in the presence of PSA - CTLL2 STAT5 phosphorylation

CTLL2 cells were either left untransduced (WT) or transduced with a vector expressing the anti-PSA CCR (D11-CD8STK-IL2Rg_A5-Hinge-IL2Rb) or an equivalent construct having a rigid linker in place of the common γ chain (D11-CD8STK-RL_A5-Hinge-IL2Rb). Cells were incubated with 500 μM pervanadate or 500 ng/ml PSA for 1 or 4 h. Y694 STAT5 phosphorylation was then examined using flow cytometry.

ESSENCE OF THE ASPECTS OF THE INVENTION

The present inventors have developed “chimeric cytokine receptors” (CCRs) that graft the binding specificity of a non-cytokine binding molecule onto the endodomain of a cytokine receptor. Coexpression of such a CCR with a chimeric antigen receptor (CAR) helps the CAR T cell to enter and spread into the tumor microenvironment. The requirement that a ligand for the CCR as well as a ligand for the CAR adds another level of selectivity and helps prevent off-target toxicity.

For example, a cell has been engineered that co-expresses a CAR with a chimeric cytokine receptor that detects PSA and transmits an IL2/15 or IL7 signal to the CAR T cell. In this way, the CAR T cell is selectively stimulated to proliferate only in the prostate cancer microenvironment, and in the absence of PSA (i.e., after the patient is in remission), the cytokine stimulation is lost.

In a first aspect, the present invention relates to a chimeric cytokine receptor (CCR) comprising:

an exodomain that binds to a ligand selected from a tumor-secreted factor, a chemokine, and a cell surface antigen; and

cytokine receptor endodomain.

In a first embodiment of the first aspect of the invention, the chimeric cytokine receptor comprises two polypeptides:

(i) a first polypeptide that comprises:

(a) the first antigen-binding domain that binds the first epitope of the ligand

(b) the first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide that comprises:

(a) a second antigen-binding domain that binds a second epitope of the ligand

(b) the second chain of the cytokine receptor endodomain. Fig. 2b shows such a scheme.

Each of the first and second antigen-binding domains may be, for example, single-chain variable fragments (scFvs) or single-domain binders.

In a second embodiment of the first aspect of the invention, a chimeric cytokine receptor comprises two polypeptides:

(i) a first polypeptide that comprises:

(a) variable domain of the heavy chain (VH)

(b) the first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide that comprises:

(a) variable domain of the light chain (VL)

(b) the second chain of the cytokine receptor endodomain.

Fig. 2c shows such a scheme.

The first and second chains for the cytokine receptor endodomains may be different and may be selected from the endodomain of the α-, β-, and γ-chains of the type I cytokine receptor.

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

For example, the endodomain of a cytokine receptor may include:

(i) IL-2 receptor β-chain endodomain

(ii) the endodomain of the IL-7 receptor α-chain;

(iii) the endodomain of the IL-15 receptor α-chain; or

(iv) the common endodomain of the receptor γ-chain.

The endodomain of a cytokine receptor may comprise (i), (ii), or (iii); and (iv).

The ligand may be a tumor secreted factor, such as a tumor secreted factor selected from prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), and CA125.

The ligand may be a chemokine, such as a chemokine selected from a chemokine selected from: CXCL12, CCL2, CCL4, CCL5, and CCL22.

The ligand may be a cell surface molecule such as a transmembrane protein. The ligand may be, for example, CD22.

In a second aspect, the present invention relates to a cell that comprises a chimeric cytokine receptor according to the first aspect of the invention.

The cell may contain a first chimeric cytokine receptor and a second chimeric cytokine receptor that bind different epitopes on a single ligand.

The cell may comprise a first chimeric cytokine receptor that includes an endodomain of an α- or β-chain of a type I cytokine receptor, and a second chimeric cytokine receptor that includes an endodomain of a γ-chain of a type I cytokine receptor, such that when the first chimeric cytokine receptor and the second chimeric cytokine receptor bind a ligand, combined signaling occurs through the α-/β-chain and the γ-chain.

The cell may also contain a chimeric antigen receptor, such as a chimeric antigen receptor that binds a tumor-associated cell surface antigen.

The chimeric antigen receptor can bind a cell surface antigen associated with prostate cancer, such as prostate stem cell antigen (PSCA) or prostate-specific membrane antigen (PSMA).

If the CCR recognizes a cell surface antigen, the CCR and CAR can recognize cell surface antigens that are co-expressed on the same target (e.g., tumor) cell. For example, for B-cell malignancies, the CAR can recognize a cell surface antigen such as CD19 and the CCR can recognize a molecule that is co-expressed on the surface of the target cell such as CD22, thereby enhancing engraftment.

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

In a fourth aspect, the present invention relates to a nucleic acid construct that comprises a first nucleic acid sequence encoding a first CCR and a second nucleic acid sequence encoding a second CCR, the nucleic acid construct having the structure:

AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR;

spacer 1 is a nucleic acid sequence encoding the spacer of the first CCR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR;

endo 1 is a nucleic acid sequence encoding the endodomain of the first CCR;

coexpr is a nucleic acid sequence that enables coexpression of both CCRs;

AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR;

spacer 2 is a nucleic acid sequence encoding the spacer of the second CCR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR;

endo 2 is a nucleic acid sequence encoding the endodomain of the second CCR.

The nucleic acid construct may also encode a chimeric antigen receptor (CAR). In this embodiment, the nucleic acid construct may have the structure:

(i) CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2-coexpr2-CARAgB-CARspacer-CARTM-CARendo;

(ii) CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CARAgB-CARspacer-CARTM-CARendo-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2; or

(iii) CARAgB-CARspacer-CARTM-CARendo-coexpr1-CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2;

in which

CCRAgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR;

CCRspacer1 is a nucleic acid sequence encoding the spacer of the first CCR;

CCRTM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR;

CCRendo1 is a nucleic acid sequence encoding the endodomain of the first CCR;

CCRAgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR;

CCRspacer2 is a nucleic acid sequence encoding the spacer of the second CCR;

CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR;

CCRendo2 is a nucleic acid sequence encoding the endodomain of the second CCR;

Coexpr1 and coexpr2 are nucleic acid sequences that provide coexpression of two flanking sequences;

CARAgB is a nucleic acid sequence encoding the antigen-binding domain of CAR;

CARspacer is a nucleic acid sequence encoding the CAR spacer;

CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR; and

CARendo is a nucleic acid sequence encoding the CAR endodomain.

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

Alternative codons may be used in sequence regions encoding the same or similar amino acid sequences to avoid homologous recombination.

In a fifth aspect, the present invention relates to a vector comprising a nucleic acid construct according to the fourth aspect of the invention.

The vector may be, for example, a retroviral vector or a lentiviral vector, or a transposon.

In a sixth aspect, the present invention relates to a kit that comprises:

i) a vector comprising a nucleic acid sequence encoding a first CCR according to the first aspect of the invention; and

ii) a vector comprising a nucleic acid sequence encoding a second CCR according to the second aspect of the invention.

The kit may also contain a vector containing a nucleic acid sequence encoding a chimeric antigen receptor.

The set may contain:

i) a vector comprising a nucleic acid sequence encoding a CCR according to the first aspect of the invention; and

ii) a vector containing a nucleic acid sequence encoding a chimeric antigen receptor.

In a seventh aspect, the present invention relates to a method for producing a cell according to the second aspect of the invention, which comprises the step of introducing: a nucleic acid sequence according to the third aspect of the invention; a nucleic acid construct according to the fourth aspect of the invention; a vector according to the fifth aspect of the invention; or a set of vectors according to the sixth aspect of the invention, into the cell.

The cell may be from a sample obtained from the subject.

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

In a ninth aspect, a method for treating and/or preventing a disease is provided, which comprises the step of administering a pharmaceutical composition according to the eighth aspect of the invention to a subject.

The method may contain the following steps:

(i) isolating a sample containing cells from the subject;

(ii) transducing or transfecting cells with a nucleic acid sequence according to the third aspect of the invention; a nucleic acid construct according to the fourth aspect of the invention; a vector according to the fifth aspect of the invention; or a set of vectors according to the sixth aspect of the invention; and

(iii) introducing cells from (ii) into the subject.

The sample may be a T cell containing sample.

The disease could be cancer.

Also provided is a pharmaceutical composition according to the eighth aspect of the invention for use in the treatment and/or prevention of a disease.

Also provided is the use of a cell according to the second aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

Further aspects of the invention are summarized in the following numbered claims:

1. A chimeric transmembrane protein containing:

dimerization domain; and

cytokine receptor endodomain.

2. A chimeric transmembrane protein according to paragraph 1, wherein the dimerization domain comprises a dimerization region of the constant domain of the heavy chain ( _CH ) and the constant domain of the light chain ( _CL ).

3. A chimeric transmembrane protein according to any of the preceding paragraphs, which comprises two polypeptides:

(i) a first polypeptide that comprises:

(a) the first dimerization domain; and

(b) the first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide that comprises:

(a) a second dimerization domain that dimerizes with the first dimerization domain; and

(b) the second chain of the cytokine receptor endodomain.

4. A chimeric transmembrane protein according to paragraph 3, wherein the first and second dimerization sites either dimerize spontaneously or in the presence of a chemical inducer of dimerization (CID).

5. A chimeric transmembrane protein according to paragraph 2, 3 or 4, which comprises two polypeptides:

(i) a first polypeptide that comprises:

(a) heavy chain constant domain (CH)

(b) the first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide that comprises:

(a) light chain constant domain (CL)

(b) the second chain of the cytokine receptor endodomain.

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

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

8. A chimeric transmembrane protein according to any of the preceding paragraphs, wherein the cytokine receptor endodomain comprises:

(i) IL-2 receptor β-chain endodomain

(ii) the endodomain of the IL-7 receptor α-chain; or

(iii) the endodomain of the IL-15 receptor α-chain; and/or

(iv) the common endodomain of the receptor γ-chain.

9. A chimeric transmembrane protein according to claim 5, wherein the first polypeptide comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH); and the second polypeptide comprises a light chain variable domain (VL) and a light chain constant domain (CL).

10. A chimeric transmembrane protein according to paragraph 9, which comprises a Fab exodomain.

11. A cell that contains a chimeric transmembrane protein as defined in any of the preceding paragraphs.

12. A cell according to paragraph 11, which also includes a chimeric antigen receptor.

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

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

15. A nucleic acid construct that comprises a first nucleic acid sequence encoding a first polypeptide as defined in paragraph 3 and a second nucleic acid sequence encoding a second polypeptide as defined in paragraph 3, the nucleic acid construct having the structure:

Dim1-TM1-endo1-coexpr-Dim2-TM2-endo2

in which

Dim1 is a nucleic acid sequence encoding the first dimerization domain;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide;

endo 1 is a nucleic acid sequence encoding the endodomain of the first polypeptide;

coexpr is a nucleic acid sequence that enables coexpression of both CCRs;

Dim2 is a nucleic acid sequence encoding the second dimerization domain;

TM2 is a nucleic acid sequence encoding the transmembrane endodomain of the second polypeptide;

endo 2 is a nucleic acid sequence encoding the endodomain of a second polypeptide.

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

17. A nucleic acid construct according to paragraph 15 or 16, wherein the coexpr encodes a sequence containing a self-cleaving peptide.

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

19. A vector containing a nucleic acid construct according to any one of paragraphs 15-18.

20. A retroviral vector or lentiviral vector, or transposon, in accordance with paragraph 19.

21. A set that includes:

i) a vector comprising a nucleic acid sequence encoding a first polypeptide as defined in paragraph 3; and

ii) a vector comprising a nucleic acid sequence encoding a second polypeptide as defined in paragraph 3.

22. The kit according to paragraph 21, which also includes a vector containing a nucleic acid sequence encoding a chimeric antigen receptor.

23. A set that includes:

i) a vector comprising a nucleic acid sequence encoding a chimeric transmembrane protein according to any one of paragraphs 1 to 10; and

ii) a vector containing a nucleic acid sequence encoding a chimeric antigen receptor.

24. A method for producing a cell according to any one of paragraphs 11-13, which comprises the step of introducing: a nucleic acid sequence according to paragraph 14; a nucleic acid construct according to any one of paragraphs 15-18; a vector according to paragraph 19 or 20; or a set of vectors according to any one of paragraphs 21-23, into the cell.

25. The method according to paragraph 24, wherein the cell is from a sample obtained from the subject.

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

27. A method for treating and/or preventing a disease, which includes the step of administering a pharmaceutical composition, in accordance with paragraph 26, to a subject.

28. The method according to item 27, which includes the following steps:

(i) isolation of a cellular sample from a subject;

(ii) transducing or transfecting cells with a nucleic acid sequence according to paragraph 14; a nucleic acid construct according to any of paragraphs 15 to 18; a vector according to paragraph 19 or 20; or a set of vectors according to any of paragraphs 21 to 23; and

(iii) introducing cells from (ii) into the subject.

29. The method according to paragraph 28, wherein the sample is a sample containing T cells.

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

31. A pharmaceutical composition, in accordance with paragraph 26, for use in the treatment and/or prevention of a disease.

32. Use of a cell, in accordance with any of paragraphs 11-13, in the manufacture of a medicinal product for the treatment and/or prevention of a disease.

DETAILED DESCRIPTION

CHIMERIC CYTOKINE RECEPTOR (CCR)

A chimeric cytokine receptor (CCR) is a molecule that includes a cytokine receptor endodomain and a heterologous ligand-binding exodomain. The heterologous exodomain binds a ligand other than the cytokine for which the cytokine receptor from which the endodomain was derived is selective. It is thus possible to modify the ligand specificity of a cytokine receptor by grafting a heterologous binding specificity.

The chimeric cytokine receptor contains:

(i) ligand-binding exodomain;

(ii) optional spacer;

(iii) a transmembrane domain; and

(iv) cytokine receptor endodomain.

CYTOKINE RECEPTORS AND SIGNALING

Many cellular functions are regulated by members of the cytokine receptor superfamily. Signaling through these receptors depends on their association with Janus kinases (JAKs), which couple ligand binding to tyrosine phosphorylation of signaling proteins involved in the receptor complex. Among these are the signal transducers and activators of transcription (STATs), a family of transcription factors that promote a variety of cytokine responses.

When the chimeric cytokine receptor of the invention binds its ligand, one or more of the following intracellular signaling pathways may be initiated:

(i) JAK-STAT pathway

(ii) MAP kinase pathway; and also

(iii) phosphoinositide 3-kinase (PI3K) pathway.

The JAK-STAT system consists of three main components: (1) the Janus kinase (JAK) receptor and (3) the signal transducer and activator of transcription (STAT).

JAKs that possess tyrosine kinase activity bind to cell surface cytokine receptors. Ligand binding to the receptor triggers activation of the JAKs. With enhanced kinase activity, they phosphorylate tyrosine residues on the receptor and create sites for interaction with proteins that contain phosphotyrosine-binding SH2 domains. STATs that possess SH2 domains capable of binding these phosphotyrosine residues are recruited to the receptors and are themselves tyrosine-phosphorylated JAKs. These phosphotyrosines then act as binding sites for the SH2 domains of other STATs, mediating their dimerization. Different STATs form hetero- or homodimers. Activated STAT dimers accumulate in the cell nucleus and activate transcription of their target genes.

CYTOKINE RECEPTOR ENDODOMAINE

The chimeric cytokine receptor of the present invention comprises an endodomain that elicits "cytokine-type" cellular signaling (either alone or in the presence of another chimeric cytokine receptor) when the exodomain binds its ligand.

The endodomain may represent the endodomain of a cytokine receptor.

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

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

Cytokine receptors type I:

(i) interleukin receptors, such as receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL13, IL-15, IL-21, IL-23 and IL-27;

(ii) colony-stimulating factor receptors such as receptors for erythropoietin, GM-CSF and G-CSF; and

(iii) hormone receptor/neuropeptide receptor, such as hormone receptor and prolactin receptor.

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

The common IL-2 receptor gamma chain (also known as CD132) is shared between the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, and IL-15 receptor.

IL-2

IL-2 binds to the IL-2 receptor, which has three forms generated by different combinations of three different proteins, often called "chains": α, β, and γ; these subunits are also parts of receptors for other cytokines. The β and γ chains of IL-2R are members of the type I cytokine receptor family.

The three receptor chains are expressed separately and differentially on different cell types and can assemble in different combinations and orders to produce low-, intermediate-, and high-affinity IL-2 receptors.

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

The three chains of the IL-2 receptor span the cell membrane and extend into the cell, thereby delivering biochemical signals to the cell's interior. The alpha chain is not involved in signaling, but the beta chain is complexed with the tyrosine phosphatase JAK1. Similarly, the gamma chain complexes with another tyrosine kinase called JAK3. These enzymes are activated by the binding of IL-2 to the outer domains of the IL-2R.

IL-2 signaling promotes the differentiation of T cells into effector and memory T cells when naïve T cells are also stimulated by antigen. Through their role in the development of immunological T cell memory, which depends on the expansion and function of antigen-dependent T cell clones, they also play a key role in long-term cell-mediated immunity.

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

The amino acid sequences for the IL-2 β-chain and common γ-chain endodomains are shown as SEQ ID Nos. 1 and 2.

SEQ ID No. 1: endodomain derived from human common gamma chain:

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

SEQ ID No. 2: endodomain derived from human IL-2Rβ:

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGP PTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

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

A "variant" sequence having at least 80, 85, 90, 95, 98, or 99% sequence identity to a wild-type sequence (e.g., SEQ ID No. 1 or 2), provided that the variant sequence retains the function of the wild-type sequence, i.e., the ability to form a complex with JAK-1 or JAK-3 and activate, for example, the JAK-STAT signaling pathway.

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

IL-7

The interleukin-7 receptor consists of two chains: the interleukin-7 receptor α chain (CD127) and the common receptor γ chain (CD132). The common receptor γ chain is shared by various cytokines, including interleukin-2, -4, -9, and -15. The interleukin-7 receptor is expressed on a variety of cell types, including naïve and memory T cells.

The interleukin-7 receptor plays a critical role in lymphocyte development, particularly in V(D)J recombination. IL-7R also controls the accessibility of the genomic region containing the T-cell receptor gamma gene via STAT5 and histone acetylation. Mouse knockout studies suggest that blocking apoptosis is an important function of this protein in T-cell differentiation and activation.

The chimeric cytokine receptor of the present invention may comprise the IL-7 receptor α-chain and/or the IL-7 receptor γ-chain (i.e., common), or a corresponding variant.

The amino acid sequence for the IL-7 α-chain endodomain is shown as SEQ ID No. 3.

SEQ ID No. 3 - endodomain derived from human IL-7Rα:

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

IL-15

Interleukin 15 (IL-15) is a cytokine with structural similarity to IL-2. IL-15, like IL-2, binds to and signals through a complex consisting of the IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following viral infection. IL-15 induces cellular proliferation of natural killer cells.

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

SPACER

The chimeric cytokine receptor of the present invention may include a spacer for connecting the antigen-binding domain to the transmembrane domain and spatially separating the antigen-binding domain from the endodomain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to ensure antigen binding.

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

The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a CD8 stem. The linker may also comprise an alternative sequence that has similar length and/or interdomain spacing properties as the IgG1 Fc region, the IgG1 hinge, or the CD8 stem.

The human IgG1 spacer can be modified to remove Fc-binding motifs.

Examples of amino acid sequences for these spacers are given below:

SEQ ID No. 4 (Human IgG1 hinge-CH2CH3)

AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD

SEQ ID No. 5 (human CD8 stem):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

SEQ ID No. 6 (human IgG1 hinge):

AEPKSPDKTHTCPPCPKDPK

TRANSMEMBRANE DOMAIN

The transmembrane domain is a CCR sequence that spans the membrane. It may include a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which provides sufficient stability to the receptor.

Alternatively, the transmembrane domain may be derived from a cytokine receptor, such as the same cytokine from which the endodomain is derived.

The transmembrane domain may be, for example, derived from IL-2R, IL-7R, or IL-15R.

SEQ ID No. 7 - transmembrane derived from human common gamma chain:

VVISVGSMGLIISLLCVYFWL

SEQ ID No. 8 - transmembrane derived from human IL-2Rβ:

IPWLGHLLVGLSGAFGFIILVYLLI

SEQ ID No. 9 - transmembrane derived from human IL-7Rα:

PILLTISILSFFSVALLVILACVLW

SEQ ID No. 10 - transmembrane derived from human IL-15Rα:

AISTSTVLLCGLSAVSLLACYL

LIGAND-BINDING EXODOMAIN

The ligand-binding domain contains an antigen-binding domain. The antigen-binding domain binds the target ligand for CCR, i.e., a tumor-secreted factor or chemokine, or a cell-surface antigen.

A variety of antigen-binding domains are known in the art, including those based on the antigen-binding site of an antibody, 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 binding domain from a natural receptor for a target antigen; a peptide with sufficient affinity for a target ligand; a single-domain binder such as camelid; an artificial single-binder such as DARPin; or a single-chain derived from a T-cell receptor.

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

When the ligand is a tumor-secreted factor, the antigen-binding domain may comprise an immunoglobulin-based antigen-binding site such as an scFv or a single-domain binder.

When the ligand is a chemokine, the antigen-binding domain may include the chemokine-binding portion of a natural receptor for the chemokine.

LIGAND

The CCR of the present invention binds a ligand.

The ligand may be a soluble ligand such as a tumor-secreted factor or chemokine.

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

The term "soluble ligand" is used to refer to a ligand or antigen that is not part of or attached to a cell, but is freely mobile in the extracellular space, such as in the physiological fluid of the tissue of interest. A soluble ligand may exist in a cell-free state in the serum, plasma, or other physiological fluid of an individual.

A soluble ligand may be associated with the presence or pathology of a particular disease, such as cancer.

A soluble ligand may be part of a cancer secretome, i.e. a group of factors secreted by a tumor, whether from cancer stem cells, non-stem cells, or the surrounding stroma. A soluble ligand may be secreted or distributed by tumor cells (see next section).

The soluble ligand may be characteristic of a disease or diseased tissue. It may be found exclusively or at a higher level in a subject having a disease compared to a healthy subject; or in diseased tissue versus healthy tissue. The soluble ligand may be expressed at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold higher than the level in a subject having a disease versus a healthy subject; or in diseased tissue versus healthy tissue.

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

The cell on which the cell surface ligand is detected may be a target cell, such as a cancer cell.

A cell surface ligand may be associated with the presence or pathology of a particular disease, such as cancer. Alternatively, a cell surface ligand may be characteristic of a cell type of the target cell (e.g., a B cell) that is not necessarily associated with the disease state.

If the cell surface ligand is characteristic of a disease or diseased tissue, it may be detected exclusively or at a higher level in the corresponding cells of a subject having a disease versus a healthy subject; 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 higher than the level in the cells of a subject having a disease versus a healthy subject; or in diseased tissue versus healthy tissue.

TUMOR-SECRETED FACTOR

The ligand recognized by CCR may be a soluble ligand secreted or diffused from the tumor.

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).

The tumor secreted factor may be a soluble ligand that is not a cytokine. Thus, the CCR of the present invention grafts binding specificity for a ligand other than a cytokine to the endodomain of a cytokine receptor.

PROSTATE SPECIFIC ANTIGEN (PSA)

The soluble ligand may be the antigen prostate-specific antigen (PSA).

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

PSA is present in low levels in the serum of men with healthy prostates, but is elevated in individuals with prostate cancer and other prostate disorders.

PSA is a glycoprotein formed by 237 residues and activated by KLK2. Its physiological role is to liquefy components of sperm clots, leading to the release of spermatozoa. In cancer, PSA may be involved in the processes of neoplastic growth and metastasis.

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

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.

The CCR of the present invention may, for example, comprise 6 CDRs or VH and/or VL domain(s) from a PSA-binding mAb such as 8G8F5, 5D5A5 or 5D3D11.

In cases where the CCR contains two antigen binding specificities binding different epitopes on PSA, one could use, for example, 5D3D11, and one could be based on, for example, 5D5A5.

The amino acid sequences for 5D3D11 and 5D5A5 VH and VL are shown below. Complementarity determining regions (CDRs) are shown in bold.

5D3D11 VH (SEQ ID No. 11)

QVQLQQSGPELVKPGASVKISCKVSGYAIS SSWMN WVKQRPGQGLEWIG RIYPGDGDTKYNGKFKD KATLTVDKSSSTAYMQLSSLTSVDSAVYFCAR DGYRYYFDY WGQGTSVTVSS

5D3D11 VL (SEQ ID No. 12)

DIVMTQTAPSVFVTPGESVSISC RSSKSLLHSNGNTYLY WFLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTDFTLRISRVEAEDVGVYYC MQHLEYPVT FGAGTKVEIK

5D5A5 VH (SEQ ID No. 13)

QVQLQQSGAELAKPGASVKMSCKTSGYSFS SYWMH WVKQRPGQGLEWIG YINPSTGYTENNQKFKD KVTLTADKSSNTAYMQLNSLTSEDSAVYYCAR SGRLYFDV WGAGTTVTVSS

5D5A5 VL (SEQ ID No. 14)

DIVLTQSPPSLAVSLGQRATISC RASESIDLYGFTFMH WYQQKPGQPPKILIY RASNLES GIPARFSGSGSRTDFTLTINPVEADDVATYYC QQTHEDPYT FGGGTKLEIK

ScFv based on 5D5A5 (SEQ ID No. 15)

QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIY RASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKLEIK

ScFv based on 5D3D11 (SEQ ID No. 16)

QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY RMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGTKVEIK

When a cell contains two CCRs, the antigen-binding domain of the first CCR may contain 6 CDRs from 5D5A5, and the antigen-binding domain of the second CCR may contain 6 CDRs from 5D3D11.

The antigen binding domain of the first CCR may comprise the VH and/or VL domain(s) of 5D5A5, or a corresponding variant; and the antigen binding domain of the second CCR may comprise the VH and/or VL domain(s) of 5D3D11, or a corresponding variant. The VH and VL domain variants may have at least 80, 90, 95 or 99% identity to the above sequences, provided that they retain PSA binding activity.

A cell expressing CCR that binds PSA may be useful in treating prostate cancer.

CARCINOEMBRYOGEN (CEA)

The soluble ligand may be CEA.

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

CEAs are cell surface glycosyl phosphatidylinositol (GPI)-anchored glycoproteins whose specialized sialofucosylated glycoforms serve as functional colon carcinoma L-selectin and E-selectin ligands that may be critical for the metastatic dissemination of colon carcinoma cells. They are immunologically characterized as members of the CD66 cluster of differentiation.

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 that encode carcinoembryonic antigen-associated cell adhesion proteins: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21.

Various antibodies that target CEA are described in patent document WO 2011/034660.

A cell expressing CCR against CEA could be useful in treating, for example, colorectal cancer.

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)

The soluble ligand may be VEGF.

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

When VEGF is overexpressed, it can contribute to disease. Solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that can express VEGF are able to grow and metastasize.

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

In mammals, the VEGF family contains five members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D.

Various antibodies to VEGF are known, such as bevacizumab (Avastin) and ranibizumab (Lucentis).

CANCER ANTIGEN 125 (CA-125)

CA-125 is associated with ovarian cancer and is the most commonly used biomarker for detecting ovarian cancer. While CA-125 is best known as a marker for ovarian cancer, it can also be elevated in other cancers, including endometrial cancer, fallopian tube cancer, lung cancer, breast cancer, and gastrointestinal cancer.

The sequence of human CA-125 (also known as mucin-16) is available from NCBI, Accession No. 078966.

A number of CA125-binding monoclonal antibodies are known, including OC125 and M11 (Nustad et al 1996, Tumour Biol. 17:196-329). This study examined the specificity of 26 monoclonal antibodies against the CA 125 antigen. It was found that the CA 125 antigen contains only two major antigenic domains, which classify the antibodies as OC125-like (group A) or M11-like (group B).

The chimeric cytokine receptor of the present invention may comprise an antigen-binding domain from such an antibody. A cell comprising such a CCR may be useful in the treatment of, for example, ovarian cancer.

The tumor-secreted factor (or, if in membrane-bound form, transmembrane protein) may be selected from the following open list:

ALK gene rearrangements and overexpression resulting in mutated forms of ALK proteins

Alpha-fetoprotein (AFP)

Beta-2-microglobulin (B2M)

Beta-human chorionic gonadotropin (beta-hCG)

BRAF V600 mutations resulting in a mutated B-REF protein

C-kit/CD117

CA15-3/CA27,29

CA19-9

Calcitonin

CD20

Chromogranin A (CgA)

Cytokeratin fragment 21-1

EGFR gene mutation analysis

Estrogen receptor (ER)/progesterone receptor (PR)

Fibrin/fibrinogen

HE4

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)

CHEMOKINE

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 mediate cancer cell targeting and immune cell homing. The effect on T cells appears to be inhibitory to cytotoxic T cell homing, while regulatory T cells appear to be recruited.

Chemokines are approximately 8-10 kilodaltons in mass and have four cysteine residues in conserved regions that are key to forming their three-dimensional shape.

Some chemokines are considered proinflammatory and may be induced during an immune response to recruit immune cells to the site of infection, while others are considered homeostatic and are involved in the control of cell migration during normal tissue maintenance or development.

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

The primary role of chemokines is to act as chemoattractants, directing cell migration. Cells that are attracted to chemokines follow the signal of increasing chemokine concentrations toward the source of the chemokine. Some chemokines monitor immune cells during immune surveillance processes, such as directing lymphocytes to lymph nodes so that they can screen for pathogens by interacting with antigen-presenting cells residing in these tissues. Other chemokines are inflammatory and are released from a wide range of cells in response to bacterial infection, viruses, and other agents. Their secretion is often stimulated by proinflammatory cytokines such as interleukin 1. Inflammatory chemokines function primarily as leukocyte chemoattractants, recruiting monocytes, neutrophils, and other effector cells from the blood to sites of infection or tissue injury. Some inflammatory chemokines activate cells to initiate an immune response or promote wound healing. They are secreted by many different cell types and serve to direct cells of both the innate immune system and the adaptive immune system.

CC chemokines

CC chemokine (or β-chemokine) proteins have two flanking cysteines (amino acids) near the amino terminus. At least 27 different members of this subgroup, called CC chemokine ligands (CCL) -1 through -28, have been reported in mammals; CCL10 is exactly the same as CCL9. Chemokines in this subfamily typically contain four cysteines (C4-CC chemokines), but a small number of CC chemokines have six cysteines (C6-CC chemokines). C6-CC chemokines include CCL1, CCL15, CCL21, CCL23, and CCL28. CC chemokines induce the migration of monocytes and other cell types such as NK cells and dendritic cells.

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

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

CXC chemokines

The two N-terminal cysteines of CXC chemokines (or α-chemokines) are separated by a single amino acid, represented in this notation as "X". Seventeen different CXC chemokines have been described in mammals, which fall into two categories: those with 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 without an ELR motif (ELR-negative). ELR-positive CXC chemokines specifically induce neutrophil migration and interact with the chemokine receptors CXCR1 and CXCR2.

C-chemokines

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

CX3C chemokine

CX3C chemokines have three amino acids between two cysteines. The only CX3C chemokine discovered to date is called fractalkine (or CX3CL1). It is secreted and binds to the surface of the cell that expresses it, thereby serving as both a chemoattractant and an adhesion molecule.

Chemokine receptors are G protein-coupled receptors containing 7 transmembrane domains that are found on the surface of leukocytes. Approximately 19 different chemokine receptors have been characterized to date, which are divided into four families based on the type of chemokine they bind; CXCR, which bind CXC chemokines, CCR, which bind CC chemokines, CX3CR1, which bind 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-terminal end, seven helical transmembrane domains with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus containing serine and threonine residues important for receptor regulation. The first two extracellular loops of chemokine receptors have a conserved cysteine residue that allows the formation of a disulfide bridge between these loops. G proteins bind to the C-terminal end of the chemokine receptor to mediate intracellular signaling upon receptor activation, while the N-terminal domain of the chemokine receptor determines the specificity of ligand binding.

CXCL12

CXCL12 is highly chemotactic for lymphocytes. CXCL12 plays an important 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 very important factor in carcinogenesis and neovascularization associated with tumor progression. CXCL12 also plays a role in tumor metastasis, where cancer cells that express the CXCR4 receptor are attracted to metastatic target tissues that secrete CXCL12 ligand.

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

CXCR4-linked IL2 expression will support the engraftment of a therapeutic T cell for cancer treatment. In multiple myeloma, a cell expressing such a CCR can recruit cells and change the bone marrow environment. Such cells are also used in the treatment of solid cancers by modifying the solid tumor microenvironment.

The amino acid sequence for CXCR4 is shown below as SEQ ID No. 17.

SEQ ID No. 17

1 msiplpllqi ytsdnyteem gsgdydsmke pcfreenanf nkiflptiys iifltgivgn

61 glvilvmgyq kklrsmtdky rlhlsvadll fvitlpfwav davanwyfgn flckavhviy

121 tvnlyssvli lafisldryl aivhatnsqr prkllaekvv yvgvwipall ltipdfifan

181 vseaddryic drfypndlwv vvfqfqhimv glilpgivil scyciiiskl shskghqkrk

241 alkttvilil affacwlpyy igisidsfil leiikqgcef entvhkwisi tealaffhcc

301 lnpilyaflg akfktsaqha ltsvsrgssl kilskgkrgg hssvsteses ssfhss

CXCR7 also binds CXCL12.

CCL2

Chemokine (C-C motif) ligand 2 (CCL2) is also referred to as monocyte chemotactic protein 1 (MCP1) and small inducible cytokine A2. CCL2 recruits monocytes, memory T cells, and dendritic cells to sites of inflammation caused by either tissue injury or infection.

CCR2 and CCR4 are two cell surface receptors that bind CCL2.

CCR2 has the amino acid sequence shown as SEQ ID No. 18

SEQ ID No. 18

1 mlstsrsrfi rntnesgeev ttffdydyga pchkfdvkqi gaqllpplys lvfifgfvgn

61 mlvvlilinc kklkcltdiy llnlaisdll flitlplwah saanewvfgn amcklftgly

121 higyfggiff iilltidryl aivhavfalk artvtfgvvt svitwlvavf asvpgiiftk

181 cqkedsvyvc gpyfprgwnn fhtimrnilg lvlpllimvi cysgilktll rcrnekkrhr

241 avrviftimi vyflfwtpyn ivillntfqe ffglsncest sqldqatqvt etlgmthcci

301 npiiyafvge kfrslfhial gcriaplqkp vcggpgvrpg knvkvttqgl ldgrgkgksi

361 grapeaslqd kega

CCR4 has the amino acid sequence shown as SEQ ID No. 19.

SEQ ID No. 19

1 mnptdiadtt ldesiysnyy lyesipkpct kegikafgel flpplyslvf vfgllgnsvv

61 vlvlfkykrl rsmtdvylln laisdllfvf slpfwgyyaa dqwvfglglc kmiswmylvg

121 fysgiffvml msidrylaiv havfslrart ltygvitsla twsvavfasl pgflfstcyt

181 ernhtycktk yslnsttwkv lssleinilg lviplgimlf cysmiirtlq hcknekknka

241 vkmifavvvl flgfwtpyni vlfletlvel evlqdctfer yldyaiqate tlafvhccln

301 piiyfflgek frkyilqlfk tcrglfvlcq ycgllqiysa dtpsssytqs tmdhdlhdal

The CCR of the present invention may comprise a CCL2 binding site in CCR2 or CCR4 in the ligand binding domain.

CELL SURFACE ANTIGEN

The ligand may be a cell surface antigen such as a transmembrane protein.

The cell surface antigen may be CD22.

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

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

CD22 is overexpressed in non-Hodgkin's and other lymphomas. Various monoclonal antibodies targeting CD22 are known, including epratuzumab, inotuzumab ozogamicin, m971, and m972.

CHIMERIC ANTIGEN RECEPTORS (CAR)

The cell of the present invention may also comprise one or more chimeric antigen receptors. The CAR(s) may be specific for a tumor-associated antigen.

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

Early CAR designs had endodomains derived from the intracellular portions of either the FcεR1 γ chain or CD3ζ. Consequently, these first-generation receptors transduced immune signal 1, which was sufficient to trigger T cell killing of cognate target cells, but failed to fully activate the T cell for proliferation and survival. To overcome this limitation, composite endodomains were engineered: fusing the intracellular portion of the T cell costimulatory molecule with the CD3ζ molecule results in second-generation receptors that can simultaneously transduce an activating and costimulatory signal upon antigen recognition. The most common costimulatory domain is CD28. This provides the most potent costimulatory signal, namely immune signal 2, which induces T cell proliferation. Some receptors that include TNF receptor family endodomains have also been described, such as the closely related OX40 and 41BB, which transduce survival signals. Even more potent third-generation CARs have now been described that have endodomains capable of transducing activation, proliferation, and survival signals.

Nucleic acids encoding CARs can be transferred into T cells using, for example, retroviral vectors. In this way, large numbers of antigen-specific T cells can be generated for adoptive cell transfer. When a CAR binds a target antigen, this results in the transmission of an activating signal to the T cell on which it is expressed. In this way, the CAR directs T cell specificity and cytotoxicity to cells expressing the target antigen.

A cell of the present invention may comprise one or more CAR(s).

CAR(s) may include an antigen-binding domain, a spacer domain, a transmembrane domain, and an endodomain. The endodomain may include or be associated with a domain that transmits T-cell activation signals.

CAR ANTIGEN BINDING DOMAIN

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

A variety of antigen-binding domains are known in the art, including those based on the antigen-binding site of an antibody, 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 a target antigen; a peptide with sufficient affinity for the target; a single-domain binder such as camelid; an artificial single-binder such as DARPin; or a single-chain derived from a T-cell receptor.

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

CELL SURFACE ANTIGEN

A CAR can recognize a cell surface antigen, which is an object such as a transmembrane protein that is expressed on the surface of a target cell, such as a tumor cell.

CAR can specifically bind tumor-associated cell surface antigen.

Various tumor associated antigens (TAAs) are known, some of which are shown in Table 1. The antigen binding domain used in the present invention may be a domain that is capable of binding TAAs as defined herein.

Table 1

Type of cancer TAA Diffuse large B-cell lymphoma CD19, CD20, CD22 Breast cancer ErbB2, MUC1 OML CD13, CD33 Neuroblastoma GD2, NCAM, ALK, GD2 HLL CD19, CD52, CD160 Colorectal cancer Folate binding protein, CA-125 Chronic lymphocytic leukemia CD5, CD19 Glioma EGFR, vimentin Multiple myeloma BCMA, CD138 Renal cell carcinoma Carbonic anhydrase IX, G250 Prostate cancer PSMA Colon cancer A33

When a CAR recognizes a B-cell lymphoma or leukemia antigen (such as CD19, CD20, CD52, CD160, or CD5), a CCR can recognize another B-cell antigen, such as CD22.

PROSTATE CANCER ASSOCIATED ANTIGENS

CAR can specifically bind a cell surface antigen associated with cancer, such as prostate stem cell antigen (PSCA) or prostate-specific membrane antigen (PSMA).

PSCA is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein. It is upregulated in most prostate cancers and is also found in bladder and pancreatic cancers.

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).

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

PSMA is a zinc metalloenzyme that is found in membranes. PSMA is highly expressed in the human prostate gland, which is one hundred times higher than the expression in most other tissues. It is upregulated in cancer, and has been reported as the second most upregulated gene in prostate cancer, with an increase of 8-12 times compared to noncancerous prostate. In addition to being expressed in human prostate gland and prostate cancer, PSMA is also found to be highly expressed in tumor neovascularization, but not normal vascularization, of all types of solid tumors, such as kidney, breast, colon, etc.

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

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

For example, the CCR may comprise a J591-based scFv having the sequence shown as SEQ ID No. 20.

SEQ ID No. 20 (J591 scFv)

EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVP DRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR

CAR-TRANSMEMBRANE DOMAIN

The transmembrane domain is the membrane-spanning sequence of the CAR. It may contain a hydrophobic alpha helix. The CAR transmembrane domain may be derived from CD28, which provides sufficient stability to the receptor.

CAR SIGNALING PEPTIDE

The CAR and CCR described herein may contain a signal peptide so that when it/they are expressed in a cell, such as a T cell, the resulting protein is directed to the endoplasmic reticulum and then to the cell surface where it is expressed.

The core of the signal peptide may contain a long stretch of hydrophobic amino acids that tends to form a single alpha helix. The signal peptide may begin with a short, positively charged stretch of amino acids that helps ensure the correct topology of the polypeptide during translocation. The signal peptide usually ends with a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave during or after translocation to generate free signal peptide and mature protein. Free signal peptides are then cleaved by specific proteases.

The signal peptide may be located at the N-terminus of the molecule.

The signal peptide may comprise the sequence shown as SEQ ID No. 21, 22 or 23, or a corresponding variant having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions), as long as the signal peptide still functions to induce cell surface expression of the CAR.

SEQ ID No. 21:MGTSLLCWMALCLLGADHADG

The signal peptide of SEQ ID No. 21 is compact and highly efficient and is derived from the beta chain of the TCR. It is predicted that approximately 95% of the cleavage will occur after the terminal glycine, allowing for efficient removal by signal peptidase.

SEQ ID No. 22: MSLPVTALLPLALLLHAARP

The signal peptide SEQ ID No. 22 is derived from IgG1.

SEQ ID No. 23: MAVPTQVLGLLLLWLTDARC

The signal peptide SEQ ID No. 23 is derived from CD8a.

CAR-ENDODOMAIN

The endodomain is the portion of a typical CAR located on the inner side of the cell membrane.

The endodomain is the signaling portion of a typical CAR. Upon recognition of an antigen by the antigen-binding domain, individual CAR molecules of the cluster, native CD45 and CD148, are excluded from the synapse and the signal is transmitted into the cell.

The CAR endodomain may be or include an intracellular signaling domain. In an alternative embodiment of the invention, the endodomain of the proposed CAR may be capable of interacting with an intracellular signaling molecule that is present in the cytoplasm, resulting in signaling.

The intracellular signaling domain or a separate intracellular signaling molecule may be or contain a T cell signaling domain.

The most commonly used domain signaling component is the endodomain component CD3-zeta, which contains 3 ITAMs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal, and additional costimulatory signaling may be required. For example, chimeric CD28 and OX40 may be used with CD3-zeta to transmit a proliferation/survival signal, or all three may be used together.

A CAR may comprise the CD3-zeta endodomain alone, the CD3-zeta endodomain plus either CD28 or OX40, or the CD28 and OX40 endodomain and the CD3-zeta endodomain.

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

The endodomain may comprise a sequence shown as SEQ ID No. 24-32 or a corresponding variant having at least 80% sequence identity.

SEQ ID No. 24 - endodomain CD3-Z RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 25 - endodomains CD28 and CD3-zeta

SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 26 - endodomains CD28, OX40 and CD3-zeta

SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 27 - ICOS endodomain

CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

SEQ ID No. 28 - CD27 endodomain

QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

SEQ ID No. 29 - BTLA endodomain

RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS

SEQ ID No. 30 - endodomain CD30

HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK

SEQ ID No. 31 - GITR endodomain

QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

SEQ ID No. 32 - HVEM endodomain

CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH

The variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 24-32, provided that the sequence provides an effective intracellular signaling domain.

NUCLEIC ACID

The present invention also relates to a nucleic acid encoding a CCR of the invention.

Nucleic acid can have the structure:

AgB-spacer-TM-endo

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of CCR;

spacer 1 is a nucleic acid sequence encoding the CCR spacer;

TM1 is a nucleic acid sequence encoding the transmembrane domain of CCR;

endo 1 is a nucleic acid sequence encoding the CCR endodomain.

NUCLEIC ACID STRUCTURE

The present invention also relates to a nucleic acid construct that comprises a first nucleic acid sequence encoding a first CCR as defined in connection with the first aspect of the invention; and a second nucleic acid sequence encoding a second CCR as defined in connection with the first aspect of the invention.

The nucleic acid construct may have the following structure:

AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR;

spacer 1 is a nucleic acid sequence encoding the spacer of the first CCR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR;

endo 1 is a nucleic acid sequence encoding the endodomain of the first CCR;

coexpr is a nucleic acid sequence that enables coexpression of both CCRs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR;

spacer 2 is a nucleic acid sequence encoding the spacer of the second CCR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR;

endo 2 is a nucleic acid sequence encoding the endodomain of the second CCR.

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

The first and second CCRs can bind different epitopes to the same antigen.

The first and second CCRs may have complementary endodomains, such as one derived from the α- or β-chain of a cytokine receptor and one derived from the γ-chain of the same cytokine receptor.

The present invention also relates to a nucleic acid construct encoding a CCR of the invention and a CAR. Such a construct may have the structure:

CCRAgB-CCRspacer-CCRTM-CCRendo-coexpr-CARAgB-CARspacer-CARTM-CARendo

or

CARAgB-CARspacer-CARTM-CARendo-coexpr-CCRAgB-CCRspacer-CCRTM-CCRendo

in which

CCRAgB is a nucleic acid sequence encoding the antigen-binding domain of CCR;

The CCR spacer is a nucleic acid sequence encoding the CCR spacer;

CCRTM is a nucleic acid sequence encoding the transmembrane domain of CCR;

CCRendo is a nucleic acid sequence encoding the CCR endodomain;

coexpr is a nucleic acid sequence that enables coexpression of both CCR and CAR

CARAgB is a nucleic acid sequence encoding the antigen-binding domain of CAR;

CARspacer is a nucleic acid sequence encoding the CAR spacer;

CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR; and

CARendo is a nucleic acid sequence encoding the CAR endodomain.

The present invention also relates to a nucleic acid construct encoding a first and a second CCR of the invention and a CAR. The first and second CCR may bind separate epitopes to the same antigen. Such a construct may have the structure:

(i) CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2-coexpr2-CARAgB-CARspacer-CARTM-CARendo;

(ii) CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CARAgB-CARspacer-CARTM-CARendo-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2; or

(iii) CARAgB-CARspacer-CARTM-CARendo-coexpr1-CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2;

in which

CCRAgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CCR;

CCRspacer1 is a nucleic acid sequence encoding the spacer of the first CCR;

CCRTM1 is a nucleic acid sequence encoding the transmembrane domain of the first CCR;

CCRendo1 is a nucleic acid sequence encoding the endodomain of the first CCR;

CCRAgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CCR;

CCRspacer2 is a nucleic acid sequence encoding the spacer of the second CCR;

CCRTM2 is a nucleic acid sequence encoding the transmembrane domain of the second CCR;

CCRendo2 is a nucleic acid sequence encoding the endodomain of the second CCR;

Coexpr1 and coexpr2 are nucleic acid sequences that provide coexpression of two flanking sequences;

CARAgB is a nucleic acid sequence encoding the antigen-binding domain of CAR;

CARspacer is a nucleic acid sequence encoding the CAR spacer;

CARTM is a nucleic acid sequence encoding the transmembrane domain of CAR; and

CARendo is a nucleic acid sequence encoding the CAR endodomain.

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

It will be appreciated by one skilled in the art that multiple different polynucleotides and nucleic acids may encode the same polypeptide as a result of degeneracy of the genetic code. It will also be appreciated that skilled artisans may, using accepted techniques, 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 polypeptides are to be expressed.

Nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that include synthetic or modified nucleotides. A number of different types of modification of oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. It should be understood that for the purposes of use as described herein, the polynucleotides may be modified by any method available in the art. Such modifications may be made to increase the in vivo activity or half-life of the target polynucleotides.

The terms "variant", "homolog" or "derivative" with respect to a nucleotide sequence include any substitution, alteration, modification, replacement, deletion or addition of one (or more) nucleic acids from or to the sequence.

In the above structure, "coexpr" is a nucleic acid sequence that provides for coexpression of both the first and second CARs. This may be a sequence encoding a cleavage site, such that the nucleic acid construct comprises two or more CCRs, or a CCR and a CAR joined by a cleavage site(s). The cleavage site may be self-cleaving, such that upon production of the polypeptide, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

The cleavage site can be any sequence that allows separation of the first and second CCRs or the CCR and CAR.

The term "cleavage" is used herein for convenience, but the cleavage site may result in peptides being separated into separate entities by a mechanism other than classical cleavage. For example, for the foot-and-mouth disease virus (FMDV) self-cleaving peptide 2A (see below), various models have been proposed to account for the "cleavage" activity: proteolysis by a host cell proteinase, autoproteolysis, or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041). The precise mechanism of such "cleavage" is not important for the purposes of the present invention, as long as the cleavage site, when placed between nucleic acid sequences that encode the proteins, results in the expression of the proteins as separate entities.

The cleavage site may be a furin cleavage site.

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

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

TEV protease is a highly sequence-specific cysteine protease that is a chymotrypsin-like protease. It is highly specific for its target cleavage site and is therefore often used for the controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (where "\" denotes the cleaved peptide bond). Mammalian cells, such as human cells, do not express TEV protease. Thus, in embodiments in which the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell, the exogenous TEV protease must also be expressed in a mammalian cell.

The cleavage site may encode a self-cleaving peptide.

"Self-cleaving peptide" refers to a peptide that functions such that when a polypeptide containing proteins and a self-cleaving peptide is produced, it is immediately "cleaved" or separated into separate and discrete first and second polypeptides without the need for any external cleaving activity.

The self-cleaving peptide may be self-cleaving peptide 2A from an aphtho- or cardiovirus. Primary cleavage of 2A/2B of aphtho- 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 virus A, the 2A region is a short stretch of about 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), constitutes an autonomous element capable of mediating "cleavage" at its own C-terminus (Donelly et al (2001), as above).

"2A-like" sequences have been found in picornaviruses other than aphtho- or cardioviruses, insect viruses similar to picoraviruses, rotavirus type C, and repetitive sequences within trypanosomes, and a bacterial sequence (Donelly et al (2001), as above). The cleavage site may contain one of these 2A-like sequences, such as:

YHADYYKQRLIHDVEMNPGP (SEQ ID No. 33)

HYAGYFADLLIHDIETNPGP (SEQ ID No. 34)

QCTNYALLKLAGDVESNPGP (SEQ ID No. 35)

ATNFSLLKQAGDVEENPGP (SEQ ID No. 36)

AARQMLLLLSGDVETNPGP (SEQ ID No. 37)

RAEGRGSLLTCGDVEENPGP (SEQ ID No. 38)

TRAEIEDELIRAGIESNPGP (SEQ ID No. 39)

TRAEIEDELIRADIESNPGP (SEQ ID No. 40)

AKFQIDKILISGDVELNPGP (SEQ ID No. 41)

SSIIRTKMLVSGDVEENPGP (SEQ ID No. 42)

CDAQRQKLLLSGDIEQNPGP (SEQ ID No. 43)

YPIDFGGFLVKADSEFNPGP (SEQ ID No. 44)

The cleavage site may contain a 2A-like sequence shown as SEQ ID No. 38 (RAEGRGSLLTCGDVEENPGP).

The present invention also relates to a kit comprising one or more nucleic acid sequences encoding first and second CCRs according to the first aspect of the present invention, or one or more CCR(s) according to the invention and one or more CAR(s).

SEQ ID NOS 45 and 46 provide the complete amino acid sequences of the fusion between the anti-PSMA CAR and the anti-PSA CCR. Headers are provided to identify each portion of the sequence, but in practice the various elements are joined to provide a single contiguous sequence.

The nucleic acid construct of the invention may encode a fusion protein as shown in SEQ ID No. 45 or 46.

SEQ ID No. 45 - Exemplary construct with IL-2R beta chain

Signal sequence derived from human CD8a:

MSLPVTALLLPLALLLHAA

scFv aPSMA (J591 H/L)

EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVP DRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR

Linker

SDPA

Human IgG1Fc spacer (HCH2CH3pvaa):

EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Transmembrane, derived from human CD28:

FWVLVVVGGVLACYSLLVTVAFIIFWV

TCRz-derived endodomain:

RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

2A peptide from the capsid protein of Thosea asigna virus:

RAEGRGSLLTCGDVEENPGP

Signal sequence derived from mouse kappa VIII:

METDTLILWVLLLLVPGSTG

scFv aPSA (5D5A5 H/L):

QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIY RASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKLEIK

Linker:

SDPA

Human CD8aSTK spacer:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

Transmembrane, derived from human common gamma chain:

VVISVGSMGLIISLLCVYFWL

Endodomain derived from human common gamma chain:

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

2A peptide from equine rhinitis virus A polyprotein:

QCTNYALLKLAGDVESNPGP

Signal sequence derived from mouse kappa VIII:

METDTLILWVLLLLVPGSTG

scFv aPSA (5D3D11 H/L):

QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY RMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGTKVEIK

Linker:

SDPA

Human CD28STK spacer:

KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

Transmembrane, derived from human IL-2Rβ:

IPWLGHLLVGLSGAFGFIILVYLLI

Endodomain derived from human IL-2Rβ:

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGP PTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

SEQ ID No. 46 - Exemplary construct with IL-7R alpha chain

Signal sequence derived from human CD8a:

MSLPVTALLLPLALLLHAA

scFv aPSMA (J591 H/L)

EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVP DRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR

Linker

SDPA

Human IgG1Fc spacer (HCH2CH3pvaa):

EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Transmembrane, derived from human CD28:

FWVLVVVGGVLACYSLLVTVAFIIFWV

TCRz-derived endodomain:

RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

2A peptide from the capsid protein of Thosea asigna virus:

RAEGRGSLLTCGDVEENPGP

Signal sequence derived from mouse kappa VIII:

METDTLILWVLLLLVPGSTG

scFv aPSA (5D5A5 H/L):

QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIY RASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKLEIK

Linker:

SDPA

Human CD8aSTK spacer:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

Transmembrane, derived from human common gamma chain:

VVISVGSMGLIISLLCVYFWL

Endodomain derived from human common gamma chain:

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

2A peptide from equine rhinitis virus A polyprotein:

QCTNYALLKLAGDVESNPGP

Signal sequence derived from mouse kappa VIII:

METDTLILWVLLLLVPGSTG

scFv aPSA (5D3D11 H/L):

QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY RMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGTKVEIK

Linker:

SDPA

Human CD28STK spacer:

KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

Transmembrane, derived from human IL-7Rα:

PILLTISILSFFSVALLVILACVLW

Endodomain derived from human IL-7Rα:

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

VECTOR

The present invention also relates to a vector or a set of vectors that comprises one or more nucleic acid sequence(s) encoding one or more CCR(s) according to the first aspect of the invention and, additionally, one or more CAR(s). Such a vector can be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses a CCR according to the first aspect of the invention.

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

The vector may be capable of transfecting or transducing a T cell or an NK cell.

CELL

The present invention relates to a cell that comprises one or more CCR(s) of the invention and, additionally, one of more CAR(s).

The cell may comprise a nucleic acid or vector of the present invention.

The cell may be a cytolytic immune cell such as a T cell or an NK cell.

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

Helper T helper (Th) cells assist other leukocytes in immunologic processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. Th cells express CD4 on the surface. Th cells are 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 a variety of cytokines to promote different types of immune responses.

Cytolytic T cells (CTLs) kill virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to MHC class I-associated antigen, which is present on the surface of all nucleated cells. Using IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an inactive state of anergy, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

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

Regulatory T cells (Treg cells), previously known as suppressor T cells, are critical for maintaining immune tolerance. Their primary role is to stop T cell-mediated immunity toward the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

Two main classes of CD4+ Treg cells have been described: natural Treg cells and adaptive Treg cells.

Natural Tregs (also known as CD4+CD25+FoxP3+ Tregs) arise in the thymus and are associated with interactions between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated by TSLP. Natural Tregs can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can impair the development of regulatory T cells, causing the fatal autoimmune disease IPEX.

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

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

NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third cell type differentiated from the common lymphoid progenitor that produces B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus, where they are then released into the bloodstream.

The CCR-expressing cells of the invention may be any of the cell types mentioned above.

T or NK cells, according to the first aspect of the invention, can be created ex vivo either from the patient's own peripheral blood (group 1), or from a hematopoietic stem cell transplant from a donor's peripheral blood (group 2), or from peripheral blood from an unrelated donor (group 3).

Alternatively, T or NK cells according to the first aspect of the invention may be obtained from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into T or NK cells. Alternatively, an immortalized T cell line may be used, which retains its lytic function and can act as a therapeutic one.

In all of these embodiments, CCR-expressing cells are generated by introducing DNA or RNA encoding one or each CCR(s) by one of a variety of methods, including transduction with a viral vector, transfection with DNA or RNA.

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

A T or NK cell according to the invention can be produced by:

(i) isolation of a sample containing a T or NK cell from a subject or other sources listed above; and

(ii) transducing or transfecting T or NK cells with one or more nucleic acid sequence(s) encoding a CCR.

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

PHARMACEUTICAL COMPOSITION

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

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

METHOD OF TREATMENT

The present invention relates to a method for treating and/or preventing a disease, which comprises the step of administering cells of the present invention (e.g., in a pharmaceutical composition as described above) to a subject.

The method of treating a disease refers to the therapeutic use of the cells of the present invention. In this case, the cells can be administered to a subject already having a disease or prerequisite in order to alleviate, reduce or improve at least one symptom associated with the disease and/or slow down, reduce or block the progression of the disease.

The method of preventing a disease refers to the prophylactic use of the cells of the present invention. In this case, such cells can be administered to a subject that is not yet infected with the disease and/or that does not show any symptoms of the disease in order to reduce or prevent the cause of the disease, or to reduce or prevent the development of at least one symptom associated with the disease. The subject may have a predisposition or is considered to be at risk of developing the disease.

The method may include the following stages:

(i) isolation of a sample containing T or NK cells;

(ii) transducing or transfecting such cells with a nucleotide sequence or vector provided by the present invention;

(iii) introducing cells from (ii) into the subject.

The sample containing T or NK cells may be obtained from the subject or from other sources, such as described above. T or NK cells may be isolated from the subject's own peripheral blood (Group 1) or from a hematopoietic stem cell transplant from a donor's peripheral blood (Group 2), or from peripheral blood from an unrelated donor (Group 3).

The present invention relates to a cell expressing the CCR of the present invention for use in the treatment and/or prevention of a disease.

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

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

When the ligand recognized by CCR is PSA, the cancer is likely to be prostate cancer.

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

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

The cells and pharmaceutical compositions of the present invention can be used for any of the methods described above.

CHIMERIC TRANSMEMBRANE PROTEIN

The present invention also relates to a chimeric transmembrane protein comprising a dimerization domain; and a cytokine receptor endodomain.

Dimerization may occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active. Alternatively, dimerization may only occur in the presence of a chemical inducer of dimerization (CID), in which case the transmembrane protein will only elicit cytokine signaling in the presence of the CID.

Suitable dimerization domains and CIDs are described in patent document WO025/50771, the contents of which are incorporated herein by reference.

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

One dimerization domain may comprise the Fc506 (tacrolimus)-binding domain of FK-binding protein 12 (FKBP12), and the other dimerization domain may comprise the cyclosporin-binding domain of cilcophilin A; and the CID may be FK506/cyclosporin or a derivative thereof.

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

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

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; and the CID may be an O6-benzylguanine/methotrexate derivative fusion protein or a derivative thereof.

One dimerization domain may include a retinoic acid receptor domain, and the other dimerization domain may include an ecodysone receptor domain; and the CID may be RSL1 or a derivative thereof.

If the dimerization domain spontaneously heterodimerizes, it may be based on the dimerization domain of an antibody. Specifically, it may contain a dimerization site of the constant domain of the heavy chain (CH) and the constant domain of the light chain (CL). The "dimerization site" of the constant domain is the part of the sequence that forms the interchain disulfide bond.

The chimeric cytokine receptor may comprise the Fab region of an antibody as an exodomain, for example as shown schematically in Fig. 5.

A chimeric transmembrane protein may include two polypeptides:

(i) a first polypeptide that comprises:

(a) the first dimerization domain; and

(b) the first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide that comprises:

(a) a second dimerization domain that dimerizes with the first dimerization domain; and

(b) the second chain of the cytokine receptor endodomain.

The above sections defining the cytokine receptor endodomain of the chimeric cytokine receptor also apply to the chimeric transmembrane protein of the present invention.

The above sections relating to nucleic acids, vectors, kits, cells, pharmaceutical compositions and methods also relate to the chimeric transmembrane protein of the present invention.

The invention will now be described by means of Examples, which are intended to assist one skilled in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1 - In vitro testing

T cells are transduced with either a PSMA-specific CAR or a construct that coexpresses a PSMA-specific CAR with a PSA-specific CCR. T cells are co-cultured with PSMA-expressing target cells that secrete or do not secrete PSA. This co-culture is performed in the presence or absence of exogenous IL2. This co-culture is performed at different effector-to-target ratios. This co-culture is repeated sequentially with T cells stimulated with repeat target cells. T cell proliferation and target cell killing are determined. In this way, the promotion of CCR to T cell proliferation and survival can be measured. In addition, the possibility of sequential action of repeated drug administration can be used to repeat the co-culture.

Example 2 - In vivo testing

NSG mice are engrafted with a human prostate cancer cell line that expresses PSMA and secretes PSA, and that expresses firefly luciferase. T cells are transduced with either a PSMA-specific CAR or a construct that coexpresses a CARMA-specific CAR with a PSA-specific CCR. T cells are injected into mice. Tumor burden can be measured serially using bioluminescence imaging and the T cell response to CAR is assessed. Mice within each group can be sacrificed at different time points and tumor burden is directly measured by macroscopic measurements and immunohistochemistry. Additionally, T cell engraftment/expansion in the tumor bed or in lymphoid tissues such as lymph nodes, spleen, and bone marrow is measured by flow cytometry of said tissues.

Example 3 - Creation and testing of a constitutively active cytokine signaling molecule

A constitutively active cytokine signaling chimeric transmembrane protein was obtained by linking cytokine receptor endodomains to a Fab-type exodomain (Fig. 5). This structure utilizes components of natural antibody dimerization, namely the dimerization domain of the constant regions of the heavy and light chains. The chimeric transmembrane protein has two chains; a first polypeptide that includes the antibody κ light chain and the common γ chain of the IL2 receptor as an endodomain; and a second polypeptide that includes the antibody CH1 heavy chain and an endodomain that includes either: the β chain of the IL2 receptor (yielding a constitutively active IL2 signaling molecule); or the IL7 receptor (yielding a constitutively active IL7 signaling molecule). The constitutively active cytokine signaling chimeric transmembrane proteins tested in this study included variable regions of the heavy and light chains of an scFv. These domains are not required for dimerization. The signal is independent of antigen binding, and the structure can equally well be "no-top" (as shown in Fig. 5) or contain another entity such as a protein tag.

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

CTLL-2 (ATCC® TIB-214™) are murine cytotoxic T lymphocytes that are dependent on IL-2 for growth. In the absence of IL-2, the cells undergo apoptosis. CTLL-2 cells were transduced with a vector expressing a chimeric protein comprising the IL2 receptor endodomain (Fab_IL2endo) or a vector expressing a chimeric protein comprising the IL7 receptor endodomain (Fab_IL7endo), or were left untransduced (WT). As a positive control, all three cell types were cocultured with 100 U/ml murine IL2. Cell proliferation was assessed after 3 and 7 days of culture, and the results are shown in Fig. 6.

Untransduced CTLL2 cells, along with CTLL2 cells transduced with either the construct (Fab_IL2endo or Fab_IL7endo), were expanded in the presence of 100 U/ml murine IL2 (Fig. 6, left panel). However, in the absence of exogenously added IL2, only cells transduced with the construct containing the IL2R endodomain (Fab_IL2endo) survived and proliferated, indicating that the chimeric transmembrane receptor provides the required IL2 signal to CTLL2 cells.

Example 4 - Production and testing of a chimeric cytokine receptor against PSA

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

Schematic drawings illustrating some of the group of CCRs are shown in Fig. 7.

The group included the following designs:

A5-CD8stk-IL2Rg_D11-Hinge-IL2Rb : CCR with the IL-2R endodomain having A5 on the chain with the common γ-chain and D11 on the chain with the IL2R β-chain;

D11-CD8stk-IL2Rg_A5-Hinge-IL2Rb : CCR with the IL-2R endodomain having D11 on the chain with the common γ-chain and A5 on the chain with the IL2R β-chain;

D11-CD8stk-RL_A5-Hinge-IL2Rb : a negative control construct that is equivalent to D11-CD8stk-IL2Rg_A5-Hinge-IL2Rb but in which the IL2Rγ chain is replaced by a rigid linker;

D11-CD8stk-IL2Rg_A5-Hinge-IL7Ra : CCR with the IL-7R endodomain, having D11 on the chain with the common γ-chain and A5 on the chain with the IL7R α-chain; and

D11-CD8stk-RL_A5-Hinge-IL7Ra : a negative control construct that is equivalent to D11-CD8stk-IL2Rg_A5-Hinge-IL7Ra but in which the IL2Rγ chain is replaced by a rigid linker;

CTLL2 cells were transduced with vectors expressing these constructs. Cells were cultured in the presence or absence of IL2 (IL2 as a positive control) and in the presence or absence of 5 ng/ml or 5 μg/ml PSA. CTLL2 cell proliferation was assessed at 3 and 7 d, and the results are shown in Fig. 8.

CTLL2 cells expressing CCR with the IL7 endodomain did not support CTLL2 cell survival and proliferation (Fig. 8, last two panels). The presence of mouse IL-2 in these cells supported CTLL2 cell growth and proliferation at day 3, but by day 7 most cells had undergone apoptosis.

Anti-PSA chimeric cytokine receptors with the ILR2 endodomain supported CTLL2 cell survival and proliferation in the absence of IL2 and the presence of PSA at both 5 ng/mL and 5 μg/mL (Fig. 8, first panel), with 5 μg/mL giving greater survival and proliferation, especially at day 7.

Both anti-PSA chimeric cytokine receptors bind to the IL2R endodomain, i.e., A5-CD8stk-IL2Rg_D11-Hinge-IL2Rb and D11-CD8stk-IL2Rg_A5-Hinge-IL2Rb, indicating that the relative positioning of the two PSA-binding domains: 5D5A5 and 5D3D11 is not important for function.

Replacement of the common γ-chain with a rigid linker abolished the ability of CCR to support CTLL2 cell survival and proliferation (Fig. 8, third panel).

As another measurable indicator of IL2 signaling, STAT5 Y694 phosphorylation was examined using flow cytometry.

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

The results are shown in Fig. 9. In cells expressing PSA CCR, the presence of PSA resulted in an increase in STAT5 phosphorylation over time (Fig. 9, center panel). This increase in phosphorylation was not observed in untransduced CTLL2 cells or CTLL2 cells transduced with an equivalent construct in which the IL2Rγ chain was replaced by a rigid linker (Fig. 9, right panel).

These results are consistent with the CTLL2 survival/proliferation data shown in Fig. 8 and demonstrate that a chimeric cytokine receptor against a soluble ligand (here PSA) can be used to activate cytokine signaling in T cells.

All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be obvious 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 specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Moreover, various modifications of the described methods for carrying out the invention that are obvious to those skilled in the art of molecular biology or related fields are intended to be covered by the following claims.

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LIST OF SEQUENCES

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Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 14

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> sequence 5D5A5 VL

<400> 14

Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Ile Asp Leu Tyr

20 25 30

Gly Phe Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Ile Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 15

<211> 248

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ScFv based on 5D5A5

<400> 15

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Ser Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Asn Asn Gln Lys Phe

50 55 60

Lys Asp Lys Val Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

130 135 140

Pro Pro Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys

145 150 155 160

Arg Ala Ser Glu Ser Ile Asp Leu Tyr Gly Phe Thr Phe Met His Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Ile Leu Ile Tyr Arg Ala

180 185 190

Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

195 200 205

Arg Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Thr His Glu Asp Pro Tyr Thr Phe Gly

225 230 235 240

Gly Gly Thr Lys Leu Glu Ile Lys

245

<210> 16

<211> 250

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ScFv based on 5D3D11

<400> 16

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ala Ile Ser Ser Ser

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Gly Tyr Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

130 135 140

Thr Ala Pro Ser Val Phe Val Thr Pro Gly Glu Ser Val Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu

165 170 175

Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

180 185 190

Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu

210 215 220

Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Val Thr

225 230 235 240

Phe Gly Ala Gly Thr Lys Val Glu Ile Lys

245 250

<210> 17

<211> 356

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CXCR4 sequence

<400> 17

Met Ser Ile Pro Leu Pro Leu Leu Gln Ile Tyr Thr Ser Asp Asn Tyr

1 5 10 15

Thr Glu Glu Met Gly Ser Gly Asp Tyr Asp Ser Met Lys Glu Pro Cys

20 25 30

Phe Arg Glu Glu Asn Ala Asn Phe Asn Lys Ile Phe Leu Pro Thr Ile

35 40 45

Tyr Ser Ile Ile Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val Ile

50 55 60

Leu Val Met Gly Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Lys Tyr

65 70 75 80

Arg Leu His Leu Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro

85 90 95

Phe Trp Ala Val Asp Ala Val Ala Asn Trp Tyr Phe Gly Asn Phe Leu

100 105 110

Cys Lys Ala Val His Val Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val

115 120 125

Leu Ile Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His

130 135 140

Ala Thr Asn Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Val Val

145 150 155 160

Tyr Val Gly Val Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe

165 170 175

Ile Phe Ala Asn Val Ser Glu Ala Asp Asp Arg Tyr Ile Cys Asp Arg

180 185 190

Phe Tyr Pro Asn Asp Leu Trp Val Val Val Phe Gln Phe Gln His Ile

195 200 205

Met Val Gly Leu Ile Leu Pro Gly Ile Val Ile Leu Ser Cys Tyr Cys

210 215 220

Ile Ile Ile Ser Lys Leu Ser His Ser Lys Gly His Gln Lys Arg Lys

225 230 235 240

Ala Leu Lys Thr Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cys Trp

245 250 255

Leu Pro Tyr Tyr Ile Gly Ile Ser Ile Asp Ser Phe Ile Leu Leu Glu

260 265 270

Ile Ile Lys Gln Gly Cys Glu Phe Glu Asn Thr Val His Lys Trp Ile

275 280 285

Ser Ile Thr Glu Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pro Ile

290 295 300

Leu Tyr Ala Phe Leu Gly Ala Lys Phe Lys Thr Ser Ala Gln His Ala

305 310 315 320

Leu Thr Ser Val Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Lys Gly

325 330 335

Lys Arg Gly Gly His Ser Ser Val Ser Thr Glu Ser Glu Ser Ser Ser

340 345 350

Phe His Ser Ser

355

<210> 18

<211> 374

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cell surface receptor CCR2 sequence

<400> 18

Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg Asn Thr Asn Glu Ser

1 5 10 15

Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cys

20 25 30

His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro Leu

35 40 45

Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val

50 55 60

Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr

65 70 75 80

Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro

85 90 95

Leu Trp Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met

100 105 110

Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile

115 120 125

Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His

130 135 140

Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr

145 150 155 160

Ser Val Ile Thr Trp Leu Val Ala Val Phe Ala Ser Val Pro Gly Ile

165 170 175

Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro

180 185 190

Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg Asn Ile

195 200 205

Leu Gly Leu Val Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly

210 215 220

Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg

225 230 235 240

Ala Val Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp

245 250 255

Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Phe

260 265 270

Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Thr Gln

275 280 285

Val Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile

290 295 300

Tyr Ala Phe Val Gly Glu Lys Phe Arg Ser Leu Phe His Ile Ala Leu

305 310 315 320

Gly Cys Arg Ile Ala Pro Leu Gln Lys Pro Val Cys Gly Gly Pro Gly

325 330 335

Val Arg Pro Gly Lys Asn Val Lys Val Thr Thr Gln Gly Leu Leu Asp

340 345 350

Gly Arg Gly Lys Gly Lys Ser Ile Gly Arg Ala Pro Glu Ala Ser Leu

355 360 365

Gln Asp Lys Glu Gly Ala

370

<210> 19

<211> 360

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cell surface receptor sequence CCR4

<400> 19

Met Asn Pro Thr Asp Ile Ala Asp Thr Thr Leu Asp Glu Ser Ile Tyr

1 5 10 15

Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Lys Pro Cys Thr Lys Glu

20 25 30

Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pro Pro Leu Tyr Ser Leu

35 40 45

Val Phe Val Phe Gly Leu Leu Gly Asn Ser Val Val Val Leu Val Leu

50 55 60

Phe Lys Tyr Lys Arg Leu Arg Ser Met Thr Asp Val Tyr Leu Leu Asn

65 70 75 80

Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Ser Leu Pro Phe Trp Gly

85 90 95

Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Leu Gly Leu Cys Lys Met

100 105 110

Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr Ser Gly Ile Phe Phe Val

115 120 125

Met Leu Met Ser Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe

130 135 140

Ser Leu Arg Ala Arg Thr Leu Thr Tyr Gly Val Ile Thr Ser Leu Ala

145 150 155 160

Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pro Gly Phe Leu Phe Ser

165 170 175

Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cys Lys Thr Lys Tyr Ser

180 185 190

Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Ser Leu Glu Ile Asn Ile

195 200 205

Leu Gly Leu Val Ile Pro Leu Gly Ile Met Leu Phe Cys Tyr Ser Met

210 215 220

Ile Ile Arg Thr Leu Gln His Cys Lys Asn Glu Lys Lys Asn Lys Ala

225 230 235 240

Val Lys Met Ile Phe Ala Val Val Val Leu Phe Leu Gly Phe Trp Thr

245 250 255

Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Leu Val Glu Leu Glu Val

260 265 270

Leu Gln Asp Cys Thr Phe Glu Arg Tyr Leu Asp Tyr Ala Ile Gln Ala

275 280 285

Thr Glu Thr Leu Ala Phe Val His Cys Cys Leu Asn Pro Ile Ile Tyr

290 295 300

Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Ile Leu Gln Leu Phe Lys

305 310 315 320

Thr Cys Arg Gly Leu Phe Val Leu Cys Gln Tyr Cys Gly Leu Leu Gln

325 330 335

Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Tyr Thr Gln Ser Thr Met

340 345 350

Asp His Asp Leu His Asp Ala Leu

355 360

<210> 20

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv based on J591

<400> 20

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser

130 135 140

Val Gly Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly

145 150 155 160

Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu

165 170 175

Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe

180 185 190

Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val

195 200 205

Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr

210 215 220

Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys Arg

225 230 235

<210> 21

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> signal peptide derived from the beta chain of TCR

<400> 21

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Gly

20

<210> 22

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> signal peptide derived from IgG1

<400> 22

Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 23

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> signal peptide derived from CD8a

<400> 23

Met Ala Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys

20

<210> 24

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> endodomain CD3 Z

<400> 24

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 25

<211> 152

<212> PROTEIN

<213> Artificial sequence

<220>

<223> endodomains CD28 and CD3-zeta

<400> 25

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

1 5 10 15

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

20 25 30

Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala

35 40 45

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

50 55 60

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

65 70 75 80

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

85 90 95

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

100 105 110

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

115 120 125

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

130 135 140

His Met Gln Ala Leu Pro Pro Arg

145 150

<210> 26

<211> 188

<212> PROTEIN

<213> Artificial sequence

<220>

<223> endodomains CD28, OX40 and CD3-zeta

<400> 26

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

1 5 10 15

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

20 25 30

Arg Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp

35 40 45

Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu

50 55 60

Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe

65 70 75 80

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

85 90 95

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

100 105 110

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

115 120 125

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

130 135 140

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

145 150 155 160

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

165 170 175

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

180 185

<210> 27

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ICOS endodomain

<400> 27

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 5 10 15

Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg

20 25 30

Leu Thr Asp Val Thr Leu

35

<210> 28

<211> 48

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD27 endodomain

<400> 28

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 29

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> BTLA endodomain

<400> 29

Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg

1 5 10 15

Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr Glu Ala

20 25 30

Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly Ile Tyr

35 40 45

Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser Glu Val

50 55 60

Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val Tyr Ala

65 70 75 80

Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu Ala Arg Asn

85 90 95

Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg Ser

100 105 110

<210> 30

<211> 188

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD30 endodomain

<400> 30

His Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys

1 5 10 15

Tyr Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg

20 25 30

Pro Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu

35 40 45

Pro Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr

50 55 60

Cys His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp

65 70 75 80

Ala Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro

85 90 95

Arg Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile

100 105 110

Met Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro

115 120 125

Glu Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu

130 135 140

Leu Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro

145 150 155 160

Pro Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly

165 170 175

Lys Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys

180 185

<210> 31

<211> 58

<212> PROTEIN

<213> Artificial sequence

<220>

<223> GITR endodomain

<400> 31

Gln Leu Gly Leu His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro

1 5 10 15

Arg Glu Thr Gln Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala

20 25 30

Arg Ser Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu

35 40 45

Glu Lys Gly Arg Leu Gly Asp Leu Trp Val

50 55

<210> 32

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HVEM endodomain

<400> 32

Cys Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val

1 5 10 15

Ser Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile

20 25 30

Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu

35 40 45

Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His

50 55 60

<210> 33

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 33

Tyr His Ala Asp Tyr Tyr Lys Gln Arg Leu Ile His Asp Val Glu Met

1 5 10 15

Asn Pro Gly Pro

20

<210> 34

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 34

His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu Thr

1 5 10 15

Asn Pro Gly Pro

20

<210> 35

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 35

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 36

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 36

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 37

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 37

Ala Ala Arg Gln Met Leu Leu Leu Leu Ser Gly Asp Val Glu Thr Asn

1 5 10 15

Pro Gly Pro

<210> 38

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 38

Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

1 5 10 15

Asn Pro Gly Pro

20

<210> 39

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 39

Thr Arg Ala Glu Ile Glu Asp Glu Leu Ile Arg Ala Gly Ile Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 40

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 40

Thr Arg Ala Glu Ile Glu Asp Glu Leu Ile Arg Ala Asp Ile Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 41

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 41

Ala Lys Phe Gln Ile Asp Lys Ile Leu Ile Ser Gly Asp Val Glu Leu

1 5 10 15

Asn Pro Gly Pro

20

<210> 42

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 42

Ser Ser Ile Ile Arg Thr Lys Met Leu Val Ser Gly Asp Val Glu Glu

1 5 10 15

Asn Pro Gly Pro

20

<210> 43

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 43

Cys Asp Ala Gln Arg Gln Lys Leu Leu Leu Ser Gly Asp Ile Glu Gln

1 5 10 15

Asn Pro Gly Pro

20

<210> 44

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 44

Tyr Pro Ile Asp Phe Gly Gly Phe Leu Val Lys Ala Asp Ser Glu Phe

1 5 10 15

Asn Pro Gly Pro

20

<210> 45

<211> 1722

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IL-2R beta chain fusion construct

<400> 45

Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Thr Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Glu Tyr Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn

65 70 75 80

Gln Lys Phe Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys Phe Met

145 150 155 160

Ser Thr Ser Val Gly Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln

165 170 175

Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser

180 185 190

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro

195 200 205

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

210 215 220

Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr

225 230 235 240

Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys

245 250 255

Arg Ser Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys

260 265 270

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

275 280 285

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val

290 295 300

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

305 310 315 320

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

325 330 335

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

340 345 350

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

355 360 365

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

370 375 380

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

385 390 395 400

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

405 410 415

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

420 425 430

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

435 440 445

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

450 455 460

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

465 470 475 480

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu

485 490 495

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

500 505 510

Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg Ser Ala

515 520 525

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

530 535 540

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

545 550 555 560

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

565 570 575

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

580 585 590

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

595 600 605

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

610 615 620

His Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly Ser Leu

625 630 635 640

Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Thr Asp

645 650 655

Thr Leu Ile Leu Trp Val Leu Leu Leu Leu Val Pro Gly Ser Thr Gly

660 665 670

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

675 680 685

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Ser Phe Ser Ser Tyr

690 695 700

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

705 710 715 720

Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Asn Asn Gln Lys Phe

725 730 735

Lys Asp Lys Val Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

740 745 750

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

755 760 765

Ala Arg Ser Gly Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr

770 775 780

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

785 790 795 800

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

805 810 815

Pro Pro Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys

820 825 830

Arg Ala Ser Glu Ser Ile Asp Leu Tyr Gly Phe Thr Phe Met His Trp

835 840 845

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Ile Leu Ile Tyr Arg Ala

850 855 860

Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

865 870 875 880

Arg Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val

885 890 895

Ala Thr Tyr Tyr Cys Gln Gln Thr His Glu Asp Pro Tyr Thr Phe Gly

900 905 910

Gly Gly Thr Lys Leu Glu Ile Lys Ser Asp Pro Ala Thr Thr Thr Pro

915 920 925

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

930 935 940

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

945 950 955 960

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Val Val Ile Ser Val Gly

965 970 975

Ser Met Gly Leu Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu

980 985 990

Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val

995 1000 1005

Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys

1010 1015 1020

Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys

1025 1030 1035

Leu Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly

1040 1045 1050

Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro

1055 1060 1065

Pro Cys Tyr Thr Leu Lys Pro Glu Thr Gln Cys Thr Asn Tyr Ala

1070 1075 1080

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met

1085 1090 1095

Glu Thr Asp Thr Leu Ile Leu Trp Val Leu Leu Leu Leu Val Pro

1100 1105 1110

Gly Ser Thr Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

1115 1120 1125

Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser Gly

1130 1135 1140

Tyr Ala Ile Ser Ser Ser Trp Met Asn Trp Val Lys Gln Arg Pro

1145 1150 1155

Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly

1160 1165 1170

Asp Thr Lys Tyr Asn Gly Lys Phe Lys Asp Lys Ala Thr Leu Thr

1175 1180 1185

Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

1190 1195 1200

Thr Ser Val Asp Ser Ala Val Tyr Phe Cys Ala Arg Asp Gly Tyr

1205 1210 1215

Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

1220 1225 1230

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

1235 1240 1245

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Ala

1250 1255 1260

Pro Ser Val Phe Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys

1265 1270 1275

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu

1280 1285 1290

Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile

1295 1300 1305

Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

1310 1315 1320

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val

1325 1330 1335

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu

1340 1345 1350

Tyr Pro Val Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Ser

1355 1360 1365

Asp Pro Ala Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp

1370 1375 1380

Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His

1385 1390 1395

Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Ile Pro

1400 1405 1410

Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly Phe

1415 1420 1425

Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro

1430 1435 1440

Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys

1445 1450 1455

Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp Val Gln Lys

1460 1465 1470

Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly

1475 1480 1485

Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys

1490 1495 1500

Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala

1505 1510 1515

Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln

1520 1525 1530

Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala

1535 1540 1545

Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro

1550 1555 1560

Asp Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro

1565 1570 1575

Leu Gln Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro

1580 1585 1590

Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly

1595 1600 1605

Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu

1610 1615 1620

Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro Arg Asp Trp

1625 1630 1635

Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro Asp Leu

1640 1645 1650

Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala Gly

1655 1660 1665

Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro

1670 1675 1680

Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala

1685 1690 1695

Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu

1700 1705 1710

Gln Gly Gln Asp Pro Thr His Leu Val

1715 1720

<210> 46

<211> 1631

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IL-7R alpha chain fusion construct

<400> 46

Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Thr Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Glu Tyr Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn

65 70 75 80

Gln Lys Phe Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys Phe Met

145 150 155 160

Ser Thr Ser Val Gly Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln

165 170 175

Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser

180 185 190

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro

195 200 205

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

210 215 220

Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr

225 230 235 240

Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys

245 250 255

Arg Ser Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys

260 265 270

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

275 280 285

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val

290 295 300

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

305 310 315 320

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

325 330 335

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

340 345 350

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

355 360 365

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

370 375 380

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

385 390 395 400

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

405 410 415

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

420 425 430

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

435 440 445

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

450 455 460

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

465 470 475 480

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu

485 490 495

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

500 505 510

Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg Ser Ala

515 520 525

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

530 535 540

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

545 550 555 560

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

565 570 575

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

580 585 590

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

595 600 605

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

610 615 620

His Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly Ser Leu

625 630 635 640

Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Thr Asp

645 650 655

Thr Leu Ile Leu Trp Val Leu Leu Leu Leu Val Pro Gly Ser Thr Gly

660 665 670

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

675 680 685

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Ser Phe Ser Ser Tyr

690 695 700

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

705 710 715 720

Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Asn Asn Gln Lys Phe

725 730 735

Lys Asp Lys Val Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

740 745 750

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

755 760 765

Ala Arg Ser Gly Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr

770 775 780

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

785 790 795 800

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

805 810 815

Pro Pro Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys

820 825 830

Arg Ala Ser Glu Ser Ile Asp Leu Tyr Gly Phe Thr Phe Met His Trp

835 840 845

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Ile Leu Ile Tyr Arg Ala

850 855 860

Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

865 870 875 880

Arg Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val

885 890 895

Ala Thr Tyr Tyr Cys Gln Gln Thr His Glu Asp Pro Tyr Thr Phe Gly

900 905 910

Gly Gly Thr Lys Leu Glu Ile Lys Ser Asp Pro Ala Thr Thr Thr Pro

915 920 925

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

930 935 940

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

945 950 955 960

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Val Val Ile Ser Val Gly

965 970 975

Ser Met Gly Leu Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu

980 985 990

Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val

995 1000 1005

Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys

1010 1015 1020

Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys

1025 1030 1035

Leu Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly

1040 1045 1050

Pro Gly Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro

1055 1060 1065

Pro Cys Tyr Thr Leu Lys Pro Glu Thr Gln Cys Thr Asn Tyr Ala

1070 1075 1080

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met

1085 1090 1095

Glu Thr Asp Thr Leu Ile Leu Trp Val Leu Leu Leu Leu Val Pro

1100 1105 1110

Gly Ser Thr Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

1115 1120 1125

Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser Gly

1130 1135 1140

Tyr Ala Ile Ser Ser Ser Trp Met Asn Trp Val Lys Gln Arg Pro

1145 1150 1155

Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly

1160 1165 1170

Asp Thr Lys Tyr Asn Gly Lys Phe Lys Asp Lys Ala Thr Leu Thr

1175 1180 1185

Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

1190 1195 1200

Thr Ser Val Asp Ser Ala Val Tyr Phe Cys Ala Arg Asp Gly Tyr

1205 1210 1215

Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

1220 1225 1230

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

1235 1240 1245

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Ala

1250 1255 1260

Pro Ser Val Phe Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys

1265 1270 1275

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu

1280 1285 1290

Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile

1295 1300 1305

Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

1310 1315 1320

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val

1325 1330 1335

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu

1340 1345 1350

Tyr Pro Val Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Ser

1355 1360 1365

Asp Pro Ala Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp

1370 1375 1380

Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His

1385 1390 1395

Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Pro Ile

1400 1405 1410

Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu

1415 1420 1425

Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile

1430 1435 1440

Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu

1445 1450 1455

Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu

1460 1465 1470

Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala

1475 1480 1485

Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln

1490 1495 1500

Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser

1505 1510 1515

Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser Phe

1520 1525 1530

Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala

1535 1540 1545

Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg

1550 1555 1560

Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu

1565 1570 1575

Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu

1580 1585 1590

Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro

1595 1600 1605

Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr

1610 1615 1620

Met Ser Ser Phe Tyr Gln Asn Gln

1625 1630

<210> 47

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> signal sequence derived from human CD8a

<400> 47

Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala

<210> 48

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> linker

<400> 48

Ser Asp Pro Ala

1

<210> 49

<211> 231

<212> PROTEIN

<213> Artificial sequence

<220>

<223> human IgG1Fc spacer (HCH2CH3pvaa)

<400> 49

Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 50

<211> 27

<212> PROTEIN

<213> Artificial sequence

<220>

<223> transmembrane, derived from human CD28

<400> 50

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 51

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TCRz-derived endodomain

<400> 51

Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 52

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> signal sequence derived from mouse kappa VIII

<400> 52

Met Glu Thr Asp Thr Leu Ile Leu Trp Val Leu Leu Leu Leu Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 53

<211> 40

<212> PROTEIN

<213> Artificial sequence

<220>

<223> human CD28STK spacer

<400> 53

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

1 5 10 15

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

20 25 30

Leu Phe Pro Gly Pro Ser Lys Pro

35 40

<210> 54

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> furin cleavage site

<220>

<221> other_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> OTHER_SIGN

<222> (3)..(3)

<223> Xaa can be Arg or Lys

<400> 54

Arg Xaa Xaa Arg

1

<210> 55

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> tobacco etch virus (TEV) consensus cleavage site

<400> 55

Glu Asn Leu Tyr Phe Gln Ser

1 5

<---

Claims (40)

1. A nucleic acid construct for producing a cell that expresses a chimeric transmembrane protein, wherein said nucleic acid construct encodes a chimeric transmembrane protein and comprises: (i) a first nucleic acid sequence encoding a first polypeptide comprising: (a) the dimerization domain of the heavy chain constant domain (CH); and (b) the first chain of the endodomain of the cytokine receptor type I; and (ii) a second nucleic acid sequence encoding a second polypeptide comprising: (a) a dimerization domain of a light chain constant domain (CL) that spontaneously heterodimerizes with said dimerization domain of a heavy chain constant domain (CH); and (b) the second chain of the endodomain of the cytokine receptor type I, wherein said chimeric transmembrane protein provides a constitutively active cytokine signal in said cell. 2. A nucleic acid construct according to paragraph 1, characterized in that said nucleic acid construct has the following structure: Dim1-TM1-endo1-coexpr-Dim2-TM2-endo2, in which Dim1 is a nucleic acid sequence encoding the dimerization domain of the heavy chain constant domain (CH); TM1 is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo 1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence that provides coexpression of both polypeptides; Dim2 is a nucleic acid sequence encoding the dimerization domain of the light chain constant domain (CL); TM2 is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo 2 is a nucleic acid sequence encoding the endodomain of a second polypeptide. 3. A nucleic acid construct according to claim 1 or 2, which further comprises a nucleic acid sequence that encodes a chimeric antigen receptor (CAR). 4. A nucleic acid construct according to any one of claims 1-3, wherein the coexpr encodes a sequence containing a self-cleaving peptide. 5. A nucleic acid construct according to any one of claims 1 to 4, wherein alternative codons are used in sequence regions encoding the same or similar amino acid sequences to avoid homologous recombination. 6. A nucleic acid construct according to any one of paragraphs 1-5, characterized in that the endodomain of the cytokine receptor in the chimeric transmembrane protein contains: (i) IL-2 receptor β-chain endodomain (ii) the endodomain of the IL-7 receptor α-chain; or (iii) the endodomain of the IL-15 receptor α-chain; and/or (iv) the common endodomain of the receptor γ-chain. 7. A nucleic acid construct according to any one of claims 1-6, characterized in that the first polypeptide in the chimeric transmembrane protein comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH); and the second polypeptide comprises a light chain variable domain (VL) and a light chain constant domain (CL). 8. A nucleic acid construct according to claim 7, characterized in that the encoded chimeric transmembrane protein includes a Fab exodomain. 9. A vector for producing a cell that expresses a chimeric transmembrane protein, wherein said vector contains a nucleic acid construct according to any one of claims 1-8, wherein said chimeric transmembrane protein is capable of providing a cytokine signal in said cell. 10. The vector of claim 9, wherein said vector is a retroviral vector, or a lentiviral vector, or a transposon. 11. A cytolytic immune cell for adoptive cell transfer that comprises a nucleic acid construct according to any one of claims 1-8 or a vector according to claim 9 or 10, wherein said cell expresses a chimeric transmembrane protein that provides a constitutively active cytokine signal in said cell. 12. A cell according to paragraph 11, which additionally contains a chimeric antigen receptor. 13. The cell according to paragraph 12, wherein the chimeric antigen receptor binds a tumor-associated cell surface antigen. 14. A pharmaceutical composition for adoptive cell transfer comprising an effective amount of cells according to any one of paragraphs 11-13. 15. A method for treating cancer, which comprises the step of administering a pharmaceutical composition according to claim 14 or an effective amount of cells according to any of claims 11-13 to a subject. 16. The method according to item 15, which includes the following steps: (i) isolating a sample containing cells from a subject; (ii) transducing or transfecting cells: with a nucleic acid construct according to any of paragraphs 1 to 8 or with a vector according to paragraph 9 or 10; and (iii) introducing cells from (ii) into the subject. 17. The method of claim 16, wherein the sample is a sample containing T cells.

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