Patent application title: T CELL WHICH EXPRESSES A GAMMA-DELTA T CELL RECEPTOR (TCR) AND A CHIMERIC ANTIGEN RECEPTOR (CAR)
Inventors:
IPC8 Class: AA61K3517FI
USPC Class:
1 1
Class name:
Publication date: 2018-05-10
Patent application number: 20180125890
Abstract:
The present invention provides a T cell which expresses a gamma-delta T
cell receptor (TCR) and a chimeric antigen receptor (CAR), wherein the
CAR comprises: an antigen binding domain; a transmembrane domain; and a
co-stimulatory intracellular signalling domain; wherein the intracellular
signalling domain provides a co-stimulatory signal to the T cell
following binding of antigen to the antigen binding domain.Claims:
1. A T cell which expresses a gamma-delta T cell receptor (TCR) and a
chimeric antigen receptor (CAR), wherein the CAR comprises; (i) an
antigen binding domain; (ii) a transmembrane domain; and (iii) a
co-stimulatory intracellular signalling domain; wherein the intracellular
signalling domain provides a co-stimulatory signal to the T cell
following binding of antigen to the antigen binding domain.
2. A cell according to claim 1 wherein the antigen binding domain is capable of binding to a tumour-associated antigen (TAA).
3. A cell according to claim 1 wherein the antigen binding domain is capable of binding to GD2, CD33, CD19 or EGFR.
4. A cell according to any preceding claim wherein the transmembrane domain comprises a CD8 stalk or a CD28 transmembrane domain.
5. A cell according to any of claims 1 to 4 wherein the intracellular signalling domain comprises the DAP10, CD28, CD27, 41BB, OX40, CD30, IL2-R, IL7-R, IL21-R, NKp30, NKp44 or DNAM-1 (CD226) signalling domain.
6. A cell according to any of claims 1 to 4 wherein the intracellular signalling domain comprises the DAP10 signalling domain.
7. A cell according to any preceding claim wherein the binding of a first antigen to the gamma-delta TCR results in signal 1 production and binding of a second antigen to the antigen binding domain of the CAR results in signal 2 production.
8. A cell according to any preceding claim wherein the CAR further comprises a spacer domain between the antigen binding domain and the transmembrane domain, for example a CD8 stalk or an Fc region.
9. A cell according to any preceding claim wherein the gamma-delta TCR is capable of binding to a phosphoantigen; major histocompatibility complex class I chain-related A (MICA); major histocompatibility complex class I chain-related B (MICB); NKG2D ligand 1-6 (ULBP 1-6); CD1c; CD1d; endothelial protein C receptor (EPCR); lipohexapeptide; phycoreythrin or histidyl-tRNA-synthase.
10. A CAR comprising; (i) an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signalling domain; wherein the intracellular signalling domain comprises a co-stimulatory intracellular signalling domain but does not comprise a CD3 endodomain.
11. A CAR according to claim 10 wherein the co-stimulatory intracellular signalling domain is selected from a DAP10, CD28, CD27, 41BB, OX40, CD30, IL2-R, IL7-R, IL21-R, NKp30, NKp44 or DNAM-1 (CD226) signalling domain.
12. A CAR comprising; (i) an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signalling domain; wherein the intracellular signalling domain comprises a DAP10 signalling domain.
13. A CAR according to claim 12 wherein the intracellular signalling domain does not comprise a CD3 endodomain.
14. A CAR according to any of claims 10 to 13 which is a CAR as defined in any of claims 2 to 9.
15. A nucleic acid sequence encoding a CAR as defined in any preceding claim.
16. A vector comprising a nucleic acid sequence as defined in claim 15.
17. A vector according to claim 16 which is a retroviral vector, a lentiviral vector or a transposon.
18. A method for making a cell according to any of claims 1 to 9, which comprises the step of introducing: a nucleic acid sequence according to claim 15 or a vector according to claim 16 or 17 into a cell.
19. A method according to claim 18 wherein the cell is stimulated with a gamma delta T cell stimulating agent.
20. A method according to claim 19 wherein the gamma-delta T cell stimulating agent is selected from isopentenyl pyrophosphate (IPP); analogs of IPP; and inhibitors of farnesyl pyrophosphate synthase (FPPS).
21. A method according to claim 19 or 20, wherein the cell is from a sample isolated from a subject.
22. A pharmaceutical composition comprising a cell according to any of claims 1 to 9, a CAR according to any of claims 10 to 14, a nucleic acid sequence according to claim 15 or a vector according to claim 16 or 17.
23. A method for treating a disease, which comprises the step of administering a pharmaceutical composition according to claim 22 to a subject.
24. A method according to claim 23 which comprises the step of administering a gamma-delta T cell stimulating agent to the subject.
25. A method according to claim 24 wherein the gamma-delta T cell stimulating agent is selected from isopentenyl pyrophosphate (IPP); analogs of IPP; and inhibitors of farnesyl pyrophosphate synthase (FPPS).
26. A method according to any of claims 23 to 25, which comprises the following steps: (i) isolation of a cell-containing sample from a subject; (ii) transduction or transfection of cells with: a nucleic acid according to claim 15 or a vector according to claim 16 or 17; and (iii) administering the cells from (ii) to the subject.
27. A pharmaceutical composition according to claim 22 for use in treating and/or preventing a disease.
28. The use of a cell according to any of claims 1 to 9 in the manufacture of a medicament for treating and/or preventing a disease.
29. A method according to any of claims 23 to 26 or a use according to claim 24 or 25 wherein the disease is cancer, microbial infection or viral infection.
30. A method according to any of claims 23 to 26 or a use according to claim 27 or 28 wherein the disease is cancer.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to immunotherapeutic T cells. In particular, the invention provides immunotherapeutic gamma-delta T cells comprising a chimeric antigen receptor (CAR).
BACKGROUND TO THE INVENTION
[0002] Chimeric antigen receptors (CARs) developed for cancer immunotherapy combine an extracellular antigen recognition domain with signalling domains specific for effector cells within a single molecule. The most common CAR system involves an antigen recognition domain derived from a monoclonal antibody fused to signalling domains which provide activating signals for T cells.
[0003] Typically, the signalling domains of a CAR provides cytotoxicity, proliferation and survival signals to activate the effector cell upon binding of antigen to the antigen recognition domain (Signals 1 and 2).
[0004] A limitation of this technology is potential `on target-off tumour toxicity`. This toxicity is caused by the recognition of low levels of a cancer-associated antigen recognised by a CAR on normal tissues. For instance GD2 is a target for neuroblastoma but also is expressed on nerves; and PSMA is a target for prostate cancer cells but is also found on normal kidney, liver and colon cells, and brain astrocytes. This problem is more profound in solid tumours where there is a dearth of highly selective targets.
[0005] Thus there is a need for cancer immunotherapies which address the above problems.
SUMMARY OF ASPECTS OF THE INVENTION
[0006] The present inventors have determined a mechanism of reducing `on target-off tumour toxicity` by using CARs in gamma delta (.gamma..delta.) T-cells. In the system described herein, a CAR is used to provide a co-stimulatory signal (signal 2) to a .gamma..delta. T-cell upon binding of antigen to the antigen recognition domain of the CAR. In this way, signal 2 is only provided to the T-cell upon binding of the CAR to its target antigen (FIG. 2A). Signal 1 for .gamma..delta. T-cell activation is provided by the endogenous TCR, which is activated by danger signals, such as phosphoantigens.
[0007] A .gamma..delta. T-cell requires both signal 1 and signal 2 for optimal effector function. Thus, in the present system the .gamma..delta. T-cell will only be fully activated for cytotoxicity, proliferation and cytokine secretion if the target cell: (i) expresses the antigen recognised by the CAR; and (ii) expresses danger signals recognised by the endogenous .gamma..delta. TCR.
[0008] Thus, in a first aspect the present invention provides a T cell which expresses a gamma-delta T cell receptor (TCR) and a chimeric antigen receptor (CAR), wherein the CAR comprises;
[0009] (i) an antigen binding domain;
[0010] (ii) a transmembrane domain; and
[0011] (iii) a co-stimulatory intracellular signalling domain; wherein the intracellular signalling domain provides a co-stimulatory signal to the T cell following binding of antigen to the antigen binding domain.
[0012] As such, binding of a first antigen to the .gamma..delta. TCR results in signal 1 production and binding of a second antigen to the antigen binding domain of the CAR results in signal 2 production.
[0013] The antigen binding domain may be capable of binding to a tumour-associated antigen (TAA).
[0014] The antigen binding domain may be capable of binding to GD2, CD33, CD19 or EGFR.
[0015] The intracellular signalling domain may comprise the DAP10, CD28, CD27, 41BB, OX40, CD30, IL2-R, IL7-R, IL21-R, NKp30, NKp44 or DNAM-1 (CD226) signalling domain.
[0016] The transmembrane domain of the CAR may comprise a CD8 stalk or a CD28 transmembrane domain.
[0017] The intracellular signalling domain of the CAR may comprise the DAP10 signalling domain.
[0018] The CAR may further comprise a spacer domain between the antigen binding domain and the transmembrane domain.
[0019] The .gamma..delta. TCR may be capable of binding to a phosphoantigen/butyrophilin 3A1 complex; major histocompatibility complex class I chain-related A (MICA); major histocompatibility complex class I chain-related B (MICB); NKG2D ligand 1-6 (ULBP 1-6); CD1c; CD1d; endothelial protein C receptor (EPCR); lipohexapeptides; phycoreythrin or histidyl-tRNA-synthase.
[0020] The CAR may comprise one of the following amino acid sequences:
TABLE-US-00001 (aCD33-Fc-DAP10 CAR) SEQ ID NO: 1 MAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIY FNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQP EDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSR SEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVS SISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQ DAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVF LFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVCARPRRSPAQEDG KVYINMPGRG (aGD2-Fc-DAP10 CAR) SEQ ID NO: 2 METDTLLLWVLLLWVPGSTGQVQLQESGPGLVKPSQTLSITCTVSGFSLA SYNIHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFL KMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLVTVSSGGGGSGGGGSGG GGSENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKV WIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPI TFGQGTKVEIKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDT LMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDP KFWVLVVVGGVLACYSLLVTVAFIIFWVCARPRRSPAQEDGKVYINMPGR G
[0021] In a further aspect the present invention provides a CAR comprising; (i) an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signalling domain; wherein the intracellular signalling domain comprises a co-stimulatory intracellular signalling domain but does not comprise a CD3 endodomain.
[0022] The co-stimulatory intracellular signalling domain may be selected from a DAP10, CD28, CD27, 41BB, OX40, CD30, IL2-R, IL7-R, IL21-R, NKp30, NKp44 or DNAM-1 (CD226) signalling domain.
[0023] In a second aspect the present invention provides a CAR comprising, an antigen-binding domain; a transmembrane domain; and an intracellular signalling domain; wherein the intracellular signalling domain comprises a DAP10 signalling domain. The intracellular signalling domain may consist of or consist essentially of a DAP10 signalling domain.
[0024] In a particular embodiment the intracellular signalling domain of the CAR according to the second aspect of the invention does not comprise a CD3 endodomain.
[0025] The CAR according to the second aspect of the invention may be a CAR as defined in the first aspect of the invention.
[0026] In a third aspect the present invention provides a nucleic acid sequence encoding a CAR as defined in the first or second aspects of the invention.
[0027] In a fourth aspect the present invention provides a vector comprising a nucleic acid sequence as defined by the third aspect of the invention.
[0028] The vector may be a retroviral vector, a lentiviral vector or a transposon.
[0029] In a fifth aspect the present invention relates to method for making a cell according to the first aspect of the invention, which comprises the step of introducing: a nucleic acid sequence according to the third aspect of the invention or a vector according to fourth aspect of the invention into a cell.
[0030] The method may comprise the step of stimulating the cell with a gamma delta T cell stimulating agent.
[0031] The .gamma..delta. T cell stimulating agent may be selected from, for example, isopentenyl pyrophosphate (IPP); analogs of IPP such as bromohydrin pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; and inhibitors of farnesyl pyrophosphate synthase (FPPS) such as aminobisphosphonates (e.g. zoledronate or pamidronate).
[0032] The cell may be from a sample isolated from a subject.
[0033] In a sixth aspect the present invention provides a pharmaceutical composition comprising a cell according to the first aspect of the present invention.
[0034] In a seventh aspect the present invention relates to a method for treating a disease, which comprises the step of administering a pharmaceutical composition according to the sixth aspect of the invention to a subject.
[0035] The method may comprise the step of administering a .gamma..delta. T cell stimulating agent to the subject.
[0036] The .gamma..delta. T cell stimulating agent may be selected from, for example, isopentenyl pyrophosphate (IPP); analogs of IPP such as bromohydrin pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; and inhibitors of farnesyl pyrophosphate synthase (FPPS) such as aminobisphosphonates (e.g. zoledronate or pamidronate).
[0037] The method may comprise the following steps:
[0038] (i) isolation of a cell-containing sample from a subject;
[0039] (ii) transduction or transfection of cells with: a nucleic acid sample according to the third aspect of the present invention or a vector according to the fourth aspect of the present invention; and
[0040] (iii) administering the cells from (ii) to the subject.
[0041] In an eighth aspect the present invention relates to a pharmaceutical composition according to the sixth aspect of the present invention for use in treating a disease.
[0042] In a ninth aspect the present invention relates to the use of a cell according to the first aspect of the present invention in the manufacture of a medicament for treating and/or preventing a disease.
[0043] The disease described herein may be cancer, microbial infection or viral infection.
[0044] The present invention therefore provides a .gamma..delta. T cell which is only fully activated by, and therefore capable of killing, a target cell which expresses a first antigen which is capable of binding to the endogenous .gamma..delta. TCR (and thus stimulating productive signal 1) and a second antigen which is capable of binding to the CAR (and thus stimulating productive signal 2).
[0045] The .gamma..delta. T cells of the invention are therefore useful for reducing unwanted `on target-off tumour` effects. In particular, a normal cell which expresses low levels of a TAA will not activate the .gamma..delta. T cell of the invention as it will not express a danger signal recognised by the endogenous .gamma..delta. TCR and thus will not provide signal 1, which is required for full activation of the .gamma..delta. T cell.
DESCRIPTION OF THE FIGURES
[0046] FIG. 1--Diagram of the signalling required for full activation of a .gamma..delta. T cell which results in killing of the target cell. A) and B) Signalling via the .gamma..delta. TCR or co-receptors alone does not result in full activation of the .gamma..delta. T cell. C) A combination of .gamma..delta. TCR and co-receptor signalling results in full activation of the .gamma..delta. T cell
[0047] FIG. 2--Illustrative diagram of a .gamma..delta. T cell of the present invention. A) Normal activation of a .gamma..delta. T cell by a target cell. B) Blocking of signal 2 by soluble NKG2D ligands secreted by cancer cells prevents full activation of .gamma..delta. T cells. C) Full activation of a .gamma..delta. T cell of the present invention by a transformed cell. D) Normal healthy cells do not express danger signals recognised by endogenous .gamma..delta. T cell receptors and do not fully activated .gamma..delta. T cells of the present invention.
[0048] FIG. 3--Examples of illustrative CARs which may be used in the present invention
[0049] FIG. 4--Representative flow cytometric dot plots to illustrate co-expression of a .gamma..delta. TCR (V.delta.2) and GD2-DAP10 CAR (Fc, CD20 marker and CD34 marker) in a .gamma..delta. T cell
[0050] FIG. 5--Killing of GD2+ cell lines LAN1 and TC71 by V.delta.2 .gamma..delta.T cells transduced with the aGD2-Fc-DAP10 CAR
[0051] (A) Significant killing of GD2+ neuroblastoma cell line LAN1 is only seen when CAR transduced cells are used and not when non-transduced (NT) V.delta.2 are used as effectors. (B) Additive effect of aGD2-Fc-DAP10 CAR when combined with 24h zoledronic acid exposure which increases phosphoantigen production, against the GD2+ Ewing sarcoma cell line TC71. (C) Addition of the CAR to .alpha..beta.T cells, which lack the signal 1 provided by the .gamma..delta.TCR in response to cellular stress, has no effect on cytotoxicity, unlike the effect of the CAR in V.delta.2+ .gamma..delta.T cells. This indicates that the CAR signal alone is insufficient for T-cell activation. Error bars denote SEM for 3-6 independent donors.
[0052] FIG. 6--Killing of GD2+ cell line LAN1 and no killing of GD2- cell line SKNSH. Error bars denote SEM for 3-6 independent donors.
[0053] FIG. 7--Preservation of CAR expression following prolonged co-culture and GD2 specific expansion
[0054] (A) Co-culture was started 24 days after transduction (labelled DO). Serial analyses of cells for presence of CAR (Y axis) and TCRV.delta.2 (X axis) were taken in the presence of irradiated GD2+ (LAN1) and GD2- (SK-N-SH) neuroblastoma cells.
[0055] Representative data from 1 of 3 donors is shown. (B) Expansion of aGD2-Fc-DAP10 transduced V.delta.2+ cells was only seen in the presence of irradiated GD2+ target cells (graphical representation, n=3 independent donors, error bars denote SEM).
[0056] FIG. 8--Flow cytometric staining for CD33 expression of AML cell lines (Nomo1, Sh1 and MV4; 11) and freshly isolated monocytes is equivalent.
[0057] FIG. 9--A) aCD33-DAP10-transduced V.delta.2 cells spare monocytes in the absence of ZOL but aCD33-CD28z-transduced V.delta.2 cells do not. B) aCD33-DAP10-transduced V.delta.2 cells kill AML better than NT V.delta.2 cells, but spare monocytes. Error bars indicate SEM for 3 independent donors.
[0058] FIG. 10--Nucleic acid and amino acid sequences of an anti-GD2-Fc-DAP10 CAR
[0059] FIG. 11--Nucleic acid and amino acid sequences of an anti-CD33-Fc-DAP10 CAR
[0060] FIG. 12--aCD33-DAP10-transduced V.delta.2 cells spare haemopoietic stem cells but aCD33-CD28z-transduced V.delta.2 cells do not. Normal human bone marrow was cultured overnight with the indicated CAR T cells. Surviving haemopoietic stem cells were assayed by myeloid colony formation in soft agar. Data is derived using transduced V.delta.2 cells from three independent donors.
[0061] FIG. 13--Differential cross-linking of "costimulation-only" CAR and V.gamma.9v.delta.2 TCR leads to differential cytokine responses. Top; Schematic of experimental design. Biotinylated beads are coated with (A) no/irrelevant antibodies, or (B) antibodies to bind either the TCR (anti-CD3) or the CAR (anti-Ig binding the spacer region of the CAR); C) following cross linking, intracellular cytokine secretion is used to measure activation. As a control, stimulatory anti-CD3/CD28 beads (Miltenyi) are used. Bottom-left: representative FACS plots; bottom-right: cytokine responses to cross linking show that the "costimulation-only" CAR cross linking leads to a TNF-.alpha. response but that additional TCR engagement is required for full response comprising both interferon gamma and TNF-.alpha.. Data is means+/-SD of 5 donors.
DETAILED DESCRIPTION
.gamma..delta. T Cell
[0062] T-cells are divided into two groups based on their T-Cell Receptor (TCR) components. The TCR heterodimer consists of an .alpha. and .beta. chain in 95% of T cells. These recognise foreign antigens via peptides presented by MHC molecules on antigen presenting cells and are essential for adaptive immunity.
[0063] 5% of T cells have TCRs consisting of .gamma. and .delta. chains. .gamma..delta. TCRs are MHC independent and detect markers of cellular stress expressed by tumours.
[0064] .gamma..delta. T cells recognize pathogens and transformed cells in an HLA-unrestricted manner. They respond to markers of cellular stress (e.g. phosphoantigens released by transformed cells as by-products of the mevalonate biosynthetic pathway). .gamma..delta. T cells display both innate cytotoxic functions and antigen-presenting capability, particularly in the presence of antibody-opsonized target cells.
[0065] .gamma..delta. T-cells are responsible for "lymphoid stress surveillance," i.e., sensing and responding immediately to infections or non-microbial stress without the need of clonal expansion or de novo differentiation.
[0066] The activation of .gamma..delta. T cells is regulated by a balance between stimulatory and inhibitory signals. They are activated by .gamma..delta. TCR ligands (e.g. phosphoantigens) in combination with MHC-associated ligands of the activatory receptor killer cell lectin-like receptor subfamily K, member 1 (KLRK1), also known as NKG2D, such as MHC class I polypeptide-related sequence A (MICA), MICB, and various members of the UL16-binding protein (ULBP) family.
[0067] .gamma..delta. cells also express killer-cell immunoglobulin-like receptors (KIRs), which can be either activatory or inhibitory, including killer cell immunoglobulin-like receptor, 2 domains, long cytoplasmic tail, 1 (KIR2DL1) and killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1 (KIR3DL1).
[0068] Full activation of a .gamma..delta. T cell which results in the effective killing of a target cell requires productive signal 1 and signal 2 generation (FIGS. 1 and 2A).
[0069] .gamma..delta. T-cells derive signal 1 of T cell activation from danger signal antigens present on transformed or infected cells. These danger signal antigens are recognised through the .gamma..delta. TCR. Signal 2 of T cell activation for .gamma..delta. T-cells is also commonly derived by danger signal molecules (such as MICA) present on transformed or infected cells. Signal 2 may be transduced, for example, through the NKG2D receptor and DAP 10 (FIG. 2A).
[0070] As a means of avoiding immune detection, cancer cells frequently secrete soluble NKG2D ligands effectively blocking signal 2 in .gamma..delta. T-cells, thus preventing their activation and facilitating tumour infiltration (FIG. 2B).
[0071] In a first aspect, the present invention provides a T cell which expresses a .gamma..delta. TCR and a CAR, wherein the intracellular signalling domain of the CAR provides a co-stimulatory signal to the T cell.
[0072] Thus, the arrangement of the .gamma..delta. TCR and the CAR is such that the .gamma..delta. TCR provides signal 1 and the CAR provides signal 2 upon binding to each receptor, respectively.
[0073] As used herein, co-stimulatory signal is synonymous with signal 2, which is required for full .gamma..delta. T cell activation.
[0074] Thus, a .gamma..delta. T cell according to the first aspect of the present invention will only be fully activated and capable of killing a target cell which expresses a first antigen which is capable of binding to the .gamma..delta. TCR (and thus stimulating productive signal 1) and a second antigen which is capable of binding to the CAR (and thus stimulating productive signal 2) (FIG. 2C).
[0075] In the absence of antigen binding to the .gamma..delta. TCR, signal 1 is not generated and full .gamma..delta. T cell activation is not achieved. In other words, in the absence of antigen binding to the .gamma..delta. TCR, the .gamma..delta. T cell is not stimulated to kill the target cell (FIG. 2D).
[0076] In the absence of antigen binding to the CAR, signal 2 is not generated and full .gamma..delta. T cell activation is not achieved. In other words, in the absence of antigen binding to the CAR, the .gamma..delta. T cell is not stimulated to kill the target cell.
[0077] The .gamma..delta. T cell of the present invention may express any .gamma..delta. TCR. Examples of .gamma..delta. TCR ligands are known in the art (see Vantourout, P. & Hayday, A. Nat. Rev. Immunol. 13, 88-100 (2013), for example).
[0078] By way of example, the .gamma..delta. TCR expressed by a cell of the present invention may recognise phosphoantigens (e.g. Isopentenyl pyrophosphate (IPP), Bromohydrin Pyrophosphate (BrHPP) and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP)); major histocompatibility complex class I chain-related A (MICA); major histocompatibility complex class I chain-related B (MICB); NKG2D ligand 1-6 (ULBP 1-6); CD1c; CD1d; endothelial protein C receptor (EPCR); lipohexapeptides; phycoreythrin or histidyl-tRNA-synthase.
[0079] One advantage of the cell of the present invention is that it comprises a CAR comprising (i) an antigen binding domain which binds a specific antigen and (ii) a particular co-stimulatory endodomain. As such, the cell of the present invention will have a greater propensity towards activation in an environment comprising an antigen which can be bound by the CAR, as the binding of antigen by the CAR will result is signalling through the co-stimulatory endodomain and signal 2 production. For example, if the antigen-binding domain of the CAR is specific for a TAA, the cell of the present invention will have an increased propensity towards activation in a tumour environment where the TAA is expressed due to the co-stimulatory signal provided by the CAR.
Chimeric Antigen Receptor
[0080] The T cell according to the present invention expresses a chimeric antigen receptor (CAR).
[0081] Chimeric antigen receptors (CARs) are engineered receptors which graft an arbitrary specificity onto an immune effector cell. In a classical CAR, the specificity of a monoclonal antibody is grafted on to a T cell. CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. In this way, a large number of cancer-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach show efficacy.
[0082] The target-antigen binding domain of a CAR is commonly fused via a spacer and transmembrane domain to a signaling endodomain. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.
[0083] Early CAR designs had endodomains derived from the intracellular parts of either the .gamma. chain of the Fc R1 or CD3.zeta.. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3.zeta. results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal--namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related OX40 and 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.
[0084] The .gamma..delta. T cell of the present invention comprises a CAR which comprises a co-stimulatory signalling endodomain which transmits signal 2 to the .gamma..delta. T cell upon the binding of target antigen.
[0085] The CARs of the T cell of the present invention may comprise a signal peptide so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
[0086] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.
[0087] The signal peptide may be at the amino terminus of the molecule.
[0088] The signal peptide may comprise the SEQ ID NO: 6, 7 or 8 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.
TABLE-US-00002 SEQ ID NO: 6: MGTSLLCVVMALCLLGADHADG
[0089] The signal peptide of SEQ ID NO: 6 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.
TABLE-US-00003 SEQ ID NO: 7: MSLPVTALLLPLALLLHAARP
[0090] The signal peptide of SEQ ID NO: 7 is derived from IgG1.
TABLE-US-00004 SEQ ID NO: 8: MAVPTQVLGLLLLWLTDARC
[0091] The signal peptide of SEQ ID NO: 8 is derived from CD8.
Co-Stimulatory Intracellular Signalling Domain
[0092] The intracellular domain/endodomain is the signal-transmission portion of a classical CAR.
[0093] The .gamma..delta. T cell of the present invention comprises a CAR which comprises a co-stimulatory signalling endodomain which transmits signal 2 to the .gamma..delta. T cell upon the binding of target antigen. Accordingly, .gamma..delta. T cell of the present invention comprises a CAR which does not transmit signal 1 to the .gamma..delta. T cell upon the binding of target antigen.
[0094] T-cell costimulatory receptors are known to induce qualitative and quantitative changes that lower activation thresholds and prevent T cell energy and enhance T cell function.
[0095] A number of co-receptors for .gamma..delta. T cells are known in the art. Productive signalling via one or more of these receptors can result in full activation of the .gamma..delta. T cell and target cell killing.
[0096] The .gamma..delta. T cell of the present invention comprises an intracellular signalling domain from a .gamma..delta. T cell co-receptor, such that binding of antigen to the antigen-binding domain of the CAR generates productive signal 2 signalling in the .gamma..delta. T cell.
[0097] The intracellular signalling domain may, for example, comprise the DAP10, CD28, CD27, 41BB, OX40, CD30, IL2-R, IL7-R, IL21-R, NKp30, NKp44 or DNAM-1 (CD226) signalling domain.
[0098] The intracellular signalling domain may comprise the DAP10 signalling domain.
[0099] DAP10 is a signalling subunit which associates with the NKG2D receptor (see FIG. 1). It is the exclusive binding partner and signalling intermediate for NKG2D and contains a YxxM activation motif that triggers the lipid kinase cascade.
[0100] An example of an amino acid sequence for a DAP10 signalling domain is shown below:
TABLE-US-00005 SEQ ID NO: 3 CARPRRSPAQEDGKVYINMPGRG
[0101] Further illustrative co-stimulatory domains are shown as SEQ ID NO: 9-19
TABLE-US-00006 (CD28 endodomain) SEQ ID NO: 9 KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY (CD27 endodomain) SEQ ID NO: 10 QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (41BB endodomain) SEQ ID NO: 11 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (OX40 endodomain) SEQ ID NO: 12 RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (CD30 endodomain) SEQ ID NO: 13 HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPV AEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVST EHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHT PHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (IL2-R endodomain) SEQ ID NO: 14 TWQRRQRKSRRTI (IL7-R endodomain) SEQ ID NO: 15 KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDI QARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRD SSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNST LPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ (IL21-R endodomain) SEQ ID NO: 16 SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLEL GPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWP TAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALD LDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPL ADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPV ECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (NKp30 endodomain) SEQ ID NO: 17 GSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPGG (NKp44 endodomain) SEQ ID NO: 18 WWGDIWWKTMMELRSLDTQKATCHLQQVTDLPWTSVSSPVEREILYHTVA RTKISDDDDEHTL (DNAM-1 (CD226) endodomain) SEQ ID NO: 19 NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNY PTFSRRPKTRV
[0102] The intracellular signalling domain may comprise, consist essentially of or consist of a co-stimulatory signalling domain as described herein.
[0103] The intracellular signalling domain may comprise a sequence shown as SEQ ID NO: 3 or 9-19 or a variant thereof.
[0104] The variant may comprise a sequence which shares at least 75% sequence identity with SEQ ID NO: 3 or 9-19 provided that the sequence provides an effective co-stimulatory signaling domain.
[0105] The variant may comprise a sequence which shares at least 80% sequence identity with SEQ ID NO: 3 or 9-19 provided that the sequence provides an effective co-stimulatory signaling domain.
[0106] The variant may comprise a sequence which shares at least 85% sequence identity with SEQ ID NO: 3 or 9-19 provided that the sequence provides an effective co-stimulatory signaling domain.
[0107] The variant may comprise a sequence which shares at least 90% sequence identity with SEQ ID NO: 3 or 9-19 provided that the sequence provides an effective co-stimulatory signaling domain.
[0108] The variant may comprise a sequence which shares at least 95% sequence identity with SEQ ID NO: 3 or 9-19 provided that the sequence provides an effective co-stimulatory signaling domain.
[0109] The variant may comprise a sequence which shares at least 99% sequence identity with SEQ ID NO: 3 or 9-19 provided that the sequence provides an effective co-stimulatory signaling domain.
[0110] In one embodiment, the intracellular signalling domain may comprise a sequence shown as SEQ ID NO: 3 or a variant thereof which shares at least 75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 3, provided that the sequence provides an effective co-stimulatory signaling domain.
[0111] In one embodiment, the endodomain does not comprise the CD3 endodomain. For example, the endodomain does not comprise the CD3 epsilon chain, the CD3 gamma chain and/or the CD3 delta chain. In a particular embodiment, the endodomain does not comprise the CD3-zeta endodomain.
[0112] An illustrative CD3-zeta endodomain is shown as SEQ ID NO: 26.
TABLE-US-00007 (CD3 zeta endodomain) SEQ ID NO: 26 RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR
[0113] The CD3-zeta endodomain as described herein may comprise or consist of SEQ ID NO: 26 or a variant thereof which has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 26 and provides an effective transmembrane domain/intracellular T cell signaling domain.
Antigen Binding Domain
[0114] The antigen binding domain is the portion of the CAR which recognizes antigen. Numerous 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, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.
[0115] The antigen binding domain may comprise a domain which is not based on the antigen binding site of an antibody. For example the antigen binding domain may comprise a domain based on a protein/peptide which is a soluble ligand for a tumour cell surface receptor (e.g. a soluble peptide such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand or a receptor for which the binding pair counterpart is expressed on the tumour cell.
[0116] By way of example, the examples described herein relate to CARs which bind GD2 and CD33, respectively.
[0117] The antigen binding domain may be based on a natural ligand of the antigen.
[0118] The antigen binding domain may comprise an affinity peptide from a combinatorial library or a de novo designed affinity protein/peptide.
Tumour-Associated Antigen (TAA)
[0119] The antigen binding domain may bind to a tumour-associated antigen (TAA).
[0120] An extensive range of TAAs are known in the art and the CAR used in the present invention may comprise any antigen binding domain which is capable of specifically binding to any TAA.
[0121] By way of example, the CAR for use in the present invention may be capable of specifically binding to a TAA listed in Table 1.
TABLE-US-00008 TABLE 1 Antigen Tumour of interest CD20 B-cell lymphomas, CLL CD19 Pre-B ALL, B-cell lymphoma, CLL CD22 Pre-B ALL, B-cell lymphomas, CLL CD30 Hodgkin's lymphoma, ALCL CD52 T-cell AML, Pre-B ALL CD70 Hodgkins Lymphoma, DLCL, Renal cell carcinoma, EBV+ glioblastoma, undifferentiated nasopharyngeal sarcoma CD33 AML, MDS, APL, CML, JMML, ALL (18% only) CD47 Pre-B ALL, T cell ALL, AML IL7 receptor .alpha. Pre-B ALL, B cell lymphomas TSLPR Pre-B ALL (7%), Pre-B aLL in Down's syndrome (60%) ROR1 Pre-B ALL, CLL mantle cell lymphoma GD2 Neuroblastoma, osteosarcoma, Ewing sarcoma, soft tissue sarcomas, melanoma IL13R.alpha.2 Glioblastoma, DIPG, melanoma, various carcinomas, mesothelioma VEGFR2 Tumour vasculature HER2 Osteosarcoma, colon cancer, breast cancer ALK Neuroblastoma, neuroectodermal tumours, glioblastoma, rhabdomyosarcoma, melanoma EGFRvIII Glioma FGFR4 Rhabdomyosarcoma B7-H3 Neuroblastoma Glypican- Wilm's tumour, neuroblastoma, rhabdomyosarcoma, 3/Glypican-5 hepatic carcinaoma, melanoma FOLR1 Rhabdomyosarcoma, osteosarcoma
[0122] A problem associated with the targeting of TAAs in cancer immunotherapy is that low levels of the TAAs may be expressed on normal tissues. For instance GD2 is a neuroblastoma TAA, but it is also expressed on nerves; PSMA is a prostate cancer TAA but also is found on normal kidney, liver and colon cells, and brain astrocytes. This problem is more profound in solid tumours where there is a dearth of highly selective targets.
[0123] The expression of TAAs on normal, healthy cells may result in `on-target, off-tumour` side effects. The present invention mitigates these effects because the .gamma..delta. T cell of the present invention is only activated by cells which express a ligand for both the .gamma..delta. TCR and the CAR. Normal, healthy cells which express the TAA at low levels will therefore not activate the .gamma..delta. T cell of the present invention because they do not express a danger signal antigen capable of binding to the .gamma..delta. TCR (FIG. 2D).
[0124] The antigen binding domain of the CAR may be capable of binding GD2, CD33, CD19 or EGFR.
[0125] Disialoganglioside (GD2, for example as shown by pubchem: 6450346) is a sialic acid-containing glycosphingolipid expressed primarily on the cell surface. The function of this carbohydrate antigen is not completely understood; however, it is thought to play an important role in the attachment of tumour cells to extracellular matrix proteins. GD2 is densely, homogenously and almost universally expressed on neuroblastoma. In normal tissues, GD2 expression is largely limited to skin melanocytes, and peripheral pain fibre myelin sheaths. Within the CNS, GD2 appears to be an embryonic antigen but is found dimly expressed in scattered oligodendrocytes and within the posterior pituitary.
[0126] The antigen binding domain may comprise a sequence shown as SEQ ID NO: 20 or a variant thereof, providing that the variant retains the ability to bind to GD2.
TABLE-US-00009 SEQ ID NO: 20 METDTLLLWVLLLWVPGSTGQVQLQESGPGLVKPSQTLSITCTVSGFSLA SYNIHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFL KMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLVTVSSGGGGSGGGGSGG GGSENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKV WIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPI TFGQGTKVEIKRS
[0127] The antigen binding domain may comprise a sequence shown as SEQ ID NO: 20 or a variant thereof which shares at least 75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 20, providing that the variant retains the ability to bind to GD2.
[0128] CD33 (for example as shown by Uniprot accession number P20138) is a putative adhesion molecule of myelomonocytic-derived cells that mediates sialic-acid dependent binding to cells. It is usually considered myeloid-specific, but it can also be found on some lymphoid cells.
[0129] The antigen binding domain may comprise a sequence shown as SEQ ID NO: 21 or a variant thereof, providing that the variant retains the ability to bind to GD2.
TABLE-US-00010 SEQ ID NO: 21 MAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIY FNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQP EDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSR SEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVS SISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQ DAYTGGYFDYWGQGTLVTVSSM
[0130] The antigen binding domain may comprise a sequence shown as SEQ ID NO: 21 or a variant thereof which shares at least 75, 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 21, providing that the variant retains the ability to bind to GD2.
[0131] The human CD19 antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily (for example as shown by Uniprot P15391). CD19 is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. Consequently, CD19 is expressed on all B-cell malignancies apart from multiple myeloma. CD19 is also expressed by the normal B cell compartment.
[0132] EGFR (for example as shown by Uniprot accession number P00533) is a receptor tyrosine kinase which binds ligands of the EGF family and activates several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. EGFR is expressed at high levels by many cancer cells. However, it is also expressed by normal, healthy cells.
Spacer Domain
[0133] CARs may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
[0134] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. A human IgG1 spacer may be altered to remove Fc binding motifs.
[0135] Examples of amino acid sequences for these spacers are given below:
TABLE-US-00011 (hinge-CH.sub.2CH.sub.3 of human IgG1) SEQ ID NO: 22 AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD (human CD8 stalk) SEQ ID NO: 23 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (human IgG1 hinge) SEQ ID NO: 24 AEPKSPDKTHTCPPCPKDPK
[0136] The spacer may be a variant of any of SEQ ID NO: 22 to 24 which shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 22 to 24 and retains the functional activity of the amino acid sequence shown as SEQ ID NO: 9 to 11.
Transmembrane Domain
[0137] The transmembrane domain is the sequence of the CAR that spans the membrane.
[0138] A transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (U.S. Pat. No. 7,052,906 B1 describes synthetic transmembrane components).
[0139] The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.
[0140] The transmembrane domain may be derived from CD28, which gives good receptor stability.
[0141] The transmembrane domain may comprise the sequence shown as SEQ ID NO: 25.
TABLE-US-00012 (CD28 transmembrane domain) SEQ ID NO: 25 FWVLVVVGGVLACYSLLVTVAFIIFWV
Nucleic Acid
[0142] The present invention further provides a nucleic acid sequence which encodes a CAR as described herein.
[0143] The nucleic acid sequence may be capable of encoding a CAR having the amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2.
TABLE-US-00013 (aCD33-Fc-DAP10 CAR) SEQ ID NO: 4 ATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGA TGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTAT TTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCT GATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTG GCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCC GAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCAC GTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGT CAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGA TCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGG GTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATG GCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCG TCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGG CCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAA TGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAG GACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGT CACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTC ACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGT CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCA ACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT GCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTG AGTCTGAGCCCAGGCAAGAAGGACCCCAAGTTCTGGGTCCTGGTGGTGGT GGGAGGCGTGCTGGCCTGTTACTCTCTCCTGGTGACCGTGGCCTTCATCA TCTTCTGGGTGTGCGCCAGACCACGGCGGAGCCCAGCCCAGGAGGACGGC AAGGTGTACATCAACATGCCCGGCCGCGGCTGA (aGD2-Fc-DAP10 CAR) SEQ ID NO: 5 ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGG CAGCACCGGCCAGGTGCAGCTGCAGGAGTCTGGCCCAGGCCTGGTGAAGC CCAGCCAGACCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGGCC AGCTACAACATCCACTGGGTGCGGCAGCCCCCAGGCAAGGGCCTGGAGTG GCTGGGCGTGATCTGGGCTGGCGGCAGCACCAACTACAACAGCGCCCTGA TGAGCCGGCTGACCATCAGCAAGGACAACAGCAAGAACCAGGTGTTCCTG AAGATGAGCAGCCTGACAGCCGCCGACACCGCCGTGTACTACTGCGCCAA GCGGAGCGACGACTACAGCTGGTTCGCCTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGA GGCGGCAGCGAGAACCAGATGACCCAGAGCCCCAGCAGCTTGAGCGCCAG CGTGGGCGACCGGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCA GCAGCTACCTGCACTGGTACCAGCAGAAGAGCGGCAAGGCCCCAAAGGTG TGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCCGGTTCAG CGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCGGCTACCCCATC ACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGTCGGATCCCGCCGA GCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTC CCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACA ATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCC AAGTTCTGGGTCCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCTCT CCTGGTGACCGTGGCCTTCATCATCTTCTGGGTGTGCGCCAGACCACGGC GGAGCCCAGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGC GGCTGA
[0144] The nucleic acid sequence may encode the same amino acid sequence as that encoded by SEQ ID NO: 1 or 2, but may have a different nucleic acid sequence, due to the degeneracy of the genetic code. The nucleic acid sequence may have at least 80, 85, 90, 95, 98 or 99% identity to the sequence shown as SEQ ID NO: 4 or SEQ ID NO: 5, provided that it encodes a CAR as defined in the first aspect of the invention.
Variant
[0145] Sequence comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.
[0146] Sequence identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
[0147] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
[0148] However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible--reflecting higher relatedness between the two compared sequences--will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap.
[0149] This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
[0150] Calculation of maximum % sequence identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid--Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
[0151] Although the final sequence identity can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix--the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
[0152] Once the software has produced an optimal alignment, it is possible to calculate % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
[0153] The terms "variant" according to the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence.
[0154] Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
TABLE-US-00014 ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R AROMATIC H F W Y
[0155] It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
[0156] A nucleic acid sequence or amino acid sequence as described herein may comprise, consist of or consist essentially of a nucleic acid sequence or amino acid sequence as shown herein.
Vector
[0157] The present invention also provides a vector which comprises a nucleic acid sequence according to the present invention. Such a vector may be used to introduce the nucleic acid sequence into a host cell so that it expresses and produces a molecule according to the first aspect of the invention.
[0158] The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector.
[0159] The vector may be capable of transfecting or transducing a T cell.
[0160] The vector may also comprise a nucleic acid sequence encoding a suicide gene, such as iCasp9 or RQR8.
[0161] A suicide-gene is a genetically encoded mechanism which allows selective destruction of adoptively transferred cells, such as T-cells, in the face of unacceptable toxicity.
[0162] Activation of Caspase 9 results in cell apoptosis. The activation mechanism behind Caspase 9 was exploited by the iCasp9 molecule. All that is needed for Caspase 9 to become activated, is overcoming the energic barrier for Caspase 9 to homodimerize. The homodimer undergoes a conformational change and the proteolytic domain of one of a pair of dimers becomes active. Physiologically, this occurs by binding of the CARD domain of Caspase 9 to APAF-1. In iCasp9, the APAF-1 domain is replaced with a modified FKBP12 which has been mutated to selectively bind a chemical inducer of dimerization (CID). Presence of the CID results in homodimerization and activation. iCasp9 is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) (Straathof et al (2005) Blood 105:4247-4254). It enables conditional dimerization in the presence of a small molecule CID, known as AP1903.
[0163] Expression of RQR8 renders T-cells susceptible to anti-CD20 antibody Rituximab but is more compact than the full-length CD20 molecule (Philip, B. et al. (2014) Blood doi: 10.1182/blood-2014-01-545020).
Pharmaceutical Composition
[0164] The present invention also relates to a pharmaceutical composition containing a vector or a CAR-expressing T cell of the invention together with a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.
Method
[0165] The present invention also relates to a method for making a cell according to the present invention, which comprises the step of introducing a nucleic acid sequence or vector according to the present invention into a cell.
[0166] CAR-expressing cells according to the present invention may either be created ex vivo either from a patient's own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party). Alternatively, CAR T-cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T-cells. In these instances, CAR T-cells are generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.
[0167] The method may further comprise stimulating the cell with a .gamma..delta. T cell stimulating agent. As used herein, a `.gamma..delta. T cell stimulating agent` refers to any agent which selectively stimulates the proliferation and/or survival of .gamma..delta. T cells from a mixed starting population of cells.
[0168] Thus, the resulting cell population is enriched with an increased number of .gamma..delta. T cells--for example particular .gamma..delta. T cells expressing a particular .gamma..delta. TCR receptor--compared with the starting population of cells.
[0169] .gamma..delta. T cell populations produced in accordance with the present invention may be enriched with .gamma..delta. T cells, for example particular .gamma..delta. T cells expressing a particular .gamma..delta. TCR receptor. That is, the .gamma..delta. T cell population that is produced in accordance with the present invention will have an increased number of .gamma..delta. T cells. For example, the .gamma..delta. T cell population of the invention will have an increased number of .gamma..delta. T cells expressing a particular .gamma..delta. TCR receptor compared with the .gamma..delta. T cells in a sample isolated from a subject. That is to say, the composition of the .gamma..delta. T cell population will differ from that of a "native" T cell population (i.e. a population that has not undergone expansion steps discussed herein), in that the percentage or proportion of .gamma..delta. T cells will be increased.
[0170] The .gamma..delta. T cell population according to the invention may have at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% .gamma..delta. T cells.
[0171] The .gamma..delta. T cell population according to the invention may have at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% .gamma..delta. T cells expressing a particular .gamma..delta. TCR receptor.
[0172] By way of example, the .gamma..delta. T cell stimulating agent may be isopentenyl pyrophosphate (IPP); an analog of IPP (e.g. bromohydrin pyrophosphate or (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate); an inhibitor of farnesyl pyrophosphate synthase (FPPS) or an aminobisphosphonate such as zoledronate or pamidronate.
[0173] The .gamma..delta. T cell stimulating agent may be used in combination with a general T cell mitogen, for example a mitogenic cytokine such as IL-2.
[0174] Additional methods of stimulating .gamma..delta. T cells are known in art and include, for example, the use of Concanavalin A (Siegers, G. M. et al. PLoS ONE 6, e16700 (2011)), anti-.gamma..delta. TCR antibodies immobilized on plastic; engineered artificial antigen presenting cells as feeders and engineered artificial antigen presenting cells coated in anti-.gamma..delta. TCR antibody (Fisher, J. et al.; Clin. Cancer Res. (2014)).
Method of Treatment
[0175] A method for the treatment of disease relates to the therapeutic use of a vector or T cell of the invention. In this respect, the vector or T cell may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
[0176] CAR-expressing T cells may either be created ex vivo either from a patient's own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party). Alternatively, CAR T-cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T-cells. In these instances, CAR T-cells are generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.
[0177] In one embodiment, the sample comprising .gamma..delta. T cell may have been previously isolated from the subject.
[0178] A CAR T cell according to the present invention may be generated by a method as described herein. In particular, a CAR-expressing T cell for use in a method for the treatment of a disease may be generated by a method comprising the steps of transduction of the T cell with a viral vector or transfection with DNA or RNA encoded the co-stimulatory CAR as described herein and expansion of .gamma..delta. T cells using a .gamma..delta. T cell stimulating agent.
[0179] The .gamma..delta. T cell stimulating agent may be isopentenyl pyrophosphate (IPP); an analog of IPP (e.g. bromohydrin pyrophosphate or (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate); an inhibitor of farnesyl pyrophosphate synthase (FPPS) or aminobisphosphonates such as zoledronate or pamidronate, for example.
[0180] T cells expressing a CAR molecule of the present invention may be used for the treatment of a various diseases including, for example, cancer, microbial infection and viral infection.
[0181] The cancer may be, for example, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), lung cancer, brain cancer, melanoma, leukaemia, lymphoma, pancreatic cancer, prostate cancer or thyroid cancer.
[0182] The methods and uses according to the present invention may be practiced in combination with additional compositions. For example, where the disease to be treated is cancer, the composition of the present invention may be administered in combination with additional cancer therapies such as chemotherapy and/or radiotherapy.
[0183] A composition of the present invention may be administered in combination with a .gamma..delta. T cell stimulating agent such as isopentenyl pyrophosphate (IPP); an analog of IPP (e.g. bromohydrin pyrophosphate or (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate); an inhibitor of farnesyl pyrophosphate synthase (FPPS) or aminobisphosphonates such as zoledronate or pamidronate.
[0184] In particular, Zoledronate and Pamidronate can be used for in vivo expansion of V.delta.2+.gamma..delta. T cells in combination with IL-2. There are a number of Phase I clinical trials that have used this approach (see Fisher et al.; Oncolmmunology; 3; e27572).
[0185] `In combination` may refer to administration of the additional therapy or .gamma..delta. T cell stimulating agent before, at the same time as or after administration of the composition according to the present invention.
[0186] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill 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--Generation of .gamma..delta. T Cells Expressing a Co-Stimulatory CAR
[0187] PBMCs were extracted from the blood of healthy donors using Ficoll density gradient separation. They were cultured in RPMI 1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin, 100u/ml human IL-2 and 5 .mu.M zoledronic acid for 5 days.
[0188] After 5 days they were transduced with retrovirus containing the CAR construct fused to RQR8, which acts as a marker gene and also provides a Rituximab (.alpha.CD20) sensitive suicide gene.
[0189] The illustrative CAR described herein includes aGD2-specific scFv, a linker based on the Fc portion of IgG1, a transmembrane domain derived from CD28 and the endodomain of DAP10 (see FIG. 10).
[0190] A second illustrative CAR includes a CD33-specific scFv, a linker based on the Fc portion of IgG1, a transmembrane domain derived from CD28 and the endodomain of DAP10 (see FIG. 11).
[0191] Co-expression of an anti-GD2-Fc-DAP10 CAR with the endogenous TCR of a .gamma..delta. T cell was demonstrated (FIG. 4).
Example 2--Killing of GD2+ Cell Lines LAN1 and TC71 by V.delta.2 .gamma..delta.T Cells Transduced with the aGD2-Fc-DAP10 CAR
[0192] Both the LAN1 and TC71 cells lines are known to express GD2.
[0193] Significant killing of GD2+ neuroblastoma cell line LAN1 was only seen when CAR transduced cells were used and not when non-transduced (NT) V.delta.2 cells were used as effectors (FIG. 5A).
[0194] There was an additive effect against the GD2+ Ewing sarcoma cell line TC71 when the aGD2-Fc-DAP10 CAR was used in combination with 24h zoledronic acid treatment (FIG. 5B).
[0195] Addition of the CAR to .alpha..beta.T cells, which lack the signal 1 provided by the .gamma..delta.TCR in response to cellular stress, had no effect on cytotoxicity, unlike the effect of the CAR in V.delta.2+.gamma..delta.T cells (FIG. 5C). This indicates that the CAR signal alone is insufficient for T-cell activation.
[0196] Expression of the aGD2-Fc-DAP10 CAR in .gamma..delta. T cells did not result in GD2-specific killing of GD2 negative SK-N-SH cells (FIG. 6).
Example 3--Preservation of CAR Expression Following Prolonged Co-Culture and GD2 Specific Expansion
[0197] Co-culture was started 24 days after transduction and serial analyses of cells for the presence of CAR and TCRV.delta.2 were taken in the presence of irradiated GD2+(LAN1) and GD2- (SK-N-SH) neuroblastoma cells (FIG. 7A).
[0198] The expansion of aGD2-Fc-DAP10 transduced V.delta.2+ cells was only seen in the presence of irradiated GD2+ target cells (FIG. 7B).
Example 4--Specific Killing of CD33+ AML Cells but not CD33+ Monocytes by .gamma..delta. T Cells Expressing an Anti-CD33-DAP10 CAR
[0199] Equivalent levels of CD33 expression were demonstrated in three AML cell lines and monocytes (FIG. 8).
[0200] V.delta.2 .gamma..delta.T cells were transduced with either an anti-CD33-Fc-DAP10 or anti-CD33-Fc-CD28-CD3z CAR construct.
[0201] The anti-CD33-Fc-CD28-CD3z CAR construct provides signal 1 and signal 2 in the presence of CD33. The anti-CD33-Fc-DAP10 provides signal 2 in the presence of CD33.
[0202] Cells transduced with the aCD33-CD28-CD3z CAR killed any CD33 positive cell and did not spare healthy monocytes. Cells transduced with the aCD33-Fc-DAP10 CAR do not kill monocytes (FIG. 9A).
[0203] There was significant enhancement of killing of the AML but no enhancement of the killing of monocytes by V.delta.2 .gamma..delta.T cells transduced with the aCD33-Fc-DAP10 CAR compared to non-transduced controls (FIG. 9B).
[0204] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cellular immunology or related fields are intended to be within the scope of the following claims.
Sequence CWU
1
1
271560PRTArtificial SequenceaCD33-Fc-DAP10 CAR (chimeric antigen receptor)
1Met Ala Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1
5 10 15 Asp Ala Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20
25 30 Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Glu Asp 35 40
45 Ile Tyr Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro 50 55 60
Lys Leu Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser 65
70 75 80 Arg Phe Ser Gly Ser
Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser 85
90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln His Tyr Lys 100 105
110 Asn Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 115 120 125 Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130
135 140 Gly Gly Gly Gly Ser Arg
Ser Glu Val Gln Leu Val Glu Ser Gly Gly 145 150
155 160 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser 165 170
175 Gly Phe Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro
180 185 190 Gly Lys
Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly Gly Ser 195
200 205 Thr Tyr Tyr Arg Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp 210 215
220 Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu 225 230 235
240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr Thr Gly Gly
245 250 255 Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met 260
265 270 Asp Pro Ala Glu Pro Lys Ser Pro
Asp Lys Thr His Thr Cys Pro Pro 275 280
285 Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro 290 295 300
Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys 305
310 315 320 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 325
330 335 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 340 345
350 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 355 360 365
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 370
375 380 Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 385 390
395 400 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu 405 410
415 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 420 425 430 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 435
440 445 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 450 455
460 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn 465 470 475
480 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
485 490 495 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe Trp 500
505 510 Val Leu Val Val Val Gly Gly
Val Leu Ala Cys Tyr Ser Leu Leu Val 515 520
525 Thr Val Ala Phe Ile Ile Phe Trp Val Cys Ala Arg
Pro Arg Arg Ser 530 535 540
Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 545
550 555 560
2551PRTArtificial SequenceaGD2-Fc-DAP10 CAR 2Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro 1 5
10 15 Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val 20 25
30 Lys Pro Ser Gln Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser 35 40 45 Leu
Ala Ser Tyr Asn Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly 50
55 60 Leu Glu Trp Leu Gly Val
Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn 65 70
75 80 Ser Ala Leu Met Ser Arg Leu Thr Ile Ser Lys
Asp Asn Ser Lys Asn 85 90
95 Gln Val Phe Leu Lys Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val
100 105 110 Tyr Tyr
Cys Ala Lys Arg Ser Asp Asp Tyr Ser Trp Phe Ala Tyr Trp 115
120 125 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135
140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Gln
Met Thr Gln Ser 145 150 155
160 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys
165 170 175 Arg Ala Ser
Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln 180
185 190 Lys Ser Gly Lys Ala Pro Lys Val
Trp Ile Tyr Ser Thr Ser Asn Leu 195 200
205 Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp 210 215 220
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225
230 235 240 Tyr Cys Gln Gln
Tyr Ser Gly Tyr Pro Ile Thr Phe Gly Gln Gly Thr 245
250 255 Lys Val Glu Ile Lys Arg Ser Asp Pro
Ala Glu Pro Lys Ser Pro Asp 260 265
270 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro 275 280 285
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala 290
295 300 Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 305 310
315 320 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 325 330
335 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 340 345 350 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355
360 365 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 370 375
380 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 385 390 395
400 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
405 410 415 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 420
425 430 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 435 440
445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 450 455 460
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 465
470 475 480 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485
490 495 Lys Lys Asp Pro Lys Phe Trp Val
Leu Val Val Val Gly Gly Val Leu 500 505
510 Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
Phe Trp Val 515 520 525
Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr 530
535 540 Ile Asn Met Pro
Gly Arg Gly 545 550 323PRTArtificial SequenceDAP10
signalling domain 3Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly
Lys Val Tyr 1 5 10 15
Ile Asn Met Pro Gly Arg Gly 20
41683DNAArtificial Sequencenucleic acid sequence which encodes a CAR,
aCD33-Fc-DAP10 CAR 4atggccgtgc ccactcaggt cctggggttg ttgctactgt
ggcttacaga tgccagatgt 60gacatccaga tgacacagtc tccatcttcc ctgtctgcat
ctgtcggaga tcgcgtcacc 120atcacctgtc gagcaagtga ggacatttat tttaatttag
tgtggtatca gcagaaacca 180ggaaaggccc ctaagctcct gatctatgat acaaatcgct
tggcagatgg ggtcccatca 240cggttcagtg gctctggatc tggcacacag tatactctaa
ccataagtag cctgcaaccc 300gaagatttcg caacctatta ttgtcaacac tataagaatt
atccgctcac gttcggtcag 360gggaccaagc tggaaatcaa aagatctggt ggcggagggt
caggaggcgg aggcagcgga 420ggcggtggct cgggaggcgg aggctcgaga tctgaggtgc
agttggtgga gtctgggggc 480ggcttggtgc agcctggagg gtccctgagg ctctcctgtg
cagcctcagg attcactctc 540agtaattatg gcatgcactg gatcaggcag gctccaggga
agggtctgga gtgggtctcg 600tctattagtc ttaatggtgg tagcacttac tatcgagact
ccgtgaaggg ccgattcact 660atctccaggg acaatgcaaa aagcaccctc taccttcaaa
tgaatagtct gagggccgag 720gacacggccg tctattactg tgcagcacag gacgcttata
cgggaggtta ctttgattac 780tggggccaag gaacgctggt cacagtctcg tctatggatc
ccgccgagcc caaatctcct 840gacaaaactc acacatgccc accgtgccca gcacctcccg
tggccggccc gtcagtcttc 900ctcttccccc caaaacccaa ggacaccctc atgatcgccc
ggacccctga ggtcacatgc 960gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt
tcaactggta cgtggacggc 1020gtggaggtgc ataatgccaa gacaaagccg cgggaggagc
agtacaacag cacgtaccgt 1080gtggtcagcg tcctcaccgt cctgcaccag gactggctga
atggcaagga gtacaagtgc 1140aaggtctcca acaaagccct cccagccccc atcgagaaaa
ccatctccaa agccaaaggg 1200cagccccgag aaccacaggt gtacaccctg cccccatccc
gggatgagct gaccaagaac 1260caggtcagcc tgacctgcct ggtcaaaggc ttctatccca
gcgacatcgc cgtggagtgg 1320gagagcaatg ggcaaccgga gaacaactac aagaccacgc
ctcccgtgct ggactccgac 1380ggctccttct tcctctacag caagctcacc gtggacaaga
gcaggtggca gcaggggaac 1440gtcttctcat gctccgtgat gcatgaggcc ctgcacaatc
actataccca gaaatctctg 1500agtctgagcc caggcaagaa ggaccccaag ttctgggtcc
tggtggtggt gggaggcgtg 1560ctggcctgtt actctctcct ggtgaccgtg gccttcatca
tcttctgggt gtgcgccaga 1620ccacggcgga gcccagccca ggaggacggc aaggtgtaca
tcaacatgcc cggccgcggc 1680tga
168351656DNAArtificial Sequencenucleic acid
sequence which encodes a CAR, aGD2-Fc-DAP10 CAR 5atggagaccg
acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc 60caggtgcagc
tgcaggagtc tggcccaggc ctggtgaagc ccagccagac cctgagcatc 120acctgcaccg
tgagcggctt cagcctggcc agctacaaca tccactgggt gcggcagccc 180ccaggcaagg
gcctggagtg gctgggcgtg atctgggctg gcggcagcac caactacaac 240agcgccctga
tgagccggct gaccatcagc aaggacaaca gcaagaacca ggtgttcctg 300aagatgagca
gcctgacagc cgccgacacc gccgtgtact actgcgccaa gcggagcgac 360gactacagct
ggttcgccta ctggggccag ggcaccctgg tgaccgtgag ctctggcgga 420ggcggctctg
gcggaggcgg ctctggcgga ggcggcagcg agaaccagat gacccagagc 480cccagcagct
tgagcgccag cgtgggcgac cgggtgacca tgacctgcag agccagcagc 540agcgtgagca
gcagctacct gcactggtac cagcagaaga gcggcaaggc cccaaaggtg 600tggatctaca
gcaccagcaa cctggccagc ggcgtgccca gccggttcag cggcagcggc 660agcggcaccg
actacaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 720tactgccagc
agtacagcgg ctaccccatc accttcggcc agggcaccaa ggtggagatc 780aagcggtcgg
atcccgccga gcccaaatct cctgacaaaa ctcacacatg cccaccgtgc 840ccagcacctc
ccgtggccgg cccgtcagtc ttcctcttcc ccccaaaacc caaggacacc 900ctcatgatcg
cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 960cctgaggtca
agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 1020ccgcgggagg
agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1080caggactggc
tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1140cccatcgaga
aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1200ctgcccccat
cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1260ggcttctatc
ccagcgacat cgccgtggag tgggagagca atgggcaacc ggagaacaac 1320tacaagacca
cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1380accgtggaca
agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1440gccctgcaca
atcactatac ccagaaatct ctgagtctga gcccaggcaa gaaggacccc 1500aagttctggg
tcctggtggt ggtgggaggc gtgctggcct gttactctct cctggtgacc 1560gtggccttca
tcatcttctg ggtgtgcgcc agaccacggc ggagcccagc ccaggaggac 1620ggcaaggtgt
acatcaacat gcccggccgc ggctga
1656621PRTArtificial Sequencesignal peptide 6Met 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
721PRTArtificial Sequencesignal peptide derived from IgG1 7Met 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 820PRTArtificial Sequencesignal peptide derived from CD8 8Met Ala
Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5
10 15 Asp Ala Arg Cys
20 937PRTArtificial Sequenceco-stimulatory domain, CD28 endodomain 9Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 1
5 10 15 Arg Pro Gly Pro Thr Arg
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 20
25 30 Asp Phe Ala Ala Tyr 35
1048PRTArtificial Sequenceco-stimulatory domain, CD27 endodomain 10Gln
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 1142PRTArtificial Sequenceco-stimulatory domain, 41BB
endodomain 11Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
Met 1 5 10 15 Arg
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30 Pro Glu Glu Glu Glu
Gly Gly Cys Glu Leu 35 40
1237PRTArtificial Sequenceco-stimulatory domain, OX40 endodomain 12Arg
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 1
5 10 15 Gly Ser Phe Arg Thr Pro
Ile Gln Glu Glu Gln Ala Asp Ala His Ser 20
25 30 Thr Leu Ala Lys Ile 35
13188PRTArtificial Sequenceco-stimulatory domain, CD30 endodomain 13His
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 1413PRTArtificial Sequenceco-stimulatory
domain, IL2-R endodomain 14Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg
Thr Ile 1 5 10
15195PRTArtificial Sequenceco-stimulatory domain, IL7-R endodomain 15Lys
Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys 1
5 10 15 Lys Thr Leu Glu His Leu
Cys Lys Lys Pro Arg Lys Asn Leu Asn Val 20
25 30 Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys
Gln Ile His Arg Val Asp 35 40
45 Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp
Thr Phe 50 55 60
Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val 65
70 75 80 Gln Ser Pro Asn Cys
Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser 85
90 95 Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu
Ala Gly Asn Val Ser Ala 100 105
110 Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg
Glu 115 120 125 Ser
Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu 130
135 140 Gly Thr Thr Asn Ser Thr
Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly 145 150
155 160 Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln
Pro Ile Leu Thr Ser 165 170
175 Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr
180 185 190 Gln Asn
Gln 195 16285PRTArtificial Sequenceco-stimulatory domain, IL21-R
endodomain 16Ser Leu Lys Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp
Ala 1 5 10 15 Val
Pro Ser Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser
20 25 30 Gly Asp Phe Lys Lys
Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu 35
40 45 Glu Leu Gly Pro Trp Ser Pro Glu Val
Pro Ser Thr Leu Glu Val Tyr 50 55
60 Ser Cys His Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln
Leu Thr Glu 65 70 75
80 Leu Gln Glu Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro
85 90 95 Ser Phe Trp Pro
Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu 100
105 110 Glu Arg Asp Arg Pro Tyr Gly Leu Val
Ser Ile Asp Thr Val Thr Val 115 120
125 Leu Asp Ala Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu
Asp Asp 130 135 140
Gly Tyr Pro Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly 145
150 155 160 Leu Glu Asp Pro Leu
Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly 165
170 175 Cys Val Ser Ala Gly Ser Pro Gly Leu Gly
Gly Pro Leu Gly Ser Leu 180 185
190 Leu Asp Arg Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala
Gly 195 200 205 Gly
Leu Pro Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu 210
215 220 Ala Gly Ser Pro Leu Ala
Gly Leu Asp Met Asp Thr Phe Asp Ser Gly 225 230
235 240 Phe Val Gly Ser Asp Cys Ser Ser Pro Val Glu
Cys Asp Phe Thr Ser 245 250
255 Pro Gly Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val
260 265 270 Ile Pro
Pro Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 275
280 285 1745PRTArtificial Sequenceco-stimulatory domain,
NKp30 endodomain 17Gly Ser Thr Val Tyr Tyr Gln Gly Lys Cys Leu Thr Trp
Lys Gly Pro 1 5 10 15
Arg Arg Gln Leu Pro Ala Val Val Pro Ala Pro Leu Pro Pro Pro Cys
20 25 30 Gly Ser Ser Ala
His Leu Leu Pro Pro Val Pro Gly Gly 35 40
45 1863PRTArtificial Sequenceco-stimulatory domain, NKp44
endodomain 18Trp Trp Gly Asp Ile Trp Trp Lys Thr Met Met Glu Leu Arg Ser
Leu 1 5 10 15 Asp
Thr Gln Lys Ala Thr Cys His Leu Gln Gln Val Thr Asp Leu Pro
20 25 30 Trp Thr Ser Val Ser
Ser Pro Val Glu Arg Glu Ile Leu Tyr His Thr 35
40 45 Val Ala Arg Thr Lys Ile Ser Asp Asp
Asp Asp Glu His Thr Leu 50 55 60
1961PRTArtificial Sequenceco-stimulatory domain, DNAM-1 (CD226)
endodomain 19Asn Arg Arg Arg Arg Arg Glu Arg Arg Asp Leu Phe Thr Glu
Ser Trp 1 5 10 15
Asp Thr Gln Lys Ala Pro Asn Asn Tyr Arg Ser Pro Ile Ser Thr Ser
20 25 30 Gln Pro Thr Asn Gln
Ser Met Asp Asp Thr Arg Glu Asp Ile Tyr Val 35
40 45 Asn Tyr Pro Thr Phe Ser Arg Arg Pro
Lys Thr Arg Val 50 55 60
20263PRTArtificial Sequenceantigen binding domain 20Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5
10 15 Gly Ser Thr Gly Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val 20 25
30 Lys Pro Ser Gln Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser 35 40 45 Leu
Ala Ser Tyr Asn Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly 50
55 60 Leu Glu Trp Leu Gly Val
Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn 65 70
75 80 Ser Ala Leu Met Ser Arg Leu Thr Ile Ser Lys
Asp Asn Ser Lys Asn 85 90
95 Gln Val Phe Leu Lys Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val
100 105 110 Tyr Tyr
Cys Ala Lys Arg Ser Asp Asp Tyr Ser Trp Phe Ala Tyr Trp 115
120 125 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135
140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Gln
Met Thr Gln Ser 145 150 155
160 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys
165 170 175 Arg Ala Ser
Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln 180
185 190 Lys Ser Gly Lys Ala Pro Lys Val
Trp Ile Tyr Ser Thr Ser Asn Leu 195 200
205 Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp 210 215 220
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 225
230 235 240 Tyr Cys Gln Gln
Tyr Ser Gly Tyr Pro Ile Thr Phe Gly Gln Gly Thr 245
250 255 Lys Val Glu Ile Lys Arg Ser
260 21272PRTArtificial Sequenceantigen binding domain
21Met Ala Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1
5 10 15 Asp Ala Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20
25 30 Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Glu Asp 35 40
45 Ile Tyr Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro 50 55 60
Lys Leu Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser 65
70 75 80 Arg Phe Ser Gly Ser
Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser 85
90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln His Tyr Lys 100 105
110 Asn Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 115 120 125 Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130
135 140 Gly Gly Gly Gly Ser Arg
Ser Glu Val Gln Leu Val Glu Ser Gly Gly 145 150
155 160 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser 165 170
175 Gly Phe Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro
180 185 190 Gly Lys
Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly Gly Ser 195
200 205 Thr Tyr Tyr Arg Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp 210 215
220 Asn Ala Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu 225 230 235
240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr Thr Gly Gly
245 250 255 Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met 260
265 270 22234PRTArtificial
Sequencespacer sequence, hinge-CH2CH3 of human IgG1 22Ala Glu Pro Lys Ser
Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5
10 15 Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 20 25
30 Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val
Val 35 40 45 Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50
55 60 Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70
75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 85 90
95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110 Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115
120 125 Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135
140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser 145 150 155
160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175 Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180
185 190 Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 195 200
205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp 225 230
2346PRTArtificial Sequencespacer sequence, human CD8 stalk 23Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1
5 10 15 Ser Gln Pro Leu Ser Leu
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20
25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe
Ala Cys Asp Ile 35 40 45
2420PRTArtificial Sequencespacer sequence, human IgG1 hinge 24Ala Glu Pro
Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5
10 15 Lys Asp Pro Lys 20
2527PRTArtificial SequenceCD28 transmembrane domain 25Phe 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 26114PRTArtificial
SequenceCD3 zeta endodomain 26Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
Ala Pro Ala Tyr Gln 1 5 10
15 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
20 25 30 Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 35
40 45 Gly Lys Pro Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu 50 55
60 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly 65 70 75
80 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
85 90 95 Thr Ala Thr
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 100
105 110 Pro Arg 274PRTArtificial
Sequenceactivation motifmisc_feature(2)..(3)Xaa can be any naturally
occurring amino acid 27Tyr Xaa Xaa Met 1
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