PEPTIDE-INDUCED NK CELL ACTIVATION

Abstract

The invention relates to a method of treatment or prophylaxis of cancer, wherein the cancer overexpresses exportin-1, the method comprising the administration of: a peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or administration of one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide.

Claims

1. A method of treatment or prophylaxis of cancer, wherein the cancer overexpresses exportin-1, the method comprising the administration of: a peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or administration of one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide.

2. The method of treatment according to claim 1, wherein the peptide comprises or consist of the amino acid sequence X.sup.nHATX.sup.y wherein X.sup.n is an amino acid sequence of between 5 and 11 residues and wherein X.sup.y is any amino acid, L or F.

3. The method of treatment according to claim 1, wherein the peptide comprises or consists of the sequence NAPLVHATL, IVDLMCHATF, or LNPSVAATL.

4. The method of treatment according to any preceding claim, wherein the peptide is between about 8 and about 15 amino acid residues in length.

5. The method of treatment according to any preceding claim, wherein the treatment further comprise the administration of an MHC class I molecule, or nucleic acid encoding an MHC class I molecule.

6. The method of treatment according to claim 5, wherein the MHC class I molecule is: provided as a complex with the peptide; encoded in the nucleic acid encoding the peptide; provided in the immunogenic composition comprising the peptide; provided in the vesicle comprising the peptide or encoded in the nucleic acid encoding the peptide in the vesicle; provided in the dendritic cell comprising the peptide and/or encoded in the nucleic acid encoding the peptide in the dendritic cell; or provided in the virus or virus like particle comprising the peptide and/or encoded in the nucleic acid encoding the peptide in the virus or virus like particle.

7. The method of treatment according to any preceding claim, wherein the activated NK cell, has been activated by the peptide complexed with an MHC class I molecule.

8. The method of treatment according to any of claims 5 to 7, wherein the MHC class I molecule comprises an MHC class I truncated at the stem region of the α3 domain.

9. The method of treatment according to any of claims 5 to 8, wherein the MHC class I molecule comprises HLA-C, or part thereof.

10. The method of treatment according to any preceding claim, wherein the cancer is a cancer selected from the group comprising liver cancer, melanoma, lymphoma, pancreatic cancer, breast cancer, lung cancer, renal cancer, leukaemia, acute myeloid/lymphoid leukaemia, chronic myeloid/lymphoid leukemia, multiple myeloma, osteosarcoma, glioma, ovarian cancer, cervical carcinoma, oesophageal carcinoma, gastric carcinoma, hepatocellular carcinoma, mantle cell lymphoma, plasma cell leukemia, and blood cancer.

11. A method of treatment or prophylaxis of a patient for cancer, wherein the cancer overexpresses exportin-1, the method comprising determining if a patient produces KIR2DS2-expressing NK cells and/or a ligand for KIR2DS2, and determining if the cancer overexpresses exportin-1; wherein if the patient produces KIR2DS2-expressing NK cells and/or a ligand for KIR2DS2, and the cancer overexpresses exportin-1, administering: a peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or administration of one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide; and optionally wherein if the patient does not produce KIR2DS2-expressing NK cells and/or a ligand for KIR2DS2, administering to the patient activated NK cells that have been activated by the peptide.

12. A method of selecting a patient for treatment or prophylaxis with an agent arranged to activate NK cell mediated protection from cancer, wherein the cancer overexpresses exportin-1, wherein the agent is selected from: a peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide; the method comprising the step of determining if the patient produces KIR2DS2-expressing NK cells and/or a ligand for KIR2DS2, and determining if the cancer overexpresses exportin-1, wherein a patient is selected for the treatment or prophylaxis with the agent if they produce KIR2DS2-expressing NK cells and/or a ligand for KIR2DS2, and the cancer overexpresses exportin-1; and optionally wherein a patient not producing KIR2DS2-expressing NK cells and/or a ligand for KIR2DS2, and the cancer does not overexpress exportin-1, the patient is not selected for the treatment or prophylaxis with the agent, and/or is selected for an alternative cancer treatment.

13. A method of producing activated NK cells comprising exposing an NK cell expressing KIR2DS2 receptor to a peptide comprising or consisting of the sequence NAPLVHATL and optionally an MHC class I molecule.

14. A method for activating an NK cell mediated immune response of a patient for recognition of exportin-1 comprising administration of: a peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide;

15. Use of: a peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide; for activating an NK cell mediated immune response of a patient for recognition and killing of cells overexpressing exportin-1, or a part thereof.

16. A peptide capable of activating NK cell-mediated immunity to cancer cells that overexpress exportin-1, the peptide comprising or consisting of the amino acid sequence X.sup.nAX.sup.2X.sup.1, wherein X.sup.n is an amino acid sequence of between 5 and 12 residues, and X.sup.1 is any amino acid; or leucine or phenylalanine; and X.sup.2 is alanine, threonine or serine; or one or more of a nucleic acid encoding the peptide; an immunogenic composition comprising the peptide; a complex comprising the peptide; a vesicle comprising the peptide or nucleic acid encoding the peptide; a dendritic cell comprising the peptide and/or comprising nucleic acid encoding the peptide; an activated NK cell, that has been activated by the peptide; or a virus or virus like particle comprising the peptide and/or comprising nucleic acid encoding the peptide; for use as a vaccine against exportin-1, or cells that overexpress exportin-1, or for use to treat cancer, wherein the cancer cells overexpress exportin-1.

17. A nucleic acid encoding a peptide comprising or consisting of the amino acid sequence NAPLVHATL and an MHC class I molecule.

18. An immunogenic composition comprising a peptide comprising or consisting of the amino acid sequence NAPLVHATL and an MHC class I molecule.

19. A complex comprising a peptide comprising or consisting of the amino acid sequence NAPLVHATL and an MHC class I molecule.

20. A vesicle comprising a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally an MHC class I molecule; and/or comprising nucleic acid encoding a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally an MHC class I molecule.

21. A dendritic cell comprising a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally an MHC class I molecule; and/or comprising nucleic acid encoding a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally nucleic acid encoding the MHC class I molecule.

22. An activated NK cell, that has been activated by a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally an MHC class I molecule.

23. A virus or virus like particle comprising a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally an MHC class I molecule; and/or comprising nucleic acid encoding a peptide comprising or consisting of the amino acid sequence NAPLVHATL and optionally nucleic acid encoding the MHC class I molecule.

Description

[0246] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0247] FIG. 1—Exportin-1 is recognised by natural killer cells expressing KIR2DS2.

[0248] A flow cytometry plots of CD107a on KIR2DS2+ or KIR2DS2− NK cells are shown when they are incubated in the absence of targets, or in the presence of HUH-7:C*0102 cells transfected with control siRNA or an siRNA that targets exportin1. CD3-CD56+ NK cells are shown.

[0249] B, a comparison is made of the degranulation (CD107a expression) of KIR2DS2+ or KIR2DS2− NK cells when cultured with the HUH7-C*0102 cell lines transfected with either control siRNA or siRNA specific for exportin-1. The data are from six experiments and are expressed as the relative ratios of CD107a expression on the KIR2DS2+ and KIR2DS2− NK cells from six experiments.

[0250] FIG. 2—Vaccination with HLA-C*0102-T2A-E3/19k-IVDVLMCHATF induces an anti-cancer response to HUH-7 cells.

[0251] FIG. 3—Vaccination with HLA-C*0102-T2A-E3/19k-LNPSVAATL gives similar results to vaccination with HLA-C*0102-T2A-E3/19k-IVDVLMCHATF.

[0252] FIG. 4—Vaccination with HLA-C*0102-T2A-E3/19k-IVDVLMCHATF induces an anti-cancer response to lymphoma.

[0253] FIG. 5—Vaccination with HLA-C*0102-T2A-E3/19k-IVDVLMCHATF induces an anti-cancer response to melanoma.

[0254] FIG. 6—Knockdown of Exportin-1 decreases killing of HUH-7 cells by KIR2DS2-positive NK cells but not KIR2DS2-negative NK cells.

[0255] FIG. 7—NAPLVHATL, a peptide derived from Exportin-1 binds KIR2DS2.

EXAMPLE 1

Exportin-1 is Recognised by Natural Killer Cells Expressing KIR2DS2

[0256] With Reference to FIG. 1, natural killer (NK) cells were incubated with the liver cancer cell line HUH-7 transfected with HLA-C*0102 (HUH-7:C*0102) and then stained for expression of CD107a, a marker of NK cell degranulation/activation. We observed a significant decrease if the HUH-7:C*0102 were transfected with an interfering RNA (siRNA) specific for exportin-1 as compared to a control siRNA.

EXAMPLE 2

Vaccination with HLA-C*0102-T2A-E3/19K-IVDVLMCHATF Induces an Anti-Cancer Response to HUH-7 Cells

[0257] KIR-transgenic mice were vaccinated with two 50 microgram doses of the DNA vaccine HLA-C*0102-T2A-E3/19k-IVDVLMCHATF, or a control vaccine in which the peptide was mutated to prevent HLA-C binding HLA-C*0102-T2A-E3/19k-IVDVLMCHAAA. Vaccine doses were given one week apart. NOD-SCID-gamma chain knockout (NSG) mice were inoculated with HUH7 cells expressing HLA-C*0102. On days 0 and 21 after tumour challenge NK cells from the vaccinated mice they were infused with purified NK cells from the vaccinated mice. Tumour growth was monitored by measuring and mice sacrificed at a humane endpoint, when the tumour volume exceeds 2000 mm.sup.3. Tumour growth was significantly attenuated in the vaccine group (Panel A: tumor volume measurement, p<0.02) and survived significantly longer in the vaccinated group (Panel B: Kaplan-Meier plot, p<0.02) (5 mice per group)

EXAMPLE 3

Vaccination with HLA-C*0102-T2A-E3/19K-LNPSVAATL Gives Similar Results to Vaccination with HLA-C*0102-T2A-E3/19K-IVDVLMCHATF

[0258] KIR-transgenic mice were vaccinated with two 50 microgram doses of the DNA vaccine HLA-C*0102-T2A-E3/19k-LNPSVAATL or HLA-C*0102-T2A-E3/19k-IVDVLMCHATF, or a control vaccine containing only the HLA-C construct (HLA-C*0102). Tumour growth was then monitored. Each line indicates a different mouse. LNPSVAATL is shown by the dashed line and open circles.

EXAMPLE 4

Vaccination with HLA-C*0102-T2A-E3/19K-IVDVLMCHATF Induces an Anti-Cancer Response to Lymphoma

[0259] KIR-transgenic mice were vaccinated with two 50 microgram doses of the DNA vaccine HLA-C*0102-T2A-E3/19k-IVDVLMCHATF, or a control vaccine containing only the HLA-C construct (HLA-C*0102) and simultaneously challenged with RMA-S lymphoma cells. Vaccine doses were given on day 0 and day 7. Tumour volume was then monitored. Mice from the vaccinate group had significantly less tumour growth (p<0.01). (3 mice per group)

EXAMPLE 5

Vaccination with HLA-C*0102-T2A-E3/19K-IVDVLMCHATF Induces an Anti-Cancer Response to Melanoma

[0260] KIR-transgenic mice were vaccinated with two 50 microgram doses of the DNA vaccine HLA-C*0102-T2A-E3/19k-IVDVLMCHATF, or a control vaccine in which the peptide was mutated to prevent HLA-C binding HLA-C*0102-T2A-E3/19k-IVDVLMCHAAA. and simultaneously challenged with B16 melanoma cells. Vaccine doses were given on day 0 and day 7. Tumour volume was then monitored. Results from days 9 and 12 are shown in panel A (p<0.01 vs control vaccine or versus no vaccine) and demonstrated that there is significant attenuation of overall survival in panel B. Mice were sacrificed at a human endpoint when tumour volume exceeded 2000 mm.sup.3.

EXAMPLE 6

Knockdown of Exportin-1 Decreases Killing of HUH-7 Cells by KIR2DS2-Positive NK Cells but not KIR2DS2-Negative NK Cells

[0261] HUH-7-C*0102 cells were transfected with siRNA to reduce expression of Exportin-1. These cells were then tested for killing by KIR2DS2+NK cells or KIR2DS2− NK cells as measured using a CD107a degranulation assay. Knockdown of exportin-1 reduced degranlulation of KIR2DS+NK cells from 40.6% to 26.5%, whereas there was a minimal change for the KIR2DS2− population

EXAMPLE 7

NAPLVHATL, a Peptide Derived from Exportin-1 Binds KIR2DS2

[0262] 721.174 cells were loaded with peptide and then stained using a tetramer of KIR2DS2 conjugated to phycoerythrin. Shown is a flow cytometry histogram plot of the staining of the 721.174 cells in the presence or absence of peptide. Mean fluorescence intensitites are indicated: 14811 for NAPLVHATL versus 10503 for no peptide.

EXAMPLE 8

HLA Binding

[0263]

TABLE-US-00005 TABLE 1 Amino acid sequences of NAPLVHATL, LNPSVAATL and IVDLMCHATF with homology at carboxy-terminal -1 and -2 positions indicated in bold. The Rank score from a NetMHCpan 4.0 Server analysis is shown, with a Rank score of <2 is predictive of binding to the HLA-C allele indicated. Source of Amino acid Rank (Predicted peptide sequence HLA allele binding stren XPO1 NAPLVHATL HLA-C*0102 0.138 XPO1 NAPLVHATL HLA-C*0801 0.494 XPO1 NAPLVHATL HLA-C*1601 0.728 XPO1 NAPLVHATL HLA-C*1801 1.574 XPO1 NAPLVHATL HLA-C*1402 0.959 Hepatitis C virus LNPSVAATL HLA-C*0102 1.812 Dengue virus IVDLMCHATF HLA-C*0102 3.839

[0264] Discussion

[0265] We have previously described a DNA vaccine in the formation HLA-C:T2A:E3/19k:peptide conformation where peptide is a 5-12 amino acid peptide in the conformation X(n)ATX where n=2-9. We now describe the use of such vaccine using the exemplar HLA-C*0102-T2A-E3/19k-IVDVLMCHATF to activate natural killer (NK) cells expressing the receptor KIR2DS2 as a cancer therapy targeting cancers that express Exportin 1. We have vaccinated KIR-transgenic mice with DNA encoding HLA-C*0102-T2A-E3/19k-IVDVLMCHATF and then adoptively transferred the NK cells from these mice into NOD-SCID-gamma chain deficient mice (NSG). We have challenged these mice with the liver cancer cell line HUH7 expressing HLA-C*0102 (HUH7-C*0102). This has resulted in significant impairment of tumour growth in mice given NK cells from the vaccinated mice, as opposed to mice given NK cells from control vaccinated mice. We have repeated this experiment 3 times with similar results in attenuation of tumour growth. We also show improved survival in the mice given NK cells from vaccinated mice (see FIG. 2B). We also have shown that the vaccine HLA-C*0102-T2A-E3/19k-LNPSVAATL also reduces tumour growth.

[0266] We have identified that the tumor cell line HUH7-C*0102 expresses Exportin-1 (XpoI) and that the peptide NAPLVHATL derived from this protein is presented by HLA-C*0102 to NK cells. In vitro assays using human NK cells knock-down of this peptide reduces the killing of the cell line by human NK cells expressing the receptor KIR2DS2, but not by NK cells not expressing the receptor KIR2DS2. Therefore Exportin-1 provides a peptide NAPLVHATL that is recognised by KIR2DS2. Therefore our vaccine specifically activates NK cells expressing KIR2DS2 and these can kill cancer cells over expressing Exportin-1. Therefore we propose that a KIR2DS2 targeting vaccine can target cancers over expressing Exportin-1.

[0267] We have also shown that using the vaccine HLA-C*0102-T2A-E3/19k-IVDVLMCHATF we can attenuate the growth of murine melanoma and lymphoma cells. We thus propose that a KIR2DS2 targeting vaccine has utility against liver cancer, melanoma and lymphoma. In general these are exemplars of tumours expressing Exportin-1, but we have not demonstrated that the NAPLVHATL is presented by these specific tumour models (unlike for the HUH7 cells). Exportin-1 has however been noted to be overexpressed in lung cancer, osteosarcoma, glioma, pancreatic cancer, ovarian cancer, cervical carcinoma, renal cell carcinoma, esophageal carcinoma, gastric carcinoma, hepatocellular carcinoma, acute myeloid/lymphoid leukemia, chronic myeloid/lymphoid leukemia, mantle cell lymphoma, plasma cell leukemia and multiple myeloma. Thus, these represent targets for this approach.