PEPTIDE-INDUCED NK CELL ACTIVATION

Abstract

The invention relates to a peptide capable of activating NK cell-mediated immunity, the peptide comprising or consisting of the amino acid sequence XnAX2X1, Wherein Xn is an amino acid sequence of between 5 and 12 residues, and X1 is any amino acid; or leucine or phenylalanine; and X2 is alanine, threonine or serine. The invention further relates to an MHC class I molecule and the peptide, nucleic acids encoding the peptide, activated NK cells, and related compositions and methods, including use in methods of treatment.

Claims

1. A peptide capable of activating NK cell-mediated immunity, 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.

2. The peptide according to claim 1, wherein X.sup.n is an amino acid sequence of 7 residues.

3. The peptide according to claim 1 or claim 2, wherein X.sup.1 is leucine.

4. The peptide according to claim 1 or claim 2, wherein X.sup.1 is phenylalanine.

5. The peptide according to any preceding claim, wherein X.sup.2 is threonine.

6. The peptide according to any of claims 1 to 4, wherein X.sup.2 is serine.

7. The peptide according to any preceding claim, wherein the peptide amino acid sequence comprises or consists of any sequence selected from the group comprising: IVDLMCHATF (SEQ ID NO: 1); VIDAMCHATL (SEQ ID NO: 2); AANVMAASL (SEQ ID NO: 3); and FLSDVPVATL (SEQ ID NO: 4).

8. The peptide according to claim 1, wherein the peptide comprises or consists of the amino acid sequence IVDLMCHATF (SEQ ID NO: 1).

9. The peptide according to claim 1, wherein the peptide comprises or consists of the amino acid sequence VIDAMCHATL (SEQ ID NO: 2).

10. The peptide according to claim 1, wherein the peptide comprises or consists of the amino acid sequence AANVMAASL (SEQ ID NO: 3).

11. The peptide according to claim 1, the peptide comprises or consists of the amino acid sequence FLSDVPVATL (SEQ ID NO: 4).

12. The peptide according to any one of claims 1, and 3 to 6, wherein the peptide is between about 8 and about 15 amino acid residues in length.

13. The peptide according to any preceding claim, wherein the peptide is an isolated peptide.

14. A complex comprising an MHC class I molecule and the peptide according to any of the preceding claims.

15. The complex according to claim 14, wherein the MHC class 1 molecule comprises HLA-C.

16. The complex according to claim 15, wherein the HLA-C is a group 1 HLA-C.

17. The complex according to any of claims 14 to 16, wherein the MHC class I molecule comprises HLA-Cw*0102 or HLA-C*304.

18. The complex according to any of claims 14 to 17, wherein the MHC class I molecule is an MHC class I truncated at the stem region of the ?3 domain.

19. The complex according to any of claims 14 to 18, wherein the complex comprises a fusion protein of the MHC class I molecule and the peptide.

20. The complex according to any of claims 14 to 19, wherein the complex is an isolated complex.

21. A vesicle comprising the complex according to any of claims 14 to 20.

22. An activated NK cell, wherein the NK cell expresses KIR2DS2 receptor, and wherein the NK cell is activated by exposure to the peptide according to any of claims 1 to 13.

23. A nucleic acid comprising a sequence encoding a peptide according to any of claims 1 to 13.

24. The nucleic acid according to claim 23, wherein the nucleic acid further comprises a sequence encoding an MHC class I molecule.

25. The nucleic acid according to claim 23 or claim 24, wherein the nucleic acid is a plasmid vector for vaccination.

26. A virus comprising the nucleic acid according to claim 23 or claim 24.

27. A dendritic cell expressing, or capable of expressing, the complex according to any of claims 14 to 20.

28. An immunogenic composition comprising one or more of: the peptide according to any of claims 1 to 13; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; and the virus according to claim 26.

29. A method of treatment or prophylaxis of an NK cell regulated disease comprising the administration of: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 30; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the activated NK cell according to claim 22; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26.

30. An agent for use in the prophylaxis or treatment of an NK cell regulated disease, the agent comprising or consisting of: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the activated NK cell according to claim 22; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26.

31. A method of treatment or prophylaxis of a patient for an NK cell regulated disease comprising determining if a patient produces KIR2DS2-expressing NK cells; wherein if the patient produces KIR2DS2-expressing NK cells, administering: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26; and optionally wherein if the patient does not produce KIR2DS2-expressing NK cells, administering to the patient the activated NK cell according to claim 22.

32. A method of treatment or prophylaxis of a patient for an NK cell regulated disease comprising determining if a patient produces a ligand for KIR2DS2; wherein if the patient produces a ligand for KIR2DS2, administering: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26; and optionally wherein if the patient does not produce a ligand for KIR2DS2, administering to the patient the activated NK cell according to claim 22.

33. A method of selecting a patient for treatment or prophylaxis with an agent arranged to activate NK cell mediated protection from a disease, wherein the agent is selected from: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26; the method comprising the step of determining if the patient produces KIR2DS2-expressing NK cells, wherein a patient producing KIR2DS2-expressing NK cells is selected for the treatment or prophylaxis with the agent; and optionally wherein a patient not producing KIR2DS2-expressing NK cells is not selected for the treatment or prophylaxis with the agent, and/or is selected for an alternative treatment.

34. A method of selecting a patient for treatment or prophylaxis with an agent arranged to activate NK cell mediated protection from a disease, wherein the agent is selected from: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26; the method comprising the step of determining if the patient produces a ligand for KIR2DS2, wherein a patient producing a ligand for KIR2DS2 is selected for the treatment or prophylaxis with the agent; and optionally wherein a patient not producing a ligand for KIR2DS2 is not selected for the treatment or prophylaxis with the agent, and/or is selected for an alternative treatment.

35. The method for selecting a patient according to claim 33 or 34 further comprises administering the agent, or the alternative treatment, to the selected patient.

36. The method for selecting a patient according to any of claims 33 to 35, wherein the alternative treatment comprises administering to the patient, the activated NK cell according to claim 18.

37. A method of producing activated NK cells comprising exposing an NK cell expressing KIR2DS2 receptor to a peptide according to any of claims 1 to 13.

38. A method for activating an NK cell mediated immune response of a patient for recognition of an antigen comprising administration of: the peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26.

39. The peptide according to any of claims 1 to 13; the immunogenic composition according to claim 28; the complex according to any of claims 14 to 20; the vesicle according to claim 21; the dendritic cell according to claim 27; the nucleic acid according to any of claims 23 to 25; or the virus according to claim 26; for use in, or as, a vaccine.

40. The peptide, immunogenic composition, the complex, the vesicle, the dendritic cell, the nucleic acid, the virus, the activated NK cell, the use, or the methods substantially described herein, with reference to the accompanying drawings.

Description

[0201] FIG. 1 shows that the peptide LNPSVAATL (SEQ ID NO: 5) binds HLA-Cw*0102. The HLA-Cw*0102-positive cell line L721.174 was incubated with increasing concentrations of the LNPSVAATL (SEQ ID NO: 5) peptide or a control peptide VAPWNSLSL (SEQ ID NO: 11). Cells were stained for cell surface of MHC class I and analysed by flow cytometry. The mean fluorescence intensity of cell surface MHC Class I was plotted against the peptide concentration.

[0202] FIG. 2 shows that LNPSVAATL (SEQ ID NO: 5) is recognized by KIR2DS2-positive NK cells and leads to NK cell activation (CD107a degranulation), and killing of target cells expressing HLA-Cw*0102. A. cells were incubated in the presence or absence of peptides, and then cultured with NK cells from either a KIR2DS2-positive or KIR2DS2-negative donor. The level of CD107a (LAMP) expression was measured on NK cells positive for the CH-L antibody. This antibody binds to KIR2DS2, KIR2DL2 and KIR2DL3. B. Summary data from 5 KIR2DS2-positive and 6 KIR2DS2 [0203] negative donors tested as per panel A of FIG. 2. To compare the different donors the data are normalized to 100% for the no peptide condition.

[0204] FIG. 3 shows that KIR2DS2-positive NK cell clones kill target cells expressing HLA-Cw*0102 and the LNPSVAATL (SEQ ID NO: 5) peptide. L721.174 cells were incubated in the presence or absence of the peptides VAPWNSFAL (SEQ ID NO: 12) and the LNPSVAATL (SEQ ID NO: 5). The cells were then labeled with the cell tracker orange dye, and then cultured with KIR2DS2-positive NK cell clones (clones 4,6,9) or a KIR2DS2-negative NK cell clone (clone 27). Cytotoxicity was then determined using Live/Dead? fixable far-red stain.

[0205] FIG. 4 shows that KIR2DS2 binds to HLA-C*0102 and the LNPSVAATL (SEQ ID NO: 5) peptide. A. The KIR2DS2 protein was expressed with a biotinylation C-terminal tag. Following biotinylation with the BirA enzyme, the KIR2DS2 protein was incubated with streptavidin-phycoerythrin to form fluorescent KIR2DS2-tetramers (KIR2DS2-PE). L721.174 cells were then incubated with indicated peptides. Cells were then stained with KIR2DS2-PE and analysed by flow cytometry. Shown are histogram plots. The bold numbers represent the mean fluorescent staining intensity of the KIR2DS2-PE staining of the cells in the presence of peptide and the non-bold numbers the staining in the absence of peptide. B. Represents the mean and standard error fluorescent intensities of the experiment in A following replicate experiments.

[0206] FIG. 5 shows that KIR2DS2 recognises peptides with XXXXXAAL (SEQ ID NO: 6) and XXXXXATL (SEQ ID NO: 7) motifs. A. L721.174 cells were incubated with indicated peptides. Cells were then stained with KIR2DS2-PE (see FIG. 4) and analysed by flow cytometry. Shown are histogram plots. The bold numbers represent the mean fluorescent staining intensity of the KIR2DS2-PE staining of the cells in the presence of peptide and the non-bold numbers the staining in the absence of peptide. B. Represents the mean and standard error fluorescent intensities of the experiment in A following replicate experiments.

[0207] FIG. 6 LNPSVAATL (SEQ ID NO: 5) activates NK cells expressing KIR2DS2. A. L721.174 cells were incubated with indicated peptides. The cells were then incubated with the NK cell line transfected with KIR2DS2. The cells were lysed and the KIR2DS2 was then immunoprecipitated from the mixture using the GL183 antibody coupled to protein-G beads. The immune precipitate was analysed by Western blotting using a phosphotyrosine antibody. The membrane was then stripped and reprobed with an antibody to DAP12 the molecule that transduces a positive signal from KIR2DS2. Shown are the regions on the blots at the level of DAP12 (12-15 kDa). B. L721.174 cells were incubated with indicated peptides. The cells were then incubated with the NK cell line transfected with KIR2DS2 or transfected with KIR2DL2. The cells were lysed in 1% NP-40 and then analysed by Western blotting using antibodies to pVav1 and then following stripping of the membrane to Vav1. Shown are Western blots around the 98 kDa region, and below them the corresponding densitometries for 3 independent experiments.

[0208] FIG. 7 1?10.sup.6 L721.221-HLA-C*0304-ICP47 cells were incubated overnight at 26? C. with indicated peptides at a concentration of 2004. The cells were then incubated with the NKL cell line transfected with KIR2DS2 or transfected with KIR2DL2 for 5 minutes. The cells were lysed in 1% NP-40 and then analysed by Western blotting using antibodies to pVav1 and then following stripping of the membrane to Vav1. Shown are Western blots around the 98 kDa region, and below them the corresponding densitometries for 2 independent experiments.

[0209] FIG. 8 The MHC class I-negative 721.221 cell line was transfected with a construct expressing HLA-C*0102:T2A:E19/3K:LNPSVAATL (SEQ ID NO: 5). The cells were washed and then the HLA-C binding peptides eluted using 10% acetic acid. The eluate was analysed by HPLC using a C18 column and a 4-60% acetonitrile gradient. The resulting profile was compared to the profile of the LNPSVAATL (SEQ ID NO: 5) peptide alone. The peptide elution peak is indicated with an arrow.

[0210] FIG. 9shows KIR2DS2 tetramer staining on 174 cells with and without the peptides. Staining comparison with the unstained cells, MFI of stained cells (indicated by *) is shown.

[0211] FIG. 10Peptide sequence (SEQ ID NO: 10) of a DNA construct of the sequence HLA-C*0102-T2A-E19/K-LNPSVAATL used to sensitize 721.221 cells for lysis by KIR2DS2-positive NK cells

[0212] FIG. 11Shows that a DNA construct of the sequence HLA-C*0102-T2A-E19/K-LNPSVAATL can sensitize cells to lysis by KIR2DS2-positive NK cells. The DNA construct of the sequence C*0102-T2A-E19/K-LNPSVAATL (FIG. 10) was transfected into 721.221 cells. A DNA construct containing only HLA-C*0102 was transfected into another batch of 721.221 cells. Killing by NKL cells either untransfected (NKL) or transfected with KIR2DL2 (2DL2) or KIR2DS2 (2DS2) was tested at the indicated effector to target ratios (1:1 or 10:1). Killing of the HLA-C*0102-T2A-E19/K-LNPSVAATL cell line was significantly greater (p<0.001) than the HLA-C*0102 cell line for the KIR2DS2-positive NK cells, but not the other two cell lines

[0213] FIG. 12shows that the peptide sequences IVDLMCHATF (SEQ ID NO: 1); VIDAMCHATL (SEQ ID NO: 2); AANVMAASL (SEQ ID NO: 3); and FLSDVPVATL (SEQ ID NO: 4) bind HLA-Cw*0102. The HLA-Cw*0102 [0214] positive cell line L721.174 was incubated with increasing concentrations of the peptides IVDLMCHATF (SEQ ID NO: 1); VIDAMCHATL (SEQ ID NO: 2); AANVMAASL (SEQ ID NO: 3); and FLSDVPVATL (SEQ ID NO: 4) or a control peptide LADKRPTAWF. Cells were stained for cell surface of MHC class I and analysed by flow cytometry. The mean fluorescence intensity of cell surface MHC Class I was plotted against the peptide concentration.

[0215] FIG. 13KIR2DS2 tetramer staining on 174 cells with and without peptides. Peptides tested include IVDLMCHATF (SEQ ID NO: 1); VIDAMCHATL (SEQ ID NO: 2); AANVMAASL (SEQ ID NO: 3); and FLSDVPVATL (SEQ ID NO: 4). A negative control peptide VAPWNSFAL (SEQ ID NO: 12) and positive control peptide LNPSVAATL (SEQ ID NO: 5) were included. The mean fluorescence intensity of the KIR2DS2 tetramer staining is shown in the right hand corner of the graph.

[0216] FIG. 14The Dengue virus peptide IVDLMCHATF (SEQ ID NO: 1) activates KIR2DS2 positive NK cells. 174 cells were incubated with the IVDLMCHATF (SEQ ID NO: 1) and then KIR2DS2-positive transfected NKL cells or KIR2DL2-positve transfected NKL cells. Activation of NK cells was assessed by western blotting for phosphorylated Vav1, and compared to total Vav1. At high peptide concentrations the peptide IVDLMCHATF (SEQ ID NO: 1) activated KIR2DS2, but not KIR2DL2-positive NK cells.

EXAMPLE 1KIR2DS1 AND ITS GROUP 1 HLA-C LIGANDS PROVIDE PROTECTION AGAINST Chronic Hepatitis C Virus (HCV) Infection

[0217] Table 1 shows a logistic regression analysis of the outcome of HCV infection and its association with KIR and HLA in 272 individuals exposed to HCV. 180 individuals had chronic infection and 92 cleared infection. An odds ratio (OR)>1 indicates protection against chronic HCV infection. (HLA-C*0102 is one of the group 1 HLA-C alleles).

TABLE-US-00001 TABLE 1 KIR2DS1 and its group 1 HLA-C ligands provide protection against chronic hepatitis C virus (HCV) infection. P OR 95% CI KIR2DL3:HLA-C1C1 0.006 2.56 1.31-5.02 KIR2DS3 0.027 0.50 0.28-0.92 KIR2DS2:group 1 HLA-C 0.033 1.83 1.05-3.19 KIR3DS1:HLA-Bw4 0.076 1.83 0.94-3.55 KIR2DS1:HLA-C2 0.176 0.60 0.29-1.25

EXAMPLE 2THE PEPTIDE LNPSVAATL (SEQ ID NO: 5) BINDS HLA-CW*0102

[0218] With reference to FIG. 1, the HLA-Cw*0102-positive cell line L721.174 was incubated with increasing concentrations of the LNPSVAATL (SEQ ID NO: 5) peptide or a control peptide VAPWNSLSL (SEQ ID NO: 11) (known to bind HLA-Cw*0102) overnight at 26? C. 3?10.sup.5 cells were used per condition. Following washing in PBS the cells were stained for cell surface of MHC class I and analysed by flow cytometry. The mean fluorescence intensity of cell surface MHC Class I was plotted against the peptide concentration.

EXAMPLE 3LNPSVAATL (SEQ ID NO: 5) IS RECOGNIZED BY KIR2DS2-POSITIVE NK CELLS AND LEADS TO NK CELL ACTIVATION (CD107A DEGRANULATION), AND KILLING OF TARGET CELLS EXPRESSING HLA-CW*0102

[0219] With reference to FIG. 2A, 3?10.sup.5 L721.174 cells were incubated at 26? C. overnight in the presence or absence of peptides, and then cultured for four hours with NK cells from either a KIR2DS2-positive or KIR2DS2-negative donor. The level of CD107a (LAMP) expression was measured on NK cells positive for the CH-L antibody. This antibody binds to KIR2DS2, KIR2DL2 and KIR2DL3. NK cells from both donors have lower levels of degranulation when exposed to the VAP-FA (VAPWNSFAL) (SEQ ID NO: 12) peptide as compared to no peptide. NK cells from the KIR2DS2 [0220] positive donors have higher levels of degranulation when exposed to the LNPSVAATL (SEQ ID NO: 5) peptide as compared to no peptide. NK cells from the KIR2DS2-positive donors have higher levels of degranulation when exposed to the VAPWNSFAL (SEQ ID NO: 12) and the LNPSVAATL (SEQ ID NO: 5) peptide in combination as compared to the VAPWNSFAL (SEQ ID NO: 12) peptide alone. With reference to FIG. 2B, summary data from 5 KIR2DS2-positive and 6 KIR2DS2 [0221] negative donors were tested as per panel A of FIG. 2. To compare the different donors the data are normalized to 100% for the no peptide condition.

EXAMPLE 4KIR2DS2-POSITIVE NK CELL CLONES KILL TARGET CELLS EXPRESSING HLA-CW*0102 AND THE LNPSVAATL (SEQ ID NO: 5) PEPTIDE

[0222] With reference to FIG. 3, 3?10.sup.5 L721.174 cells were incubated at 26? C. overnight in the presence or absence of the peptides VAPWNSFAL (SEQ ID NO: 12) and the LNPSVAATL (SEQ ID NO: 5). The cells were then labeled with the cell tracker orange dye, and then cultured for four hours with KIR2DS2-positive NK cell clones (clones 4,6,9) or a KIR2DS2-negative NK cell clone (clone 27). Cytotoxicity was then determined using Live/Dead? fixable far-red stain. Only clones 4, 6, 9 killed the L721.174 cell line to greater extent in the presence of the LNPSVAATL (SEQ ID NO: 5) peptide as compared to no peptide or the VAPWNSFAL (SEQ ID NO: 12) peptide.

EXAMPLE 5KIR2DS2 BINDS TO HLA-C*0102 AND THE LNPSVAATL (SEQ ID NO: 5) PEPTIDE

[0223] With reference to FIG. 4A, the KIR2DS2 protein was expressed as a recombinant protein with a biotinylation C-terminal tag. Following biotinylation with the BirA enzyme, the KIR2DS2 protein was incubated with streptavidin-phycoerythrin to form fluorescent KIR2DS2-tetramers (KIR2DS2-PE). 3?10.sup.5 L721.174 cells were then incubated overnight at 26? C. with indicated peptides at a concentration of 20004. Cells were then stained with KIR2DS2-PE and analysed by flow cytometry. Shown are histogram plots. The bold numbers represent the mean fluorescent staining intensity of the KIR2DS2-PE staining of the cells in the presence of peptide and the non-bold numbers the staining in the absence of peptide. With reference to FIG. 4B, panel B represents the mean and standard error fluorescent intensities of the experiment in A following replicate experiments.

EXAMPLE 6KIR2DS2 RECOGNISES PEPTIDES WITH XXXXXAAL (SEQ ID NO: 6) AND XXXXXATL (SEQ ID NO: 7) MOTIFS

[0224] With reference to FIG. 5A, 3?10.sup.5 L721.174 cells were incubated overnight at 26? C. with indicated peptides at a concentration of 200 ?M. Cells were then stained with KIR2DS2-PE (see FIG. 4) and analysed by flow cytometry. Shown are histogram plots. The bold numbers represent the mean fluorescent staining intensity of the KIR2DS2-PE staining of the cells in the presence of peptide and the non-bold numbers the staining in the absence of peptide. With reference to FIG. 5B, panel B represents the mean and standard error fluorescent intensities of the experiment in A following replicate experiments.

EXAMPLE 7LNPSVAATL (SEQ ID NO: 5) ACTIVATES NK CELLS EXPRESSING KIR2DS2

[0225] With reference to FIG. 6A, 1?10.sup.6 L721.174 cells were incubated overnight at 26? C. with indicated peptides at a concentration of 2004. The cells were then incubated with the NK cell line transfected with KIR2DS2 for 5 minutes. The cells were lysed in 1% digitonin buffer and the KIR2DS2 was then immunoprecipitated from the mixture using the GL183 antibody coupled to protein-G beads. The immune precipitate was analysed by Western blotting using a phosphotyrosine antibody. The membrane was then stripped and reprobed with an antibody to DAP12 the molecule that transduces a positive signal from KIR2DS2. Shown are the regions on the blots at the level of DAP12 (12-15 kDa). The LNPSVAATL (SEQ ID NO: 5) peptide induces phosphorylation of DAP12. With reference to FIG. 6B, 1?10.sup.6 L721.174 cells were incubated overnight at 26? C. with indicated peptides at a concentration of 2004. The cells were then incubated with the NK cell line transfected with KIR2DS2 or transfected with KIR2DL2 for 5 minutes. The cells were lysed in 1% NP-40 and then analysed by Western blotting using antibodies to pVav1 and then following stripping of the membrane to Vav1. Shown are Western blots around the 98 kDa region, and below them the corresponding densitometries for 3 independent experiments.

EXAMPLE 8KIR2DS2 IS ACTIVATED BY THE PEPTIDE GAVPDLAWL (SEQ ID NO: 8) AND GAVPDLATL (SEQ ID NO: 9)

[0226] With reference to FIG. 7, 1?10.sup.6 HLA-C*0304:ICP47:721.221 cells were incubated overnight at 26? C. with indicated peptides at a concentration of 2004. The cells were then incubated with the NK cell line transfected with KIR2DS2 for 5 minutes. The cells were lysed in 1% NP-40 and then analysed by Western blotting using antibodies to pVav1 and then following stripping of the membrane to Vav1. Shown is a Western blots around the 98 kDa region, and below them the corresponding densitometries for 3 independent experiments.

EXAMPLE 9

[0227] FIG. 8 shows the elution profile of the peptide in a construct HLA-C:T2A:E19/3K:Peptide (FIG. 8). The cell line 721.221 expressing HLA-C*0102:T2A:E19/3K:LNPSVAATL. Following elution of the HLA-C binding peptides using 10% acetic acid the peptides were analysed by HPLC using a C18 column and an acetonitrile gradient. When compared to the control LNPSVAATL (SEQ ID NO: 5) peptide a peak is seen in the peptide eluate that corresponds to the retention time of the LNPSVAATL (SEQ ID NO: 5) peptide indicating that it is endogenously processed and presented and that this is a suitable vaccine construct.

EXAMPLE 10: A CONSTRUCT IN THE SEQUENCE HLA-C*0102-T2A-E19/K-LNPSVAATL CAN BE USED TO ACTIVATE NK CELLS

[0228] With reference to FIG. 11, 721.221 HLA class I-negative cells were transduced with a construct expressing HLA-C*0102-T2A-E19/K-LNPSVAATL or with a construct expressing HLA-C*0102. Cells were then used in cytotoxicity assays at different effector to target ratios. At effector to target ratios of 1:1 or 10:1 KIR2DS2-positive NKL cells killed the cells expressing HLA-C*0102-T2A-E19/K-LNPSVAATL more efficiently than those expressing only HLA-C*0102. This demonstrated that a peptide (LNPSVAATL) can be sued to activate NK cells through the route of intracellular expression and endogenous presentation.

EXAMPLE 11: PEPTIDES IVDLMCHATF (SEQ ID NO: 1), VIDAMCHATL (SEQ ID NO: 2), AANVMAASL (SEQ ID NO: 3), AND FLSDVPVATL (SEQ ID NO: 4) CAN BIND HLA-C*0102

[0229] With reference to FIG. 12, the HLA-Cw*0102-positive cell line L721.174 was incubated with increasing concentrations of the peptides IVDLMCHATF (SEQ ID NO: 1), VIDAMCHATL (SEQ ID NO: 2), AANVMAASL (SEQ ID NO: 3); and FLSDVPVATL (SEQ ID NO: 4) overnight at 26? C. 3?10.sup.5 cells were used per condition. Following washing in PBS the cells were stained for cell surface of MHC class I and analysed by flow cytometry. The mean fluorescence intensity of cell surface MHC Class I was plotted against the peptide concentration.

EXAMPLE 12 KIR2DS2 BINDS TO HLA-C*0102 AND THE EXAMPLE 11KIR2DS2 BINDS TO HLA-C*0102 AND THE PEPTIDES IVDLMCHATF (SEQ ID NO: 1), VIDAMCHATL (SEQ ID NO: 2), AANVMAASL (SEQ ID NO: 3), AND FLSDVPVATL (SEQ ID NO: 4)

[0230] With reference to FIG. 13, the KIR2DS2 protein was expressed as a recombinant protein with a biotinylation C-terminal tag. Following biotinylation with the BirA enzyme, the KIR2DS2 protein was incubated with streptavidin-phycoerythrin to form fluorescent KIR2DS2-tetramers (KIR2DS2-PE). 3?10.sup.5 L721.174 cells were then incubated overnight at 26? C. with indicated peptides at a concentration of 20004. Cells were then stained with KIR2DS2-PE and analysed by flow cytometry. Shown are histogram plots. The numbers in the top right-hand corners represent the mean fluorescent staining intensity of the KIR2DS2-PE staining of the cells in the presence of peptide.

EXAMPLE 13IVDLMCHATF (SEQ ID NO: 1) ACTIVATES NK CELLS EXPRESSING KIR2DS2

[0231] With reference to FIG. 14, 1?10.sup.6 L721.174 cells were incubated overnight at 26? C. with indicated peptides at increasing concentrations. The cells were then incubated with the NK cell line transfected either with KIR2DS2 or KIR2DL2 for 5 minutes. The cells were lysed in 1% NP-40 and then analysed by Western blotting using antibodies to pVav1 and then following stripping of the membrane to Vav1.

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