WO2012169911A1 - Peptides, constructs and uses therefor - Google Patents

Abstract

The invention relates to novel peptides comprising the core amino acid motif DCV as well as constructs containing same and uses for the constructs, particularly but not exclusively for treatment of disorders where the reduction or removal of a specific type of cells is of benefit.

Description

PEPTIDES, CONSTRUCTS AND USES THEREFOR

FIELD

The present invention relates to novel peptides, constructs containing same and uses therefor.

BACKGROUND

The human hepatitis B virus (HBV) contains four ORFs encoding the viral envelope, core and E antigen, a polymerase protein and the X-protein. The X-protein of HBV is a complex pleiotropic molecule, generally divided into six domains denoted A-F, based on homology to other X-proteins in the hepadnaviridae family (Kumar, Jayasuryan, & Kumar, 1996; Misra, Mukherji, & Kumar, 2004). ' The various functions of the X-protein have not been fully elucidated, but it is believed to confer some survival advantage to the virus. The X-protein is a multifunctional protein having a number of complex and often competing functions. For example, it has both pro- and anti-apoptotic effects on hepatocytes. The region involved in apoptosis lies in the C-terminal end of the X-protein. However, the exact nature and location of the site has not previously been elucidated and it has been suggested that more than one site may be involved, for example transfection of HuH7 cells with a construct encoding residues 68 to 117 caused cell apoptosis, and a Kunitz-like peptide (residues 131-142) was shown to cause cell apoptosis when added to HepG2 cells.³'⁴ The opposing functions of the X-protein on apoptosis are partly responsible for the balance between chronic infection and progression to hepatocellular carcinoma (HCC). X-protein-induced apoptosis of hepatocytes can lead to an increased turnover and proliferation of cells and a larger virus reservoir as there are constantly new cells ready to be infected. This also increases the chance of integration of the HBV genome, which can facilitate chronic hepatitis. X-protein-mediated inhibition of apoptosis is typical of an oncogene as inhibition of apoptosis allows genetically mutated transformed cells to proliferate unhindered leading to increased progression of cancer, and eventual systemic spread of tumors. There are two distinct caspase-dependent apoptotic pathways. They include the extrinsic pathway involving death receptors on the cell surface and death signals in the form of soluble ligands, and the intrinsic pathway mediated via the mitochondria which release the apoptotic effector cytochrome C in response to stress such as oxidative and UV radiation (Gupta, 2003). The X-protein can induce both pathways to cause apoptosis.

Agents which induce or promote apoptosis and necrosis are known and are considered to be useful to treat, for example, diseases associated with aberrant cell growth and proliferation by promoting the death of those cells. However, such agents are often selective in that they can only inhibit specific cell types or specific cancer cell types due to interference by signalling pathways or tumour survival pathways. Bibliographic details of the publications referred to herein are collected at the end of the description.

OBJECT

It is an object of the present invention to provide novel peptides, conjugates comprising same, and/or uses therefor, or at least to provide the public with a useful choice.

STATEMENT OF INVENTION

In a first broad aspect, the invention provides a peptide comprising the amino acid sequence DCV (SEQ ID 1) or a functionally equivalent variant thereof.

In one embodiment, the invention provides a peptide comprising the amino acid sequence KDCV (SEQ ID 2) or a functionally equivalent variant thereof.

In one embodiment, the invention provides a peptide comprising the amino acid sequence DCVF (SEQ ID 3) or a functionally equivalent variant thereof.

In one embodiment, the invention provides a peptide comprising the amino acid sequence KDCVF (SEQ ID 4) or a functionally equivalent variant thereof. In one embodiment, the peptide or functionally equivalent variant further comprises at its N-terminus, one or more amino acids which correspond consecutively to amino acids 81 to 112 of a native X-protein, and/or at its C-terminus, one or more amino acids which correspond consecutively to amino acids 116 to 120 of a native X-protein. In another embodiment, the peptide or functionally equivalent variant further comprises at its N- terminus, one or more amino acids which correspond consecutively to amino acids 80 to 111 of a native X-protein, and/or at its C-terminus, one or more amino acids which correspond consecutively to amino acids 117 to 120 of a native X-protein. In one embodiment, there is provided a peptide comprising the amino acid sequence FKDCVFTYWR (SEQ ID 5) or a functionally equivalent variant thereof.

In one embodiment, there is provided a peptide comprising the amino acid sequence TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR (SEQ ID 6) or a functionally equivalent variant thereof.

In certain embodiments, the peptide consists of the amino acid sequence DCV (SEQ ID 1), KDCV (SEQ ID 2), DCVF (SEQ ID 3), KDCVF (SEQ ID 4), FKDCVFTYWR (SEQ ID 5) or TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR (SEQ ID 6).

In a second broad aspect, the invention provides a peptide comprising the amino acid sequence AMSTTDPEAY (SEQ ID 7) or a functionally equivalent variant thereof.

In one embodiment, the peptide or functionally equivalent variant further comprises at its N-terminus, one or more amino acids which correspond consecutively to amino acids 81 to 100 of a native X-protein, and/or at its C-terminus, one or more amino acids which correspond consecutively to amino acids 111 to 120 of a native X-protein.

In one embodiment, the peptide consists of the amino acid sequence AMSTTDPEAY (SEQ ID 7).

In a third broad aspect, the invention provides a nucleic acid encoding a peptide or functionally equivalent variant of the first or second broad aspects.

In a fourth broad aspect, the invention provides a nucleic acid vector comprising a nucleic acid of the third broad aspect.

In a related aspect, the invention provides a host cell comprising a nucleic acid or vector of the third or fourth broad aspects of the invention. In a fifth broad aspect the invention provides the use of a peptide or functionally equivalent variant of the first or second broad aspect, a nucleic acid of the third aspect and/or a vector of the fourth aspect as an agent for inducing or promoting apoptosis or necrosis in one or more cell.

In a sixth broad aspect, the invention provides a construct comprising a peptide or functionally equivalent variant of the first or second broad aspect and at least one other compound.

In one embodiment the at least one other compound is a cell membrane translocation compound. In one embodiment, the at least one other compound is a cell targeting compound. In one embodiment, the construct comprises both a cell membrane

translocation compound and a cell targeting compound.

In one embodiment, the cell membrane translocation compound is a peptide. In one embodiment, the cell membrane translocation compound is a polyArg peptide or penetratin or Tat. In another embodiment, the cell membrane translocation compound is a peptide comprising the amino acid sequence LCLRP (SEQ ID 8), LCLRPVG (SEQ ID 9),

LCLRP VGAE (SEQ ID 10), LCLRP VGAESR (SEQ ID 11 ), LCLRPVGAESRGRP V (SEQ ID 12), LCLRPVGAESRGRPVSGPFG (SEQ ID 13),

MAARLCCQLDPARDVLCLRP (SEQ ID 14), MAARLCCQ (SEQ ID 15),

MAARLCCQLDPARDV (SEQ ID 16) or a functionally equivalent variant of any one thereof.

In one embodiment, the cell targeting compound is a ligand specific to a particular cell type, for example YIGSR (SEQ ID 17) or a-melanocyte stimulating hormone for melanoma, peptide-MHC-tetramer for a clonotypic T cell, anti-idiotype antibody for a clonotypic B cell, antibody, scFV, phagemid, or aptamer recognizing a cell-type-specific marker.

In a seventh broad aspect, the invention provides a nucleic acid encoding a construct of the sixth broad aspect. In an eighth broad aspect, the invention provides a vector comprising a nucleic acid of the seventh broad aspect.

In a related aspect, the invention provides a host cell comprising a nucleic acid of the seventh aspect or a vector of the eighth aspect of the invention.

The invention also provides the use of a construct of the sixth aspect, a nucleic acid of the seventh aspect and/or a vector of the eighth aspect as an agent for inducing or promoting apoptosis or necrosis in one or more cell.

In a ninth broad aspect, the invention provides a composition comprising one or more peptide of the first or second broad aspects, one or more nucleic acid of the third aspect and/or one or more vector of the fourth aspect. In a tenth broad aspect, the invention provides a composition comprising one or more construct of the sixth broad aspect, one or more nucleic acid of the seventh aspect, and/or one or more vector of the eighth aspect.

The invention also provides compositions comprising any combination of one or more peptide, construct, nucleic acid and vector of the invention.

In an eleventh broad aspect, the invention provides a method of promoting or inducing apoptosis in a cell, the method comprising contacting a peptide or a functionally equivalent variant thereof of the first or second broad aspect, a nucleic acid or vector of the third or fourth broad aspect, a construct of the sixth broad aspect, a nucleic acid or vector of the seventh or eighth broad aspects and/or a composition comprising any one or more thereof with the cell or a composition comprising the cell.

In a twelfth broad aspect, the invention provides a method of promoting or inducing necrosis in a cell, the method comprising contacting a peptide or a functionally equivalent variant thereof of the first or second broad aspect, a nucleic acid or vector of the third or fourth broad aspect, a construct of the sixth broad aspect, a nucleic acid or vector of the seventh or eighth broad aspects and/or a composition comprising any one or more thereof with the cell or a composition comprising the cell. In one particular embodiment of the twelfth broad aspect, the construct comprises a peptide of the invention in combination with a peptide membrane translocation compound comprising at least the amino acid sequence LCLRPVG (SEQ ID 9) or a functionally equivalent variant thereof. In a further embodiment, the peptide membrane translocation compound comprises the amino acid sequence LCLRPVGAE (SEQ ID 10),

LCLRPVGAESR (SEQ ID 11), LCLRPVGAESRGRPV (SEQ ID 12),

LCLRPVGAESRGRPVSGPFG (SEQ ID 13), MAARLCCQLDPARDVLCLRP (SEQ ID 14), MAARLCCQ (SEQ ID 15), MAARLCCQLDPARDV (SEQ ID 16) or a functionally equivalent variant of any one thereof.

In a related broad aspect, the invention provides a method for the treatment of a condition or disorder where reduction in the number of specific cells or removal of specific cells is of therapeutic benefit, the method comprising administering to a subject a peptide or a functionally equivalent variant thereof of the first or second broad aspect, a nucleic acid or vector of the third or fourth broad aspect, a construct of the sixth broad aspect, a nucleic acid or vector of the seventh or eighth broad aspects and/or a composition comprising any one or more thereof. In one embodiment, the disorder is cancer, an inflammatory disease, hypersensitivity, a hyper-proliferative disease, a metabolic disease, a neurodegenerative disease, a skin disease, infected cells, obesity, and/or a disorder mediated by pathogenic cells.

In another broad aspect, the invention provides the use of a peptide or a functionally equivalent variant thereof of the first or second broad aspect, a nucleic acid or vector of the third or fourth broad aspect, a construct of the sixth broad aspect, a nucleic acid or vector of the seventh or eighth broad aspects and/or a composition comprising any one or more thereof in the manufacture of a medicament for the treatment of a condition or disorder where reduction in the number of specific cells or removal of specific cells is of therapeutic benefit.

In one embodiment, the disorder is cancer, an inflammatory disease, hypersensitivity, a hyper-proliferative disease, a metabolic disease, a neurodegenerative disease, a skin disease, infected cells, obesity, and/or a disorder mediated by pathogenic cells. In another broad aspect, the invention provides compositions comprising at least one peptide, construct, nucleic acid or vector of the invention. In one embodiment, the composition comprises one or more pharmaceutically acceptable diluents, carriers and/or excipients.

In another aspect, the invention provides a method of deleting or at least reducing the number of cells of a selected cell type in an animal by administering a peptide, construct and/or nucleic acid of the invention to the animal.

In another aspect, the invention also provides an animal model of a disease, the animal characterised in that a selected cell type has been deleted or at least reduced by delivery of a peptide, construct and/or nucleic acid of the invention. The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

FIGURES

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures.

A number of the figures illustrate cells or nuclei which have been labelled or stained in different colours. When reproduced in black and white, all the spots visible in these Figures represent cells or nuclei, in accordance with the label or stain used, unless otherwise stated. In addition, in these figures, the "merge" results illustrate cell staining coinciding with incidence of nucleus staining. Generally, the central areas represent the nucleus and the halo represents the cell as a whole (including the cytoplasm). Figure 1. Screening X-peptides for their ability to cause the apoptosis of HepG2 cells. A panel of 9 peptides that encompass the entire X-protein sequence were incubated for 3 h with HepG2 cells. The cells were simultaneously stained with Annexin-V fluos and propidium iodide to detect cell apoptosis and necrosis, respectively.

Figure 2. Screening peptide fragments of the proapoptotic peptide aa 81-120 for their ability to cause the apoptosis of HepG2 cells. The cells were stained with Annexin-V fluos to detect cell apoptosis, and the nuclei were stained with DAPI. Annexin-V and DAPI images were merged. The merged photos illustrate the total number of cells, and the number of cells undergoing apoptosis ie the proportion of cells undergoing apoptosis. Peptides 81-120, 101-110, and 111-120 caused HepG2 cells to undergo apoptosis. A and B, repeat experiments.

Figure 3. The minimal proapoptotic peptide aa 112-116 induces DNA fragmentation in HepG2 cells. HepG2 cells were incubated for 3 h with three cell-permeable peptides encompassing aa 106-116, 112-118 and 112-116. The cells were stained with the FITC- labelled TUNEL agent to reveal DNA strand breaks. Cell nuclei were stained with DAPI.

Figure 4. Cell-permeable peptide aa 16-22 carries the proapoptotic peptide aa 112-116 into HepG2 cells to cause apoptosis. HepG2 cells were incubated in the absence of peptide, or were incubated with the cell-permeable X-protein peptide aa 16-22 fused to the

proapoptotic X-protein peptide aa 112-116, peptide aa 112-116 fused to the polyarginine carrier peptide, or the non-apoptotic X-protein peptide aa 1-50. Cells were stained with annexin-V, propidium iodide, and DAPI.

Figure 5. Dose-response of apoptosis mediated by the proapoptotic peptide aa 112-116, and its parental peptide 111-120. The proapoptotic peptide 112-116 (A) and its parental peptide 111-120 (B) fused to a polyarginine carrier peptide were incubated at increasing concentrations, as indicated, with HepG2 cells. Cells were stained with annexin-V and DAPI, and images were merged. The merged photos illustrate the total number of cells, and the number of cells undergoing apoptosis ie the proportion of cells undergoing apoptosis. The cells were sensitive to low concentrations of peptide 112-116 and 111-120. Figure 6. Time-scale of apoptosis mediated by proapoptotic peptide aa 112-116 fused to a polyarginine carrier peptide (A) and X-peptide 16-22 carrier peptide (B). The proapoptotic peptide 112-116 fused to a polyarginine carrier peptide or to X-peptide 16-22 causes HepG2 cells to undergo apoptosis within 30 min of incubation. HepG2 cells were incubated for different time periods with the 112-116 fusion peptides, as indicated. Cells were stained with annexin-V, propidium iodide, and DAPL Annexin-V and DAPI images were merged. The merged photos illustrate the total number of cells, and the number of cells undergoing apoptosis ie the proportion of cells undergoing apoptosis. Apoptosis mediated by proapoptotic peptide aa 112-116 was rapid with cells undergoing apoptosis within 30 min of addition of peptide. Proapoptotic peptide aa 112-116 fused to a polyarginine carrier peptide caused apoptosis only, whereas peptide aa 112-116 fused to the X-peptide 16-22 carrier peptide caused apoptosis and necrosis.

Figure 7. A cell-permeable form of the proapoptotic peptide aa 112-116 is able to kill multiple cell types. The proapoptotic peptide 112-116 fused to a polyarginine carrier peptide was incubated for 3 h with an array of different cell types, including (A) HepG2, DU145, Caco2, (C) SK-N-SH, THP-1, BT549, (E) A375, and (F) COS-1

tumour/immortalized cells; and (G) normal peripheral blood mononuclear cells, human keratinocytes, and aortic smooth muscle cells. The cells were also treated with a cell- permeable non-apoptotic peptide (B, D-F, H). Cells were stained with annexin-V and DAPI, and images were merged. The merged photos illustrate the total number of cells, and the number of cells undergoing apoptosis ie the proportion of cells undergoing apoptosis. The proapoptotic peptide 112-116 killed multiple cell types. Figure 8. The proapoptotic peptides aa 111-120 and 112-116 induce apoptosis via a caspase 3/7 dependent pathway. HepG2 cells were treated with the X-protein proapoptotic peptides aa 111-120 and 112-116 for 3 h and tested for caspase 3/7 activity using the Magic Red dye. Cell nuclei were stained with DAPI. Figure 9. A D-isomeric form of the peptide aa 112-116, and L-isomers of the proapoptotic peptides aa 112-116, 113-116 and 112-115 induce apoptosis of HepG2 cells. HepG2 cells were treated with a D-isomer of the X-protein proapoptotic peptide 112-116, and with L- isomers of the peptides aa 112-116, 113-116 and 112-115 for 3 h and examined for apoptosis using Annexin-V fluos. Cell nuclei were stained blue with DAPI. PREFERRED EMBODIMENT(S)

The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the heading "Examples" herein below, which provides experimental data supporting the invention, specific examples of various aspects of the invention, and means of performing the invention.

The inventors have surprisingly identified peptide motifs derived from the X-protein of the hepatitis B Virus (HBV) which are proapoptotic. These peptides may be used to induce apoptosis and/or necrosis in cells, and accordingly used to delete or at least reduce the number of cells in an in vitro or in vivo setting, including in the treatment of a number of diseases, including for example those associated with aberrant cell growth and

proliferation, such as cancers. They may also find use in a number of research

applications, including, for example, making animal models of diseases to be studied.

Peptides and functionally equivalent variants

In one embodiment, the invention provides peptides comprising the amino acid sequence DCV or functionally equivalent variants of said peptides. This core amino acid sequence maps to amino acid position 113 to 115 of the mature X-protein of HBV (GenBank accession number Y18857). However, it should be appreciated that there are a number of variants of HBV and the inventors contemplate equivalent peptides derived from these variants, while differing in their amino acid sequence, as being of use in the invention. Such peptides are encompassed by reference to the term "functionally equivalent variants".

Peptides comprising the core sequence DCV (SEQ ID 1) may further comprise at the N- terminus, one or more amino acids which correspond to amino acids 81 to 112 of a native X-protein, and/or at the C-terminus one or more amino acids corresponding to amino acids 116 to 120 of a native X-protein, such that the peptide sequence corresponds to a region of consecutive amino acids from the native protein. They may also include heterologous amino acids at the N- or C-terminus.

In one embodiment, the invention provides peptides comprising the amino acid sequence KDCV (SEQ ID 2), DCVF (SEQ ID 3), KDCVF (SEQ ID 4) or functionally equivalent variants of said peptides. The amino acid sequence KDCVF (SEQ ID 4) maps to amino acid position 112 to 116 of the mature X-protein of HBV (GenBank accession number Y18857). In other embodiments, a peptide of the invention comprises the amino acid sequence FKDCVFTYWR (SEQ ID 5), or the amino acid sequence

TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR (SEQ ID 6).

The invention also relates to peptides comprising the amino acid sequence

AMSTTDPEAY (SEQ ID 7) or functionally equivalent variants thereof. This core amino acid sequence maps to amino acid position 101 to 110 of the mature X-protein of HBV (GenBank accession number Y18857). However, it should be appreciated that there are a number of variants of HBV and the inventors contemplate equivalent peptides derived from these variants, while differing in their amino acid sequence, as being of use in the invention. Such peptides are encompassed by reference to the term "functionally equivalent variants".

In one embodiment, the peptide or functionally equivalent variant further comprises at its N-terminus, one or more amino acids which correspond consecutively to amino acids 81 to 100 of a native X-protein, and/or at its C-terminus, one or more amino acids which correspond consecutively to amino acids 111 to 120 of a native X-protein, such that the peptide sequence corresponds to a region of consecutive amino acids from the native protein. They may also include heterologous amino acids at the N- or C-terminus.

Skilled persons will readily appreciate amino acids at positions 81 to 120 of a native X- protein, having regard to the information herein and other published sequence information. By way of example, see GenBank accession number Yl 8857 also provides exemplary sequence information. In addition Gunther S, Fischer L, Pult I, Sterneck M, Will H.

Naturally occurring variants of hepatitis B virus. Adv Virus Res. 1999;52:25-137 provides sequence information for a number of X-proteins. Further, examples of useful sequence information is provided in Table 1 , below.

Table 1:

Protein Locus Sequence SEQ ID No

Accession

No.

Q81163 HBVC8 TV AHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 18 P0C689 HBVC5 TVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCVFKDWE 19

P12936 HBVC3 TVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 20

P0C686 HBVC1 TVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 21

Q9YZR6 HBVC2 TVNAHQVLPKVLYKRTLGLSAMSTTDLEAYFKDCLFKDWE 22

093195 HBVD7 TVNAHQFLPKVLYKRTLGLSVMSTTDLEAYFKDCLFKDWE 23

Q67863 HBVC4 TVNADQVLPKVLHKRTLGLSAMSTSDLEAYF DCLFKDWE 24

Q67877 HBVD6 TVNAHQILPKVLHKRTLGLPAMSTTDLEAYFKDCVFKDWE 25

P24026 HBVD2 TVNAHQFLPKVLHKRTLGLSVMSTTDLEAYFKDCLFKDWE 26

P0C687 HBVC9 TV THMILPKVLHKRTLGLPAMSTIDLEAYFKDCLFKDWE 27

P0C681 HBVD5 TVNAHQFLPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 28

Q913A9 HBVC7 TVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCVFKDWE 29

091531 HBVA7 TVNAHQSLPKVLHKRTLGLPAMSTTDLEAYFKDCVFKDWE 30

Q9E6S8 HBVCO TVNANQVLPKVLHKRTLGLSALSTTDLEAYFKDCVFI DWE 31

Q9PX75 HBVB7 TVNAHRNLPKVLFiKRTLGLSVMSTTDLEAYFKDCVFTEWE 32

P20975 HBVB2 TVNAHRNLPKVLFiKRTLGLSAMSTTDLEAHF DCVFTEWE 33

P0C685 HBVB3 T TsTAHWNLPKVLHKRTLGLSAMSTTDLEAYFKDCVFTEWE 34

P20976 HBVB1 TVNAHRNLPKVLHKRTLGLSAMSTTDLEAYFKDCVFNEWE 35

P17102 HBVA4 TVNAHQILPKVLHKRTLGLPAMSTTDLEAYFKDCVFKDWE 36

Q9PXA2 HBVB5 T TvTAPGNLPKVLHKRTLGLSVMSTTDLEAYFKDCVFTEWE 37

P03165 HBVD3 TVNAHQILPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 38

P20977 HBVB4 TWAHGNLPKVLHKRTLGLSAMSTTDLEAYFKDCVFNEWE 39

Q99HR6 HBVF4 TWAPWSLPTVLHKRTIGLSGRSMTWIEEYIKDCVFKDWE 40

Q67923 HBVB6 TVNAHGNLPKVLHKRTLGLSAMSTTDLEAYFKDCVFNEWE 41

P69714 HBVA2 TVNAHQILPKVLHKRTLGLPAMSTTDLEAYFKDSVFKDWE 42

P69713 HBVA3 TVNAHQILPKVLHKRTLGLPAMSTTDLEAYFKDCVFKDWE 43

Q9H3I5 HBVG3 AM TSHHLPRQLYKWTLGLFVMSTTGVEKYFKDCVFAEWE 44

P0C678 HBVB8 TWAHRNLPKVLHKRTLGLSAMSTTDLEAYFKDCVFTEWE 45

Q05499 HBVF1 TWAPWSLPTVLHKRTLGLSGWSMTWIEEYIKDCVFKDWE 46

Q80IU5 HBVE4 TVNAHQILPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 47

Q8JMY3 HBVF2 TVNAPRSLPTVLHKRTLGLSGRSMTWIEDYIKDCVFKDWE 48

Q9J5S3 HBVOR TVNAPRNLPKVLHKRTLGLSTMSTTGIETYFKDCVFKDWE 49

Q9QAX0 HBVE3 TVNAHQILPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWE 50

' Q69604 HBVE1 TV AHQILPKVLH RTLGLSAMSTTDLEAYFKDCLFKDWE 51

Q4R1S9 HBVA8 TVNAHQILPKVLHKRTLGLPAMSTTDLEAYFKDCLFKDWE 52

Q91C38 HBVA6 TVNAHQILPKVLFn RTLGLPAMSTTDLEAYFKDCVFKDWE 53

Q8JMY5 HBVH1 TVTSTAPWNLPTTLFiKRTLGLSPRSTTWIEEYIKDCVFKDWE 54

Q80IU8 HBVE2 TV AHQILPKVLHKRTLGLSAMSTTDLEAYFKACLFKDWE 55

P12912 HBVCP TVTSIAPR LPKVLHKRTLGLSAMSTTKIETYFKDCVFKDWE 56

Q69607 HBVF6 TVTSJAPWSLPTVLHKRTLGLSGRSMTWIEDYIKX)CVFKDWE 57

Q8JMZ5 HBVH3 TV APQSLPTTLHKRTLGLSPRSTTWIEEYIKDCVF DWE 58

Q8JN06 HBVH2 TVTSiAPQSLPTPLHKRTLGLSPRSTTWIEEYIKDCVFKDWE . 59

P87743 HBVGB TVNAPRSLPTVLHKRTLGLPAMSTTGIETYFKDCVFKDWE 60

Q9YJT2 HBVGO TVNAPRNLPTVLHKRTLGLSA STTKIETYFKDCVFKDWE 61

Q4R1S1 HBVA9 TVNAHQELPKVLYKRTLGLPAMSTTDLEAYFKDCVFKDWE 62

By way of example, in one embodiment, a peptide of the invention consists of the amino acid sequence DCV (SEQ ID 1). In another embodiment, a peptide of the invention consists of the amino acid sequence KDCV (SEQ ID 2). In another embodiment, a peptide of the invention consists of the amino acid sequence DCVF (SEQ ID 3). In another embodiment, a peptide of the invention consists of the amino acid sequence KDCVF (SEQ ^■ ID 4). In another embodiment, a peptide of the invention consists of the amino acid sequence FKDCVFTYWR (SEQ ID 5). In another embodiment, the peptide consists of the amino acid sequence

TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR (SEQ ID 6). In another embodiment, the peptide consists of the amino acid sequence AMSTTDPEAY (SEQ ID 7). As noted herein before, the invention includes functionally equivalent variants of the peptides of the invention. The phrase "functionally equivalent variants" as used herein, includes those peptides in which one or more conservative amino acid substitutions have been made, while substantially retaining the desired function of the peptide. By way of example, the peptide and a functionally equivalent variant thereof will have the ability to promote or induce apoptosis in a cell. In certain embodiments, the peptide and

functionally equivalent variant thereof will have the ability to promote or induce necrosis in a cell.

A peptide(s) of the invention and its functionally equivalent variant(s) may be referred to herein collectively as "peptide(s)". Accordingly, where not specifically mentioned, references to a "peptide" or "peptides" of the invention herein should be taken to include reference to functionally equivalent variants thereof.

It should be appreciated that a "functionally equivalent variant" may have a level of activity higher or lower than the peptide of which it is a variant. In various embodiments of the invention a functionally equivalent variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the level of activity of the peptide of which it is a variant. Skilled persons will readily appreciate the desired function and be able to assess function and determine the level of activity of a peptide or functionally equivalent variant thereof peptide of the invention, based on the information contained herein, and using techniques known in the art. However, by way of example, one can determine whether a peptide or variant has the desired pro-apoptotic or pro-necrotic function, using an assay as herein after described in the examples section.

As used herein "conservative amino acid substitution(s)" should be taken broadly to mean substitution of amino acids that have similar biochemical properties. Persons skilled in the art will appreciate appropriate conservative amino acid substitutions based on the relative similarity between different amino acids, including the similarity of the amino-acid side chain substituents (for example, their size, charge, hydrophilicity, hydrophobicity and the like). By way of example, a conservative substitution includes substitution of one aliphatic amino acid for another aliphatic amino acid, substitution of an amino acid with a hydroxyl- or sulphur-containing side chain with another amino acid with a hydroxyl- or sulphur- containing side chain, substitution of an aromatic amino acid with another aromatic amino acid, substitution of a basic amino acid with another basic amino acid, or substitution of an acidic amino acid with another acid amino acid. By way of further example, "conservative amino acid substitution(s)" include:

- substitution of Glycine, Alanine, Valine, Leucine, or Isoleucine, one for another substitution of Serine, Cysteine, Theronine, or Methionine, one for another

- substitution of Phenylalanine, Tyrosine, or Tryptophan, one or another

- substitution of Histidine, Lysine, or Arginine, one for another

- substitution of Aspartic acid, Glutamic acid, Asparagine or Glutamine, one for another

Functionally equivalent variants containing amino acid substitutions in accordance with this aspect of the invention will preferably retain at least 70%, 80%, 90%, 95% or 99% amino acid sequence similarity to the original peptide. In one embodiment, the functionally equivalent variant has at least 70%, 80% 90%, 95% or 99% sequence identity with the original peptide.

Peptides of the invention (including functionally equivalent variants) may be composed of L-amino acids, D-amino acids or a mixture thereof and may include non-naturally occurring amino acids.

It should be understood that peptides of the invention (including functionally equivalent variants), are "isolated" or "purified" peptides. An "isolated" or "purified" peptide is one which has been identified and separated from the environment in which it naturally resides, or artificially synthesized. It should be appreciated that these terms do not reflect the extent to which the peptide has been purified or separated from an environment in which it naturally resides. A peptide of the invention may be isolated from natural sources, or preferably derived by chemical synthesis (for example, fmoc solid phase peptide synthesis as described in Fields GB, Lauer-Fields JL, Liu RQ and Barany G (2002) Principles and Practice of Solid-Phase peptide Synthesis; Grant G (2002) Evaluation of the Synthetic Product. Synthetic Peptides, A User's Guide, Grant GA, Second Edition, 93-219; 220-291, Oxford University Press, New York) or genetic expression techniques, methods for which are readily known in the art to which the invention relates. Standard recombinant DNA and molecular cloning techniques are described for example in: Sambrook, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and, Ausubel et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). The inventor's also contemplate production of a peptide of the invention by an appropriate transgenic animal, microbe, or plant.

Constructs

The peptides of the invention may be connected to one or more additional compounds. For example, they may be connected to one or more additional compound that aids the function or activity of the peptide, protects the peptide from degradation, otherwise improves its half life, aids in isolation and/or purification of the peptide during

manufacture (for example ubiquitin, a his-tag, or biotin), or assists with cell membrane translocation or cell-specific targeting. Peptides of the invention which are so connected to one or more other compounds may be referred to herein as "constructs" comprising the peptides.

The one or more additional compounds may be of any desirable nature and include, for example, peptides, nucleic acids, lipids, carbohydrates, and natural and synthetic chemicals. In certain particular embodiments, the peptides are connected to one or more cell membrane translocating compound and/or one or more cell targeting compound.

A "cell membrane translocating compound" as used herein is a compound which assists a peptide of the invention to move or translocate across a cell membrane into a cell. As used herein the phrases "move across a cell membrane", "translocate across a cell membrane", "cell membrane translocation" and like phrases, should be taken broadly to encompass transport of the peptide and/or a conjugate comprising such peptide from the outside of a cell to the inside of the cell. It should not be taken to imply a particular mode or mechanism of transport across or through the cell membrane.

A "cell targeting compound" is one which allows the peptide to be delivered with a level of specificity to a particular cell type. The phrase should be taken broadly and not taken to imply the compound allows for 100% specificity for a particular cell type, although this may be preferred. In certain embodiments, the compound allows for a specificity of at least approximately 10%, at least approximately, 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, or at least approximately 90%. In one embodiment, the cell membrane translocating compound is a peptide. Skilled persons will readily appreciate appropriate peptide compounds of use. However, by way of example, they include poly-arginine, Tat and penetratin. Additional examples can be found in the following references: Bitler BG, Schroeder JA. Anti-cancer therapies that utilize cell penetrating peptides. Recent Pat Anticancer Drug Discov. 2010 Jun;5(2):99- 108; Lindgren M, Langel U. Classes and prediction of cell-penetrating peptides. Methods Mol Biol. 2011 ;683 :3-19; Fonseca SB, Pereira MP, Kelley SO. Recent advances in the use of cell-penetrating peptides for medical and biological applications. Adv Drug Deliv Rev. 2009 Sep 30;61(11):953-64; Lindgren M, Langel U. Classes and prediction of cell- penetrating peptides. Methods Mol Biol. 2011;683:3-19; Sebbage V. Cell-penetrating peptides and their therapeutic applications. BioSci Horizons 2009;2: 64-72;

Gautam A, Singh H, Tyagi A, Chaudhary K, Kumar R, Kapoor P, Raghava GP.

CPPsite: a curated database of cell penetrating peptides. Database (Oxford). 2012 Mar 7;2012:bas015. In one particular example, the cell membrane translocation compound is a peptide comprising the amino acid sequence LCLRP (SEQ ID 8) or a functionally equivalent variant thereof. This core amino acid sequence maps to amino acid position 16-20 of the mature X-protein of HBV (GenBank accession number Yl 8857). Cell membrane translocation compounds of this embodiment of the invention may further comprise at the N-terminus, one or more amino acids which correspond to amino acids 1 to 15 of a native X-protein, and/or at the C-terminus one or more amino acids corresponding to amino acids 21 to 35 of a native X-protein, such that the peptide sequence corresponds to a region of consecutive amino acids from the native protein. They may also include heterologous amino acids at the N- or C-terminus. In certain embodiments, the cell membrane translocation compound comprises the amino acid sequence LCLRPVG (SEQ ID 9), LCLRPVGAE (SEQ ID 10), LCLRPVGAESR (SEQ ID 11), LCLRPVGAESRGRPV (SEQ ID 12), LCLRPVGAESRGRPVSGPFG (SEQ ID 13), or

MAARLCCQLDPARDVLCLRP (SEQ ID 14).

In another particular example, the cell membrane translocation compound is a peptide comprising the amino acid sequence MAARLCCQ (SEQ ID 15) or functionally equivalent variants of said peptides. This core amino acid sequence maps to the N-terminal amino acids 1-8 of the mature X-protein of HBV (GenBank accession number Y18857). Cell membrane translocation compounds of this embodiment of the invention may further comprise at the C-terminus one or more amino acids corresponding to amino acids 9 to 35 of a native X-protein, such that the peptide sequence corresponds to a region of

consecutive amino acids from the native protein. They may also include heterologous amino acids at the N- or C- terminus. In one embodiment the peptide comprises the amino acid sequence MAARLCCQLDPARDV (SEQ ID 16).

Skilled persons will readily appreciate amino acids at positions 1 to 35 of a native X- protein, having regard to the information herein and other published sequence information. By way of example, see GenBank accession number Y18857 also provides exemplary sequence information. In addition Gunther S, Fischer L, Pult I, Sterneck M, Will H.

Naturally occurring variants of hepatitis B virus. Adv Virus Res. 1999;52:25-137 provides sequence information for a number of X-proteins. Further, examples of useful sequence information is provided in Table 2, below. Table 2:

Protein Locus Sequence SEQ ID No

Accession

No.

Q81163 HBVC8 MAARVCCQLDPARDVLCLRPVGAESRGRPVSGPFG 63

P0C689 HBVC5 As above

P12936 HBVC3 As above P0C686 HBVC1 As above

Q9YZR6 HBVC2 MAARMCCQLDPARDVLCLRPVGAESRGRPVSGPFG 64

093195 HBVD7 MAARLCCQLDPARDVLCLRPVGAESRGRPFSGPFG 65

067863 HBVC4 MAARVCCQLDPARDVLCLRPVGAESRGRPVSRPFG 66

Q67877 HBVD6 MAARLCCQLDPARDVLCLRPVGAESRGRPFSGPLG 67

P24026 HBVD2 MAARLCCQLDPARDVLCLRPVGAESRGRPFSGPLG 68

P0C687 HBVC9 MAARLCCQLDPTRDVLCLRPVGAESRGRPVSGPLG 69

P0C681 HBVD5 MAARLCCQLDPARDVLCLRPVGAESRGRPFSGPLG 70

Q913A9 HBVC7 MAARLCCQLDPARDVLCLRPVGAESRGRPFSGPLG 71

091531 HBVA7 MAARLCCQLDPSRDVLCLRPVGAESRGRPLSGPLG 72

Q9E6S8 HBVCO MAARLCCQLDPARDVLCLRPVGAESRGRPVSGSLG 73

Q9PX75 HBVB7 MAARLCCQLDPARDVLCLRPVGAESRGRPLPGPLG 74

P20975 HBVB2 MAARLCCQLDPARDVLCLRPVGAESRGRPLPGPLG 75

P0C685 HBVB3 MAARLCCQLDPARDVLCLRPVGAESRGRPLPGPLG 76

P20976 HBVB1 iVLAARLCCQLDPARDVLCLRPVGAESRGRPLPGPLG 77

P17102 HBVA4 MATRLCCQLDPSRDVLCLRPVGAESRGRPLSGPLG 78

Q9PXA2 HBVB5 MAARLCCQLDPARDVLCLRPVGAESRGRPLPGPLG 79

P03165 HBVD3 MAARLCCQLDPARDVLCLRPVGAESRGRPFSGSLG 80

P20977 HBVB4 MAARLCCQLDPARDVLCLRPVGAESRGRPFPGPLG 81

Q99HR6 HBVF4 MAARMCCQLDPARDVLCLRPVGAESRGRPLPGPLG 82

Q67923 HBVB6 MAARVCCQLDPARDVLCLRPVGAESRGRPLPGPLG 83

P69714 HBVA2 MAARLYCQLDPSRDVLCLRPVGAESRGRPLSGPLG 84

P69713 HBVA3 MAARLYCQLDPSRDVLCLRPVGAESRGRPLSGPLG 85

Q9IBI5 HBVG3 MAARLCCQLDPSRDVLCLRPVSAESSGRPLPGPFG 86

P0C678 HBVB8 MAARLCCQLDTARDVLCLRPVGAESRGRPLPGPLG 87

Q05499 HBVF1 MAARMCCKLDPARDVLCLRPIGAESRGRPLPGPLG 88

Q80IU5 HBVE4 MAARLCCQLDPARDVLCLRPVGAESCGRPVSGSLG 89

Q8JMY3 HBVF2 MAARLCCQLDPARDVLCLRPVGAESRGRSLSGSLG 90

Q9J5S3 FfflVOR MAARLCCQLDTARDVLCLRPVGAESRGRPFSGSVG 91

Q9QAX0 HBVE3 MAARLCCQLDPARDVLCLRPVSAESCGRPVSGSLG 92

Q69604 HBVE1 MAARLCCQLDPARDVLCLRPVSAESCGRPVSGSLG 93

Q4R1S9 HBVA8 MAARLYCQLDSSRDVLCLRPVGAESRGRPFSGPLG 94

Q91C38 HBVA6 MAARLYCQLDSSRDVLCLRPVGAESRGRPLAGPLG 95

Q8JMY5 HBVH1 MAARLCCQLDPARDVLCLRPVGAESCGRPLSWSLG 96

Q80IU8 HBVE2 MAARLCCQLDPARDVLCLRPVSAESCGRSVSGSLG 97

P12912 HBVCP MAARLCCQLDTSRDVLCLRPVGAESCGRPFSGPL 98

Q69607 HBVF6 MAARLCCQLDPARDVLCLRPVGAESSGRTLPGSLG 99

Q8J Z5 HBVH3 MAARLCCQLDPARDVLCLRPVGAESCGRPLS 100

Q8JN06 HBVH2 MAARLCCQLDPARDVLCLRPVGAESCGRPLS 101

P87743 HBVGB MAARMCCQLDPSQDVLCLRPVGAESRGRP 102

Q9YJT2 HBVGO MAARLCCQLDPARDVLCLRPVGAEPCRRPVSG 103

Q4R1S1 HBVA9 MAARLYCQLDSSRNVLCLRPVGAESCGRPLSGPVG 104

In one particular embodiment, the peptide is connected to a cell targeting compound. Persons of skill in the art to which the invention relates will readily appreciate cell targeting compounds of use in the invention, having regard to the desired use of the peptide of the invention, for example in the treatment of a particular disease or disorder. However, by way of example a ligand specific to a particular cell type, for example YIGSR or cc-melanocyte stimulating hormone for melanoma, peptide-MHC-tetramer for a clonotypic T cell, anti-idiotype antibody for a clonotypic B cell, an antibody, and scFV, a phagemid, or an aptamer recognizing a cell-type-specific marker. Additional examples of targeting compounds can be found in the following references: Laakkonen P, Vuorinen K. Homing peptides as targeted delivery vehicles. Integr Biol (Camb). 2010 Aug;2(7-8):326- 37; Shadidi M, Sioud M. Selective targeting of cancer cells using synthetic peptides. Drug Resist Updat. 2003 Dec;6(6):363-71; Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004

Jun;10(6):625-32; Kapoor P, Singh H, Gautam A, Chaudhary K, Kumar R, Raghava GP. TumorHoPe: a database of tumor homing peptides. PLoS One. 2012;7(4):e35187; Svensen N, Walton JG, Bradley M. Peptides for cell-selective drug delivery. Trends Pharmacol Sci. 2012 Apr;33(4): 186-92.

In certain embodiments, the peptides of the invention may be targeted to various specific cell types using cell targeting compounds as follows: to melanoma cells by fusion to the peptides TAASGVRSMH (SEQ ID 105), LTRWVGLMS (SEQ ID 106), CLSDGKRKC (SEQ ID 107), CGKRK (SEQ ID 108), CDTRL (SEQ ID 109); to breast tumours by fusion to the peptides CGNKRTRGC (SEQ ID 110), CREAGRKAC (SEQ ID 111), CGKRK (SEQ ID 112), CDTRL (SEQ ID 113); to cervical cancers by fusion to the peptide CNRRTKAGC (SEQ ID 114); to bone cancers by fusion to the peptides CGNKRTRGC (SEQ ID 115) and CLSDGKRKC (SEQ ID 116); to skin cancers by fusion to the peptides CNRRTKAGC (SEQ ID 117), CRGRRST (SEQ ID 118), CGKRK (SEQ ID 119), CDTRL (SEQ ID 120), CGTKRKC (SEQ ID 121); to prostate cancer by fusion to the peptides CREAGRKAC (SEQ ID 122) and CAGRRSAYC (SEQ ID 123); to pancreatic tumours by fusion to the peptides CKAAKNK (SEQ ID 124) and CRGRRST (SEQ ID 125). By way of example the peptides of the invention could be targeted to the brain by fusion to the peptide CLSSRLDAC (SEQ ID 126); to the kidney by fusion to the peptide CLPVASC (SEQ ID 127); to the lung by fusion to the peptides CGFERVRQCPERC (SEQ ID 128) and CGFELETC (SEQ ID 129); to the skin by fusion to the peptide CVALCREACGEGC (SEQ ID 130); to the pancreas and uterus by fusion to the peptide SWCEPGWCR (SEQ ID 131); to the intestine by fusion to the peptide YSGKWGW (SEQ ID 132); to the uterus by fusion to the peptide GLSGGRS (SEQ ID 133); to the adrenal gland by fusion to the peptide LMLPRAD (SEQ ID 134); to white fat by fusion to the peptides CKCCRAKDC (SEQ ID 135) and CSWKYWFGEC (SEQ ID 136); to muscle by fusion to the peptide ASSLNIA (SEQ ID 137); to the prostate by fusion to the peptide SMSIARL (SEQ ID 138); to the breast by fusion to the peptide CPGPEGAGC (SEQ ID 176), and to the heart by fusion to the peptides CRPPR (SEQ ID 139), CKRAVR (SEQ ID 140), CPKTRRVPC (SEQ ID 141), CRSTRANPC (SEQ ID 142), and CARPAR (SEQ ID 143).

In other embodiments, the cell targeting peptides CKGGRAKDC (SEQ ID 144) and CSWKYWFGEC (SEQ ID 145) could be used to target the peptides of the invention to white adipose cells.

The compounds may be connected to the peptide, or synthesised as a part of the construct, using any appropriate means which allows the peptide to retain at least a level of its desired function. The word "connected" or like terms should be taken broadly to encompass any form of attachment, bonding, fusion or association between the peptide and the at least one compound (for example, but not limited to, covalent bonding, ionic bonding, hydrogen bonding, aromatic stacking interactions, amide bonds, disulfide bonding, chelation) and should not be taken to imply a particular strength of connection. The peptide and the at least one compound may be connected in an irreversible or a reversible manner, such that upon entry into a cell the peptide is released from the compound.

The at least one compound may be connected to the carrier peptide at its N-terminus, its C- terminus or at any other location. In one particular embodiment, a compound is connected to the carrier peptide at its N-terminus. In another particular embodiment, a compound is connected to the carrier peptide at its C-terminus.

Persons skilled in the art will readily appreciate methodology for manufacturing constructs of the invention, having regard to the nature of the at least one compound to be included in the construct. Such methods include manufacturing the peptide and compound separately and then connecting them, chemical synthesis of the construct, recombinant expression of the construct, and the like. By way of example, in embodiments of the invention where the at least one compound is a peptide, the constructs may be produced in the form of fusion proteins using known recombinant expression or chemical synthesis techniques (as herein before described). The peptide and the connected peptide may also be manufactured separately and later connected to one another. By way of further example, where the compound connected to the peptide is a nucleic acid, the peptide and the nucleic acid may be made separately (using chemical synthesis or recombinant techniques, for example) and then connected via one of a number of known techniques.

By way of further example, in embodiments of the invention where the at least one compound is a carbohydrate, the peptide and the carbohydrate may be made separately and then connected or the construct could be produced recombinantly.

By way of example only, the methodology described in WO 91/09958, WO 03/064459, WO 00/29427, WO 01/13957 may be used to manufacture various constructs of the invention. It should be appreciated that while the peptide and at least one compound may be connected directly to one another, constructs of the invention may also utilise linker molecules which connect the at least one compound to the peptide. Skilled persons will appreciate appropriate linker molecules of use in the invention. However, by way of example, the linker molecule may be a peptide. Examples of appropriate linker molecules are also provided in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957.

In certain embodiments, a construct of this aspect of the invention comprises the amino acid sequence:

RRRRRR RKDCVF (SEQ ID 146);

LCLRPVGKDCVF (SEQ ID 147); or,

MAARLCCQGKDCVF (SEQ ID 148).

Nucleic acids

To the extent that a peptide or construct of the present invention may be produced by recombinant cloning and expression techniques the invention provides nucleic acids encoding peptides and constructs of the invention and vectors comprising nucleic acids encoding peptides of the invention. In addition, it should be appreciated that nucleic acids encoding peptides/constructs of the invention could be used therapeutically or in vitro. For example, in the embodiment of the invention in which a peptide or construct of the invention is used in the treatment of a disease, a nucleic acid/expression vector encoding the construct could be administered, with the peptide/construct subsequently being expressed. Accordingly, the invention includes nucleic acids and nucleic acid vectors suitable for this purpose.

It should be understood that a nucleic acid in accordance with the invention, is an "isolated" or "purified" nucleic acid. An "isolated" or "purified" nucleic is one which has been identified and separated from the environment in which it naturally resides, or artificially synthesized. It should be appreciated that these terms do not reflect the extent to which the nucleic has been purified or separated from the environment in which it naturally resides. Nucleic acids of use in accordance with the invention may be isolated from natural sources, or preferably derived by chemical synthesis or recombinant techniques which will be readily known to persons skilled in the art. Those of general skill in the art to which the invention relates will readily be able to identify a variety of nucleic acids which encode the peptides, functionally equivalent variants and constructs of the invention on the basis of the amino acid sequences provided herein, the genetic code and the understood degeneracy therein and published X-protein nucleic acid sequences (for example, see Guo, Y. and Hou, J. Establishment of the consensus sequence of hepatitis B virus prevailing in the mainland of China. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi 19: 189-2000, 1999). However, by way of example, the following nucleic acids are suitable: aaagactgtgtgttc (peptide mapping to amino acids 112-116) (SEQ ID 149) accgtgaacggccgccggggcttgcccaaggtcttgcataagaggactcttggactctcagcaatgtcaacgaccgaccctgag gcatacttcaaagactgtgtgttcacatactggagg (peptide mapping to amino acids 81-120) (SEQ ID

150) ttcaaagactgtgtgttcacatactggagg (peptide mapping to amino acids 111-120) (SEQ ID 151) gcaatgtcaacgaccgaccctgaggcatac (peptide mapping to amino acids 101-110) (SEQ ID 152) Exemplary and non-limiting examples of nucleic acids encoding constructs of the invention include: agaagaagaagaagaagaagaagaaaagactgtgtgttc (SEQ ID 153) (construct comprising

RRR RRRRKDCVF) (SEQ ID 146) agaagaagaagaagaagaagaagaacCgtgaacggCCgCCggggcttgCCCaaggtcttgcataagaggactCttggactC tcagcaatgtcaacgaccgaccctgaggcatacttcaaagactgtgtgttcacatactggagg (SEQ ID I54 )

(construct comprising

RRRR RRRTVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR) (SEQ ID

159) agaagaagaagaagaagaagaagattcaaagactgtgtgttcacatactggagg (SEQ ID 155) (construct comprising RRRRRRRRFKDCVFTYWR) (SEQ ID 160) agaagaagaagaagaagaagaagagcaatgtcaacgaccgaccctgaggcatac (SEQ ID 156) (construct comprising RRRRRRRRAMSTTDPEAY) (SEQ ID 161) ctttgtctacgtcccgtcggcaaagactgtgtgttc (SEQ ID 157) (construct comprising

LCLRPVGKDCVF) (SEQ ID 162) atggctgctaggctgtgctgccaaggcaaagactgtgtgttc (SEQ ID 158) (construct comprising

MAARLCCQGKDCVF) (SEQ ID 163) Nucleic acid vectors will generally contain heterologous nucleic acid sequences; that is nucleic acid sequences that are not naturally found adjacent to the nucleic acid sequences of the invention. The constructs or vectors may be either RNA or DNA, either prokaryotic or eukaryotic, and typically are viruses or a plasmid. Suitable constructs are preferably adapted to deliver a nucleic acid of the invention into a host cell and are either capable or not capable of replicating in such cell. Recombinant constructs comprising nucleic acids of the invention may be used, for example, in the cloning, sequencing, and expression of nucleic acid sequences of the invention. Additionally, recombinant constructs or vectors of the invention may be used to a therapeutic end. Those of skill in the art to which the invention relates will recognise many constructs suitable for use in the present invention. However, the inventors contemplate the use of cloning vectors such as pUC and pBluescript and expression vectors such as pCDM8, adeno-associated virus (AAV) or lentiviruses to be particularly useful.

The constructs may contain regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other appropriate regulatory sequences as are known in the art. Further, they may contain secretory sequences to enable an expressed protein to be secreted from its host cell. In addition, expression constructs may contain fusion sequences (such as those that encode a heterologous amino acid sequence) which lead to the expression of inserted nucleic acid sequences of the invention as fusion proteins or peptides. Heterologous amino acid sequences of use may include, for example, those which can aid in subsequent isolation and purification of the peptide (for example, ubiquitin, his-tag, myc-tag or biotin), or those which assist the activity of the peptide (for example, an additional sequence which aids in transport across a cell membrane, such as a poly arginine sequence, Tat, penetratin or other cell membrane translocation peptides exemplified to herein before). Heterologous amino acid sequences may also include peptide linkers which aid in linking the peptide to another compound to form a construct of the invention

In accordance with the invention, transformation of a nucleic acid vector into a host cell can be accomplished by any method by which a nucleic acid sequence can be inserted into a cell. For example, transformation techniques include transfection, electroporation, microinjection, lipofection, adsorption, biolistic bombardment, and cell-penetrating peptide technology.

As will be appreciated, transformed nucleic acid sequences of the invention may remain extrachromosomal or can integrate into one or more sites within a chromosome of a host cell in such a manner that their ability to be expressed is retained.

The invention encompasses host cells comprising a nucleic acid or nucleic acid vector of the invention. Any number of host cells known in the art may be utilised in cloning and expressing nucleic acid sequences of the invention. For example, these include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); animal cell systems such as CHO (Chinese hamster ovary) cells using the pEE14 plasmid system; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid).

A recombinant peptide in accordance with the invention may be recovered from a transformed host cell, or culture media, following expression thereof using a variety of techniques standard in the art. For example, detergent extraction, sonication, lysis, osmotic shock treatment and inclusion body purification. The protein may be further purified using techniques such as affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, and chromatofocusing.

Additional or alternative methodology for recombinant expression of peptides of the invention may be obtained from Sambrook, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), for example.

Compositions

The invention also provides compositions comprising one or more of the peptides and/or constructs (and/or one or more nucleic acid encoding the peptides and/or constructs) of the invention optionally in association with one or more diluents, carriers and/or excipients and/or additional ingredients. To this extent, it should be appreciated that reference herein to delivery or administration of a peptide, construct, nucleic acid and/or vector of the invention is to include reference to delivery or administration of a composition comprising a peptide, construct, nucleic acid and/or vector of the invention. In one embodiment, the one or more diluents, carriers and/or excipients are suitable for use in vitro. In another embodiment, the one or more diluents, carriers and/or excipients are suitable for use in vivo (in this instance they may be referred to as "pharmaceutically acceptable"). "Pharmaceutically acceptable diluents, carriers and/or excipients" is intended to include substances that are useful in preparing a pharmaceutical composition, may be coadministered with a peptide, construct, or nucleic acid encoding a peptide or construct of the invention while allowing it to perform its intended function, and are generally safe, non-toxic and neither biologically nor otherwise undesirable. Pharmaceutically acceptable diluents, carriers and/or excipients include those suitable for veterinary use as well as human pharmaceutical use. Examples of pharmaceutically acceptable diluents, carriers and/or excipients include solutions, solvents, dispersion media, delay agents, emulsions and the like.

In addition to standard diluents, carriers and/or excipients, a composition in accordance with the invention may be formulated with one or more additional constituents, or in such a manner, so as to enhance the activity of a peptide, construct, nucleic acid encoding a peptide or construct, and/or compound to be delivered to a cell, help protect the integrity or increase the half life or shelf life of such agents, or provide other desirable benefits, for example. By way of example, the composition may further comprise constituents which provide protection against proteolytic degradation, enhance bioavailability, decrease antigenicity, or enable slow release upon administration to a subject. For example, slow release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or a hydrogel. By way of further example, the compositions may also include preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifying agents, sweetening agents, colouring agents, flavouring agents, coating agents, buffers and the like. Those of skill in the art to which the invention relates will readily identify further additives which may be desirable for a particular purpose.

Furthermore, while not necessary for the performance of the invention, cell permeability of the peptides, constructs, nucleic acids encoding the peptides or constructs of the invention may be increased, or facilitated, through formulation of the composition. For example, the peptides, constructs, nucleic acids encoding the peptides or constructs may be formulated into liposomes. Further examples are provided in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957.

Additionally, it is contemplated that a pharmaceutical composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a cell or a subject in particular instances. Persons of ordinary skill in the art to which the invention relates will readily appreciate suitable additional active ingredients having regard to the description of the invention herein and the purposes for which the delivery of the peptide, compound and/or construct is required, including, for example, the nature and progression of any disease to be treated.

As a general example, agents used to prevent or inhibit the development of HCC (Sorafenib, Aurora Kinase Inhibitor PHA-739358, lactoferrin, omega 3 fatty acids, Gefitinib an EGFR inhibitor, Urocortin, angiogenesis inhibitors (eg TNP-470), Phenyl N- tert-butyl nitrone, immunostimulants) may be used. As a further example, in the case of cancer, one or more of the agents or therapies referred to in WO 2006/054908 may be used in compositions and methods of the invention.

By way of further example, in the case of anti-inflammation therapy the following agents may be used in compositions and methods of the invention: steroids, non-steroidal antiinflammatory drugs (for example aspirin, ibuprofen, and naproxen), DMARDs (disease- modifying antirheumatic drugs, for example azathioprine, sulfasalazine, cyclosporin, methotrexate), and biologies (for example anti-TNF antagonists, β-interferon, β2 and oc4 integrin antagonists).

By way of further example, in the case of anti-infection therapy, agents such as the following may be used in compositions and methods of the invention: anti-microbials rifampicin, macrolides, quinolones, ketolides, tetracyclines, isoniazid, pyrazinamide, ampicillin gentamicin, trimethoprim/sulfamethoxazole, erythromycin, vancomycin, fluoroquinolones, azithromycin, doxycycline, cefixime, ceftriaxone, ciprofloxacin, levofloxacin, ofloxacin, and the anti-virals Acyclovir, Famciclovir, Valacyclovir, antiretro viral drugs, anti-HIV drugs.

Compositions of the invention may be formulated into any customary form such as solutions, orally administrable liquids, injectable liquids, tablets, coated tablets, capsules, pills, granules, suppositories, trans-dermal patches, suspensions, emulsions, sustained release formulations, gels, aerosols, and powders, for example. Additionally, sustained release formulations may be utilised. The form chosen will reflect the purpose for which the composition is intended and the mode of delivery or administration to a sample or a subject. By way of example only, the dosage forms exemplified in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957 may be used where the compositions are formulated for administration to a subject, for example for the treatment of a disease. Skilled persons will readily recognise appropriate formulation methods. However, by way of example, certain methods of formulating compositions may be found in Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins, 2000. Methods

As mentioned herein before, in one embodiment, the invention provides methods of inducing or promoting apoptosis in a cell using a peptide, construct of the invention, and/or a nucleic acid encoding either one or both thereof. In another embodiment, the invention provides methods of inducing or promoting necrosis in a cell using one or more peptide, construct, and/or a nucleic acid encoding either one or both thereof. The inventors have found that using a construct comprising a peptide of the invention in combination with a peptide membrane translocation compound comprising at least the amino acid sequence LCLRPVG (or a functionally equivalent variant thereof) or MAARLCCQ (or a

functionally equivalent variant thereof) as described previously herein, is particularly useful in inducing or promoting necrosis.

When used herein "inducing or promoting apoptosis" and "inducing or promoting necrosis" should be taken broadly to include inducing or promoting one or more event which is required for apoptosis or necrosis to occur. Neither phrase should be taken to imply that the peptides and/or constructs of the invention are the only agents or signals required for cell death to occur, although that may be preferred.

The inventors contemplate the methods of the invention being applicable to any cell type. However, by way of example, the methods may be used to induce or promote apoptosis and/or necrosis in cells such as liver cells, kidney cells, adipose cells, cardiac cells, blood cells, endothelial cells, epithelial cells, mesechymal cells, eye cells (for example, retinal, corneal), skin cells, melanocytes, prostate cells, mammary cells, lung cells, brain cells, spinal cord cells, intestinal cells, stomach cells, salivary gland cells, esophagus cells, gallbladder cells, pancreatic cells (endocrine and exocrine), endocrine cells (for example, hypothalamus, pituitary or pituitary gland, pineal body or pineal gland, thyroid, parathyroid and adrenal), bladder cells, urethra cells, endometrial cells, ovary cells, lymph node cells (for example, peripheral lymph nodes, tonsils, adenoids, thymus and spleen), muscle cells, uterus, vagina, mammary glands, cells of the testes, vas deferens, seminal vesicles and penis, cells of the pharynx, larynx, trachea, bronchi, lungs and diaphragm, cells of the bones, cartilage, ligaments and tendons, stem cells, progenitor cells, and tumour cells.

In accordance with the above, the invention provides methods for the treatment of disorders characterised by a condition or disorder where reduction or removal of specific cells is of therapeutic benefit. Skilled persons will readily appreciate diseases falling within this definition. However by way of example, diseases characterised by aberrant cell growth and proliferation (such as cancer, tumors, obesity, inflammation, microbial infection), inflammatory disease, hypersensitivity, metabolic disease, neurodegenerative disease, skin disease, infected cells, cellular pathogenesis. In a particular embodiment the invention provides methods for the treatment of melanoma, liver cancer, kidney cancer, prostate cancer, ductal carcinoma, leukemia (such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute granulocytic leukemia, acute myelocytic leukemia such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemia and myelodysplastic syndrome, chronic leukemia such as but not limited to, chronic myelocytic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia), lymphoma (such as but not limited to Hodgkin's disease and non- Hodgkin's disease), hematopoietic tumor of myeloid lineage (such as but not limited to acute and chronic myelogenous leukemia, smoldering multiple myeloma, nonsecretory myeloma and osteosclerotic myeloma), hematopoietic tumor of lymphoid lineage

(including leukemia, acute and chronic lymphocytic leukemia, acute and chronic lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitts lymphoma), hematopoietic tumor of B lymphoid lineage, hematopoietic tumor of T cell lineage, multiple myelomas, bone and connective tissue sarcoma, brain tumor, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancer, eye cancer, vaginal cancer, vulvar cancer, cervical cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, testicular cancer, penal cancer, oral cancer, basal cancer, salivary gland cancer, pharynx cancer, skin cancer, Wilms' tumor, bladder cancer, angiogenesis, hyperproliferative disorders, cardiovascular disease (atherosclerosis and restenosis for example), chronic inflammation (rheumatoid arthritis and Crohn's disease for example), diabetes (including diabetic retinopathy), psoriasis, endometriosis, macular degeneration, obesity and adiposity, viral-induced hyperproliferation, papillomas and warts,

premalignant and nonneoplastic hyperproliferative disorders (including, for example, myelodysplastic disorders; cervical carcinoma-in-situ; familial intestinal polyposes such as Gardner syndrome; oral leukoplakias; histiocytoses; keloids; hemangiomas;

hyperproliferative arterial stenosis; EBV-induced lymphoproliferative disease,

hyperkeratoses and papulosquamous eruptions including arthritis, autoimmune disorders such as lupus, inflammatory arthritis, graft- vs-host disease, and scar formation).

By way of specific, but non-limiting, example the invention may be of use in the treatment of one or more of different cancer types as listed in WO 2006/054908 and Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,Inc, United States of America.

By way of further specific, but non-limiting, example the invention may be of use in the treatment of an inflammatory disease involving proliferation and/or pathogenesis of immune cells including but not limited to age-related macular degeneration, Crohn's disease, cirrhosis, chronic inflammatory-related disorders, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, granulomatosis, immune hyperproliferation associated with organ or tissue transplantation, an

immunoproliferative disease or disorder, e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), vascular hyperproliferation secondary to retinal hypoxia, or vasculitis; angiogenesis (proliferation of endothelial cells) which has applications in other disorders including but not limited to cancer,

cardiovascular diseases (atherosclerosis and restenosis for example) as found in Goronzy JJ, Weyand CM. The innate and adaptive immune systems. In: Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia, Pa: Saunders Elsevier;2007: chap 42. Male, Brostoff, Roth, Roit, Immunology 7th Ed, 2006.

By way of further specific, but non-limiting, example the invention may be of use in the treatment of intracellular infection including but not limited to infection with Epstein-Barr virus, herpes simplex virus (type 1 and 2), human immunodeficiency virus, Varicella zoster virus, Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Salmonella typhi/, S. Paratyphi, Legionella pneumophila. Chlamydia, Neisseria, Yersinia; as found in Murray et al.; Medical Microbiology 6th Ed, 2008; Brock Biology of

Microorganisms, 12th Ed, 2008.

In one embodiment, the disorder is cancer, an inflammatory disease, hypersensitivity, a hyper-proliferative disease, a metabolic disease, a neurodegenerative disease, a skin disease, infected cells, or a disorder mediated by pathogenic cells.

As used herein, the term "treatment" is to be considered in its broadest context. The term does not necessarily imply that a subject is treated until total recovery. Accordingly, "treatment" broadly includes, for example, the prevention, amelioration or management of one or more symptoms of a disease or the severity of one or more symptoms, and preventing or otherwise reducing the risk of developing a disease. "Prevention" of disease should not be taken to imply that disease development is completely prevented, and includes delay of disease development.

Delivery of the peptides and/or constructs (or nucleic acids encoding same) of the invention may occur in vivo or in vitro, depending on the purposes for which delivery is required.

The peptides and/or constructs (or nucleic acids encoding same) may be delivered to a cell by a number of different means, as will be readily appreciated by persons skilled in the art. However, by way of example, an in vitro method may comprise bringing the construct and/or peptide (or nucleic acids encoding same) into contact with one or more cells or a composition comprising one or more cells of interest; for example, contacting the construct or peptide (or nucleic acids encoding same) with a sample, composition or media in which the one or more cells are contained (such as mixing a composition of the invention with a liquid sample containing one or more cells). In another embodiment, a method of the invention comprises administering a construct and/or peptide (or nucleic acids encoding same) to a subject. It will be appreciated by those of general skill in the art to which the invention relates, having regard to the nature of the invention and the results reported herein, that the present invention is applicable to a variety of different animals. Accordingly, a "subject" includes any animal of interest. However, in one particular embodiment the "subject" is a mammal, more particularly human.

Administration to a subject may occur by any means capable of delivering the agents of the invention (peptides, constructs or nucleic acids encoding same) to target cells within the body of a subject. By way of example, agents of the invention may be administered by one of the following routes: oral, topical, systemic (eg. transdermal, intranasal, or by suppository), parenteral (eg. intramuscular, subcutaneous, or intravenous injection), by administration to the CNS (eg. by intraspinal or intracistemal injection), by administration to the liver (eg by intraportal injection), by implantation, and by infusion through such devices as osmotic pumps, transdermal patches, and the like. Skilled persons may identify other appropriate administration routes. Exemplary administration routes are also outlined in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957 for example.

As will be appreciated, the dose of an agent administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the reason for delivery of the agent, the target cells to which the agent is to be delivered, and the severity of any symptoms of a subject to be treated, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/or general health of a subject. It should be appreciated that administration may include a single daily dose, administration of a number of discrete divided doses, or continuous administration, as may be appropriate.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in cell cultures or animal models to achieve a cellular concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. l, p.l).

Administration could occur at any time during the progression of a disease, or prior to or after the development of a disease. In one embodiment, the agents of the invention are administered on a daily basis for an extended period to assist with ongoing management of symptoms. In another embodiment, the agents of the invention are administered on a daily basis for an extended period or life-long to prevent or delay the development of a disease. Additional examples of administration regimes are provided in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957.

It should be appreciated that a method of the invention may further comprise additional steps such as the delivery of additional agents or compositions to a sample, cell or subject.

As mentioned herein before, peptides, constructs and/or nucleic acids of the invention can be applied to the treatment of disease, but also can be used for research purposes, in vitro or in vivo. In one particular embodiment, the peptides could be connected to cell targeting compounds to induce apoptosis and/or necrosis and delete, knock out or at least reduce the number of cells of a selected cell types in an animal to study the contribution of the cell to organ system function or to generate an animal model of a disease, for example. For example, a peptide could be conjugated to a pancreatic beta cell homing peptide (ie targeting compound) to create a diabetic mouse; conjugated to a thyroid homing peptide to create a model of thyroid deficiency; conjugated to a stem cell homing peptide to examine aging; conjugated to peptide that homes to a brain cell population to examine neurodegenerative disease; conjugated to a leukocyte subset specific homing peptide to examine the contribution of a particular leukocyte subset to an immune response etc. By way of further example, a peptide, construct and/or nucleic acid of the invention could be used to create a model of Addison's disease due to adrenal insufficiency, vitiligo due to depigmentation, and immunodeficiences and autoimmunities due to loss of particular immune cell subsets. By way of further example, a construct of the invention could be used to knockout or at least reduce particular cell(s) in a mixed cell culture in order to enrich for a particular cell population. Accordingly, the invention also relates to a method of deleting or at least reducing the number of a selected cell type in an animal by administering a peptide, construct and/or nucleic acid of the invention to the animal. This could also be done in vitro. In another embodiment, the invention also provides an animal model of a disease, the animal characterised in that a selected cell type has been deleted or at least reduced by delivery of a peptide, construct and/or nucleic acid of the invention. In one embodiment, the animal is genetically modified to express a peptide or construct of the invention. Standard techniques may be used to generate genetically modified animals of the invention.

EXAMPLES

Example 1:

Materials and Methods Materials

The C32 melanoma cell line (Cat# CRL-1585), HepG2 liver cancer cell line (Cat# HB- 8065), Cos-7 green monkey kidney cell line (Cat# CRL-1651), DU-145 prostate cancer cell line (Cat# HTB-81), BT-549 ductal carcinoma cell line (Cat# HTB-122), SK-N-SH neuroblastoma cell line (Cat# HTB-11), and the THP-1 acute monocytic leukemia cell line (Cat# HIB-202) were purchased from the American Type Culture Collection. The COS-1 green monkey kidney cell line was kindly donated by Professor Phil Crosier (University of Auckland). Primary human keratinocytes were purchased from Invitrogen (Cat# 12332- 01). The HASMC human aortic smooth muscle cell line was purchased from Cascade Biologies (Cat# C-007-5C). The human amniotic stem cell line was purchased from

Celprogen (Cat# 36101-28). The buffy coat was isolated from the peripheral blood of a donor. An Annexin V-FLUOS staining kit (Roche Diagnostics cat# 11858777001) was used for visualization of apoptosis.

Methods

Testing the ability of the X-protein proapoptotic peptide to kill cancer and primary cell lines

The different cell lines were seeded into 8-well chamber slides at 1 x 10⁵ cells per well in full medium and incubated at 37° and 5% C0₂ overnight. The next day the cells were washed thrice with serum-free medium and 500 μΐ of the same medium was added to the wells. The X-protein proapototic peptide fused to a polyarginine stretch (biotin- RRRRRRRRKDCVF ) (SEQ ID 146) and a control cell-permeable peptide (biotin- MAARLCCQLDPARDVLCLRPVGAESRGRPVSGPFGPLSSPAFSVPADHGA) (SEQ ID 164), which does not cause apoptosis, were added to appropriate cells to final concentrations of 10 μΜ followed by incubation for 3 h at 37°C. Annexin-V labelling solution was prepared by adding 20 μΐ of Annexin V labelling reagent and 10 μΐ of propidium iodide in 1 ml of incubation buffer. The cells were washed with incubation buffer, and 100 μΐ of Annexin-V labelling solution was added, followed by incubation for 15 min in the dark. The cells were washed with incubation buffer, fixed with 4% formaldehyde in PBS for 30 min, washed with PBS and slide chambers removed. A drop of Prolong Gold anti-fade reagent with DAPI was added to each sample, the slides dried overnight, and then examined by fluorescence microscopy.

Visualization of cell apoptosis by the TUNEL assay

The cells were washed with PBS, fixed in 4% formaldehyde in PBS for 60 min, and incubated with permeabilization solution for 2 min on ice. TUNEL reagent (50 μΐ) was added to each well, and the slides incubated in the dark at 37°C for 60 min. Cells were washed with PBS, the chambers removed, and a drop of Prolong Gold antifade reagent with DAPI (Invitrogen cat# P36931) was added to each well. Slides were dried overnight in the dark, and examined by microscopy by using a Nikon E600 fluorescent microscope. Photos were taken with using Nikon ACT-1 software.

Measurement of caspase-3, 7 activities

Cells were plated into eight chamber slides at 1 x 10⁵ cells per well and incubated overnight. Wells were washed thrice with MEM media, and X-protein peptides added in 250 μΐ of MEM media to a final concentration of 10 μΜ. Slides were incubated for 3 h at 37°C and 5% C(¾., and then the medium was replaced with 300 μΐ of MEM containing 10% FCS. Magic Red dye apoptosis detection solution (Kamiya Biomedical Company cat# KT-086), made according to manufacturer's instructions, was added to the cells at a ratio of 1 :31. Slides were incubated for 1 h at 37°C and 5% C0₂. Cells were washed with PBS, fixed with 4% formaldehyde in PBS for 30 min, washed twice with PBS using 1 min rinses, and the slide chambers removed. A drop of Prolong Gold antifade reagent with DAPI (Invitrogen cat# P36931) was added to each well. Slides were dried overnight in the dark, and examined by microscopy by using a Nikon E600 fluorescent microscope. Photos were taken with using Nikon ACT-1 software.

Results

Identification of a proapoptotic X-protein peptide motif

HepG2 cells were incubated for 3 h with nine peptides 1-40, 41-80, 81-120, 121-141, 140- 153, 1-20, 16-35, 21-40, and 34-53 covering the entire X-protein sequence. The peptides were rendered cell permeable by including a polyArg (R8) sequence at the N terminal of each peptide. Peptides were added at a final concentration of 10 μΜ. The cell-permeable peptide encompassing aa residues 81-120

TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR (SEQ ID 165) of the X-protein was the only peptide able to cause the apoptosis and death of HepG2 cells (Fig. 1). Four shorter cell-permeable peptides encompassing aa residues 81-120

(TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR), namely aa 81-90 (TVNGRRGLPK) (SEQ ID 166), 91-100 (VLHKRTLGLS) (SEQ ID 167), 101-110

(AMSTTDPEAY) (SEQ ID 7), and 111-120 (FKDCVFTYWR) (SEQ ID 5) were tested for their proapoptotic ability in order to identify a minimal motif (as noted above each peptide was rendered cell permeable by including of the peptide R8 at the N terminus; for example, RRRRRRRRTVNGRRGLPK (SEQ ID 168), RRRRRRRRVLHKRTLGLS (SEQ ID 169), RRRRRRRRAMSTTDPEAY (SEQ ID 170), and

RRRRRRRRFKDCVFTYWR) (SEQ ID 171). Peptides 101-110 and 111-120 both caused HepG2 cells to undergo apoptosis, whereas the other peptides were inactive (Fig. 2 A). A repeat experiment gave similar results (Fig. 2B). Three cell-permeable peptides encompassing aa 106-116 (DPEAYFKDCVF) (SEQ ID 172,) 1 12-118 (KDCVFTY) (SEQ ID 173), and 112- 116 (KDCVF) (SEQ ID 4) (rendered cell-permeable by fusion to R8) were next tested for proapoptotic ability in order to define the minimal apoptotic domain of the region encompassing aa residues 101-120. Peptide 1 12-116 was the only peptide able to cause apoptosis of HepG2 cells, as detected by TUNEL staining (Fig. 3). That peptide 112-118 was inactive, whereas peptide 111-120 was active, suggests that addition of two aa residues to the C-terminus of peptide 112-116 is inhibitory, whereas the addition of four aa residues restores activity. Alternatively, peptide 112-118 may have been synthesized incorrectly, causing it to be inactive. In the above experiments the apoptotic peptides were fused to an Arg8 peptide to render them cell-permeable. The 112-116 peptide was also fused to the X-protein carrier peptide aa 16-22 (LCLRPVG) to determine whether the carrier peptide influences its apoptotic function. HepG2 cells incubated with the fusion peptide for 3 h showed high levels of apoptosis, comparable to that of HepG2 cells treated with the apoptosis peptide aa 112-116 fused to a polyarginine tag for cell permeability (Fig. 4). Further, the cells underwent necrosis, which was not evident with cells treated with the cell-permeable peptide aa 112- 116 fused to polyarginine. Control HepG2 cells not treated with peptide or treated with a cell-permeable form of the non-apoptotic X-protein peptide aa 1-50 showed no signs of apoptosis (Fig. 4).

Dose-response and time-scale of apoptosis

HepG2 cells were incubated for 3 h with 0, 1, 5, and 10 μΜ concentrations of the parental peptide aa 111-120 (Figure 5 A) and the tuncated peptide 112-116 (Figure 5B). Both peptides induced cell apoptosis at 1 μΜ. The time taken for the polyarginine fused peptide aa 112-116 (Fig. 6 A) to induce the apoptosis of HepG2 cells was compared with that of peptide aa 112-116 fused to X-protein peptide aa 16-22 (Fig. 6B). Apoptosis, as measured by annexin-V staining, was compared at time points of 0.5 h, 1 h, 2 h and 3 h after addition of the peptides. Apoptosis was visible 30 min after addition of the peptides, and increased slightly at 2 and 3 h (Fig. 6A,B). As found above, the polyarginine-fused peptide aa 112- 116 did not cause cell necrosis. In contrast, peptide aa 112-116 fused to peptide aa 16-22 caused necrosis that was detectable after 2 h of incubation with the peptide, and increased at 3 h such that almost all cells were necrotic. As before, control HepG2 cells not treated with peptide or treated with the non-apoptotic X-protein peptide aa 1-50 showed no signs of apoptosis during a 3 h incubation (data not shown).

The proapoptotic peptide 112-116 is able to kill multiple cell types

The proapoptotic peptide 112-116 rendered cell-permeable by fusion to a polyarginine carrier peptide was tested for its ability to kill a variety of cancer and primary cell types. It caused all tumour/immortalized cell lines tested to undergo apoptosis including HepG2, DU145, Caco2 (Fig. 7A), SK-N-SH, THP-1, BT549 (Fig. 7C), A375 (Fig. 7E), and COS-1 (Fig. 7F) cells. It also caused normal peripheral blood mononuclear cells, human keratinocytes, and aortic smooth muscle cells to undergo apoptosis (Fig. 7G). In contrast, cells treated with a cell-permeable non-apoptotic peptide did not undergo apoptosis (Figs 7B, 7D, 7E, 7F, 7H).

X-protein proapoptotic peptides induce the activity of caspase(s) 3 and 7

HepG2 cells treated with the X-protein proapoptotic peptides aa 111 - 120 and 112-116 were tested for caspase 3/7 activity to establish whether the X-protein peptides induce apoptosis through a caspase-dependent pathway. The cells were incubated with Magic Red dye consisting of the fluorophore cresyl violet attached to the tetrapeptide DEVD (SEQ ID 174), which is a substrate for caspase 3/7. The fluorophore fluoresces red at the excitation wavelength of 550-590 nm when DEVD is hydrolyzed by caspase 3/7. HepG2 cells incubated with the aa 111-120 and 112-116 peptides fluoresced red establishing that caspase 3/7 activity had been induced (Fig. 8). Control cells not treated with the apoptotic peptides did not fluoresce, indicating there was no endogenous caspase 3/7 activity. D-isomeric and truncated forms of the proapoptotic peptide KDCVF are able to cause HepG2 cells to undergo apoptosis

A protease-resistant D-isomeric form of the proapoptotic peptide 112-116 (kdcvf (SEQ ID 4)) rendered cell-permeable by fusion to the carrier peptide lclrpvg was incubated at 10 μΜ with HepG2 cell for 3 h causing the cells to undergo apoptosis. The proapoptotic peptide KDCVF was truncated by removing the N- and C-terminal residues to give the peptides DCVF (SEQ ID 3) and KDCV (SEQ ID 2). The peptides as L-isomers were rendered cell-permeable by fusion to the carrier peptide LCLRPVG (SEQ ID 9). Both peptides at 10 μΜ caused HepG2 cells to undergo apoptosis following a 3 h incubation. The biotin-LCLRPVGDCVF (SEQ ID 175) peptide was only partially soluble which may account for its reduced level of activity. The results suggest the core proapoptotic peptide in the tripeptide "DCV".

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Furthermore, titles, headings, or the like are provided to enhance the reader's

comprehension of this document, and should not be read as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference.

However, the reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country in the world. Throughout this specification (and any claims which follow), unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".

REFERENCES

1. Kumar V, Jayasuryan N, Kumar R. A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function. Proc Natl Acad Sci U S A 93:5647-52, 1996.

2. Misra KP, Mukherji A, Kumar V. The conserved amino-terminal region (amino acids 1-20) of the hepatitis B virus X protein shows a transrepression function. Virus Res 105:157-65, 2004.

3. Shirakata, Y. and K. Koike (2003). Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J Biol Chem 278(24): 22071- 8

4. Liu, H., L. Ye, et al. (2009). Effect of a conserved peptide derived from Kunitz domain of hepatitis B virus x protein on the cell cycle and apoptosis of HepG2 cells via the proteasome pathway. Chinese Med J 122(4): 460-5.

5. Gupta, S. (2003). "Molecular signaling in death receptor and mitochondrial

pathways of apoptosis (Review)." Int J Oncol 22(1): 15-20.

Claims

1. A peptide comprising the amino acid sequence DCV or a functionally equivalent variant thereof.

2. A peptide as claimed in claim 1 wherein the peptide or functionally equivalent variant further comprises at its N-terminus, one or more amino acids which correspond consecutively to amino acids 81 to 112 of a native X-protein, and/or at its C-terminus, one or more amino acids which correspond consecutively to amino acids 116 to 120 of a native X-protein.

3. A peptide as claimed in claim 1 or 2 wherein the peptide comprises the amino acid sequence KDCV or is a functionally equivalent variant thereof.

4. A peptide as claimed in claim 1 or 2 wherein the peptide comprises the amino acid sequence DCVF or is a functionally equivalent variant thereof.

5. A peptide as claimed in any one of claims 1 to 4 wherein the peptide comprises the amino acid sequence KDCVF or is a functionally equivalent variant thereof.

6. A peptide as claimed in any one of claims 1 to 5 wherein the peptide comprises the amino acid sequence FKDCVFTYWR or is a functionally equivalent variant thereof.

7. A peptide as claimed in any one of claims 1 to 7 wherein the peptide comprises the amino acid sequence TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR or is a functionally equivalent variant thereof.

8. A peptide as claimed in claim 1 wherein the peptide consists of the amino acid sequence DCV, KDCV, DCVF, KDCVF, FKDCVFTYWR or

TVNGRRGLPKVLHKRTLGLSAMSTTDPEAYFKDCVFTYWR.

9. A peptide comprising the amino acid sequence AMSTTDPEAY or a functionally equivalent variant thereof.

10. A peptide as claimed in claim 9 wherein the peptide or functionally equivalent variant further comprises at its N-terminus, one or more amino acids which correspond consecutively to amino acids 81 to 100 of a native X-protein, and/or at its C-terminus, one or more amino acids which correspond consecutively to amino acids 1 11 to 120 of a native X-protein.

11. A peptide as claimed in claim 9 wherein the peptide consists of the amino acid sequence AMSTTDPEAY.

12. A nucleic acid encoding a peptide or functionally equivalent variant of any one of claims 1 to l l.

A nucleic acid vector comprising a nucleic acid of claim 12.

14. A host cell comprising a nucleic acid of claim 12 or a vector of claim 13.

15. Use of a peptide or a functionally equivalent variant thereof as claimed in any one of claim 1 to 11 aspect as an agent for inducing or promoting apoptosis or necrosis in one or more cell.

16. A construct comprising a peptide or a functionally equivalent variant thereof as claimed in any one of claims 1 to 11 and at least one other compound.

17. A construct as claimed in claim 16 wherein the at least one other compound is a cell membrane translocation compound.

18. A construct as claimed in claim 16 wherein the at least one other compound is a cell targeting compound.

19. A construct as claimed in claim 16, wherein the construct comprises both a cell membrane translocation compound and a cell targeting compound.

20. A construct as claimed in any one of claims 17 to 19 wherein the cell membrane translocation compound is a peptide.

21. A construct as claimed in claim 20 wherein the peptide is chosen from the group consisting of: polyArg peptide, penetratin, Tat, a peptide comprising the amino acid sequence LCLRP, LCLRPVG, LCLRPVGAE, LCLRPVGAESR, LCLRPVGAESRGRPV,

LCLRPVGAESRGRPVSGPFG, MAARLCCQLDPARDVLCLRP, MAARLCCQ,

MAARLCCQLDPARDV and a functionally equivalent variant of any one thereof.

22. A construct as claimed in any one of claim 18 to 21 wherein the cell targeting compound is a ligand specific to a particular cell type.

23. A construct as claimed in claim 22 wherein the cell targeting molecule is chosen from the group consisting of: YIGSR, a-melanocyte stimulating hormone, peptide-MHC-tetramer, antiidiotype antibody, an antibody, scFV, a phagemid, and an aptamer recognizing a cell-type-specific marker.

24. Use of a construct as claimed in any one of claim 16 to 23 as an agent for inducing or promoting apoptosis or necrosis in one or more cell.

25. A nucleic acid encoding a construct of any one of claims 16 to 23.

26. A vector comprising a nucleic acid of claim 25.

27. A host cell comprising a nucleic acid of claim 25 or a vector of claim 26.

28. A composition comprising one or more peptide of any one of claims 1 to 11 , one or more nucleic acid of claim 12, and/or one or more vector of claim 13.

29. A composition comprising one or more construct of any one of claims 16 to 23, one or more nucleic acid of claim 25 and/or one or more vector of claim 26.

30. A method of promoting or inducing apoptosis in a cell, the method comprising contacting a peptide or a functionally equivalent variant thereof as claimed in any one of claims 1 to 11, a nucleic acid as claimed in claim 12, a vector as claimed in claim 13, a construct as claimed in any one of claims 16 to 23, a nucleic acid as claimed in claim 25, a vector as claimed in claim 26, and/or a composition comprising any one or more thereof with the cell or a composition comprising the cell.

31. A method of promoting or inducing necrosis in a cell, the method comprising contacting a peptide or a functionally equivalent variant thereof as claimed in any one of claims 1 to 11, a nucleic acid as claimed in claim 12, a vector as claimed in claim 13, a construct as claimed in any one of claims 16 to 23, a nucleic acid as claimed in claim 25, a vector as claimed in claim 26 and/or a composition comprising any one or more thereof with the cell or a composition comprising the cell.

32. A method for the treatment of a condition or disorder where reduction in the number of specific cells or removal of specific cells is of therapeutic benefit, the method comprising administering to a subject a peptide or a functionally equivalent variant thereof as claimed in any one of claims 1 to 11, a nucleic acid as claimed in claim 12, a vector as claimed in claim 13, a construct as claimed in any one of claims 16 to 23, a nucleic acid as claimed in claim 25, a vector as claimed in claim 26 and/or a composition comprising any one or more thereof.

33. Use of a peptide or a functionally equivalent variant thereof as claimed in any one of claims 1 to 11, a nucleic acid as claimed in claim 12, a vector as claimed in claim 13, a construct as claimed in any one of claims 16 to 23, a nucleic acid as claimed in claim 25, a vector as claimed in claim 26 and/or a composition comprising any one or more thereof in the manufacture of a medicament for the treatment of a condition or disorder where reduction in the number of specific cells or removal of specific cells is of therapeutic benefit.

34. A method as claimed in claim 30 or the use as claimed in claim 31 wherein the disorder is cancer, an inflammatory disease, hypersensitivity, a hyper-proliferative disease, a metabolic disease, a neurodegenerative disease, obesity, a skin disease, infected cells, and/or a disorder mediated by pathogenic cells.

35. A method of deleting or at least reducing the number of cells of a selected cell type in an animal by administering a peptide, construct and/or nucleic acid of the invention to the animal.

36. An animal model of a disease, the animal characterised in that a selected cell type has been deleted or at least reduced by delivery of a peptide, construct and/or nucleic acid of the invention.