WO2018109771A1 - Fusion proteins for treatment of cancer - Google Patents

Abstract

Chimeric polypeptides comprising a first moiety comprising an NKp44-derived peptide fused to a second moiety comprising one or more cell penetrating peptides (CPP), and polynucleotides encoding the same. Further provided are methods for treating, or ameliorating cancer.

Description

FUSION PROTEINS FOR TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Israel Patent Application No. 249610 filed December 15, 2016, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present invention is directed to, inter alia, fusion polypeptides and use thereof such as in the therapy of cancer. BACKGROUND OF THE INVENTION

[003] Cancer is a leading cause for high mortality and morbidity rates among the human population. Proliferating Cell Nuclear Antigen (PCNA) is a key regulator of DNA replication, repair, cell cycle control and apoptosis. PCNA is over-expressed in many cancer types and PCNA-overexpression is a marker for cancer virulence. PCNA can be considered as a hub protein regulating growth of cancer cells as well as their responses to cellular stress induced by other therapies such as chemotherapies. Therefore, PCNA is considered as a potential anticancer target as a sole agent or in combination with chemotherapeutic regimens.

[004] The activity of NK cells is acquired through a delicate balance between activation and inhibitory signals. For that purpose, NK cells bear a variety of activation and inhibitory receptors. Major activating receptors include NKG2D and the natural cytotoxicity receptors (NCRs): NKp46 (NCR1), NKp44 (NCR2) and NKp30 (NCR3). Activated NK cells harbor cytolytic activity against both tumor and virus-infected cells. NKp44 is a receptor expressed by natural killer (NK) cells and PCNA is a ligand for NKp44 (Rosental et al. Journal of immunology 2011, 187:5693-702). The interaction of NKp44 with PCNA inhibits lysis and IFNy secretion by NK cells. The inhibition is mediated by an immunoreceptor tyrosine -based inhibitory motif (ITIM) on the NKp44 cytoplasmic domain, and the nuclear/cytoplasmic PCNA in the target cell is recruited to the NK immunological synapse (NKIS). SUMMARY OF THE INVENTION

[005] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[006] According to a first aspect, there is provided a chimeric polypeptide comprising a first moiety and a second moiety, said first moiety comprising 15 to 25 amino acid residues derived from SEQ ID NO: 1 (E AS ALVCIRLVTS S KPRTX i A WTS RFTIWX2X3 , wherein Xi is selected from V and M, X₂ is selected from A and D, and X3 is selected from A and D), or an analog thereof having at least 85% sequence identity thereto, and said second moiety comprises one or more cell penetrating peptides (CPP)s. [007] In some embodiments, the chimeric polypeptide is capable of penetrating a cell and binding proliferating cell nuclear antigen (PCNA). In some embodiments, the first moiety binds PCNA. In some embodiments, the second moiety is capable of penetrating a cell.

[008] In some embodiments, the chimeric polypeptide comprises one or more modifications. In one embodiment, the one or more modifications is a post-translation modification. In one embodiment, the modification is selected from the group consisting of: acetylation, amidation, cyclization, and pegylation. In some embodiments, the chimeric polypeptide further comprises an acetyl group attached to the N-terminus thereof. In some embodiments, the chimeric polypeptide comprises a D-amino acid residue at the C-termini of the first moiety thereof. In one embodiment, the c-termini alanine residue of SEQ ID NO: 3 or 4 is a D-amino acid. In one embodiment, the c- termini aspartate or alanine residue of SEQ ID NO: 5 or 6 is a D-amino acid

[009] In some embodiments, the first moiety comprises the amino acid sequence as set forth in SEQ ID NO: 2 (VTSSKPRTXiA wherein Xi is selected from V and M), at its N-terminus or its C -terminus.

[010] In some embodiments, the first moiety comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3 (E AS ALVCIRLVTS S KPRT V A) , SEQ ID NO: 4 (E AS ALVCIRLVTS S KPRTM A) , SEQ ID NO: 5 (VTSSKPRTVAWTSRFTIWX2X3, wherein X₂ is selected from D and A, and X3 is selected from D and A), and SEQ ID NO: 6 ( VTS S KPRTM A WTS RFTIWX2X3 , wherein X₂ is selected from D and A, and X₃ is selected from D and A), or an analog thereof having at least 85% sequence identity thereto.

[Oi l] In some embodiments, the second moiety comprises a nuclear localization sequence having an amino acid sequence selected from the group consisting of: SEQ ID NO: 7 (KKKRR), SEQ ID NO: 8 (PKKKRRV), SEQ ID NO: 9 (KRRMKWKK), and SEQ ID NO: 10 (KKKRK).

[012] In some embodiments, the second moiety comprises a nuclear localization sequence having the amino acid sequence as set forth in SEQ ID NO: 10 (KKKRK).

[013] In some embodiments, the second moiety further comprises between 6 and 20 contiguous arginine residues comprising the amino acid sequence as set forth in SEQ ID NO: 11 (RRRRRR). [014] In some embodiments, the contiguous arginine residues consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 12 (RRRRRRRRR), and SEQ ID NO: 13 (RRRRRRRRRRR).

[015] In some embodiments, the second moiety comprises between 6 and 20 contiguous arginine residues and one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 7 (KKKRR), SEQ ID NO: 8 (PKKKRRV), SEQ ID NO: 9 (KRRMKWKK), and SEQ ID NO: 10 (KKKRK), and any combination thereof.

[016] In some embodiments, the second moiety is attached to said first moiety via a linker. In some embodiments, the second moiety is attached to said first moiety via a peptide bond. In some embodiments, the second moiety is contiguous to the C-terminus and/or the N-terminus of said first moiety.

[017] In some embodiments, the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 19 (RRRRRRRRRKKKRKE AS ALVCIRLVTS S KPRTX A , wherein X is selected from A and D, bolded underlined residue is amidated), SEQ ID: 22 (EASALVCIRLVTSSKPRTXAWKKKRKIRRRRRRRRRRR, wherein X is selected from A and D, bolded underlined residue is amidated), SEQ ID NO: 23 (RRRRRRRRRRRIKKKRKWEASALVCIRLVTSSKPRTXA, wherein X is selected from A and D, bolded underlined residue is amidated), SEQ ID NO: 27 (RRRRRRRRRRRIKKKRKVTS S KPRT VAWTSRFTIW AD, bolded underlined residue is amidated), SEQ ID NO: 28 (RRRRRRRRRRRIKKKRKVTSSKPRTVAWTSRFTIWAD, SEQ ID NO: 29 (RRRRRRRRRRRIKKKRKVTS S KPRT A A ATS RFTIW AD ) , SEQ ID 30 (RRRRRRRRRRRIKKKRKWVCIRLVTS S KPRTVAWTSRF) , SEQ ID NO: 31 (RRRRRRRRRRRIKKKRKVTS S KPRT AAWTSRFTIW AD ), SEQ ID NO: 32 (RRRRRRRRRRRIKKKRKVTS S KPRTWAATSRFTIW AD ), and SEQ ID NO: 33 (RRRRRRRRRRRIKKKRKWEASALVCIRLVTSSKPRTVAVTSSKPRTXAWTSRFTrWAA).

[018] In some embodiments, the invention provides a pharmaceutical composition comprising any of the chimeric polypeptide of the invention, and a pharmaceutically acceptable carrier.

[019] In some embodiments, there is provided a polynucleotide molecule encoding any of the chimeric polypeptides of the invention. In some embodiments, there is provided an expression vector comprising the polynucleotide molecule.

[020] According to some aspects, the invention provides a method for treating or ameliorating cancer in a subject in need thereof, the method comprising administering to the subject any one of: (i) any of the chimeric polypeptide of the invention; (ii) an expression vector comprising a polynucleotide molecule encoding any of the chimeric polypeptides of the invention; or (iii) a pharmaceutical composition comprising any of the chimeric polypeptide of the invention, thereby treating or ameliorating cancer in said subject. In some embodiments, the cancer is selected from: breast cancer, lung cancer, and colon cancer.

[021] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[022] Fig. 1 is a graph demonstrating cell viability in 4T1 cells treated with chimeric peptides comprising NKp44-derived peptides: SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 19, and SEQ ID NO: 23;

[023] Fig. 2 is a graph demonstrating cell viability in A549 cells treated with chimeric peptides comprising NKp44-derived peptides: SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 19, and SEQ ID NO: 23;

[024] Fig. 3 is a graph demonstrating cell viability in 4T1 cells treated with chimeric peptides comprising NKp44-derived peptides: SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 27, and Ac- SEQ ID NO: 23; [025] Fig. 4 is a graph demonstrating cell viability in A549 cells treated with chimeric peptides comprising NKp44-derived peptides: SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 27, SEQ ID NO: 23;

[026] Fig. 5 is a graph demonstrating tumor progression in C57BL mice that were injected with 0.5xl0⁶ cells of murine B 16 melanoma cell line and treated with a PBS solution, which served as the vehicle control;

[027] Fig. 6 is a graph demonstrating tumor progression in C57BL mice that were injected with 0.5xl0⁶ cells of murine B 16 melanoma cell line and treated with 1 mg/Kg of SEQ ID NO: 23 three times a week; [028] Fig. 7 is a graph demonstrating tumor progression in C57BL mice that were injected with 0.5xl0⁶ cells of murine B 16 melanoma cell line and treated with 5 mg/Kg of SEQ ID NO: 23 three times a week;

[029] Fig. 8 is a graph demonstrating tumor progression in C57BL mice that were injected with 0.5xl0⁶ cells of murine B 16 melanoma cell line and treated with 5-FU, at a dose of 30 mg/Kg; [030] Fig. 9 is a graph demonstrating tumor progression in six weeks-old female BALB/c mice that were injected with 2xl0⁵ 4T1 cells in the left mammary fat pad, allowed to develop tumors and treated intravenously, with SEQ ID NO: 29, at a dose of 10 or 5 mg/Kg, 5 times a week or a PBS solution, which served as a vehicle control, consecutively, for a duration of 2 weeks, results represent means + SE of 5 mice in peptide groups, and 4 mice in the control group; [031] Figs. 10A-10B are bar graphs demonstrating (A) endpoint tumor size of tumors obtained from euthanized mice that were treated, intravenously, with a PBS solution, or with SEQ ID NO: 29, at a dose of 10 or 5 mg/Kg, 5 times a week, consecutively, for a period of 2 weeks, results represent means + SE of 5 mice in peptide groups, and 4 mice in the control group; and (B) endpoint tumor weight of tumors obtained from euthanized mice that were treated, intravenously, with a PBS solution, or with SEQ ID NO: 29, at a dose of 10 or 5 mg/Kg, 5 times a week, consecutively, for a period of 2 weeks, results represent means + SE of 5 mice in peptide groups, and 4 mice in the control group;

[032] Figs. 11A-11E are graphs of ELISA assays. (A) Binding curves of SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 6 at a peptide concentration range of 0-7.24 μΜ to plate bond hPCNA. (B) ProteOn array showing the binding of hPCNA at protein concentrations ranging of 0-250 nM to bond SEQ ID NO: 14 (E AS ALVCIRLVTS S KPRTX A) . (C) ELISA assay showing the specific recognition of SEQ ID NO: 14 (5 μ§/ηύ) to hPCNA relative to (i) TL1A, (ii) APO- E3, and (iii) HNF4. (D) ELISA blocking assay of NKp44-PCNA interaction via SEQ ID NO: 14. SEQ ID NO: 36 was used as a negative control. No peptide represents the maximal binding capacity of NKp44 to hPCNA. (E) ELISA assay representing the binding of biotinylated NKp44- derived pep8 to NKp44-hFc. NKp46-hFc and hFc were used as negative controls; [033] Figs. 12A-12F are graphs demonstrating cell viability in murine 4T1 breast cancer and human 549 lung cancer lines used for screening different CPP fused to NKp44-derivedd pep8 and their viability in a PrestoBlue assay. The results are shown in respect to the basal viability indicated using equivalent %DMSO (background). CPP-NKp44-derived pep8 were divided according to their effect on cell viability and the cancer model. (A) 4T1; low effect: pep8 (SEQ ID NO: 5), Ac-Rl l-NLS-pep8short (SEQ ID NO: 24), Ac-Tf-NLS-pep8 (SEQ ID NO: 18), Ac- Tf-pep8 (SEQ ID NO: 17), and Ac-TfTf-pep8 (SEQ ID NO: 25); (B) 4T1; medium effect: Ac- mAntp-NLS-pep8 (SEQ ID NO: 20), Ac-mAntp-pep8 (SEQ ID NO: 15), and Ac-NLS-pep8 (SEQ ID NO: 16); (C) 4T1; high effect: Ac-pep8-NLS-Rl l (SEQ ID NO: 22), Ac-R9-NLS-pep8 (SEQ ID NO: 19), and Ac-Rl l-NLS-pep8 (SEQ ID NO: 23); (D) A549; low effect: Ac-NLS- pep8, Ac-Rl l-NLS-pep8short, Ac-TfTf-pep8, Ac-mAntp-pep8, Ac-Tf-pep8, pep8, Ac-mAntp- NLS-pep8, and Ac-Tf-NLS-pep8; (E) A549; medium-high effect: Ac-R9-NLS-pep8, Ac-pep8- NLS-R11, and Ac-Rl l-NLS-pep8. ED50 was calculated using a sigmoidal dose-response curve only for CPPs-NKp44-derived pep8 that have shown reduced viability below 50% after 24 h; (F) Cell viability of DMSO (negative control) or selenite (positive control) treated 4T1 and A549 cells relative to complete medium. Statistical analysis was performed in comparison to DMSO. Unpaired t-test; one-tail. * p<0.05, **p<0.01.

Figs. 13A-13F depicts affinity of Rl l-NLS-pep8 (SEQ ID NO: 23) towards PCNA and its effect on cell viability. ProteOn array showing the binding of hPCNA at protein concentration range of 0-250 nM to bond Rl l-NLS-pep8 (A) and Rl l-NLS-pep8short (SEQ ID NO:24) (B). Murine B 16 melanoma, human PANC-1 (pancreas ductal adenocarcinoma), HepG2 (liver hepatocellular carcinoma), and MDA-MB-231 (breast adenocarcinoma) cancer cell lines were used for checking the effect of Rl l-NLS-pep8 and Rl l-NLS-pep8short on cell viability in a PrestoBlue assay. The results were calculated with respect to the basal viability indicated using equivalent % DMSO (background). ED50 was calculated using a sigmoidal dose-response curve. ED50 values were 7.1, 7.8, 5.9, and 5.6 μΜ respectively for B 16, PANC-1, HepG2 and MDA-MB-231 (C, D, E, and F).

[034] Figs. 14A-14D are graphs representing flow cytometry based cell death assay using PI of (A.i) gating strategy of cancer cell line for the detection of PI positive (dead) cells in culture medium; (A.ii) representative dot-plots for the effect of Ac-Rl l-NLS-pep8 (SEQ ID NO: 23) on cell death relative to equivalent DMSO percentages. The effect of Ac-Rl l-NLS-pep8 on cell viability was examined using the PrestoBlue assay in a cell death assay of (B) murine B 16 cell line, (C) murine 4T1 cell line, and (D) human MDA-MB-231 cell line. Camptothecin and APIM peptide were used as positive control; Ac-Rl l-NLS-pep8short (SEQ ID NO :24) and DMSO were used as negative controls. ED50 was calculated using a sigmoidal dose-response curve only for treatments that have shown to induce cell death above 50% after 24 h.

[035] Figs. 15A-15B are graphs demonstrating tumor progression in mice treated with Ac-Rl l- NLS-pep8 (SEQ ID NO: 23). (A.i) 4T1 cells or (B.i) B 16 cells were injected in to the nipple pad or flank of young BALB/C females or C57BL/6 males, respectively (day 0). At day 5, mice were divided to sub-groups according to weights. Treatment was initiated at day 5 (4T1) or day 6 (B 16) and ended at day 22. Treatment was administrated every 2 days/ 3 times/week; 5-FU was administered LP while Ac-Rl l-NLS-pep8 was administrated I.V (4T1) or LP (B 16). Tumor volume (mm³) was measured at the days indicated on the graphs. Mean tumor volumes (mm³) for 4T1 cells (A.ii) and B 16 cells (B.ii), were normalized to vehicle/day to show the effect of treatment on tumor volume per day. Statistical analysis was performed on original tumor volume data. Unpaired t-test; one-tail. * p<0.05.

DETAILED DESCRIPTION OF THE INVENTION [036] The present invention provides a chimera comprising a NKp44-derived peptide moiety ('first moiety') and a cell penetrating moiety ('second moiety') and composition comprising same. The invention further provides methods of treatment using said chimera. Advantageously, the chimeras of the invention reduce and/or inhibits cancer cell proliferation.

[037] The present invention is based, in part, on the surprising finding that a chimera comprising a NKp44-derived peptide and a cell penetrating moiety interact with PCNA and inhibit cancer cell proliferation. The present invention is further based, in part, on the surprising finding that the chimera disclosed herein induces cell death of cancer cells.

Chimeras

[038] According to some aspects, the invention provides a chimera comprising a first moiety and a second moiety, said first moiety has 15 to 25 amino acid residues derived from NKp44 (SEQ ID NO: 34), wherein X is selected from Val and Met, and said second moiety comprises one or more cell penetrating peptides.

[039] In some embodiments, the chimera is capable of penetrating a cell (e.g. a cancer cell) and binding PCNA. [040] The terms "chimera" and "chimeric polypeptide" are used interchangeably to refer to a polypeptide formed by the joining of two or more peptides through a peptide bond formed between the amino terminus of one peptide and the carboxyl terminus of another peptide. In some embodiments, the chimera further comprises an acetyl group. In some embodiments, the acetyl group is attached to the N-terminus of the chimera. [041] The chimera may be formed by a chemical coupling of the constituent peptides or it may be expressed as a single polypeptide fusion protein from a nucleic acid sequence encoding the single contiguous conjugate.

[042] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogs peptoids and semi-peptoids or any combination thereof. In another embodiment, the terms "peptide", "polypeptide" and "protein" apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid. [043] In some embodiments, the second moiety is attached to the first moiety via a linker. In another embodiment, the second moiety is attached to the first moiety via a covalent bond. In some embodiments, the second moiety is attached to the first moiety via a peptide bond. In some embodiments, the second moiety is contiguous to the C-terminus and/or the N-terminus of the first moiety. [044] In some embodiments, the chimeric polypeptide of the invention comprises or consists of an amino acid sequence selected from the amino acid sequences presented in Table 1.

Table 1 : chimeric polypeptides of the invention

[045] In some embodiments, the chimeric polypeptide of the invention comprises or consists of an amino acid sequence selected from SEQ ID NOs: 19, 22, and 23. In some embodiments, the chimeric polypeptide of the invention comprises or consists of an amino acid sequence selected from SEQ ID NOs: 27, 28, 29, 30, 31, 32, and 33. In some embodiments, the chimeric polypeptide of the invention comprises or consists of an amino acid sequence selected from SEQ ID NOs: 27, 28, 29, 31, 32, and 33. In some embodiments, the chimeric polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 19, or a sequence derived therefrom. In some embodiments, the polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 22, or a sequence derived therefrom. In some embodiments, the polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 23, or a sequence derived therefrom. In some embodiments, the polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 27, or a sequence derived therefrom. In some embodiments, the polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 28, or a sequence derived therefrom. In some embodiments, the chimeric polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 29, or a sequence derived therefrom. In some embodiments, the chimeric polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 30, or a sequence derived therefrom. In some embodiments, the chimeric polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 31, or a sequence derived therefrom. In some embodiments, the chimeric polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 32, or a sequence derived therefrom. In some embodiments, the chimeric polypeptide of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 33, or a sequence derived therefrom. [046] In one embodiment, the chimera comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23. In one embodiment, the chimera comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, 27 and 28. In one embodiment, the chimera comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, and 33. In one embodiment, the chimera comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, 27, 28, 29, 30, 31, 32, and 33.

Cell penetrating moiety

[047] In some embodiments, the second moiety is capable of penetrating a cell. In some embodiments, the second moiety is capable of penetrating a nucleus of a cell. In some embodiments, the second moiety comprises a cell penetrating peptide sequence (CPP). In some embodiments, the second moiety comprises a nuclear localization sequence (NLS).

[048] In some embodiments, the nuclear localization sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 7 (KKKRR), SEQ ID NO: 8 (PKKKRRV), SEQ ID NO: 9 (KRRMKWKK), and SEQ ID NO: 10 (KKKRK). [049] In some embodiments, the second moiety comprises a nuclear localization sequence having the amino acid sequence as set forth in SEQ ID NO: 10 (KKKRK).

[050] The term "nuclear localization signal" and "NLS" sequence as described herein are used interchangeably and refers to an amino acid sequence which is capable of inducing transport of molecules comprising such sequence(s) or linked to such sequences into the nucleus of cells. The NLS can, for example, direct transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier. The NLS is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier. NLSs are capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide.

[051] The term "cell penetrating peptide sequence" and "CPP" sequence as described herein are used interchangeably and refers to an amino acid sequence which is capable of inducing transport of molecules comprising such sequence(s) or linked to such sequences into cells. The CPP can, for example, direct transport of a protein with which it is associated from the extracellular domain across the cell membrane. The CPP is intended to encompass not only the cell penetration sequence of a particular peptide, but also derivatives thereof that are capable of directing importation of any given polypeptide across the cell membrane. CPPs are capable of directing importation across the cell membrane of a polypeptide when attached to the N-terminus, the C- terminus, or both the N- and C-termini of the polypeptide.

[052] In some embodiments, the second moiety further comprises between 3 and 20 contiguous arginine residues. In some embodiments, the second moiety further comprises between 6 and 20 contiguous arginine residues. In some embodiments, the second moiety further comprises contiguous arginine residues as set forth in formula NO: 1 : (Rb), wherein R is arginine and b is an integer ranging from 3 to 20. In some embodiments, b is an integer ranging from 6 to 20, from 6 to 19, from 6 to 18, from 6 to 17, from 6 to 16, from 6 to 15, from 6 to 14, from 6 to 13, from 6 to 12, from 6 to 11, from 7 to 20, from 7to 19, from 7 to 18, from 7 to 17, from 7 to 16, from 7 to 15, from 7 to 14, from 7 to 13, from 7 to 12, from 7 to 11, from 8 to 20, from 8 to 19, from 8 to 18, from 8 to 17, from 8 to 16, from 8 to 15, from 8 to 14, from 8 to 13, from 8 to 12, from 8 to 11, from 9 to 20, from 9 to 19, from 9 to 18, from 9 to 17, from 9 to 16, from 9 to 15, from 9 to 14, from 9 to 13, from 9 to 12, or from 9 to 11. Each possibility represents a separate embodiment of the present invention. [053] In some embodiments, the second moiety further comprises between 6 and 20 contiguous arginine residues comprising the amino acid sequence as set forth in SEQ ID NO: 11 (RRRRRRRRR). In some embodiments, the contiguous arginine residues consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 12 (RRRRRRRRR), and SEQ ID NO: 13 (RRRRRRRRRRR). [054] In some embodiments, the second moiety comprises between 6 and 20 contiguous arginine residues and a nuclear localization sequence (NLS). In some embodiments, the second moiety comprises between 6 and 20 contiguous arginine residues and one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 7 (KKKRR), SEQ ID NO: 8 (PKKKRRV), SEQ ID NO: 9 (KRRMKWKK), and SEQ ID NO: 10 (KKKRK), and any combination thereof.

[055] In some embodiments, the second moiety comprises at least 50% hydrophilic amino acid residues. In some embodiments, the second moiety comprises at least 60% hydrophilic amino acid residues. In some embodiments, the second moiety comprises at least 70% hydrophilic amino acid residues. In some embodiments, the second moiety comprises at least 80% hydrophilic amino acid residues. In some embodiments, the second moiety comprises at least 90% hydrophilic amino acid residues. In some embodiments, the second moiety comprises at least 100% hydrophilic amino acid residues. In some embodiments, the second moiety comprises Arginine (R) as a hydrophilic residue. In some embodiments, the second moiety comprises Lysine (K) as a hydrophilic residue. In some embodiments, the second moiety comprises a mixture of Arginine (R) and Lysine as hydrophilic residues. In some embodiments, the second moiety is an amphipathic molecule. In some embodiments, the second moiety is an amphiphilic molecule. In some embodiments, the second moiety comprises both hydrophilic and hydrophobic amino acid residues. In some embodiments, the second moiety comprises at least 5% hydrophobic amino acid residues. In some embodiment, the second moiety comprises at least 10% hydrophobic amino acid residues. In some embodiments, the second moiety comprises at least 15% hydrophobic amino acid residues. In some embodiments, the second moiety comprises at least 20% hydrophobic residues. In some embodiments, the second moiety comprises 10% hydrophobic amino acid residues and 90% hydrophilic residues. In some embodiments, the second moiety comprises 15% hydrophobic amino acid residues and 85% hydrophilic residues. In some embodiments, the second moiety comprises 20% hydrophobic amino acid residues and 80% hydrophilic residues. In some embodiments, the second moiety comprises 25% hydrophobic amino acid residues and 75% hydrophilic residues. In some embodiments, the second moiety comprises at least 70% hydrophilic amino acid residues and the remaining amino acid residues are hydrophobic. In some embodiments, the second moiety comprises at least 80% hydrophilic amino acid residues and the remaining amino acid residues are hydrophobic. In some embodiments, the second moiety comprises at least 85% hydrophilic amino acid residues and the remaining amino acid residues are hydrophobic. In some embodiments, the second moiety comprises at least 90% hydrophilic amino acid residues and the remaining amino acid residues are hydrophobic. In some embodiments, the second moiety comprises at least 95% hydrophilic amino acid residues and the remaining amino acid residues are hydrophobic. NKp44 derived peptide moiety

[056] In some embodiments, the first moiety has 15 to 25 amino acid residues derived from amino acid 56 to amino acid 86 of NKp44.

[057] In some embodiments, the NKp44 is a human NKp44 receptor (GenBank accession No: NP001186438.1) as set forth in SEQ ID NO: 34 MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEA SALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNS VSKSVRFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSHPS S PLP VPLPS RPQNS TLRPGP A APIALVP VFCGLLV AKS LVLS ALLVWW VLRNRHMQHQG RSLLHPAQPRPQAHRHFPLSHRAPGGTYGGKP.

[058] As used herein, the term "derived from" or "corresponding to" refers to construction of an amino acid sequence based on the knowledge of a sequence using any one of the suitable means known to one skilled in the art, e.g. chemical synthesis in accordance with standard protocols in the art. [059] In some embodiments, the first moiety comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids derived from amino acid 56 to amino acid 86 of SEQ ID NO: 34. Each possibility represents a separate embodiment of the present invention.

[060] In some embodiments, the first moiety comprises 15 to 25 amino acid residues derived from SEQ ID NO: 1 (E AS ALVCIRLVTS S KPRTX i A WTS RFTIWX2X3 , wherein Xi is selected from V and M, X₂ is selected from D and A, and X3 is selected from D and A.

[061] In some embodiments, the first moiety comprises 15 to 25 amino acid residues derived from SEQ ID NO: 1 with one or more conservative substitutions. In some embodiments, the first moiety comprises 15 to 25 amino acid residues derived from SEQ ID NO: 1 with at most 1, 2, 3, or 4 conservative substitutions. Each possibility represents a separate embodiment of the present invention.

[062] According to another embodiment of the invention, the first moiety comprises 15 to 25 amino acid residues derived from a sequence homologous to SEQ ID NO: 1. According to another embodiment of the invention, the polypeptide of the invention comprises a sequence having greater than 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention. [063] In some embodiments, the first moiety comprises or consists of 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, or 25 amino acids residues derived from SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention. In some embodiments, the first moiety comprises the amino acid sequence as set forth in SEQ ID NO: 2 (VTSSKPRTXA, wherein X is selected from V and M). In some embodiments, the first moiety comprises the amino acid sequence as set forth in SEQ ID NO: 2 (VTSSKPRTXiA wherein Xi is selected from V and M) at its N-terminus or its C-terminus.

[064] In some embodiments, the first moiety comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 3 (EASALVCIRLVTSSKPRTVA), SEQ ID NO: 4 (E AS ALVCIRL VTS S KPRTM A) , SEQ ID NO: 5 (VTSSKPRTVAWTSRFTIWX2X3), and SEQ ID NO: 6 (VTSSKPRTMAWTSRFTIW X2X3), or an analog thereof.

[065] One of skill in the art will recognize that individual substitutions, deletions or additions to a peptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a similar charge, size, and/or hydrophobicity characteristics, such as, for example, substitution of a glutamic acid (E) to aspartic acid (D).

[066] In some embodiments, the first moiety comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6 with one or more conservative substitutions. In some embodiments, the mutant polypeptide of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6 with at most 1, 2, 3, or 4 conservative substitutions. Each possibility represents a separate embodiment of the present invention. According to another embodiment of the invention, the first moiety comprises a sequence homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6. According to another embodiment of the invention, the first moiety comprises a sequence having greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6. Each possibility represents a separate embodiment of the present invention. [067] In one embodiment, the first moiety comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6. [068] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.

[069] As used herein, the phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function as specified herein.

[070] In one embodiment, the chimeras and/or peptides of the invention encompass variant thereof. As used herein, the term "variant" refers to a polypeptide or nucleotide sequence which comprises a modification of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively. In some embodiments, the modifications are substitution, deletion, and/or insertion of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively. In some embodiments, the changes may be of minor nature, such as conservative amino acid substitutions or for nucleotide sequence resulting in conservative amino acid substitutions that do not significantly affect the activity of the polypeptide. In some embodiments, the changes may be substitution of an amino acid molecule, resulting in an addition of a glycosylation site, thereby increasing glycosylation of the polypeptide. [071] Typically, the present invention encompasses derivatives of the polypeptides (chimeras and each of the moieties). The term "derivative" or "chemical derivative" includes any chemical derivative of the polypeptide having one or more residues chemically derivatized by reaction of side chains or functional groups. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p- toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O- alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine. Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine.

[072] In addition, a peptide derivative can differ from the natural sequence of the peptides of the invention by chemical modifications including, but are not limited to, terminal-NH2 acylation, acetylation, or thioglycolic acid amidation, and by terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like. Peptides can be either linear, cyclic or branched and the like, which conformations can be achieved using methods well known in the art.

[073] The peptide derivatives and analogs according to the principles of the present invention can also include side chain bond modifications, including but not limited to -CH2-NH-, -CH2-S-, -CH2-S=0, OC-NH-, -CH2-0-, -CH2-CH2-, S=C-NH-, and -CH=CH-, and backbone modifications such as modified peptide bonds. Peptide bonds (-CO-NH-) within the peptide can be substituted, for example, by N-methylated bonds (-N(CH3)-CO-); ester bonds (-C(R)H-C-O-O- C(R)H-N); ketomethylene bonds (-CO-CH2-); a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)-CH2-); thioamide bonds (-CS-NH); olefmic double bonds (-CH=CH-); and peptide derivatives (-N(R)- CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at one or more of the bonds along the peptide chain and even at several (e.g., 2-3) at the same time.

[074] The present invention also encompasses peptide derivatives and analogs in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.

[075] As used herein the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid. Salts describe here also ionic components added to the peptide solution to enhance hydrogel formation and /or mineralization of calcium minerals.

[076] The peptide analogs can also contain non-natural amino acids. Examples of non-natural amino acids include, but are not limited to, sarcosine (Sar), norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala).

[077] Furthermore, the peptide analogs can contain other derivatized amino acid residues including, but not limited to, methylated amino acids, N-benzylated amino acids, O-benzylated amino acids, N-acetylated amino acids, O-acetylated amino acids, carbobenzoxy-substituted amino acids and the like. Specific examples include, but are not limited to, methyl- Ala (Me Ala), MeTyr, MeArg, MeGlu, MeVal, MeHis, N-acetyl-Lys, O-acetyl-Lys, carbobenzoxy-Lys, Tyr-O- Benzyl, Glu-O-Benzyl, Benzyl-His, Arg-Tosyl, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, and the like. [078] The invention further includes peptide analogs, which can contain one or more D-isomer forms of the amino acids. Production of retro-inverso D-amino acid peptides where at least one amino acid and perhaps all amino acids are D-amino acids is well known in the art. When all of the amino acids in the peptide are D-amino acids, and the N- and C-terminals of the molecule are reversed, the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule. However, the molecule is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein. Diastereomeric peptides may be highly advantageous over all L- or all D-amino acid peptides having the same amino acid sequence because of their higher water solubility, lower immunogenicity, and lower susceptibility to proteolytic degradation. The term "diastereomeric peptide" as used herein refers to a peptide comprising both L-amino acid residues and D-amino acid residues. The number and position of D-amino acid residues in a diastereomeric peptide of the preset invention may be variable so long as the peptide is capable of displaying the function of disclosed chimera of the invention.

Synthesizing the polypeptide [079] According to one embodiment, the polypeptides of the present invention may be synthesized or prepared by any method and/or technique known in the art for peptide synthesis. According to another embodiment, the polypeptides may be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). According to another embodiment, the polypeptides of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984). [080] In general, the synthesis methods comprise sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain bound to a suitable resin. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions conductive for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by the solid phase method, is cleaved from the solid support to afford the final peptide.

[081] In the solid phase peptide synthesis method, the alpha-amino group of the amino acid is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain. Suitable protecting groups are t- butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t- amyloxycarbonyl, isobornyloxycarbonyl, (alpha,alpha)-dimethyl-3 ,5 dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (FMOC) and the like. In the solid phase peptide synthesis method, the C-terminal amino acid is attached to a suitable solid support. Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the solvent media used. Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like. The coupling reaction is accomplished in a solvent such as ethanol, acetonitrile, Ν,Ν-dimethylformamide (DMF), and the like. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art.

[082] In another embodiment, polypeptides of the invention may be synthesized such that one or more of the bonds, which link the amino acid residues of the peptides are non-peptide bonds. In another embodiment, the non-peptide bonds include, but are not limited to, imino, ester, hydrazide, semicarbazide, and azo bonds, which can be formed by reactions well known to one skilled in the art.

[083] The term "linker" refers a molecule or macromolecule serving to connect different moieties of a peptide or a polypeptide. In one embodiment, said linker may also facilitated other functions, including, but not limited to, preserving biological activity, maintaining sub-units and domains interactions, and others.

[084] In some embodiments, the different moieties of the chimeric peptides of the present invention may be attached or linked via a chemical linker. Chemical linkers are well known in the art, and include but are not limited to dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), maleiimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-ethyloxycarbonyl-2-ethyloxy- 1 ,2-dihydroquinoline (EEDQ), N-isobutyloxy-carbonyl-2-isobutyloxy- 1 ,2-dihydroquinoline (IIDQ). In another embodiment, linkers may also be monomeric entities such as a single amino acid. In another embodiment, amino acids with small side chains are especially preferred, or a small polypeptide chain, or polymeric entities of several amino acids. In another embodiment, preferred polypeptide linkers are fifteen amino acids or less, more preferred are polypeptide linkers of ten or less amino acids. In another embodiment, even more preferred are polypeptide linkers of five or less amino acids. In an alternative embodiment, the linker may be a nucleic acid encoding a small polypeptide chain. In another embodiment, preferred linkers encode a polypeptide of fifteen or less amino acids. In another embodiment, more preferred linkers are nucleic acids encoding a small polypeptide chains of ten or less amino acids. In another embodiment, even more preferred linkers are nucleic acid encoding a small polypeptide of five or less amino acids.

[085] Recombinant technology may be used to express the chimeric polypeptide of the present invention comprising both moieties and a linker, and is well known in the art. In another embodiment, the linker may be a cleavable linker, resulting in cleavage of the chimeric polypeptide of the invention once delivered to the tissue or cell of choice. In such an embodiment, the cell or tissue would have endogenous (either naturally occurring enzyme or be recombinantly engineered to express the enzyme) or have exogenous (e.g., by injection, absorption or the like) enzyme capable of cleaving the cleavable linker.

[086] In another embodiment, the linker may be biodegradable such that the chimeric polypeptide of the invention is further processed by hydrolysis and/or enzymatic cleavage inside cells. In one embodiment, tumors specifically-expressed proteases, can be used in the delivery of prodrugs of cytotoxic agents, with the linker being selective for a site-specific proteolysis. In some embodiments, readily-cleavable groups include acetyl, trimethylacetyl, butanoyl, methyl succinoyl, t-butyl succinoyl, ethoxycarbonyl, methoxycarbonyl, benzoyl, 3- aminocyclohexylidenyl, and the like.

[087] The invention further encompasses a polynucleotide sequence comprising a nucleic acid encoding any of the polypeptides of the invention. In another embodiment, the nucleic acid sequence encoding the polypeptide is at least 70%, or alternatively at least 80%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 99% homologous to the nucleic acid sequence encoding the nucleic acid sequence of the polypeptides of the invention or a fragment thereof.

[088] In some embodiment, the invention provides a polynucleotide encoding the polypeptides of the invention.

[089] In some embodiments, the polynucleotide of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention. In some embodiments, the cis- regulatory sequence is suitable for directing tissue-specific expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention.

[090] The term "polynucleotide" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide. In one embodiment, a polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

[091] In one embodiment, "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. In one embodiment, the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase.

[092] In one embodiment, "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome. [093] In one embodiment, "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements.

[094] In some embodiments, polynucleotides of the present invention are prepared using PCR techniques as described in Example 1, or any other method or procedure known to one skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[095] In one embodiment, polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[096] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host- expression systems to express the polypeptide of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; 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, such as Ti plasmid, containing the polypeptide coding sequence. [097] In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems) to express the polypeptide of the present invention. In one embodiment, the expression vector is used to express polynucleotides of the present invention in mammalian cells.

[098] In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[099] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[0100] In one embodiment, the expression vector of the present invention may further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES).

[0101] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+), pGL3, pZeoSV2(+), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0102] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV- 1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0103] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression of the polypeptide of the present invention. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0104] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0105] A person with skill in the art will appreciate that the polypeptide of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy). [0106] In one embodiment, in vivo gene therapy using a cytokine has been attempted in animal models such as rodents [Bohl et al., Blood. 2000; 95:2793-2798], primates [Gao et al., Blood, 2004, Volume 103, Number 9] and has proven successful in human clinical trials for patients with chronic renal failure [Lippin et al Blood 2005, 106, Number 7].

[0107] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307- 311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0108] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide. [0109] In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art. [0110] In some embodiments, depending on the vector and host system used for production, resultant polypeptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane. In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is affected.

[0111] In one embodiment, the phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. [0112] In one embodiment, polypeptides of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. [0113] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854- 15859 (1990)].

[0114] In one embodiment, the polypeptide of the present invention is retrieved in "substantially pure" form that allows for the effective use of the protein in the applications described herein.

[0115] As used herein, the term "substantially pure" describes a peptide/ polypeptide or other material which has been separated from its native contaminants. Typically, a monomeric peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence. A substantially pure peptide can comprise over about 85 to 90% of a peptide sample, and can be over 95% pure, over 97% pure, or over about 99% pure. Purity can be measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for certain purposes high resolution may be necessary and HPLC or a similar means for purification can be used. For most purposes, a simple chromatography column or polyacrylamide gel can be used to determine purity.

[0116] The term "purified" does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition. A peptide is in the "purified" state after purification of the starting material or of the natural material by at least one order of magnitude, 2 or 3, or 4 or 5 orders of magnitude.

[0117] In one embodiment, the polypeptides of the present invention are substantially free of naturally-associated host cell components. The term "substantially free of naturally-associated host cell components" describes a peptide or other material which is separated from the native contaminants which accompany it in its natural host cell state. Thus, a peptide which is chemically synthesized or synthesized in a cellular system different from the host cell from which it naturally originates will be free from its naturally-associated host cell components.

[0118] In one embodiment, the polypeptide of the present invention can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.

Pharmaceutical compositions

[0119] According to another aspect, the invention provides a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the polypeptide of the present invention, and pharmaceutically acceptable carrier and/or diluents. In some embodiments, the pharmaceutical composition facilitates administration of a compound to an organism.

[0120] In another embodiment, the pharmaceutical compositions of the invention may be formulated in the form of a pharmaceutically acceptable salt of the polypeptides of the present invention or their analogs, or derivatives thereof. In another embodiment, pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from nontoxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from nontoxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0121] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein. [0122] As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. [0123] In another embodiment, the compositions of the invention take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like. In another embodiment, the compositions of the invention can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the polypeptide of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0124] According to an embodiment of the invention, pharmaceutical compositions contain 0.1% - 95% of the polypeptide(s) of the present invention, derivatives, or analogs thereof. According to another embodiment of the invention, pharmaceutical compositions contain 1% - 70% of the polypeptide(s) derivatives, or analogs thereof. According to another embodiment of the invention, the composition or formulation to be administered may contain a quantity of polypeptide(s), derivatives, or analogs thereof, according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.

[0125] An embodiment of the invention relates to polypeptides of the present invention, derivatives, or analogs thereof, presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. In an embodiment of the invention, the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial or pre-filled syringe. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose- response curves derived from in-vitro or in-vivo animal model test bioassays or systems. [0126] According to one embodiment, the compositions of the present invention are administered in the form of a pharmaceutical composition comprising at least one of the active components of this invention (the chimeric polypeptides) together with a pharmaceutically acceptable carrier or diluent. In another embodiment, the compositions of this invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form. In some embodiments, the pharmaceutical composition further comprises at least one anticancer agent such as a chemotherapeutic agent. In some embodiments, the pharmaceutical composition is adopted for combined administration with an anticancer therapy such as chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery.

[0127] As used herein, the terms "administering," "administration," and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.

[0128] Depending on the location of the tissue of interest, the polypeptide of the present invention can be administered in any manner suitable for the provision of the polypeptides to cells within the tissue of interest. Thus, for example, a composition containing the polypeptides of the present invention can be introduced, for example, into the systemic circulation, which will distribute the peptide to the tissue of interest. Alternatively, a composition can be applied topically to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.).

[0129] In some embodiments, the pharmaceutical compositions comprising the chimeric polypeptides are administered via oral, rectal, vaginal, topical, nasal, ophthalmic, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of peptides administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer.

[0130] For topical application, a peptide of the present invention, derivative, analog or a fragment thereof can be combined with a pharmaceutically acceptable carrier so that an effective dosage is delivered, based on the desired activity. The carrier can be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.

[0131] For oral applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The tablets of the invention can further be film coated.

[0132] For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. [0133] According to some embodiments, the chimeric polypeptides of the present invention, derivatives, or analogs thereof can be delivered in a controlled release system. In another embodiment, an infusion pump can be used to administer the peptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors. In another embodiment, the peptides of the invention are administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site. Examples of preferred polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety). In yet another embodiment, a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.

[0134] The presently described peptides, derivatives, or analogs thereof may also be contained in artificially created structures such as liposomes, ISCOMS, slow -releasing particles, and other vehicles which increase the half -life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0135] The compositions also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. [0136] In one embodiment, the present invention provides combined preparations. In one embodiment, "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation. In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.

[0137] In one embodiment, it will be appreciated that the peptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies.

[0138] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.

[0139] In some embodiments, the peptides are administered in a therapeutically safe and effective amount. As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the presently described manner. In another embodiment, a therapeutically effective amount of the polypeptide is the amount of the polypeptide necessary for the in vivo measurable expected biological effect. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, preparation of effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[0140] In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, 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) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].

[0141] Pharmaceutical compositions containing the presently described polypeptide as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). See also, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0142] In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0143] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contains, one or more unit dosages forms containing the active ingredient. In one embodiment, the pack, for example, comprises metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Use of the compositions

[0144] According to some aspects, there is provided a method for treating, ameliorating, reducing and/or preventing a condition associated with increased cell proliferation in a subject in need thereof, the method comprising the step of: administering to a subject a pharmaceutical composition comprising an effective amount of the chimeric polypeptide of the invention, thereby treating, ameliorating, reducing and/or preventing a condition associated with increased cell proliferation in a subject in need thereof.

[0145] According to some embodiments, there is provided a method for treating, ameliorating, reducing and/or preventing cancer or pre-malignancy condition in a subject in need thereof, the method comprising the step of: administering to a subject a pharmaceutical composition comprising an effective amount of the chimeric polypeptide of the invention, thereby treating, ameliorating, reducing and/or preventing cancer or pre-malignancy condition in a subject in need thereof.

[0146] According to some embodiments, there is provided a method for treating or ameliorating a cancer in a subject in need thereof, the method comprising administering to the subject any one of:

(i) the chimeric polypeptide of the invention;

(ϋ) the expression vector of the invention; or

(iii) the pharmaceutical composition of the invention, thereby treating or ameliorating cancer in said subject.

[0147] In some embodiments, there is provided a method for treating cancer or pre-malignancy condition in a subject in need thereof, the method comprising the step of administering to said subject a pharmaceutical composition comprising an effective amount of one or more amino acid molecules, each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, 27, 28, 29, 30, 31, 32, and 33 and a pharmaceutical acceptable carrier, thereby treating, ameliorating, reducing and/or preventing cancer or pre-malignancy condition in a subject in need thereof. In some embodiments, the subject is further treated with an additional anticancer therapy such as chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery. [0148] In another embodiment, the chimeric polypeptide of the invention or a composition comprising the chimeric polypeptide is for use in treatment, amelioration, reduction, and/or prevention of cancer or pre-malignancy condition in a subject in need thereof. In some embodiments, there is provided a composition comprising an effective amount of chimeric polypeptide for use in the treatment or prevention of cancer or pre-malignancy condition in a subject in need thereof. In some embodiments, there is provided a composition comprising an effective amount of one or more amino acid molecules, each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, 27, 28, 29, 30, 31, 32, and 33 for use in the treatment or prevention of cancer or pre-malignancy condition in a subject in need thereof. In some embodiments, the composition further comprises at least one anticancer agent such as a chemotherapeutic agent. In some embodiments, the composition is adopted for use in combination with an anticancer therapy such as chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery.

[0149] In some embodiments, there is provided a use of a composition comprising an effective amount of the chimeric polypeptide in the preparation of a medicament for the treatment, amelioration, reduction, or prevention of a disease associated with increased cell proliferation in a subject in need thereof. In some embodiments, the invention provides use of a composition comprising an effective amount of one or more amino acid molecules, each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 23, 27, 28, 29, 30, 31, 32, and 33 in the preparation of a medicament for the treatment of a disease associated with increased cell proliferation in a subject in need thereof. [0150] In one embodiment, the chimeric polypeptide of the present invention is provided to the subject per se. In one embodiment, one or more of the chimeric polypeptide of the present invention are provided to the subject per se. In one embodiment, the chimeric polypeptide of the present invention is provided to the subject as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. In one embodiment, one or more of the chimeric polypeptides of the present invention are provided to the subject as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

[0151] In some embodiments, the disease associated with increased cell proliferation is cancer. In some embodiments, the cancer is selected from: breast cancer, lung cancer, melanoma, liver cancer and pancreatic cancer. [0152] As used herein "cancer" or "pre-malignancy" are diseases associated with cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma and germ cells tumors. In one embodiment, carcinoma refers to tumors derived from epithelial cells including but not limited to breast cancer, prostate cancer, lung cancer, pancreas cancer, and colon cancer. In one embodiment, sarcoma refers of tumors derived from mesenchymal cells including but not limited to sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas. In one embodiment, lymphoma refers to tumors derived from hematopoietic cells that leave the bone marrow and tend to mature in the lymph nodes including but not limited to Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma and immunoproliferative diseases. In one embodiment, leukemia refers to tumors derived from hematopoietic cells that leave the bone marrow and tend to mature in the blood including but not limited to acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia and adult T-cell leukemia. In one embodiment, blastoma refers to tumors derived from immature precursor cells or embryonic tissue including but not limited to hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma and glioblastoma- multiforme. In one embodiment, germ cell tumors refer to tumors derived from germ cells including but not limited to germinomatous or seminomatous germ cell tumors (GGCT, SGCT) and nongerminomatous or nonseminomatous germ cell tumors (NGGCT, NSGCT). In one embodiment, germinomatous or seminomatous tumors include but not limited to germinoma, dysgerminoma and seminoma. In one embodiment, non-germinomatous or non-seminomatous tumors refers to pure and mixed germ cells tumors including but not limited to embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, tearoom, polyembryoma, gonadoblastoma and teratocarcinoma.

[0153] As used herein, "cancer or pre-malignant cell proliferation" is a molecular process which requires the involvement of the proliferating cell nuclear antigen (PCNA). In another embodiment, the present invention refers to a cancer-associated isoform of PCNA. In some embodiments, cancer cell proliferation is PCNA-dependent. In another embodiment, PCNA is an indicator of malignancy of tissues selected from, but not limited to: breast epithelium, colorectal tissue, lung, liver, lymph, stomach, and eye. In another embodiment, PCNA is a biomarker correlating with poor patient prognosis.

[0154] In some embodiments, as known to one skilled in the art, malignancy-associated PCNA is detected by an assay, including immune-assays, western-blot, immune-histochemistry, and the like, such as for detecting K164-PCNA, and/or p211Y-PCNA. [0155] The term "subject" as used herein refers to an animal, more particularly to non-human mammals and human organism. Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses. Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, pig. In one embodiment, the subject is a human. Human subjects may also include fetuses. In one embodiment, a subject in need thereof is a subject afflicted with and/or at risk of being afflicted with a condition associated with increased cell proliferation.

[0156] As used herein, the terms "treatment" or "treating" of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject's quality of life.

[0157] As used herein, the term "prevention" of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the presently described subject matter, the term "prevention" relates to a process of prophylaxis in which a subject is exposed to the presently described peptides prior to the induction or onset of the disease/disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease/disorder to be prevented. For example, this might be true of an individual whose ancestors show a predisposition toward certain types of, for example, inflammatory disorders. The term "suppression" is used to describe a condition wherein the disease/disorder process has already begun but obvious symptoms of the condition have yet to be realized. Thus, the cells of an individual may have the disease/disorder but no outside signs of the disease/disorder have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.

[0158] As used herein, the term "condition" includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions. [0159] Any concentration ranges, percentage range, or ratio range recited herein are to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.

[0160] Any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.

[0161] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal," refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human. [0162] In the discussion unless otherwise stated, adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word "or" in the specification and claims is considered to be the inclusive "or" rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0163] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms "a," "an" and "at least one" are used interchangeably in this application.

[0164] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0165] In the description and claims of the present application, each of the verbs, "comprise," "include" and "have" and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. [0166] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0167] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0168] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

EXAMPLES

[0169] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); "Bacteriophage Methods and Protocols", Volume 1: Isolation, Characterization, and Interactions, all of which are incorporated by reference. Other general references are provided throughout this document. [0170] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Materials and methods

Peptides synthesis

[0171] Peptides were synthesized with/without biotin using PEPscreen technology, which is a peptide synthesis platform that utilizes Fmoc protective chemistry (Sigma- Aldrich). Nineteen overlapping peptides (20mer peptides with a 15-aa overlap between successive peptides) covered the NKp44 ectodomain and hinge region (amino acids: 22-131; Accession: CAB39168.1; www.ncbi.nlm.nih.gov). Single nucleotide polymorphism (NCBI; SNP; rs9471577) lead to a replacement of M- V in amino acid #75 on NKp44 and we used this valine replacement in the synthesized peptides (in peptides 8, 9 and 10). Selected peptides and CPP containing peptides were ordered from GL Biochem (Shanghai, China, purity > 95%, in TFA-salt and Acetate salt for in vitro and in vivo assays, respectively). Lyophilized peptide stocks were kept frozen in dehydrating conditions. Stock solutions of peptides (2 mM) were solubilized in DDW-5% DMSO and stored in frozen aliquots. The following cell penetrating peptides (CPP) were used in order to test the function of NKp44-derived pep8; miniAntp (KRRMKWKK), SV40 large T antigen NLS (PKKKRRV), Transferrin (HAIYPRH), R9 (RRRRRRRRR) or Rl l (RRRRRRRRRRR). Cell lines and Mice strains

[0172] Murine cell lines: 4T1; mammary carcinoma (ATCC® CRL-2539™), B16-F0; melanoma (ATCC® CRL-6322™). Human cell lines: A549; lung carcinoma (ATCC® CCL-185™), MDA- MB-23; breast adenocarcinoma (ATCC® HTB-26™), HepG2; hepatocellular carcinoma (ATCC® HB-8065™), PANC-1; pancreas ductal adenocarcinoma (ATCC® CRL-1469™). Culture medium was prepared as following; DMEM (Gibco, 41965-039) supplemented with 10% fetal calf serum (FCS) (Gibco, 12657-029), 1% L-glutamine (BI, 03-020-1 A), 1% Pen-Strep (BI, 03-031-1B), 1% sodium pyruvate (BI, 03-042-1B), 1% MEM-Eagle (BI, 01-340-1B), 1% HEPES 1M (BI, 03-025-1B). [0173] Six to eight weeks old C57BL/6 male and BALB/C female mice were purchased from Envigo/Harlan Laboratories (Rehovot, Israel). Maintenance and breading of all mice used in this study were done in the local animal care facility, approved by the Institutional Animal Care and Use Committee (IACUC) of Ben-Gurion University of the Negev (BGU). Revision and approval of all experimental procedures were done by the Institutional Animal Care and Use Committee (IACUC) of Ben-Gurion University of the Negev (BGU's IACUC) according to specified protocols that aim to ensure animal welfare and reduce suffering

Recombinant His tag and MBP '-fusion proteins production

[0174] pET-28 or pMAL-c2x vectors were used to produce soluble human PCNA (hPCNA) in Rosetta™ 2 (DE3) cells. Plasmids containing the mRNA sequence of PCNA or TL1A, APO-E3, HNF4 were transformed into Rosetta™ 2 cells via heat shock and grown on LB+Kan+CP agar plates. A fresh colony of transformed bacteria was grown overnight in a 5ml LB+Kan+CP in an incubator shaker set to 37 °C and at 250 rpm. The next day, bacteria calls were diluted 1: 100 into a 500 ml LBKan+CP (to discard dead cells) and grown to an O.D. of 0.6-0.8 (λ = 650 nm). Isopropyl β-D-l-thiogalactopyranoside (IPTG) was added for the induction of the PCNA (0.5 mM) and cells were further incubated for 4 h at 26 °C (hPCNA). Cells were then centrifuged, and the pellet was resuspended with solution A (20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 20 mM Imidazole), sonicated six times (20 seconds with 40 seconds intervals) and sieved through 0.45 μΜ (Sartorius Stedim biotech) filter before loaded on a His-tag or Amylose resin beads. Purification of His-tagged proteins was done using a Gravity-flow Column with "HisPur™ Ni- NTA Resin" Bead kit (Thermo-Scientific) [Binding Capacity < 60 mg of a 28 kDa 6xHis-tagged protein from a bacterial source per milliliter of settled resin] according to the kit's protocol. Purification of MBP-tagged proteins was done using the Amylose resin beads (Catalog #E8035S). ELISA assay

[0175] To determine the interaction between NKp44 derived peptides and PCNA, ELISA plates were coated overnight at 4 °C with 2 μg/ml of the recombinant PCNA or TLA1, APO-E3, HNF4, hFc, NKp44-hFc, NKp46-hFc, PBSxl (negative controls). Blocking buffer (PBS + 2.5% skim milk) was applied for 2 h at room temperature, after which the plates were incubated with 0-8 μΜ of biotinylated NKp44 derived peptides or PBSxl (background control) for 2 h at room temperature. NKp44 derived peptides binding to PCNA was detected using horseradish peroxidase (HRP)-conjugated to streptavidin (1 h at 1:500 dilution). Following washing, TMB (DAKO, S 1599) was added. Optical density (O.D.) was read at 650 nm (Thermo Electron Corporation Multiskan Spectrum). Between each step, wells were washed three times with PBS containing 0.05% Tween 20 (PBST).

[0176] To determine the ability of NKp44-derived pep8 to block NKp44-hFc interaction with PCNA, ELISA plates were coated overnight at 4 °C with 2 μg/ml of the recombinant PCNA, Blocking buffer (PBS + 2.5% skim milk) was applied for 2 h at room temperature, after which the plates were incubated with 0-10 μΜ of biotinylated Kp44 derived peptides or PBSxl (100 μΐ/well, background control) for 2 h at room temperature and then NKp44-hFc (80 nM, 20 μΐ/well) was added without washing for 1 h at room temperature. NKp44-hFc binding to PCNA was detected using horseradish peroxidase (HRP)-conjugated to anti-human IgG (1 h at 1:400 dilution 100 μΐ/well). Following washing, TMB (100 μΐ/well DAKO, S1599) was added. Optical density (O.D.) was read at 650 nm (Thermo Electron Corporation Multiskan Spectrum). Between each step, wells were washed three times with 200 μΐ/well PBSxl containing 0.05% Tween 20 (PBST).

Kinetic analysis by Surface Plasmon Resonance

[0177] A ProteOn™ XPR36 Protein Interaction Array System (Bio-Rad) was used to measure the affinity of NKp44 derived peptides to MBP-fused recombinant Human PCNA. For the assay, a GLC sensor chip (7CG21401) and ProteOn Manager Version 3.1.0.6 (Bio-Rad Laboratories) was employed. After activation of the chip using EDC/S-NHS amine coupling procedure the ligand immobilization process was performed with NKp44-derived pep8, Rl l-NLS-pep8, Rl l-NLS- pep8 short and BSA as a control at a flow rate of 30 μΐ/min in different flow cells. Different concentrations (0-250 nM) of analyte (MBP-PCNA) were then injected at a flow rate of 25 μΐ/min. Regeneration of the surface was done using 50 mM NaOH. Data were analyzed using equilibrium analysis model.

Cell viability assay employing PrestoBlue [0178] Target cells (2.5xl0⁴ cells/well of a 48 well plate, Croning) were seeded and incubated in 37 °C, 5% C0₂ for 24 h in 0.5 ml of fresh complete DMEM (10% FBS) culture medium. According to molecular weight peptides were diluted from 8 to 2 μΜ with 5% FBS containing DMEM culture medium just before the incubation with the target cells. Old medium was removed from each well and 250 μΐ_^ of diluted peptides were added and incubated for another 24 h in 37 °C, 5% C0₂. PrestoBlue (A13261, Invitrogen™) solution was prepared by diluting the dye 10-fold with complete DMEM. Old medium was discarded from each well and 250 μΐ_^ of PrestoBlue solution was added to each well, shake gently and incubated in 37 °C for another 1 h. Emission at 560 nm was measured in Premium Quad4 Monochromators plate reader (Tecan). Normalization was done according to appropriate control (DMEM or DMSO) to calculate the percentage of viable cells in each group. Selenite (25 μΜ) was used as a positive control.

Flow cytometry based cell proliferation assay and cell death assay

[0179] Flow cytometry was employed for analysis of CFSE based cell proliferation assay (C34554, Invitrogen™) and cell death using propidium iodide (PI: P3566, Invitrogen™). Target cells (2E5 cells/well, 12 well/plate, Craning) were seeded and incubated for 24 h in 1 ml, 10% FCS complete DMEM culture medium. For the of cell proliferation assay, target cells were labeled using CFSE according to the company protocol. Peptides were diluted prior to the incubation with the target cells with free DMEM culture medium to 0-16 μΜ according to peptide molecular weight. For adding the peptides, 0.5 mL was removed from each well and 0.5 mL of diluted peptide was added to a final volume of 1 mL, 5% FCS, duplicates. Peptides were incubated with target cells for 24 h and then the medium was collected, adherent cells were removed with trypsin-EDTA (0.5 mL, 3 min, BI: 03-052-1B), 1 mL of complete medium was added to each well and the medium was collected (final volume 2.5 mL, on ice). For the cell death assay, samples were then centrifuged (1,300 rpm, 4 °C, 5 min), the medium was removed, fresh 1 mL 10% FCS was added and then 1 μΕ of 1 mg/mL PI was added to each sample and incubated (15 min, on ice) with the target cells. Before analyzing the samples, samples were centrifuged (1,300 rpm, 4 °C, 5 min), the medium was removed and fresh 0.2 mL FCAS buffer (PBSxl, 20% FCS, 0.5% NaN₃) was added. Samples were then read and analyzed using BD FACSCanto™ II and FlowJo®. APEVI peptide and Camptothecin were used as positive controls.

In vivo tumor growth and treatments

[0180] Seven weeks old female BALB/C mice with average body weight of 20 gram were used in this experiment. 4T1 cells suspended at a density of 2xl0⁵ cells/50 μΐ, in serum- free DMEM containing 40% Geltrex, were injected in the left mammary fat pad, and allowed to develop tumors. After 5 days, mice were randomly divided into three groups, one group was treated with Rl l-NLS-pep8, at a dose of 5 mg/Kg, intravenously (IV), other group with 5-FU, at a dose of 30 mg/Kg, (IV) and mice treated with a 1.44% DMSO/150 mM NaCl solution, (IV) served as the vehicle control. Treatment was continued following every two days at least for four weeks. Each week, tumor size was evaluated by measuring their length (L) and width (W), using a Caliper device and tumor's volume (V) was calculated according to the equation: V=LW²/2. After four weeks of treatment, mice were euthanized, and tumors' volume and weight were measured. [0181] Seven weeks old male C57BL/6 mice with average body weight of 23 gram were used in the other experiment. B 16 cells suspended at a density of 0.5xl0⁶ cells/100 μΐ, in serum-free DMEM containing 40% Geltrex, were injected subcutaneously in the left dorsal flank, and allowed to develop tumors. After 6 days, mice were randomly divided into three groups, one group was treated with Rl l-NLS-pep8, at a dose of 5 mg/Kg, intraperitoneally (IP), other group with 5-FU, at a dose of 30 mg/Kg, (IP) and mice treated with a 1.44% DMSO/150 mM NaCl solution, (IP) served as the vehicle control. Treatment was done 3 times a week at least for four weeks. Each week, tumors size was evaluated by measuring their length (L) and width (W), using a Caliper device and tumor's volume (V) was calculated according to the equation: V=LW²/2. After three weeks of treatment, mice were euthanized, and tumors' volume and weight were measured.

Statistics

[0182] Graphics and statistical analysis were performed using GraphPad Prism software. Statistical analysis of the data was performed using unpaired t-test (with p-values of *<0.05, **<0.01 or ***<0.001, as indicated on the figures).

EXAMPLE 1

Cytotoxicity of the chimeric peptides in which the NKp44 moiety comprises SEQ ID NO: 5

[0183] Cytotoxicity of the peptides listed in table 2 was tested by introduction of the peptides to a number of cell-lines at increasing concentrations followed by a viability measurement using the PrestoBlue assay according to the manufacturer's instructions. Results demonstrated that peptides GL8-1: Ac-Rl l-NLS-p8 (SEQ ID NO: 23), GL7-3: Ac-p8-NLS-Rl l (SEQ ID NO: 22) and GL5- 8: Ac-R9-NLS -pep8 (SEQ ID NO: 19) were shown to cause the highest rates of cell death induction in three independently tested cell lines: 4T1, A549 and PyMT. Table 2: P8 (SEQ ID NO: 5) based peptides of the invention

EAS ALVCIRLVTS S KPRTXA

SEQ ID NO: 17 GL5-2 Ac-Transferrin-pep8 Ac-HAIYPRH-

EAS ALVCIRLVTS S KPRTXA

SEQ ID NO: 18 GL5-6 Ac-Transferrin-NLS-pep8 Ac-HAIYPRH-KKKRK- EAS ALVCIRLVTS S KPRTXA

SEQ ID NO: 19 GL5-8 Ac-R9-NLS-pep8 Ac-RRRRRRRRRKKKRK- EAS ALVCIRLVTS S KPRTXA

SEQ ID NO: 20 GL5-9 Ac-Mini-Antp/NLS -pep8 Ac-KRRMKW-KKKRK- EAS ALVCIRLVTS S KPRTXA

SEQ ID NO: 21 GL6-4 Ac-Rl l-NLS-p8short Ac-RRRRRRRRRRR-I- KKKRK- W- VTS S KPRTXA

SEQ ID NO: 22 GL7-3 Ac-p8-NLS-Rl l Ac-

EAS ALVCIRLVTS S KPRTXA- W-KKKRK-I-RRRRRRRRRRR

SEQ ID NO: 23 GL8-1 Ac-Rl l-NLS -p8 Ac-RRRRRRRRRRR-I- KKKRK-W-

EAS ALVCIRLVTS S KPRTXA

SEQ ID NO: 24 GL8-2 Ac-Rl l-NLS-p8short Ac-RRRRRRRRRRR-I- KKKRK- W- VTS S KPRTXA

SEQ ID NO: 25 GL8-3 Ac-TransferinTransferin- Ac-HAIYPR-HHAIYPR- pep8 HE AS ALVCIRLVTS S KPRTXA

SEQ ID NO: 26 GL8-4 APIM (a known Ac- MDRWLVK-W-KKKRK-I- antiPCNA peptide) RRRRRRRRRRR

Abbreviations - Underlined/Bold font indicates a D-form amino acid, Ac=acetyl group that was added to the N terminus. Rl l=eleven consecutive Arginine residues. NLS-nuclear localization signal. Mini-Antp= CPP derived from Antennapedia (Antp) transcription factor of Drosophila.

In the experiments below X=V.

Cytotoxicity in 4T1 cells using the PrestoBlue assay

[0184] The results of PrestoBlue viability assay of 4T1 cells are shown in table 3 and figure 1, while DMSO, at a cone, of 0.05% served as the vehicle control, it showed no cytotoxicity in this assay, there was a slight increase in cell viability. Selenite, which is known to induce cell apoptosis, served as the positive control for cell death (result not shown). Values were normalized to the vehicle control (0.05% DMSO) in order to calculate the percentage of viable cells for each treatment group. The peptides showed low, moderate and high cytotoxicity. Results demonstrated that pep8 in itself (SEQ ID NO: 14), does not exhibit marked cytotoxicity in 4T1 cells even after the addition of NLS or transferring sequences (SEQ ID Nos: 16, 17, 18, and 25), in order to facilitate peptide' s intake to cells (table 3). In addition, the short derivative of pep8 consisting of SEQ ID NO: 2 does not exhibit marked cytotoxicity in 4T1 cells even after the addition of NLS (SEQ ID NO: 21) and NLS element in combination with arginine residues (R9) (SEQ ID NO: 24). In contrast, results demonstrated that the addition of NLS element, in combination with arginine residues (R9 or Rl l) such as in SEQ ID Nos: 19, 22 and 23, significantly increased peptides toxicity (table 3).

Table 3: Cytotoxicity in 4T1 cells using the PrestoBlue assay

Peptide Ο μΜ 2 μΜ 3 μΜ 4 μΜ 6 μΜ 8 μΜ

GLl-1 : Biot-Pep8-PCNA 100 95 106 103 109 88

GL2-2: Ac- Mini-Antp-pep8 100 91 83 87 72 59

GL2-6: NLS-p8 100 90 83 86 65 59

GL5-2: Ac-Transferrin-pep8 100 97 100 96 87 82

GL5-6: Ac-Transferrin-NLS-pep8 100 102 99 99 89 81

GL5-8: Ac-R9-NLS -pep8 100 65 76 65 44 26

GL5-9: Ac-Mini-Antp/NLS-pep8 100 88 89 89 76 70

GL6-4: Ac-Rll-NLS-p8s 100 92 100 101 87 77

GL7-3: Ac-p8-NLS-Rll 100 66 77 66 58 39

GL8-1: Ac-Rll-NLS-p8 100 55 64 57 48 30

GL8-2: Ac-Rll-NLS -p8short 100 99 101 103 91 82

GL8 -3 : Ac-TransferrinTransferrin-pep8 100 97 96 95 93 87

GL8-4: APIM 100 89 98 92 65 46

Cytotoxicity in PyMT cells using the PrestoBlue assay

[0185] The results of PrestoBlue viability assay of 4T1 cells are shown in table 4. The cytotoxic effect of the peptides was examined using the PrestoBlue viability assay. While DMSO, at a cone, of 0.05% served as the vehicle control, it showed no cytotoxicity in this assay. Selenite, which is known to induce cell apoptosis, served as the positive control for cell death (results not shown). Values were normalized to the vehicle control (0.05% DMSO) in order to calculate the percentage of viable cells for each treatment group. Results demonstrated that pep8 in itself (SEQ ID NO: 14), does not exhibit marked cytotoxicity in 4T1 cells even after the addition of NLS or transferring sequences (SEQ ID Nos: 16, 17, 18, and 25), in order to facilitate peptide' s intake to cells (table 4). In addition, the short derivative of pep8 consisting of SEQ ID NO: 2 does not exhibit marked cytotoxicity in PyMT cells even after the addition of NLS (SEQ ID NO: 21) and NLS element in combination with arginine residues (R9) (SEQ ID NO: 24). In contrast, results demonstrated that the addition of NLS element, in combination with arginine residues (R-9 or Rl l) such as in SEQ ID Nos: 19, 22 and 23, significantly increased peptides toxicity in PyMT cells (table 4). Table 4: Cytotoxicity in PyMT cells using the PrestoBlue assay

Peptide Ο μΜ 2 μΜ 3 μΜ 4 μΜ 6 μΜ 8 μΜ

GLl -1 : Biot-Pep8-PCNA 100 100 93 99 96 89

GL2-2: Ac- Mini-Antp-pep8 100 90 87 79 77 80

GL2-6: NLS-p8 100 87 87 84 82 79

GL5-2: Ac-Transferrin-pep8 100 92 90 80 80 77

GL5-6: Ac-Transferrin-NLS-pep8 100 91 91 88 89 83

GL5-8: Ac-R9-NLS -pep8 100 87 91 85 68 49

GL5-9: Ac-Mini-Antp/NLS-pep8 100 95 96 83 90 84

GL6-4: Ac-Rll-NLS-p8s 100 90 96 90 88 73

GL7-3: Ac-p8-NLS-Rll 100 97 93 98 84 68

GL8-1 : Ac-Rll-NLS-p8 100 96 96 91 91 59

GL8-2: Ac-Rll-NLS -p8short 100 92 97 95 80 78

GL8 -3 : Ac-TransferrinTransferrin-pep8 100 93 97 90 91 88

GL8-4: APIM 100 66 58 38 34 31

Cytotoxicity in Α549 cells using the PrestoBlue assay

[0186] The results of PrestoBlue viability assay of A549 cells are shown in table 5. While DMSO, at a cone, of 0.05% served as the vehicle control, it showed no cytotoxicity in this assay, there was a very slight increase in cell viability. Selenite, which is known to induce cell apoptosis, served as the positive control for cell death (result not shown). Values were normalized to the vehicle control (0.05% DMSO) in order to calculate the percentage of viable cells for each treatment group. As demonstrated in table 5, The peptides show low, moderate and high cytotoxicity. [0187] Results demonstrated that pep8 in itself (SEQ ID NO: 14), does not exhibit marked cytotoxicity in A549 cells even after the addition of NLS or transferring sequences (SEQ ID Nos: 16, 17, 18, and 25), in order to facilitate peptide's intake to cells (table 3). In addition, the short derivative of p8 consisting of SEQ ID NO: 2 does not exhibit marked cytotoxicity in A549 cells even after the addition of NLS (SEQ ID NO: 21) and NLS element in combination with arginine residues (R9) (SEQ ID NO: 24). In contrast, results demonstrated that the addition of NLS element, in combination with arginine residues (Rl l) such as in SEQ ID Nos: 22 and 23, significantly increased peptides toxicity in PyMT cells (table 4). Contrary to the other cell lines tested, the addition of 9 arginine residues (R9) (SEQ ID NO: 19) did not show marked cytotoxicity in A549 cells.

Table 5: Cytotoxicity in A549 cells using the PrestoBlue assay

Peptide Ο μΜ 2μΜ 3μΜ 4μΜ 6μΜ 8μΜ

GLl -1 : Biot-Pep8-PCNA 100 109 106 95 91 101

GL2-2: Ac- Mini-Antp-pep8 100 99 98 97 93 92

GL2-6: NLS-p8 100 100 100 102 103 101

GL5-2: Ac-Transferrin-pep8 100 100 1 12 1 1 1 1 15 1 13

GL5-6: Ac-Transferrin-NLS-pep8 100 97 1 10 1 14 106 105

GL5-8: Ac-R9-NLS -pep8 100 92 104 95 92 74

GL5-9: Ac-Mini-Antp/NLS-pep8 100 105 1 12 1 13 1 13 97

GL6-4: Ac-Rll-NLS-p8s 100 1 13 1 10 103 105 88

GL7-3: Ac-p8-NLS-Rll 100 77 88 73 72 50

GL8-1 : Ac-Rll-NLS-p8 100 74 75 73 57 48

GL8-2: Ac-Rll-NLS -p8short 100 97 100 98 89 76

GL8 -3 : Ac-TransferrinTransferrin-pep8 100 95 96 97 95 84

GL8-4: APIM 100 103 100 45 30 36

EXAMPLE 2

Cytotoxicity of the chimeric peptides in which the NKp44 moiety comprises SEQ ID NO: 3

[0188] The cytotoxic effect of the Ac-Rl l-NLS-pl0.19 peptide (SEQ ID NO: 27 (Ac- RRRRRRRRRRR-IKKKRK-VTSSKPRTVAWTSRFTIWAD)) was examined using the PrestoBlue viability assay. Ac-R9-NLS-p8 (SEQ ID NO: 19) and Ac-Rl l-NLS-p8 (SEQ ID NO: 23) were used as positive controls and Ac-Rl l-NLS-p8short (SEQ ID NO: 24) was used as negative control. DMSO, at a cone, of 0.05%, which served as the vehicle control, showed no cytotoxicity in this assay. For this assay cells were treated with NKp44-derived peptides, at various concentrations (2, 3, 4, 6 and 8 μΜ), for 24 h. Cells were incubated with PrestoBlue dye and measured for fluorescence. Values were normalized to their appropriate controls (DMEM or 0.05% DMSO) and the percentage of viable cells was calculated for each treatment group. Results represent means + SD of duplicates.

[0189] As observed in figures 1-2, both cell lines - 4T1 mouse mammary carcinoma and A549 human lung carcinoma show similar sensitivity to Ac-Rl 1-NLS-plO.19 peptide (SEQ ID NO: 27), and exhibit a viability of 50% and 42% (respectively), after 24 h of treatment, at the highest dose examined (8 μΜ).

[0190] As expected, Ac-Rl l-NLS-p8 short peptide (SEQ ID NO: 24), which served as the negative control, did not show any cytotoxic effect on these cells. However, Ac-R9-NLS-p8 and Ac-Rl l-NLS-p8 showed different cytotoxic effect between these cell lines, revealing greater toxicity in 4T1 cells. While both peptides served as a positive control, Ac-R9-NLS-p8 (SEQ ID NO: 19) showed 46% viability for 4T1 cells and only 73% viability for A549. Similarly, Ac-Rl l- NLS-p8 (SEQ ID NO: 23) showed 42% viability for 4T1 cells and only 67% viability for A549.

[0191] Ac-Rl l-NLS-pl0.19 (SEQ ID NO: 27) showed a similar cytotoxic effect in both 4T1 mouse mammary carcinoma cells (Fig. 1) and A549 human lung carcinoma cells (Fig. 2).

[0192] In additional experiments, the cytotoxic effect of the Ac-Rl l-NLS-pl0.19 peptide (SEQ ID NO: 27) and Ac-Rl l-NLS-plO (SEQ ID NO: 28 Ac-RRRRRRRRRRR-IKKKRK- VTS S KPRT V A WTS RFTIW AD ) was examined using the PrestoBlue viability assay. In this experiment, Ac-Rl l-NLS-p8 (SEQ ID NO: 23) was used as a positive control and Ac-Rl l-NLS- p8 short (SEQ ID NO: 24) was used as a negative control. For these assays cells were treated with NKp44-derived peptides, at various concentrations (2, 3, 4, 6 and 8 μΜ), for 24 h. Cells were incubated with PrestoBlue dye and measured for fluorescence. Values were normalized to their appropriate controls (DMEM or 0.05% DMSO) and the percentage of viable cells was calculated for each treatment group. Results represent means + SD of duplicates. Results demonstrated that Ac-Rl l-NLS-pl0.19 (SEQ ID NO: 27) and Ac-Rl l-NLS-plO (SEQ ID NO: 28) showed a similar cytotoxic effect in both 4T1 mouse mammary carcinoma cells (Fig. 3) and A549 human lung carcinoma cells (Fig. 4).

EXAMPLE 3

In vivo effect of Ac-Rll-NLS-p8

[0193] To test the in vivo effect of the Ac-Rl l-NLS-p8 peptide (SEQ ID NO: 23), C57BL mice were injected with 0.5xl0⁶ cells of murine B 16 melanoma cell line. Mice weight and tumor progression were followed. Four to seven days after transplantation, mice were treated, intravenously or intraperitoneally, with Ac-Rl l-NLS-p8 at a dose of 1 and 5 mg/Kg, three times a week, for a period of at least two weeks. A PBS solution, served as the vehicle control. Five (5)- FU, at a dose of 30 mg/Kg, was used as a positive control for treatment of mammary carcinoma.

[0194] During the study, mice weight (data not shown) and tumor size (Figs. 5, 6, 7 and 8) were monitored weekly. After a three-week period of treatment, mice were euthanized, tumors were removed, weighted, and measured. In addition, lungs, spleen, and peritoneal cavity were examined for metastases.

[0195] Tumor size in mice that were treated with 5 mg/Kg with Ac-Rl l-NLS-p8 (Fig. 7) was significantly smaller than tumors of mice treated with the vehicle (Fig. 5) and comparable to the size of the tumors from the positive control (5-FU, Fig. 8) demonstrating the beneficial anti- carcinogenic property of Ac-Rl l-NLS-p8.

EXAMPLE 4

In vivo effect of Ac-Rll-plO.9-11-19

[0196] The in vivo effect of the Ac-Rl l-NLS-plO.9-11-19 (SEQ ID NO: 29 (Ac- RRRRRRRRRRR-I-KKKRK-VTSSKPRTAAATSRFTIWAD)) peptide on tumor progression was evaluated. To this end, six weeks-old female BALB/c mice were injected with 2xl0⁵ 4T1 cells in the left mammary fat pad, and allowed to develop tumors. After 3 days, mice were treated, intravenously, with Ac-Rl l-NLS-plO.9-11-19 peptide, at a dose of 10 or 5 mg/Kg, 5 times a week, consecutively, for a duration of 2 weeks. Mice treated with a PBS solution, served as the vehicle control. Each week, tumors' size was evaluated by measuring their length (L) and width (W), using a Caliper device, and tumor's volume (V) was calculated according to the equation: V=LW²/2. Data was plotted to illustrate the progression of tumor during study.

[0197] Tumors' size progression in mice that were treated with either 10 mg/Kg or 5 mg/Kg Ac- Rl l-NLS-plO.9-11-19 peptide was significantly smaller than in tumors of mice treated with the vehicle (Fig. 9).

[0198] After two weeks of treatment mice were euthanized, and tumors were removed, measured, and weighed. The size (Fig. 10A) and the weight (Fig. 10B) of the tumors obtained from mice that were treated with the Ac-Rl l-NLS-plO.9-11-19 peptide, were significantly smaller than the size and weight of tumors obtained from mice treated with the vehicle. Further, tumors obtained from mice that were treated with 10 mg/kg of Ac-Rl l-NLS-plO.9-11-19 were smaller and weighted less than tumors taken from mice that were treated with 5 mg/kg of Ac-Rl l-NLS- plO.9-11-19 (Fig. 10A-B). These results demonstrate the beneficial anti-carcinogenic property of Ac-Rl l-NLS-plO.9-11-19.

EXAMPLE 5 NKp44-derived pep8 binds to recombinant PCNA and inhibit NKp44 binding

[0199] The inventors previously showed that PCNA is a cellular ligand for NKp44. In the current study, the inventors aimed to identify the PCNA-binding NKp44 epitope by dividing NKp44 ectodomain and hinge region into 19 overlapping, 20mer peptides with a 15-aa overlap between successive peptides. The inventors first studied whether these 19 peptides could bind to recombinant PCNA. Of the 19-screened peptides, pep8 (SEQ ID NO: 3) manifested a substantial binding to PCNA, while overlapping pep 10 also bound to PCNA but to a lesser extent as compared to pep8. Table 6 shows the sequence of the NKp44-derived peptides sequences 8 and 10. The binding of titrated amounts of pep8 and peplO to human PCNA as compared to the negative binding of SEQ ID NO: 36, which is representative of the negative binding of the other screened NKp44 -derived peptides, was shown (Fig. 11 A). The 10-mer shared core sequence of pep8 and pep 10 did not bind PCNA (data not shown), either due to conformational stability or due to 3D peptide structure that did not fit the structure of the PCNA -binding motif in the NKp44 protein ectodomain. Peptide 8 displayed a characteristic binding curve to PCNA (Kd = 4.9E^"7 M), indicating binding with a very moderate affinity (Fig. IB). Binding of pep8 to PCNA was found to be specific, as pep8 did not bind other recombinant proteins, produced similarly to PCNA, such as TL1A, APO-E3 and HNF-4 (Fig. 11C). Pep8 was capable to mediate inhibition of rNKp44 binding to PCNA. The inhibition of rNKp44 binding to PCNA for titrated amounts of pep8 as compared to negative control pep5, was shown (Fig. 11D). The inventors previously showed that pep4 derived from NKp46 mediate its function through binding to NKp46 itself. However, pep8 did not bind specifically to rNKp44 as compared to rNKp46 or to hFc (Fig. 1 IE). Therefore, the inhibition of NKp44-PCNA interaction was solely due to its interaction of pep8 with PCNA (Fig. 11B-C).

Table 6: Amino acid sequence of peptides employed in this study

NKp44-derived peptides:

Modificati

Name Sequence

ons

NKp44-derived pep8 (SEQ ID NO:

EASALVCIRLVTSSKPRTVA

3)

Biotin or

NKp44-derived pep8short (SEQ ID

VTSSKPRTVA purified NO: 35)

(N-

NKp44-derived peplO (SEQ ID NO:

VTSSKPRTVAWTSRFTIWAA terminal) 5)

SEQ ID NO: 36 (SEQ ID NO: 36) YPPTGSLYEKKGWCKEASAL NKp44-derived pep8-CPP chimera:

Modificatio

Name Seauence

ns

Mini-Antp-pep8 (SEQ ID NO: 15) KRRMKWKK-EASALVCIRLVTSSKPRTVA

NLS-pep8 (SEQ ID NO: 16) PKKKRRV-EASALVCIRLVTSSKPRTVA

Transferin-pep8 (SEQ ID NO: 17) HAIYPRH-EASALVCIRLVTSSKPRTVA

Mini-Antp/NLS-pep8 (SEQ ID NO: KRRMKW-KKKRK-

20) EASALVCIRLVTSSKPRTVA

Transferrin-NLS-pep8 (SEQ ID

HAIYPRH-KKKRK-EASALVCIRLVTSSKPRTVA Ac (N-

NO: 18)

terminal),

TransferinTransferin-pep8 (SEQ ID HAIYPR-HHAIYPR- last

NO: 25) HEASALVCIRLVTSSKPRTVA

position; D

RRRRRRRRR-KKKRK-

R9-NLS-pep8 (SEQ ID NO: 19) -amino

EASALVCIRLVTSSKPRTVA

acid

RRRRRRRRRRR-I-KKKRK-W-

Rl l-NLS-pep8 (SEQ ID NO: 23)

EASALVCIRLVTSSKPRTVA

EASALVCIRLVTSSKPRTVA-W-KKKRK-I- pep8-NLS-Rl l (SEQ ID NO: 22)

RRRRRRRRRRR

Rl l-NLS-pep8short (SEQ ID NO:

RRRRRRRRRRR-I-KKKRK-W-VTSSKPRTVA

24)

Published PCNA targeting peptides

Modificati Name Seauence

ons

Ac (N- terminal), last

APIM (SEQ ID NO: 26) MDRWLVK-W-KKKRK-I-RRRRRRRRRRR

position; D

-amino acid

EXAMPLE 6

NKp44-derived pep8 conjugated to CPP reduces cell viability [0200] Disruption of intracellular PCNA activity was previously shown to affect tumor cell viability. The inventors hypothesized that introduction of PCNA-binding NKp44-derived peptides into the cell will result in inhibition of cell proliferation and/or cell death. Therefore, the inventors designed a number of combinations of cell-penetrating peptides, nuclear localization signal and NKp44-derived pep8 (Table 6: Amino acid sequence of peptides employed in this study). Effects of CPP-pep8 combinations were tested on murine 4T1 breast cancer cells and on human A549 lung adenocarcinoma cells (Fig. 12). Both murine and human tumor models were investigated since PCNA is a conserved protein as can be seen by the amino acid sequence homology among mammalian PCNAs. The inventors first assessed CPP-pep8 effect on cell viability employing the PrestoBlue reagent in a concentration range of 0 to 8 μΜ applied to cells for 24 h. the inventors defined low, medium and high effect (down to 80%, 50 to 80% and below 50%, respectively) on cell viability as compared to negative control. Since peptide stocks were solubilized in 5% DMSO, negative controls included both "medium only" and "medium with 0.005% DMSO", reflecting percent DMSO contamination in the highest concentration of applied peptide (8 μΜ). Peptides' low, medium and high manifested effects on 4T1 cells were shown in Fig 12A-C, respectively. As expected, pep8 alone (SEQ ID NO: 3), without any CPP, was shown to assert almost no effect on cell viability (Fig. 12A). The transferrin (with or w/o NLS) (SEQ ID Nos : 18 and 17, respectively) and double transferrin CPPs preceding pep8 (SEQ ID NO: 25) manifested low effect on viability of 4T1 cells, while the miniAntp (with or w/o NLS) (SEQ ID Nos :20 and 15, respectively) and NLS CPPs preceding pep8 manifested medium effect. Only Rl l or R9 CPPS preceding pep8 (SEQ ID Nos: 23 and 19, respectively) or Rl l succeeding pep8 (SEQ ID NO: 22) demonstrated high effect on cell viability of 4T1 cells. The high effect was specific to pep8-PCNA interaction, since Rl l CPP preceding the short form of pep8 (SEQ ID NO: 24), which does not bind PCNA, did not affect 4T1 cell viability (Fig. 12A).

[0201] A549 cells were less sensitive to CPP-pep8 effect as compared to 4T1. All combinations involving miniAntp and Tf/TfTf CPPs manifested negative to low effect on cell viability of A549 cells (Fig. 12D). As before, pep8 alone or CPP-pep8short did not have any effect on tumor cell viability. In accordance with 4T1, Arginine-based CPPS showed the highest potency, with medium effect for R9-based CPP and high effect for Rl l-based CPP (Fig. 12E). Controls employed for these experiments were also recorded (Fig. 12F). DMSO-containing negative control did not reduce cell viability as compared to cell culture medium control. Selenite positive control manifested similar effect on cell viability (e.g., suppression) of 4T1 and A549 cells.

[0202] Since Rl l-NLS-pep8 showed the greatest reduction in PrsetoBlue staining for both murine 4T1 and human A549 models, the inventors further tested its binding affinity to PCNA and its functional efficacy on other murine and human tumor models. Rl l-NLS-pep8 displayed a characteristic binding curve to PCNA (Kd = 8.7E^"8 M), indicating binding with a moderate affinity (Fig. 13A). This affinity was found to be higher than that of pep8 (Fig. 11F), yet in the same ballpark, and could be due to Rl l -based stabilization of the peptide. Nevertheless, binding was pep8-specific since Rl l-NLS-pep8short affinity was null (Fig. 13B), as was previously observed by the inventors for pep8short. The inventors further compared the cell viability effect of Rl l-NLS-pep8 and Rl l-NLS-pep8short on the murine B 16 melanoma, PANC-1 human pancreatic adenocarcinoma, human HEPG2 hepatocellular carcinoma and MDA-MB-231 breast adenocarcinoma. Rl l-NLS-pep8short had no to null effect on cell viability of these cell lines, while Rl l-NLS-pep8 manifested high effect (as defined above) on the cell viability of these lines (Fig. 13C-F).

EXAMPLE 7

NKp44-derived pep8 conjugated to Rll-NLS CPP mediates tumor cell death

[0203] PCNA roles extends over regulation of cell cycle, DNA synthesis, DNA repair and cellular death. However, PrestoBlue assay is unable to differentiate whether it is reduction in cell proliferation or induction of cell death, which are mediated by CPP-pep8. The inventors labeled target cells with CFSE, treated with CPP-pep8 and pep8 and monitored division rate of live cells; no difference was observed between CPP-pep8 and pep8 (data not shown). This result indicated that the reduction in PrestoBlue staining (Figs. 12 and 13) did not result from inhibition of cell proliferation. To investigate the mechanism, the inventors studied the effect of CPP-pep8 on cell death by flow cytometry analysis using the PI marker. The inventors focused on Rl l-NLS-pep8 (SEQ ID NO: 23) as this CPP-pep8 showed the strongest reduction in PrsetoBlue staining (Fig. 12C and E). The cytotoxic agent camptothecin (CPT), an inhibitor of topoisomerase I, and the previously published APIM peptide, which is interacting with intracellular PCNA (Miiller et al., (2013); PLoS One 8: e70430), were used as positive controls. Peptide Rl l-NLS-pep8short (SEQ ID NO: 24) was used as a negative control. Cells were incubated with each agent at a concentration range of 0-8 μΜ for 24 h and then assayed for cell death. Culture medium, alone or with DMSO, had no effect on cell death while Rl l-NLS-pep8 had a strong effect (Fig. 14A.i and A.ii bottom panels). Incubation of Rl l-NLS-pep8 for 24 h, with the murine cell lines B 16 (C57BL/6 origin) and 4T1 (BALB/c origin) resulted in cell death with an ED50 of 3.94 μΜ and 3.78 μΜ, respectively (Fig. 14B-C). In order to confirm that Rl l-NLS-pep8 mediated cells death not only in murine cells, the inventors tested the effect of Rl l-NLS-pep8 on MDA-MB-231 mammary gland/breast cancer cell line. Rl l-NLS-pep8 induced cell death of MDA-MB-231 cells with an ED50 of 4.07 μΜ (Fig. 14D). Cell death of B 16, 4T1 and MDA-MB-231 cell lines was not due to the CPP Rl l-NLS moiety since treatment with Rl l-NLS-pep8short did not induce any cell death. A strong induction of cell death was also seen with the positive APEVI peptide control, yet to a lesser extent as compared to Rl l-NLS-pep8. The other positive control, that is CPT, had induced lower, but stable levels of cell death, which may had resulted from the short time window (24 h) between the treatment and the cells' death recording.

EXAMPLE 8

Rll-NLS-pep8 inoculated systemically mediates suppression of tumor growth in vivo

[0204] In-vitro, Rl l-NLS-pep8 (SEQ ID NO: 23) showed promising results in various murine and human cancer cell lines. Therefore, the inventors wanted to further test if Rl l-NLS-pep8 can mediate tumor growth arrest in vivo. The inventors established that Rl l-NLS-pep8 is active when injected I.V, LP or S.C, the effective Rl l-NLS-pep8 dose (mg/Kg) that can be administered to mice, and further studied several concentrations of peptide and different injection routes. For the 4T1 murine breast cancer the inventors employed IV route of therapy. Growth suppression effect of Rl l-NLS-pep8 (5 mg/kg) was shown to be comparable to that of 5-FU injected LP at 30 mg/kg dose. For clarity, Figure 15A.H present the in vivo 4T1 results in values normalized to vehicle treatment for each measurement day. Differences between growth in vehicle-treated mice and Rl l-NLS-pep8-treated mice were statistically significant on days 19 and 23. While treatment was stopped on day 22, the measures were ceased on day 28, and showed a non- statistically significant difference (p = 0.1). Growth suppression effect of 5-FU treatment served as a positive control and was evident, yet non- significant on days 19 and 23. Interestingly, although treatment was stopped on day 22, accumulated 5-FU effect was significant on day 28 (Fig. 15A.H).

[0205] For the B 16 model the inventors compared 5-FU and Rl l-NLS-pep8 inoculated in the same anatomical site (LP), yet peptide was injected at a dose of 5 mg/kg while 5-FU was injected as before (30 mg/kg). in accordance to the 4T1 model, 5-FU and peptide treatments were similar in their effect on B 16 growth (Fig. 15B.i). Interestingly, although both treatments were stopped on day 22, measurements on day 23 showed that both treatments had still significantly suppressed B 16 growth in vivo (Fig. 15B.H).

[0206] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

A chimeric polypeptide comprising a first moiety and a second moiety, said first moiety comprising 15 to 25 amino acid residues derived from SEQ ID NO: 1 (E AS ALVCIRLVTS S KPRTX i A WTS RFTIWX2X3 , wherein Xi is selected from V and M, X2 is selected from A and D, and X3 is selected from A and D), or an analog thereof having at least 85% sequence identity thereto, and said second moiety comprises one or more cell penetrating peptides.

The chimeric polypeptide of claim 1, capable of penetrating a cell and binding proliferating cell nuclear antigen (PCNA).

The chimeric polypeptide of any one of claims 1 or 2, further comprising one or more modifications selected from the group consisting of: acetylation, amidation, cyclization, and pegylation.

The chimeric polypeptide of claim 1, further comprising an acetyl group attached to the N- terminus thereof.

The chimeric polypeptide of claim 1, comprising a D-amino acid residue at the C-termini of the first moiety.

The chimeric polypeptide of claim 1, wherein said first moiety comprises the amino acid sequence as set forth in SEQ ID NO: 2 (VTSSKPRTXiA wherein Xi is selected from V and M), at its N-terminus or its C-terminus.

The chimeric polypeptide of claim 1, wherein said first moiety comprises an amino acid sequence selected from the group consisting of:

SEQ ID NO: 3 (EAS ALVCIRLVTS S KPRT VA) ,

SEQ ID NO: 4 (EASALVCIRLVTSSKPRTMA),

SEQ ID NO: 5 ( VTS S KPRT V A WTS RFTIWX2X3 , wherein X₂ is selected from D and A, and X3 is selected from D and A)

SEQ ID NO: 6 (VTSSKPRTMAWTSRFTIWX2X3, wherein X₂ is selected from D and A, and X3 is selected from D and A), or an analog thereof having at least 85% sequence identity thereto.

8. The chimeric polypeptide of claim 2, wherein said first moiety binds PCNA.

9. The chimeric polypeptide of claim 1, wherein said second moiety is capable of penetrating a cell.

10. The chimeric polypeptide of claim 1, wherein said second moiety comprises a nuclear localization sequence having an amino acid sequence selected from the group consisting of: SEQ ID NO: 7 (KKKRR), SEQ ID NO: 8 (PKKKRRV), SEQ ID NO: 9 (KRRMKWKK) , and SEQ ID NO: 10 (KKKRK).

11. The chimeric polypeptide of claim 10, wherein said second moiety comprises a nuclear localization sequence having the amino acid sequence as set forth in SEQ ID NO: 10 (KKKRK).

12. The chimeric polypeptide of any one of claims 10 or 11, wherein said second moiety further comprises between 6 and 20 contiguous arginine residues comprising the amino acid sequence as set forth in SEQ ID NO: 11 (RRRRRR).

13. The chimeric polypeptide of claim 12, wherein said contiguous arginine residues consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 12 (RRRRRRRRR), and SEQ ID NO: 13 (RRRRRRRRRRR).

14. The chimeric polypeptide of claim 1, wherein said second moiety comprises between 6 and 20 contiguous arginine residues and one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 7 (KKKRR), SEQ ID NO: 8 (PKKKRRV), SEQ ID NO: 9 (KRRMKWKK), and SEQ ID NO: 10 (KKKRK), and any combination thereof.

15. The chimeric polypeptide of claim 1, wherein said second moiety is attached to said first moiety via a linker.

16. The chimeric polypeptide of claim 1, wherein said second moiety is attached to said first moiety via a peptide bond.

17. The chimeric polypeptide of claim 1, wherein said second moiety is contiguous to the C- terminus and/or the N-terminus of said first moiety.

18. The chimeric polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of: SEQ ID Nos: 19, 22, 23, 27, 28, 29, 30, 31, 32, and 33.

19. A pharmaceutical composition comprising the chimeric polypeptide of any one of claims 1- 18, and a pharmaceutically acceptable carrier.

20. The composition of claim 19, for use in treating or ameliorating cancer in a subject in need thereof.

21. The composition of claim 20 wherein said cancer is selected from: breast cancer, lung cancer, melanoma, liver cancer and pancreatic cancer.

22. A polynucleotide molecule encoding the chimeric polypeptide of any one of claims 1-18.

23. An expression vector comprising the polynucleotide molecule of claim 22.

24. The expression vector of claim 23, for use in treating or ameliorating cancer in a subject in need thereof.

25. The expression vector of claim 24, wherein said cancer is selected from: breast cancer, lung cancer, melanoma, liver cancer and pancreatic cancer.

26. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimeric polypeptide of any one of claims 1-18 or an expression vector encoding same.

27. The method of claim 26, wherein said cancer is selected from: breast cancer, lung cancer, melanoma, liver cancer and pancreatic cancer.