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Ilan MORAD, et al.
MuTaTo vs Cancer



https://www.jpost.com/HEALTH-SCIENCE/A-cure-for-cancer-Israeli-scientists-say-they-think-they-found-one-578939
Jerusalem Post, 28 January 2019

 A cure for cancer? Israeli scientists may have found one
“Our results are consistent and repeatable.”
By Maayan Jaffe-Hoffman

“We believe we will offer in a year’s time a complete cure for cancer,” said Dan Aridor, of a new treatment being developed by his company, Accelerated Evolution Biotechnologies Ltd. (AEBi), which was founded in 2000 in the ITEK incubator. AEBi developed the SoAP platform, which provides functional leads to very difficult targets.

“Our cancer cure will be effective from day one, will last a duration of a few weeks and will have no or minimal side-effects at a much lower cost than most other treatments on the market,” Aridor said. “Our solution will be both generic and personal.”

It sounds fantastical, especially considering that an estimated 18.1 million new cancer cases are diagnosed worldwide each year, according to reports by the International Agency for Research on Cancer. Further, every sixth death in the world is due to cancer, making it the second leading cause of death (second only to cardiovascular disease).

Aridor, chairman of the board of AEBi and CEO Dr. Ilan Morad, say their treatment, which they call MuTaTo (multi-target toxin) is essentially on the scale of a cancer antibiotic – a disruption technology of the highest order.

The potentially game-changing anti-cancer drug is based on SoAP technology, which belongs to the phage display group of technologies. It involves the introduction of DNA coding for a protein, such as an antibody, into a bacteriophage – a virus that infects bacteria. That protein is then displayed on the surface of the phage. Researchers can use these protein-displaying phages to screen for interactions with other proteins, DNA sequences and small molecules.

In 2018, a team of scientists won the Nobel Prize for their work on phage display in the directed evolution of new proteins – in particular, for the production of antibody therapeutics.

AEBi is doing something similar but with peptides, compounds of two or more amino acids linked in a chain. According to Morad, peptides have several advantages over antibodies, including that they are smaller, cheaper, and easier to produce and regulate.

When the company first started, Morad said, “We were doing what everyone else was doing, trying to discover individual novel peptides for specific cancers.” But shortly thereafter, Morad and his colleague, Dr. Hanan Itzhaki, decided they wanted to do something bigger.

To get started, Morad said they had to identify why other cancer-killing drugs and treatments don’t work or eventually fail. Then, they found a way to counter that effect.

For starters, most anti-cancer drugs attack a specific target on or in the cancer cell, he explained. Inhibiting the target usually affects a physiological pathway that promotes cancer. Mutations in the targets – or downstream in their physiological pathways – could make the targets not relevant to the cancer nature of the cell, and hence the drug attacking it is rendered ineffective.

In contrast, MuTaTo is using a combination of several cancer-targeting peptides for each cancer cell at the same time, combined with a strong peptide toxin that would kill cancer cells specifically. By using at least three targeting peptides on the same structure with a strong toxin, Morad said, “we made sure that the treatment will not be affected by mutations; cancer cells can mutate in such a way that targeted receptors are dropped by the cancer.”

“The probability of having multiple mutations that would modify all targeted receptors simultaneously decreases dramatically with the number of targets used,” Morad continued. “Instead of attacking receptors one at a time, we attack receptors three at a time – not even cancer can mutate three receptors at the same time.”
s. The cells pump out the drugs or modify them to be non-functional. But Morad said detoxification takes time. When the toxin is strong, it has a high probability of killing the cancer cell before detoxification occurs, which is what he is banking on.

Many cytotoxic anticancer treatments aim at fast-growing cells. But cancer stem cells are not fast growing, and they can escape these treatments. Then, when the treatment is over, they can generate cancer again.





WO2018061004
THERAPEUTIC MULTI-TARGETING CONSTRUCTS AND USES THEREOF

[ PDF ]
Inventor: MORAD ILAN / ITZHAKI HANAN

The present invention provides constructs comprising a plurality of peptides capable of targeting at least two different extracellular tumor antigens and a toxin, optionally connected to an organic scaffold. Use of such constructs in treating cancer are provided as well. The invention also provides particular peptides binding certain extracellular tumor antigens as well as toxins having antitumor activity.

FIELD OF THE INVENTION

[0001] The invention relates to constructs comprising a plurality of peptides capable of targeting at least two different extracellular tumor antigens and at least one toxin, optionally connected to an organic scaffold and use of such constructs in treating cancer are provided as well. The invention also relates to particular peptides binding certain extracellular tumor antigens as well as toxins having antitumor activity, and conjugates of these peptides and toxins.

BACKGROUND OF THE INVENTION

[0002] Targeted cancer therapies are drugs or other substances designed to interfere with specific molecules involved in cancer cell growth and survival. In contrast to traditional chemotherapy drugs, which usually act against all actively dividing cells, a primary goal of targeted therapies is to fight cancer cells with more precision and potentially fewer side effects. Targeted cancer therapies that have been approved for use against specific cancers include agents that prevent cell growth signaling, interfere with tumor blood vessel development, promote the death of cancer cells, stimulate the immune system to destroy cancer cells, and deliver toxic drugs to cancer cells. The latter mainly includes monoclonal antibodies that deliver toxic molecules. Once the antibody has bound to its target cell, the toxic molecule that is linked to the antibody, such as a radioactive substance, a toxic polypeptide or a poisonous chemical, is taken up by the cell, ultimately killing that cell. The toxin will not affect cells that lack the target for the antibody.

[0003] Efficient tumor targeting is challenging for a number of reasons. First, it requires identifying a target that is sufficiently specific to the tumor cells to avoid as much as possible non-specific killing of cells. In addition, cancer cells tend to be variable, both between cancer types and within the same type of cancer: the expression pattern of surface targets may vary between cells of a particular tumor. Cancer cells may also alter expression of their cell surface receptors during tumor development or become resistant to the therapy. Resistance may occur in two ways: the target itself changes through mutation so that the targeted therapy no longer interacts well with it, and/or the tumor finds a new pathway to achieve tumor growth that does not depend on the target. Most anti-cancer drugs attack a specific target on, or in, the cancer cell. Inhibiting the target usually aims to block a physiological pathway that promotes cancer. Mutations in the targets, or in their downstream physiological pathways, make the targets not relevant to the cancerous nature of the cell.

[0004] DeNardo et al, 2003, Clin Cancer Res. 9(10 Pt 2): 3854S-64S report about the synthesis of branched poly(ethylene glycol) (PEGylated) polymers (Mr 40,000, Mr 70,000, Mr 100,000, and Mr 150,000) conjugated to tumor-specific or control peptides, to assess the effect of both molecular weight and tumor specificity on pharmacokinetics and biodistribution.

[0005] Tsai et al., 2011, J Neurooncol. 103(2): 255-266, describe a bispecific ligand- directed toxin designed to simultaneously target epidermal growth factor receptor (EGFR) on human glioblastoma cells and urokinase receptor (uPAR) on tumor neovasculature. The construct is a single-chain polypeptide consisting of human epidermal growth factor (EGF), a fragment of urokinase and truncated pseudomonas exotoxin (PE38).

[0006] McGuire et al., 2014, Sci Rep., 4:4480 report about the characterization of a suite of tumor targeting peptides for non-small cell lung cancer identified from phage - display libraries. The peptides were synthesized as monomers and homo-tetramers.

[0007] US 7,947,289 discloses compositions comprising modified bacterial toxins and methods for using the modified bacterial toxins for targeting particular cell populations and for treating diseases.

[0008] US 2004/0058865 discloses synthetic multimeric ligands that provide for enhanced cell-, and organ- specific targeting, and methods of their preparation and use.

[0009] US 2009/0130105 discloses compositions that bind to multiple epitopes of IGF- 1R, for example, combinations of monospecific binding molecules or multispecific binding molecules (e.g., bispecific molecules). Methods of making the subject binding molecules and methods of using the binding molecules to antagonize IGF-1R signaling are also disclosed.

[0010] WO 2007/093373 discloses in vivo stable branched peptides, in particular derived from the sequence of Neurotensin (NT) and Luteinizing hormone-releasing hormone (LHRH), conjugated to functional units for specific targeting of cancer cells, either for tumor diagnosis or therapy.

[0011] WO 2008/088422 discloses a composition of matter comprising an OSK1 peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. Further disclosed are pharmaceutical compositions comprising the composition and a pharmaceutically acceptable carrier, DNAs encoding the composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

[0012] There still remains an unmet need for improved compositions and methods for targeted cancer therapy, with enhanced potency and reduced adverse non-specific effects.

SUMMARY OF THE INVENTION

[0013] The present invention relates to a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier. The invention is based on an unexpected observation that a construct comprising two peptides binding two different targets on cancer cells and a toxin has an advantageous and, in some cases, a synergic cytotoxic effect in comparison to constructs having only one of these peptides.

[0014] According to one aspect, the present invention provides a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier.

[0015] According to some embodiments of the invention, at least one of the peptides binds specifically to an extracellular tumor antigen selected from human epidermal growth factor receptor (EGFR) and human Programmed death- ligand 1 (PD-L1). In certain embodiments, the another one of the at least two peptides binds specifically to an extracellular tumor antigen selected from the group consisting of EGFR, PD-L1, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1 , OGFR, PD-1, PD-L2, PDGFR, and VEGFR.

[0016] According to some embodiments, the construct can comprise from 3 to 10 different peptides binding to different extracellular tumor antigens.

[0017] In some embodiments, the present invention provides a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier and wherein at least one of the peptides binds specifically to EGFR. In one embodiment, peptide comprises the amino acid sequence as set forth in SEQ ID NO: 1 (CHPGDKQEDPNCLQADK) or being an analog thereof.

[0018] In other embodiments, the present invention provides a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier and wherein at least one of the peptides binds specifically to PD-L1. In one embodiment, the peptide comprises the amino acid sequence as set forth in SEQ ID NO: 2 (CEGLPADWAAAC) or being an analog thereof.

[0019] In certain embodiment, the present invention provides a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier and wherein one of the peptides binds specifically to EGFR and one of the peptides binds specifically to PD-L1. According to one embodiments, the EGFR the peptide that binds specifically to EGFR is a peptide having SEQ ID NO: 1 or an analog thereof, and the peptide that binds specifically to PD-L1 is a peptide having SEQ ID NO: 2 or an analog thereof.

[0020] According to any one of the above embodiments, the construct comprises multiple copies of at least one or of at least two of the peptides. In some embodiments, the construct comprises from 2 to 50 copies of at least one of the peptides.

[0021] According to any one of the above embodiments, the toxin is a peptide, polypeptide or protein toxin. In some embodiments, the toxin is selected from a toxin binding to a eukaryotic elongation factor 2, BIM-BH3 consisting of SEQ ID NO: 5, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake-venom toxin, and cyanotoxin. In one embodiments, the toxin binding to eukaryotic elongation factor 2 is a toxin comprising the amino acid sequence selected from SEQ ID NO: 3 (CSARWGPTMPWC), SEQ ID NO: 4 (CRRGSRASGAHC), or an analog thereof. According to some embodiments, the construct comprises 2 to 10 different toxins. According to certain embodiments, the construct comprises a toxin having SEQ ID NO: 3 and a toxin having SEQ ID NO: 4.

[0022] According to any one of the above embodiments, the construct comprises multiple copies of at least one or of at least two of the toxins. According to one embodiment, the construct comprises from 2 to 50 copies of the at least one of the toxins. According to another embodiment, the construct comprises 2 to 50 copies of a toxin having SEQ ID NO: 3 and 2 to 50 copies of a toxin having SEQ ID NO: 4.

[0023] According to some embodiments, the present invention provides a construct, wherein one of the peptides binds specifically to EGFR and one of the peptides binds specifically to PD-L1 and the toxin is selected from a toxin binding specifically to eukaryotic elongation factor 2 and a toxin having SEQ ID NO: 5. According to one such embodiment, the construct comprises a peptide having SEQ ID NO: 1 or an analog thereof, a peptide having SEQ ID NO: 2 or an analog thereof, and at least one toxin having amino acid sequence selected from SEQ ID NO: 3, 4, and 5. According to one embodiment, the construct comprises a peptide comprising SEQ ID NO: 1 or an analog thereof, a peptide comprising SEQ ID NO: 2 or an analog thereof, a toxin comprising SEQ ID NO: 3, and a toxin comprising SEQ ID NO: 4. According to any one of such embodiments, the construct comprises multiple copies of each one of the peptides and the toxin(s).

[0024] According to any one of the above embodiments, at least one of the peptides and/or at least one toxin are covalently bound through a carrier. According to one embodiment, the carrier is an organic scaffold. According to another embodiment, each one of the peptides and of the toxin(s) are bound to a carrier, wherein the carrier is an organic scaffold. According to some embodiments, the scaffold is a polyethylene glycol (PEG) molecule or a modified PEG molecule. According to one embodiments, the PEG molecule is a branched molecule. According to another embodiment, the PEG molecules comprises a plurality of sites for binging the peptides and/or the toxin(s) of the present invention. According to one embodiment, the PEG molecule comprises 8 to 56 sites available to bind the peptides and the toxin(s).

[0025] According to some embodiments, the present invention provides a construct comprising multiple copies of each one of at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin(s) are bound to the scaffold and wherein at least one of the peptides binds specifically to the extracellular tumor antigens selected from EGFR or PD-L1. According to some embodiments, one of the peptides binds specifically to EGFR and one of the peptides binds specifically to PD-L1. According to one embodiment, the peptide that binds specifically to EGFR is a peptide having SEQ ID NO: 1 or an analog thereof, and the peptide that binds specifically to PD-L1 is a peptide having SEQ ID NO: 2 or an analog thereof. According to certain embodiments, the toxin comprises the amino acid sequence selected from SEQ ID NO: 3, 4 and 5, or an analog thereof. According to one embodiments, the scaffold is PEG scaffold. According to one embodiment, the PEG molecule comprises 8 to 56 sites available to bind the peptides and the toxin(s).

[0026] According to one embodiment, the construct comprises multiple copies of each one of the: (i) a peptide having SEQ ID NO: 1, (ii) a peptide having SEQ ID NO: 2, (iii) a toxin having SEQ ID NO: 3 and (iv) a toxin having SEQ ID NO: 4, wherein each one of the peptides and the toxins is bound to the scaffold. According to one embodiments, the scaffold is PEG scaffold. According to another embodiment, the construct comprises multiple copies of each one of the: (i) a peptide consisting of SEQ ID NO: 1 , (ii) a peptide consisting of SEQ ID NO: 2, (iii) a toxin consisting of SEQ ID NO: 3, and (iv) a toxin consisting of SEQ ID NO: 4. According to some embodiments, the stoichiometric molar ratio between the peptide having or consisting of SEQ ID NO: 1, the peptide having or consisting of SEQ ID NO: 2, the toxin having or consisting of SEQ ID NO: 3 and the toxin having or consisting of SEQ ID NO: 4 is 1 : 1 :3:3.

[0027] According to any one of the above embodiments, at least one of the peptides or of the toxins is connected to the scaffold through a linker or spacer.

[0028] According to any one of the above embodiments, the construct further comprises a permeability-enhancing moiety.

[0029] According to another aspect, the present invention provides a composition comprising a construct of the present invention. According to one embodiment, the composition is a pharmaceutical composition. Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising a construct of the present invention and a pharmaceutically acceptable excipient. According to one embodiment, the pharmaceutical composition comprises a construct comprising multiple copies of each one of at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin(s) are bound to the scaffold and wherein at least one of the peptides binds specifically to the extracellular tumor antigens selected from EGFR or PD-Ll. According to some embodiments, one of the peptides binds specifically to EGFR and one of the peptides binds specifically to PD-Ll. According to one embodiment, the peptide that binds specifically to EGFR is a peptide having SEQ ID NO: 1 or an analog thereof, and the peptide that binds specifically to PD-Ll is a peptide having SEQ ID NO: 2 or an analog thereof. According to certain embodiments, the toxin comprises the amino acid sequence selected from SEQ ID NO: 3, 4 and 5, or an analog thereof. According to one embodiments, the scaffold is PEG scaffold.

[0030] According to one embodiment, the pharmaceutical composition comprises a the construct comprising multiple copies of each one of the: (i) a peptide having SEQ ID NO: 1, (ii) a peptide having SEQ ID NO: 2, (iii) a toxin having SEQ ID NO: 3 and (iv) a toxin having SEQ ID NO: 4, wherein each one of the peptides and the toxins is bound to the scaffold. According to one embodiments, the scaffold is PEG scaffold. According to another embodiment, the construct comprises multiple copies of each one of the: (i) a peptide consisting of SEQ ID NO: 1 (ii) a peptide consisting of SEQ ID NO: 2, (iii) a toxin consisting of SEQ ID NO: 3, and (iv) a toxin consisting of SEQ ID NO: 4. According to some embodiments, the stoichiometric molar ratio between the peptide having or consisting of SEQ ID NO: 1, the peptide having or consisting of SEQ ID NO: 2, the toxin having or consisting of SEQ ID NO: 3 and the toxin having or consisting of SEQ ID NO: 4 is 1 :1 :3:3.

[0031] According to one embodiment, the pharmaceutical composition of the present invention is for use in treating cancer.

[0032] According to certain aspects, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to said subject a pharmaceutical composition of the present invention. According to one embodiment, the pharmaceutical composition comprises a construct of the present invention. According to one embodiment, the present invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the construct of the present invention.

[0033] According to one aspect, the present invention provides a peptide that binds specifically to human eukaryotic Elongation Factor 2 (eEF2), wherein the peptide comprises the amino acid sequence selected from SEQ ID NO:3, SEQ ID NO: 4 and an analogs thereof. According to one embodiment, the peptide or the analog is cyclic.

According to one embodiment, the peptide comprising or consisting of SEQ ID NO:3, or an analog thereof enhances eEF2 activity. According to another embodiment, the peptide comprising or consisting of SEQ ID NO:4, or an analog thereof enhances eEF2 activity. According to one embodiment, the peptide or analog is for use in inducing cell death.

[0034] According to another aspect, the present invention provides a peptide comprising the amino acids sequence set forth in SEQ ID NO: 1 or an analog thereof. According to one embodiment, the peptide or the analog is an antagonist of a human Epidermal Growth Factor Receptor (EGFR). According to another embodiment, the peptide or the analog is cyclic. According to one embodiment, the peptide or the analog is for use in targeting cancer cells.

[0035] According to a further embodiment, the present invention provides a peptide comprising the amino acids sequence set forth in SEQ ID NO: 2 or an analog thereof. According to one embodiment, the peptide or the analog is an antagonist of a human Programmed death-ligand 1 (PD-L1). According to another embodiment, the peptide or the analog is cyclic. According to one embodiment, the peptide or the analog is for use in targeting cancer cells.

[0036] According to certain aspects, the present invention provides a conjugate comprising at least one peptide of the present invention. According to one embodiment, the peptide is selected from a peptide comprising or consisting of SEQ ID NO:l , a peptide comprising or consisting of SEQ ID NO:2, a peptide comprising or consisting of SEQ ID NO:3, a peptide comprising or consisting of SEQ ID NO: 4 and an analog of said peptides.

[0037] According to another aspect, the present invention provides a composition comprising the peptide of the present invention or the conjugate of the present invention. According to one embodiment, the composition is a pharmaceutical composition. Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising the peptide of the present invention or the conjugate of the present invention. According to one embodiment, the peptide is selected from a peptide comprising or consisting of SEQ ID NO:l, a peptide comprising or consisting of SEQ ID NO:2, a peptide comprising or consisting of SEQ ID NO:3, a peptide comprising or consisting of SEQ ID NO: 4 and an analog of said peptides. According to another embodiment, the conjugate is a conjugate of said peptides. According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0038] According to one aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the peptides of the present invention or of the conjugates of the present invention. According to one embodiment, the method of treating cancer comprises administering a pharmaceutical composition comprising said peptides or said conjugates. According to one embodiment, the peptide is selected from a peptide comprising or consisting of SEQ ID NO: 1 , a peptide comprising or consisting of SEQ ID NO: 2, a peptide comprising or consisting of SEQ ID NO: 3, a peptide comprising or consisting of SEQ ID NO: 4 and an analog of said peptides. According to another embodiment, the conjugate is a conjugate of said peptides. According to some embodiments, the method comprises administering the pharmaceutical composition of the present invention comprising said peptides or said conjugates.

[0039] According to a further aspect, the present invention provides an isolated polynucleotide comprising a sequence encoding the peptide or analog of the present invention. According to one embodiment, the peptide is selected from a peptide comprising or consisting of SEQ ID NO: 1 , a peptide comprising or consisting of SEQ ID NO: 2, a peptide comprising or consisting of SEQ ID NO: 3, and a peptide comprising or consisting of SEQ ID NO: 4. According to another embodiment, the analog is an analog of said peptides.

[0040] According to further aspect, the present invention provides an isolated polynucleotide comprising a sequence encoding for a polypeptide comprising (i) at least one copy of SEQ ID NO: 1 ; (ii) at least one copy of SEQ ID NO: 2; (iii) at least one copy of SEQ ID NO: 3, 4 or combination thereof.

[0041] According to yet another aspect, the present invention provides a nucleic acid construct comprising the polynucleotide of the present invention. According to one embodiment, the polynucleotide is operably linked to a promoter.

[0042] According to certain aspects, the present invention provides a vector comprising at least one polynucleotide or at least one nucleic acid construct of the present invention.

[0043] According to a further aspect, the present invention provides a cell comprising at least one polynucleotide or at least one nucleic acid construct of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0044] Fig. 1 shows schematic structure of a multi-arm-PEG complex loaded with two targeting molecules such as E13.3 and PL-L1-GR peptides (solid circles and squares) and a toxin (hollow circles) such as Toxl and/or Tox2.



[0045] Fig. 2 shows the result of the ELISA experiment demonstrating the binding of several peptides (toxins) to eEF2 or BSA at two different incubation times: 1.5 min and 30 min (TB2 - Toxl, GW -Tox2). [0046] Fig. 3 shows the results of the activity of several peptides (toxins) tested in the in vitro transcription/translation system (TB2 - Toxl, GW -Tox2, GR - non-eEF2- binding control).

[0047] Figs 4-7 show the effect of PEG-E13.3-toxin construct on A431 and MCF-7 cells:

[0048] Fig. 4 - control (no treatment): Fig. 4A and 4B: A431 cell at T=0 and 48h, respectively; Fig. 4C and 4D: MCS-7 cell at T=0 and 48h, respectively.

[0049] Fig. 5 shows the effect of PEG-E13.3-BIM (Fig. 5A-C) construct and of PEG- E13.3-Toxl-Tox2 (Fig 5D-5F) on A431 cells at different concentrations: 1 μΜ (Figs. 5A and 5D), 3 μΜ (Figs. 5B and 5E) and 8 μΜ (Figs. 5C and 5F). The pictures were taken 48 hours after the treatment.

[0050] Fig. 6 shows the effect of PEG-E13.3-BIM (Fig. 6A-C) construct and of PEG- E13.3-Toxl-Tox2 (Fig 6D-6F) on MCF-7 cells at different concentrations: 1 μΜ (Figs. 6A and 6D), 3 μΜ (Figs. 6B and 6E) and 8 μΜ (Figs. 6C and 6F). The pictures were taken 48 hours after the treatment.

[0051] Fig. 7 shows treatment of A431 cells (Fig 7A-7C) and MCF-7 (Fig. 7D-7F) with a complex of PEG-BIM (without E13.3) at different concentrations: 1 μΜ (Fig. 7A and 6D), 3 μΜ (Fig. 7B and 7E) and 8 μΜ (Fig. 7C and 7F). The pictures were taken 48 hours after the treatment.

[0052] Fig. 8 shows the effect of treatment of A431 cells with different constructs: PEG-BIM (Fig. 8B-D), PEG-E13.3-BIM (Fig. 8E-G), PEG-PD-Ll -GR-BIM (Fig. 8H- J) and PEG-PD-Ll GR-E13.3-BIM (Fig. 8K-M) at different concentrations: 10 nM (Fig 8B, 8E, 8H and 8K), 100 nM (Fig 8C, 8F, 81 and 8L) and 1 μΜ (Fig 8D, 8G, 8J and 8M). Fig 8A is a control. The pictures were taken 48 hours after the treatment.

[0053] Fig. 9 shows effect of treatment of A-549 cells with PEG-E13.3-(PD-L1 -GR)- Toxl-Tox2: 3 and 10 μΜ (Fig. 9C and 9D, respectively) or PEG-E13.3-(PD-L1-GR)- BIM 10 μΜ. Fig 9A (T=0) and 9B (T=48h) are used as a control. Fig. 9B-9D were taken 48 hours after the treatment.

[0054] Fig. 10 shows the Coomassie Plus stained electrophoresis gel of selected peptides: lanes (from left to right): 1 - E7.1 ; 2 - E10.2; 3 - E13.3; 4 - E14.1.1 ; 5 - E14.1.4; 6 - E23I3; 7 - E23I5 ; 8 - E15.1.3-T; 9 - A4.3.12-T; 10 - Protein Marker (Fermentas).

[0055] Fig. 11 shows the normalized results of inhibition analysis of the selected peptides by measuring auto phosphorylation of EGFR. [0056] Fig. 12 shows the result for measurement of the stability of selected peptides incubated in bovine serum for different time periods.

[0057] Fig. 13 shows the result of assessment of inhibitory activity of the selected peptides at different concentrations (Fig. 13A general view and Fig. 13B shows E13.3 alone).

[0058] Fig. 14 shows the in vivo stability of E13.3 peptide alone or in complex with 8- armed PEG.

[0059] Fig. 15 shows the effect of E13.3 on viability of two cancer cell lines.

[0060] Fig 16. shows the accumulation of E13.3-PEG complex in kidney, liver and tumor in mice.

[0061] Fig. 17 show a picture of cancer cells that were isolated from a tumor in mice 1 hour (left panel) and 24 hour (right panel) following IV injection of fluorescently marked E13.3-PEG complex.

[0062] Fig. 18 shows the effect of the treatment of A- 549 cells with ΙμΜ of: PEG- E13.3-BIM(18B), PEG-(PD-L1-GR)-BIM (18C)and PEG-E13.3-(PD-L1-GR)-BIM (18D) using PBS as a control (18A).

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention relates to therapeutic constructs comprising a plurality of multi-target peptides and at least one toxin moiety. In particular, a construct according to the present invention comprises a plurality of peptides each directed against a different cell-target. Peptides contained in a construct according to the invention are capable of binding, blocking, inhibiting, or activating at least two different antigens expressed on the membrane of cancer cells. The present invention provides, according to one aspect, a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier.

[0064] The term "peptide" refers to a short chain of amino acid residues linked by peptide bonds, i.e., a covalent bond formed between the carboxyl group of one amino acid and an amino group of an adjacent amino acid. The term "peptide" refers to short sequences having up to 50 amino acids. A chain of amino acids monomers longer than 50 amino acid is referred as a "polypeptide". Such polypeptides, when having more than 50 amino acid residues, can also be classified as proteins, more particularly, proteins of low or medium molecular weight. [0065] The term "peptide" encompasses also the term "peptide analog". The term "peptide analog" and "analog" are used herein interchangeably and refer to an analog of a peptide having at least 70% identity with the original peptide, wherein the analog retains the activity of the original peptide. Thus, the terms "analog" and "active analog" may be used interchangeably. The term " "analog" refer to a peptide which contains substitutions, rearrangements, deletions, additions and/or chemical modifications in the amino acid sequence of the parent peptide. The term "analog" refers also to analogs of peptide toxins, i.e. toxins being peptides. According to some embodiments, the peptide analog has at least 80%, at least 90% or at least 95% sequence identity to the original peptide. According to one embodiment, the analog has about 70% to about 95%, about 80% to about 90% or about 85% to about 95% sequence identity to the original peptide. According to some embodiments, the analog of the present invention comprises the sequence of the original peptide in which 1, 2, 3, 4, or 5 substitutions were made.

[0066] The substitutions of the amino acids may be conservative or non-conservative substitution. The non-conservative substitution encompasses substitution of one amino acid by any other amino acid. In one particular embodiment, the amino acid is substituted by a non-natural amino acid.

[0067] The term "amino acid" as used herein refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non- natural amino acid. The twenty two natural amino acids are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (He), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl). Non- limiting examples of non-natural amino acids include diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Orn), aminoadipic acid, β-alanine, 1- naphthylalanine, 3-(l-naphthyl)alanine, 3-(2-naphthyl)alanine, γ-aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, p-ethynyl-phenylalanine, p-propargly-oxy- phenylalanine, m-ethynyl-phenylalanine, p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine, p-acetylphenylalanine, azidonorleucine, 6 -ethynyl- tryptophan, 5- ethynyl-tryptophan, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5- bromoindolyl)alanine, azidohomo alanine, p-chlorophenylalanine, a-aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine-a-threonine, and N-acetylgalactosamine- α-serine. According to one embodiment, the substitution is substitution with a non- natural amino acid.

[0068] According to some embodiments, the term "analog" encompasses also the term "conservative analog".

[0069] Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. One of skill will recognize that individual substitutions, is a "conservatively modified analog" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. One typical example of conservative substitution is provided below.

[0070] The following six groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). In other embodiments, the conservative substitution encompass substitution with a chemically similar non-natural amino acid.

[0071] Thus, in some embodiments, the analog is a conservative analog of the peptide. According to some embodiments, the conservative analog of the present invention comprises the sequence of the original peptide in which 1, 2, 3, 4, or 5 conservative substitutions were made. According to another embodiment, the analog consists of the amino acid sequence of the original peptide in which 1 , 2 or 3 conservative substitution were made. Thus, the analog consists of the amino acid sequence of the original peptide with 1, 2 or 3 conservative substitutions.

[0072] The term "peptide" encompasses also the term "peptide fragment". The term "fragment" refers to a fragment of the original peptide or of an analog thereof wherein said fragment retains the activity of the original peptide or analog. Thus, the terms "fragment" and "active fragment" may be used interchangeably. According to some embodiments, the fragment consists of at least 6, at least 8, at least 9, or at least 10 consecutive amino acids of the original sequence or of an analog thereof. According to one embodiment, the fragment consists of 6 to 11, 7 to 10 or 8 to 9 consecutive amino acids of the original sequence or analog thereof.

[0073] The peptides, analogs and fragments of present invention may be produced by any method known in the art, including recombinant (for peptides consisting of genetically encoded amino acids) and synthetic methods. Synthetic methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. Solid phase peptide synthesis procedures are well known to one skilled in the art. Synthetic methods to produce peptides include but are not limited to FMOC solid phase peptide synthesis described, for example in Fields G. B., Noble R., Int. J. Pept. Protein Res., 35 : 161-214, 1990. Methods for synthesizing peptides on PEG are described for example in DeNardo et al. Ibid.

[0074] In some embodiments, synthetic peptides are purified by preparative high performance liquid chromatography and the peptide sequence is confirmed via amino acid sequencing by methods known to one skilled in the art.

[0075] In some embodiments, recombinant protein techniques, well known in the art, are used to generate peptides and peptide multimers (consisting of genetically encoded amino acids) of the present invention.

[0076] As used herein, the term "toxin" refers to a peptide or polypeptide substance which is poisonous, harmful or toxic (cytotoxic) to mammalian cells, such as human cells. The toxin according to the present invention may be originated from living organism such as a microorganism, plant, or higher organism, or which may be synthetically prepared, produced, or designed using any known technique, for example as described in WO 2007/010525. The toxin typically interacts with cellular biological macromolecules such as enzymes and receptors to mediate its effect. The term encompasses biologically active subunits or fragments of a toxin. According to certain embodiments, the toxin is a peptide toxin, consisting of up to 50 amino acids. According to some embodiments, the toxin being a peptide may be a cyclic peptide. For the sake of clarity, the toxin being a cyclic peptide is referred as a "cyclotoxin" or "cyclic toxin". Within a construct of the present invention, a toxin moiety confers at least some of its properties to the construct, and the construct mediates poisonous or harmful effects on the target cells. None limiting examples of peptide toxin include cyanobacteria toxins such as Microcystins and Nodularins, ProTx-I and ProTx-II toxins, snake venom-derived disintegrins such as Viperistatin or fragments thereof, and Hm-1 and Hm-2 toxins. [0077] The terms "carrier" refers to any molecule that covalently binds or capable of binding to the at least two different peptides and/or a toxin. Several possible binding arrangements are encompassed. According to one embodiment, one peptide and one toxin are bound via a carrier and the second peptide is bound directly to the first peptide or to the toxin. According to another embodiment, two peptides are bound via a carrier, and the toxin is bound to one of the peptides. According to a further embodiment, all peptides and toxin(s) are covalently bound to a carrier.

[0078] According to any one of the above embodiment, the peptides and/or the toxin(s) are bound via a linker. The terms "linker" and "spacer" are used herein interchangeably and refer to any molecule that covalently binds and therefore linking two molecules. Non-limiting examples of the linker are amino acids, peptides, or any other organic substance that can be used to allow distance between two linked molecules.

[0079] As used herein, the terms "target" and "cell target" refer to molecules found on cancer cells that may be a marker of cancer cell and may be involved in cancer cell growth, proliferation, survival and metastasis development. Particular examples of targets include cell-surface proteins, which upon binding to their counterparts, such as ligands, initiate a cascade that promotes tumor growth and development. A target according to the present invention is optionally highly expressed on cancer cells and not found, or found in substantially lower levels, on normal non-cancerous cells. The term "target" encompass therefore the term "extracellular tumor antigen". The term "tumor antigen" or "extracellular tumor antigen" are used herein interchangeably and include both tumor associated antigens (TAAs) and tumor specific antigens (TSAs). A tumor- associated antigen means an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development. A tumor specific antigen is an antigen that is unique to tumor cells and is not expressed on normal cells. The term tumor antigen includes TAAs or TSAs that have been already identified and those that have yet to be identified and includes fragments, epitopes and any and all modifications to the tumor antigens.

[0080] As used herein, the term "cell-targeting", when referring to a moiety, particularly a peptide, that is part of a construct of the present invention, indicates that the peptide provides cell-, tissue- or organ-specific targeting. In particular, a cell-targeting peptide specifically recognizes and binds a cell target on cancer cells. By virtue of its binding, the cell-targeting peptide directs the entire construct to the cancerous tissue, to facilitate specific killing/inhibition of cancerous cells. Killing/inhibition of cancerous cells is typically affected via the toxin present in the construct, but in some embodiments it may be affected directly by the binding of the cell-targeting peptide.

[0081] In one embodiment, the present invention provides a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently connected directly or through a carrier. According to some embodiments, the construct comprises at least 3 different said peptides. According to other embodiments, the construct comprises at least 4 different said peptides. According to certain embodiments, the construct comprises two or more different peptides binding to two or more different extracellular tumor antigens. According to one embodiment, the construct comprises three or more different peptides binding to three or more different extracellular tumor antigens. According to another embodiment, the construct comprises 4 or more different peptides binding to 4 or more different extracellular tumor antigens.

[0082] Not limiting examples of the extracellular tumor antigens are EGFR, PD-L1 , HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1 , OGFR, PD-1 , PD-L2, PDGFR, and VEGFR, thus according to one embodiment, at least one of the peptides binds specifically to one such extracellular tumor antigen.

[0083] According to some embodiments, at least one of the peptides binds specifically to an extracellular tumor antigens selected from Epidermal Growth Factor Receptor (EGFR) or programmed death-ligand 1 (PD-L1). The terms "PD-L1" and "human PD- Ll" are used herein interchangeably. The terms "EGFR" and "human EGFR" are used herein interchangeably.

[0084] According to other embodiments, the other one of the at least two peptides binds specifically to an extracellular tumor antigen selected from the group consisting of EGFR, PD-L1, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1 , PD-L2, PDGFR, and VEGFR.

[0085] According to a further embodiment, at least one of the peptides binds specifically to EGFR or PD-L1 and the other one of the at least two peptides binds specifically to an extracellular tumor antigen selected from EGFR, PD-L1, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1, PD-L2, PDGFR, and VEGFR.

[0086] According to any one of the above embodiments, the peptide consists of 5 to 30 amino acids. According to other embodiments, each peptide consists of 6 to 25 amino acids. According to yet other embodiments, each peptide consists of 7 to 20 amino acids. According to some embodiments, each peptide consists of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Each possibility represents a separate embodiment of the invention.

[0087] According to any one of the above embodiments, the peptide of the present invention is a cyclic peptide. The terms "cyclic peptide" and "cyclopeptide" are used herein interchangeably and refer to a peptide having an intramolecular bond between two non-adjacent amino acids. The cyclization can be effected through a covalent or non-covalent bond. Intramolecular bonds include, but are not limited to, backbone to backbone, side-chain to backbone and side-chain to side-chain bonds. According to some embodiments, the cyclization occurs between the cysteines of the peptide, analogs of fragments. According to other embodiments, the cyclization occurs between the N- terminal and C-terminal amino acids.

[0088] According to any one of the above embodiments, the construct comprises two or more peptides binding to two or more different extracellular tumor antigens. According to some embodiments, the construct comprises 2 to 10 different peptides binding to 2 to 10 different extracellular tumor antigens. According to other embodiments, the construct comprises 3 to 8, 3 to 10, or 4 to 6 different peptides. According to one embodiment, the construct comprises 2 different peptides binding to 2 different extracellular tumor antigens. According to a further embodiment, the construct comprises 3 different peptides binding to 3 different extracellular tumor antigens. According to another embodiment, the construct comprises 4 different peptides binding to 4 different extracellular tumor antigens. According to certain embodiments, the construct comprises 5, 6, 7 or 8 different peptides binding to 5, 6, 7 or 8 different extracellular tumor antigens, respectively. According to some embodiments, at least one of the peptides bind specifically to EGFR or PD-L1.

[0089] According to one embodiment, the extracellular tumor antagonist is human EGFR. Thus, according to one embodiment, at least one of the peptides binds specifically to EGFR. According to some embodiments, the peptide is a peptide having the amino sequence set forth in SEQ ID NO: 1 (CHPGDKQEDPNCLQADK). According to other embodiments, the peptide is a peptide consisting of the amino sequence set forth in SEQ ID NO: 1. According to some such embodiments, the peptide comprising or consisting of SEQ ID NO: 2 is cyclic.

[0090] According to another embodiment, the peptide is an analog of the peptide having SEQ ID NO: 1. In yet another embodiment, the peptide is a conservative analog of SEQ ID NO: 1. According to some embodiments, the peptide is an analog having at least 70%, at least 75%, at least 80%, at least 85, at least 90% or at least 95% identity to SEQ ID NO: 1. According to other embodiments, the analog is a peptide having 70% to 95%, 75% to 90%, or 80% to 85% sequence identity to SEQ ID NO: 1. According to some embodiments, the analog of SEQ ID NO: 1 is a conservative analog of SEQ ID NO: 1 that has 1 , 2, 3, 4 or 5 conservative substitutions.

[0091] According to a further embodiment, the peptide is a fragment of SEQ ID NO: 1 or of an analog thereof. According to some embodiments, the fragment consists of at least 6, at least 8, at least 10, at least 12, at least 14 or at least 16 consecutive amino acids of SEQ ID NO: 1 or analog thereof. According to one embodiment, the fragment consists of 5 to 16, 6 to 14, 7 to 13, 8 to 12, 8 to 12, or 9 to 11 consecutive amino acids of SEQ ID NO: 1 or analog thereof. In another embodiment, the peptide fragment consists of 6 to 16, 8 to 14 or 10 to 12 consecutive amino acids of SEQ ID NO: 1

[0092] According to any one of the aspects and embodiments of the invention, the terms "peptide comprising the amino acid sequence set forth in SEQ ID NO: X", "peptide comprising SEQ ID NO: X" and "peptide having SEQ ID NO: X" are used herein interchangeably. The terms "peptide consisting of the amino acid sequence set forth in SEQ ID NO: X", "peptide consisting of SEQ ID NO: X" and "peptide of SEQ ID NO: X" are used herein interchangeably.

[0093] According to one embodiment, the extracellular tumor antagonist is human PD- Ll. Thus according to one embodiment, at least one of the peptides binds specifically to PD-L1. According to some embodiments, the peptide is a peptide having the amino sequence set forth in SEQ ID NO: 2 (CEGLP ADW AA AC) . According to certain embodiments, the peptide is a peptide consisting of SEQ ID NO: 2. According to some such embodiments, the peptide comprising or consisting of SEQ ID NO: 2 is cyclic.

[0094] According to another embodiment, the peptide is an analog of SEQ ID NO: 2. In yet another embodiment, the peptide is a conservative variant of SEQ ID NO: 2. According to some embodiments, the analog is a peptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity to SEQ ID NO: 2. According to other embodiments, the analog is a peptide having 70% to 95%, 75% to 90%, or 80% to 85% identity to SEQ ID NO: 2. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 2 that has 1, 2, 3, 4 or 5 conservative substitutions.

[0095] According to a further embodiment, the peptide is a fragment of SEQ ID NO: 2 or of an analog thereof. According to some embodiments, the fragment consists as least 6, at least 7, at least 8, at least 9, at least 10 or 11 consecutive amino acids of SEQ ID NO: 2 or analog thereof. According to one embodiment, the fragment consists of 5 to 16, 6 to 14, 7 to 13, 8 to 12, 8 to 12, or 9 to 11 consecutive amino acids of SEQ ID NO: 1 or analog thereof. In another embodiment, the peptide fragment consists of 6 to 16, 8 to 14 or 10 to 12 consecutive amino acids of SEQ ID NO: 1

[0096] According to any one of the above embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2, analogs of fragments thereof are cyclic peptides, analogs or fragments.

[0097] According to some embodiments, at least one of the peptides binds specifically to EGFR, and at least one of the peptides binds specifically to PD-Ll . According to some embodiments, the construct of the present invention has a synergistic cytotoxicity. The term "synergistic cytotoxicity" as used herein refers to a condition in which the cytotoxicity of the construct comprising two or more tumor antigen targeting peptides is higher that the cytotoxicity of 2 or more constructs, respectively, when each such construct comprises only one of the targeting peptides. Thus, the cytotoxicity of a construct comprising PD-Ll and EGFR targeting peptides is higher that a cytotoxicity of two constructs each comprising PD-Ll or EGFR targeting peptides (considering the concentrations of the constructs). According to some embodiments, the construct comprises one peptide that binds specifically to EGFR and another peptide that binds specifically to PD-Ll. According to one embodiment, the peptide that binds to EGFR is a peptide having SEQ ID NO: 1 , analog or fragment thereof. According to another embodiment, the peptide that binds specifically to PD-Ll is a peptide having SEQ ID NO: 2, analog or fragment thereof. According to certain embodiments, the construct comprises a peptide having SEQ ID NO:l , analog or fragment thereof and a peptide having SEQ ID NO: 2, analog or fragment thereof. According to certain embodiments, the construct comprises a peptide having SEQ ID NO: 1 and a peptide having SEQ ID NO: 2. According to certain embodiments, the construct comprises a peptide of SEQ ID NO: 1 and a peptide of SEQ ID NO: 2. According to some such embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2, analogs of fragments thereof are cyclic peptides, analogs or fragments. According to some embodiments, the construct of the present invention has a synergistic cytotoxicity.

[0098] According to any one of the above embodiments, the construct of the present invention comprises multiple copies of at least one of the different peptides.

[0099] According to other embodiments, the construct of the present invention comprises multiple copies of each one of the at least two of the different peptides. According to another embodiment, the construct comprises multiple copies of each one of the peptides.

[0100] The term "different peptides" refer to peptides binding to different binding site and not to two copies of the same peptide.

[0101] According to some embodiments, the construct comprises from 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 copies of a peptide. According to one embodiment, the construct comprises 2 to 50 copies of a peptide. According to another embodiment, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of a peptide. According to other embodiments, the construct comprises 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 copies of each one of the two different peptides. According to one embodiment, the construct comprises 2 to 50 copies of each one of the two different peptides. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of each one of the two different peptides. In certain embodiments, the contract comprises from 7 or from 14 to 28 copies of each one of the 3, 4 or 5 different peptides.

[0102] According to some embodiments, the construct comprises multiple copies of a peptide that binds specifically to EGFR and/or multiple copies of a peptide that binds specifically to PD-L1. According to some other embodiments, the construct comprises multiple copies of the peptide having the SEQ ID NO: 1, analog or fragment thereof and multiple copies of the peptide having the SEQ ID NO: 2, analog or fragment thereof. According to some embodiments, the construct comprises from 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 copies of the peptide having SEQ ID NO: 1 , analog or fragment thereof and/or of the peptide having the SEQ ID NO: 2, analog or fragment thereof. According to one embodiment, the construct comprises 2 to 50 copies of the peptide having SEQ ID NO: 1 , analog or fragment thereof and/or of the peptide having the SEQ ID NO: 2, analog or fragment thereof. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, or from 7 to 21 copies of the peptide having the SEQ ID NO: 1. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, or from 7 to 21 copies of the peptide having the SEQ ID NO: 2. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, or from 7 to 21 copies of the each one of the peptide having the SEQ ID NO: 1 and 2. According to any one of the above embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2, analogs of fragments thereof are cyclic peptides, analogs or fragments.

[0103] According to any one of the above embodiments, the toxin is selected from a peptide toxin, polypeptide toxin or peptide toxin.

[0104] According to some embodiments, the toxin is selected from the group consisting of a toxin binding to a eukaryotic elongation factor 2 or analog of that toxins, BIM-BH3 toxin, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake- venom toxin, and cyanotoxin. According to some embodiments, the BIM-BH3 toxin consists of the amino acid sequence MRPEIWIAQELRRIGDEFNA (SEQ ID NO: 5).

[0105] According to some embodiments, the toxin binding to eukaryotic elongation factor 2 is a toxin having the amino acid sequence selected from CSARWGPTMPWC (as set forth in SEQ ID NO: 3) or CRRGSRASGAHC (as set forth in SEQ ID NO: 4), or an analog thereof.

[0106] According to another embodiment, the toxin is selected from the group consisting a toxin having SEQ ID NO: 3, a toxin having SEQ ID NO: 4, a toxin of SEQ ID NO: 5 (BIM-BH3 toxin), Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake- venom toxin, and cyanotoxin.

[0107] According to some embodiments, the toxin is a toxin comprising SEQ ID NO: 3. According to other embodiments, the toxin is a toxin comprising SEQ ID NO: 4. According to another embodiment, the toxin consists of SEQ ID NO: 3. According to yet another embodiment, the toxin consists of SEQ ID NO: 4. According to one embodiment, the toxin consists of SEQ ID NO: 5. According to some embodiments, the toxin is an analog of a toxin comprising the SEQ ID NO: 3 or 4. According to certain embodiments, the toxin is an analog of a toxin consisting of the SEQ ID NO: 3 or 4. According to some such embodiments, the toxin or analog thereof is cyclic toxin or analog.

[0108] According to some embodiments, the analog of a toxin comprising SEQ ID NO: 3 has at least 70%, at least 75%, at least 80%, at least 85, at least 90% or at least 95% identity to SEQ ID NO: 3. According to other embodiments, the analog is a peptide having 70% to 95%, 75% to 90%, or 80% to 85% sequence identity to SEQ ID NO: 3. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 3 that has 1 , 2, 3, 4 or 5 conservative substitutions.

[0109] According to some embodiments, the analog of a toxin comprising SEQ ID NO: 4 has at least 70%, at least 75%, at least 80%, at least 85, at least 90% or at least 95% identity to SEQ ID NO: 4. According to other embodiments, the analog is a peptide having 70% to 95%, 75% to 90%, or 80% to 85% identity to SEQ ID NO: 4. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 4 that has 1 ,

2, 3, 4 or 5 conservative substitutions.

[0110] According to some such embodiments, the toxins binding to eEF2, and in particular the toxins comprising or consisting of SEQ ID NO: 3, or, analogs or fragments thereof have cyclic structure, i.e. being cyclotoxins.

[0111] According to some embodiments, the construct comprises 2 to 10 different toxins. According to one embodiment, the construct comprises 2 different toxins. According to another embodiment, the construct comprises 3 different toxins. According to a further embodiment, the construct comprises 4, 5, 6, 7, 8, 9 or 10 different toxins.

[0112] According to certain embodiments, the construct comprises a toxin having the amino acid SEQ ID NO: 3 and a toxin having the amino acid SEQ ID NO: 4.

[0113] According some embodiments, the construct of the present invention comprises multiple copies of at least one of the toxins. According to other embodiment, the construct comprises multiple copies of at least two toxins.

[0114] According to some embodiments, the construct comprises multiple copies of at least one toxin having SEQ ID NO: 3 or 4. According to other embodiments, the construct comprises multiple copies of at least one toxin having SEQ ID NO: 3, or 4.

[0115] According to some embodiments, the construct comprises from 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 copies of a toxin. According to one embodiment, the construct comprises 2 to 50 copies of a toxin. According to another embodiment, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of a toxin. According to other embodiments, the construct comprises 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 copies of each one of two different toxins. According to one embodiment, the construct comprises 2 to 50 copies of each one of two different toxins. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of each one of two different toxins. In certain embodiments, the contract comprises from 7 or from 14 to 28 copies of each one of the 3, 4 or 5 different toxins.

[0116] According to some embodiments, the construct comprises from 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25, 2 to 50, or 10 to 20 copies of a toxin having SEQ ID NO: 3, analog or fragment thereof and/or of the toxin having the SEQ ID NO: 4, analog or fragment thereof. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of the toxin having the SEQ ID NO: 3. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of the toxin having the SEQ ID NO: 4. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of the each one of the toxins having the SEQ ID NO: 3 and 4.

[0117] According to one embodiment, the molar ratio of the toxin having the amino acid SEQ ID NO: 3 to the toxin having the amino acid SEQ ID NO: 4 is about 0.1 :1 to about 10:1. According to some embodiments, the ratio is about 0.2:1 to 8:1 , about 0.4:1 to 6:1 about 0.5:1 to 5:1 about 0.6:1 to 4:1, about 0.8 to 1 to 2:1 or about 1 :1. According to one embodiment, the molar ratio of the toxin having the amino acid SEQ ID NO: 3 to the toxin having the amino acid SEQ ID NO: 4 is 1: 1.

[0118] According to some embodiments, the present invention provides a construct of the present invention comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein one of the peptides binds specifically to EGFR and one of the peptides binds specifically to PD-L1 and the toxin is selected from a toxin binding to a eukaryotic elongation factor 2, BIM- BH3 toxin having the amino acid sequence set forth in SEQ ID NO: 5, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake- venom toxin, cyanotoxin, and any combination thereof. According to some embodiments, the toxin is a toxin binding to eukaryotic elongation factor 2. According to some embodiments, the present invention provides a construct in which one of the peptides binds specifically to EGFR and one of the peptides binds specifically to PD-L1 and the toxin binds to a eukaryotic elongation factor 2 or the toxin of SEQ ID NO: 5. According to some embodiments, the peptides that binds specifically to EGFR is a peptide having SEQ ID NO:l , an analog or a fragment thereof, the peptide that binds specifically to PD-L1 is a peptide having SEQ ID NO: 2, an analog or a fragment thereof, and the toxin is selected from a toxin having SEQ ID NO: 3 or 4. According to some embodiments, the construct comprises multiple copies of (i) one peptide, (ii) two peptides, (iii) one toxin and/or (iv) two toxins. According to some embodiments, the construct comprises multiple copies of: (i) a peptide having SEQ ID NO:l , an analog or a fragment thereof, (ii) a peptide having SEQ ID NO: 2, an analog or a fragment thereof, and (iii) a toxin selected from a toxin having SEQ ID NO: 3 or 4, or combination thereof. According to other embodiments, the construct comprises multiple copies of (i) a peptide of SEQ ID NO:l , (ii) a peptide of SEQ ID NO: 2, (iii) the toxin of SEQ ID NO: 3 or 4, or a combination thereof. According to other embodiments, the construct comprises multiple copies of each one of: (i) a peptide of SEQ ID NO: 1, (ii) a peptide of SEQ ID NO: 2, (iii) the toxin of SEQ ID NO: 3, and (iv) the toxin of SEQ ID NO: 4. According to some embodiments, the construct of the present invention has a synergistic cytotoxicity. According to some such embodiments, the peptides, analogs thereof or the fragments thereof and/or the toxins, the analogs thereof or the fragments thereof are cyclic peptides, analogs or the fragments and/or cyclic toxins, analogs of fragments thereof, respectively.

[0119] According to any one of the above embodiment, the peptides of the present invention are covalently bound to each other. According to one embodiment, the peptides and the toxins are bound directly, i.e. without a carrier. According to other embodiments, the peptides of the present invention are covalently bound through a carrier. According to one embodiment, the carrier is an organic scaffold, thus the peptides are covalently bound through a scaffold.

[0120] According to some embodiments, the scaffold is a peptidic scaffold. According to other embodiments, the peptidic scaffold connects the peptides to each other on a single location in the scaffold, or to a different location on a scaffold. Each possibility represents a separate embodiment of the invention. According to some embodiments, the scaffold comprises at least one Lysine (Lys) residue. According to other embodiments, the scaffold comprises at least three Lys residues. According to further embodiments, the at least three Lys residues are connected together by amide bonds to form a branched multimeric scaffold. According to some embodiments, at least one amide bond is formed between the epsilon amine of a Lys residue and the carboxy group of another Lys residue.

[0121] According to a particular embodiment, the construct comprises a molecule according to one of the schemes presented below,

Image available on "Original document"


wherein X represents the peptide's and/or the toxin's C-terminal selected from carboxy acid, amide or alcohol group and optionally a linker or spacer, and peptide denotes a peptide according to the present invention, e.g. having 7-20 amino acids capable of binding to a cell-target. Each possibility represents a separate embodiment of the present invention.



[0122] According to some specific embodiments, at least one of the peptides and/or the toxin(s) is present in multiple copies. According to some embodiments, the multiple copies are linked thereby forming a multi-target peptide multimer. According to some embodiments, the peptide and/or the toxin(s) copies are linked through a linker. According to other embodiments, the peptides and/or the toxin(s) copies are linked directly. According to a further embodiments, the multimer comprises copies linked both directly and via a linker.

[0123] According to some embodiments, the construct comprises a peptide multimer comprising a plurality of cell-targeting peptides arranged in an alternating sequential polymeric structure B(XiX2X3...Xm)nB or in a block copolymer structure B(Xi)nz(X2)nz(X3)nZ...(Xm)n, wherein B is an optional sequence of 1 -10 amino acid residues; n is at each occurrence independently an integer of 2-50; m is an integer of 3- 50; each of Xi, X2...Xmis an identical or different peptide consisting of 5-30 amino acid residues; Z at each occurrence is a bond or a spacer of 1-4 amino acid residues. According to particular embodiments, n is at each occurrence independently an integer of 2-10; m is an integer of 3-10; each of Xi, X2.. .Xmis an identical or different peptide consisting of 7-20 amino acid residues; Z at each occurrence is a bond or a spacer of 1-4 amino acid residues. Each possibility represents a separate embodiment of the present invention.

[0124] According to some embodiments, the peptide multimer comprises 2-8 different or identical peptides. According to a particular embodiment, the peptide multimer comprises 4-10 copies of a single peptide sequence. According to yet other embodiments, the peptide multimer consists of 2-10, 3-9, 4-8, or 10-100 different or identical peptides. Each possibility represents a separate embodiment of the present invention.

[0125] According to other embodiments, the scaffold comprises or formed from a polyethylene glycol (PEG) molecule(s) or a modified PEG molecule(s). According to certain embodiments, the scaffold comprises a branched PEG molecule. According to some embodiments, the branched molecule comprises at least two sites available to bind a peptide of the present invention. According to other embodiments, the scaffold comprises from 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20, or 2 to 50 sites available to bind a peptide. According to one embodiment, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 sites available to bind a peptide. According to certain embodiment, the scaffold comprises 8 or 56 sites available to bind a peptide. According to further embodiments, the scaffold comprises 42 or 49 to 56 sites available for binding a peptide.

[0126] According to some embodiments, the PEG molecule is a branched molecule, comprising at least two separate connections to a peptide. According to some embodiments, the PEG has 8 binding sites. According to other embodiments, the PEG is bound to additional PEG molecules. According to certain embodiments, multiple PEG molecules are bound to provide a multi-armed PEG molecule. According to some embodiments, eight 8-armed PEG molecules are abound to one central 8-armed PEG molecule to provide one PEG molecules with 56 sites capable of binding the peptides of the toxins of the present invention. According certain embodiments, the peptides are connected to the PEG scaffold through amide bonds formed between amino groups of an NH2-PEG molecule. According to yet other embodiments, at least one peptide is connected to PEG scaffold though a Lys residue.

[0127] According to some embodiments, the peptides are bound to a PEG scaffold though a Lys residue. [0128] According to some embodiments, the present invention provides a construct in which at least one of the peptides bound to PEG scaffold binds specifically to an extracellular tumor antigen selected from EGFR, PD-Ll, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1, PD- L2, PDGFR, and VEGFR. According to certain embodiments, at least one of the peptides bound to PEG scaffold binds specifically to EGFR or PD-Ll . According to some embodiments, the peptide that binds specifically to EGFR and the peptide that binds specifically to PD-Ll are both bound to the scaffold. According to one embodiment, the peptide that binds to EGFR is a peptide having SEQ ID NO: 1 , analog or fragment thereof. According to another embodiment, the peptide that binds specifically to PD-Ll is a peptide having SEQ ID NO: 2, analog or fragment thereof. According to certain embodiments, the construct comprises the peptide having SEQ ID NO: 1 , analog or fragment thereof and a peptide having SEQ ID NO: 2, analog or fragment thereof both bound to the scaffold. According to certain embodiments, the construct comprises a peptide having SEQ ID NO: 1 and peptide having SEQ ID NO: 2 bound to the scaffold. According to certain embodiments, the construct comprises a peptide of SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 bound to the scaffold. According to some such embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2, analogs of fragments thereof are cyclic peptides, analogs or fragments.

[0129] According to some embodiments, the present invention provides a construct, wherein the scaffold is bound to multiple copies of at least one of the peptides. According to some embodiments, the scaffold is bound to multiple copies of each of the at least two of the peptides. According to certain embodiments, at least one of the peptides that is bound to PEG scaffold binds specifically to EGFR or PD-Ll . According to some embodiments, the scaffold is bound to multiple copies of a peptide that binds specifically to EGFR. According to other embodiments, the scaffold is bound to multiple copies of a peptide that binds specifically to PD-Ll . According to a further embodiment, the scaffold is bound to multiple copies of a peptide that binds specifically to EGFR and to multiple copies of a peptide that binds specifically to PD-Ll . According to one embodiment, the peptide that binds to EGFR is a peptide having SEQ ID NO: 1 , analog or fragment thereof. According to another embodiment, the peptide that binds specifically to PD-Ll is peptide having SEQ ID NO: 2, analog or fragment thereof. According to one embodiment, the scaffold is bound to multiple copies of the peptide having SEQ ID NO: 1 and to multiple copies of the peptide having SEQ ID NO: 2. According to some such embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2, analogs of fragments thereof are cyclic peptides, analogs or fragments.

[0130] According to some embodiments, the scaffold comprises a carbohydrate moiety.

[0131] According to other embodiments, the toxin is bound to a carrier. The carrier may be as described herein above. Thus, according to one embodiment, the carrier is a scaffold. According to certain embodiments, the carrier is a peptidic scaffold.

[0132] According to other embodiments, the scaffold is PEG scaffold, i.e. formed from PEG. According to certain embodiments, the scaffold comprises a branched PEG molecule. According to some embodiments, the branched molecule comprises at least one available site to bind a toxin.

[0133] According to other embodiments, the scaffold comprises from 2 to 100, 3 to 90, 4 to 60, 5 to 50, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20, or 2 to 50 sites available to bind a toxin. According to one embodiment, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 sites available to bind a toxin. According to certain embodiment, the scaffold comprises 8 or 56, or 42 or 49 to 56 sites available for bind a toxin.

[0134] According to some embodiments, the present invention provides as a construct, wherein the PEG scaffold is bound to multiple copies of at least one toxin. According to some embodiments, the present invention provides a construct, where the scaffold is bound to multiple copies of at least two toxins. According to some embodiments, the toxin is selected from the groups consisting of a toxin having SEQ ID NO: 3, a toxin having SEQ ID NO: 4, a toxin having SEQ ID NO: 5 (BIM-BH3 toxin), Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake-venom toxin, cyanotoxin, and any combination thereof. According to some embodiments, the toxin is a toxin of SEQ ID NO: 3 or 4.

[0135] According to one embodiment, the PEG scaffold is bound to multiple copies of a toxin having SEQ ID NO: 3, an analog or fragment thereof. According to one embodiment, the PEG scaffold is bound to multiple copies of a toxin having SEQ ID NO: 4, an analog or fragment thereof. According to one embodiment, the PEG scaffold is bound to multiple copies of a toxin having SEQ ID NO: 5. According to one embodiment, the PEG scaffold is bound to multiple copies of a toxin of SEQ ID NO: 3 or 4. [0136] According to one embodiment, the PEG scaffold is bound to multiple copies of each one of the toxins. According to one embodiment, the PEG scaffold is bound to multiple copies of a toxin having SEQ ID NO: 3 and to multiple copies of a toxin having SEQ ID NO: 4. According to one embodiment, the PEG scaffold is bound to multiple copies of the toxin of SEQ ID NO: 3 and to multiple copies of a toxin of SEQ ID NO: 4.

[0137] According to some such embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2, analogs of fragments thereof are cyclic peptides, analogs or fragments. Additionally, the peptides comprising or consisting of SEQ ID NO: 3 or 4, analogs or fragments thereof are cyclic, i.e. cyclic toxins.

[0138] According to some embodiments, the present invention provides a construct comprising a PEG scaffold bound to at least two different peptides binding to at least two different extracellular tumor antigens, and to at least one toxin, wherein at least one of peptides binds specifically to the extracellular tumor antigens is selected from EGFR, PD-L1 , HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1 , OGFR, PD-1, PD-L2, PDGFR, and VEGFR.

[0139] According to some embodiments, the present invention provides a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens and at least one toxin, wherein each of said peptides and toxin(s) is bound to a PEG scaffold and wherein at least one of peptides binds specifically to the extracellular tumor antigens selected from EGFR and PD-L1. According to one embodiment, one of the peptides binds specifically to EGFR and the another one of the at least two peptides binds specifically to an extracellular tumor antigen selected from the group consisting of PD-L1 , HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1, PD-L2, PDGFR, and VEGFR. According to another embodiment, one of the peptides binds specifically to PD-L1 and the another one of the at least two peptides binds specifically to an extracellular tumor antigen selected from the group consisting of EGFR, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1, PD- L2, PDGFR, and VEGFR. According to other embodiments, one of the peptides binds specifically to EGFR and the other one of the at least two peptides binds specifically to PD-L1. According to some embodiments, the peptides binds specifically to EGFR is a peptide having SEQ ID NO:l, an analog or fragment thereof. According to some embodiments, the peptides binds specifically to PD-L1 is a peptide having SEQ ID NO:2, an analog or fragment thereof. According to some such embodiments, the peptide is a cyclopeptide. According to some embodiments, the toxin is selected from the groups consisting of a toxin having SEQ ID NO: 3, a toxin having SEQ ID NO: 4, a toxin having SEQ ID NO: 5 (BIM-BH3 toxin), Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake-venom toxin, and cyanotoxin. According to some embodiments, the toxin is selected from the groups consisting of a toxin of SEQ ID NO: 3, a toxin of SEQ ID NO: 4, and a toxin of SEQ ID NO: 5. According to some embodiments, the scaffold is bound to 2, 3, or 4 different toxins. According to some embodiments, the PEG scaffold is bound to multiple copies of at least one of the peptides. According to other embodiments, the PEG scaffold is bound to multiple copies of each one of the at least two peptides. According to further embodiments, the PEG scaffold is bound to multiple copies of a toxin. According to certain embodiments, the PEG scaffold is bound to multiple copies of each one of two or more toxins. According to one embodiment, the scaffold is bound to multiple copies of a peptide having SEQ ID NO: l. According to another embodiment, the scaffold is bound to multiple copies of a peptide having SEQ ID NO:2. According to a further embodiment, the scaffold is bound to multiple copies of a peptide having SEQ ID NO: 1 and to multiple copies of a peptide having SEQ ID NO:2. According to one embodiment, the scaffold is bound to multiple copies of a toxin having SEQ ID NO: 3. According to another embodiment, the scaffold is bound to multiple copies of a toxin having SEQ ID NO: 4. According to a further embodiment, the PEG scaffold is bound to multiple copies of a toxin having SEQ ID NO: 3 and to multiple copies of a toxin having SEQ ID NO: 4. According to yet another embodiment, the PEG scaffold is bound to multiple copies of the toxin of SEQ ID NO: 3 and to multiple copies of a toxin of SEQ ID NO: 4. According to one embodiment, the molar ratio of the toxin having the amino acid SEQ ID NO: 3 or 5 to the toxin having the amino acid SEQ ID NO: 4 is about 0.1 :1 to about 10:1 or 1 :1. According to some embodiments, the construct of the present invention has a synergistic cytotoxicity.

[0140] According to some embodiments, the present invention provides a construct comprising multiple copies of a peptide having SEQ ID NO:l , multiple copies of a peptide having SEQ ID N0:2, multiple copies of a toxin having SEQ ID NO: 3 and multiple copies of a toxin having SEQ ID NO: 4, wherein each of the copies of the peptides and the toxins is bound to a PEG scaffold. According to some embodiments, the present invention provides a construct comprising a PEG scaffold bound to multiple copies of a peptide of SEQ ID NO:l , to multiple copies of a peptide of SEQ ID NO:2, multiple copies of a toxin of SEQ ID NO: 3 and to multiple copies of a toxin of SEQ ID NO: 4. According to one embodiment, the molar ratio of the toxin having the amino acid SEQ ID NO: 3 to the toxin having the amino acid SEQ ID NO: 4 is about 0.1 :1 to about 10:1 or 1 : 1. According to some embodiments, the stoichiometric molar ratio between the peptide having SEQ ID NO:l , the peptide of SEQ ID NO:2, the toxin having SEQ ID NO: 3 and the toxin having SEQ ID NO: 3 is 1 :1 :3:3. According to other embodiments, the stoichiometric molar ratio between the peptide having SEQ ID NO: l, the peptide of SEQ ID NO:2, the toxin having SEQ ID NO: 3 and the toxin having SEQ ID NO: 4 is selected from 1 :2:3:2, 1 :2:2:3, 2:1 :3:2, 2:1 :2:3 and 2:2:2:2. In the abovementioned embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2 are cyclopeptides and the toxins comprising or consisting of SEQ ID NO: 3 or 4 are cyclotoxins. According to some embodiments, the construct of the present invention has a synergistic cytotoxicity.

[0141] According to any one of the above embodiments, the peptides and/or the toxin(s) are bound directly or through a spacer. According to other embodiments, the peptides and/or the toxin(s) are bound to the carrier, e.g. to a scaffold, through a spacer. According to some specific embodiments, the spacer comprises at least one amino acid residue.

[0142] According to any one of the above embodiments, the construct further comprises a permeability-enhancing moiety. The permeability-enhancing moiety may be bound directly to a peptide and/or to a toxin, or may be bound to the scaffold, optionally via a spacer. The term "permeability-enhancing moiety" refers to any moiety known in the art to facilitate actively or passively or enhance permeability of the compound through body barriers or into the cells. Non-limitative examples of permeability-enhancing moiety include: hydrophobic moieties such as fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides, nanoparticles and liposomes. The term "permeability" refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, or a skin layer.

[0143] According to another aspect, the present invention provides a composition comprising a construct of the present invention. According to one embodiment, the composition is a pharmaceutical composition. Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising a construct of the present invention and a pharmaceutically acceptable excipient. All definitions, terms and embodiments of previous aspects are explicitly encompassed by this aspect.

[0144] The term "pharmaceutical composition" as used herein refers to a composition comprising the construct of the present invention as disclosed herein optionally formulated with one or more pharmaceutically acceptable excipients.

[0145] Formulation of the pharmaceutical composition may be adjusted according to applications. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration to mammals. For example, the formulation may be any one selected from among plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, ophthalmic ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluidextracts, emulsions, suspensions, decoctions, infusions, ophthalmic solutions, tablets, suppositories, injections, spirits, capsules, creams, troches, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0146] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. Non-limiting examples of suitable excipients are example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

[0147] The constructs of the present invention could be, according to some embodiments, suspended in a sterile saline solution for therapeutic uses. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebaric acid. The rate of release of the molecule according to the present invention from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.

[0148] The pharmaceutical composition of the present invention may be administered by any know method. The terms "administering" or "administration of a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitonealy, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some embodiments, the administration includes both direct administration, including self- administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self- administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

[0149] According to some embodiments, the pharmaceutical composition is administered by an invasive mode of administration such as intramuscularly, intravenously, intra-arterially, intraarticulary or parenterally.

[0150] It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician. As used herein, a "therapeutically effective amount" refers to the amount of a molecule required to alleviate one or more symptoms associated with a disorder being treated over a period of time.

[0151] Although an appropriate dosage of a molecule of the invention varies depending on the administration route, type of molecule (polypeptide, polynucleotide, organic molecule etc.) age, body weight, sex, or conditions of the patient, it will be determined by the physician in the end. Various considerations in arriving at an effective amount are described, e.g., in Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

[0152] In one particular embodiment, the pharmaceutical composition of the present invention comprises a construct comprising at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein the peptides and the toxin are covalently bound directly or through a carrier. According to some embodiments, at least one of the peptides binds specifically to an extracellular tumor antigens selected from EGFR and PD-L1. According to another embodiment, the other one of the at least two peptides binds specifically to an extracellular tumor antigen selected from the group consisting of EGFR, PD-L1 , HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1, PD- L2, PDGFR, and VEGFR. According to certain embodiments, the construct comprises from 2 to 10 different peptides. According to some embodiments, at least one of the peptides binds specifically to EGFR, and at least one of the peptides binds specifically to PD-L1. According to one embodiment, the peptide that binds to EGFR is a peptide having SEQ ID NO: 1 , analog or fragment thereof. According to another embodiment, the peptide that binds specifically to PD-L1 is a peptide having SEQ ID NO: 2, analog or fragment thereof. According to a further embodiment, the construct comprises a peptide having or consisting of SEQ ID NO: 1 and a peptide having or consisting of SEQ ID NO: 2. According to some embodiments, the pharmaceutical composition comprises a construct comprising multiple copies of one or of two of said peptides. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42, from 28 to 35, or from 7 to 21 copies of the each one of the peptide having the SEQ ID NO: 1 and 2. According to some embodiments, the toxin is selected from the group consisting of a toxin binding to a eukaryotic elongation factor 2 or analog of that toxins, BIM-BH3 toxin, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake-venom toxin, and cyanotoxin. According to some embodiments, the BIM-BH3 toxin consists of SEQ ID NO: 5. According to certain embodiments, the toxin binding to eukaryotic elongation factor 2 is a toxin having the amino acid sequence selected from SEQ ID NO: 3 or 4, or an analog thereof. According to some embodiments, the construct comprises 2 to 10 different toxins. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, or from 7 to 21 copies of one or of two toxins. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of the each one of the toxins having the SEQ ID NO: 3 or 4. According to some embodiments, the construct comprises from 7 to 56, from 14 to 48, from 21 to 42 from 28 to 35, from 7 to 21 copies of the each one of the toxins having the SEQ ID NO: 3 and 4. According to one embodiment, the molar ratio of the toxin having the amino acid SEQ ID NO: 3 to the toxin having the amino acid SEQ ID NO: 4 is about 0.1 :1 to about 10:1 or about 1 :1. According to some embodiments, the peptide(s) is a cyclic peptide(s) and the toxin(s) is a cyclic toxin(s).

[0153] According to some embodiments, the pharmaceutical composition of the present invention comprises a construct comprising a PEG scaffold, at least two different peptides binding to at least two different extracellular tumor antigens, and at least one toxin, wherein at least one of peptides binds specifically to the extracellular tumor antigens selected from EGFR and PD-L1, and each one of the peptides and the toxins are bound to the scaffold. According to other embodiments, one of the peptides binds specifically to EGFR and the other one to the at least two peptides binds specifically to PD-L1. According to some embodiments, the peptides binds specifically to EGFR is a peptide having SEQ ID NO:l, an analog or fragment thereof. According to some embodiments, the peptides binds specifically to PD-L1 is a peptide having SEQ ID NO:2, an analog or fragment thereof. According to some embodiments, the present invention provides a construct comprising a PEG scaffold, multiple copies of a peptide having SEQ ID NO:l, multiple copies of a peptide having SEQ ID NO:2, and multiple copies of a toxin having SEQ ID NO: 3, wherein each copy of each one of the peptides and each copy of the toxin are bound to the scaffold. According to one embodiment, the construct comprising a PEG scaffold, multiple copies of a peptide having SEQ ID NO: l, multiple copies of a peptide having SEQ ID NO:2, and multiple copies of a toxin having SEQ ID NO: 4, wherein each copy of each one of the peptides and each copy of the toxin are bound to the scaffold.

[0154] According to some embodiments, the present invention provides a construct comprising a PEG scaffold bound to multiple copies of a peptide having SEQ ID NO:l , to multiple copies of a peptide having SEQ ID NO:2, multiple copies of a toxin having SEQ ID NO: 3 and to multiple copies of a toxin having SEQ ID NO: 4. According to some embodiments, the present invention provides a construct comprising a PEG scaffold bound to multiple copies of a peptide of SEQ ID NO:l, to multiple copies of a peptide of SEQ ID NO:2, multiple copies of a toxin of SEQ ID NO: 3 and to multiple copies of a toxin of SEQ ID NO: 4. According to one embodiment, the molar ratio of the toxin having the amino acid SEQ ID NO: 3 to the toxin having the amino acid SEQ ID NO: 4 is about 0.1 : 1 to about 10: 1 or 1 :1. According to some embodiments, the stoichiometric molar ratio between the peptide having SEQ ID NO:l , the peptide of SEQ ID NO:2, the toxin having SEQ ID NO: 3 and the toxin having SEQ ID NO: 3 is 1 :1 :3:3. According to other embodiments, the stoichiometric molar ratio between the peptide having SEQ ID NO:l , the peptide of SEQ ID NO: 2, the toxin having SEQ ID NO: 3 and the toxin having SEQ ID NO: 4 is selected from 1 :2:3:2, 1 :2:2:3, 2:1 :3 :2, 2:1 :2:3 and 2:2:2:2. In the abovementioned embodiments, the peptides comprising or consisting of SEQ ID NO: 1 or 2 are cyclopeptides and the toxins comprising or consisting of SEQ ID NO: 3 or 4 are cyclotoxins. According to some embodiments, the construct of the present invention has a synergistic cytotoxicity, therefore such pharmaceutical composition, when administered, provides a profound therapeutic effect.

[0155] According to any one of the above embodiments, the pharmaceutical composition comprises a plurality of the constructs according to the present invention and according to the above embodiments.

[0156] According to another embodiment, the present invention provides a pharmaceutical composition according to the present invention, for use in treating a cell proliferative disease or disorder. According to some embodiments, the cell proliferative disease or disorder is cancer. Thus, according to one embodiment, the pharmaceutical composition of the present invention is for use in treating cancer.

[0157] The terms "treating" of "treatment of a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, or ameliorating abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and/or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[0158] According to some embodiments, treating cancer comprises preventing or treatment tumor metastasis. According to certain embodiments, the metastasis is decreased. According to other embodiments, the metastasis is prevented.

[0159] According to some embodiments, treating cancer comprises increasing the duration of survival of a subject having cancer, comprising administering to the subject in need thereof a composition comprising a construct defined above whereby the administration of the construct increases the duration of survival.

[0160] According to some embodiments, treating cancer comprises increasing the progression of free survival of a subject having cancer.

[0161] According to some embodiments, treating cancer comprises increasing the duration of response of a subject having cancer. According to other embodiments, treating cancer comprises preventing tumor recurrence.

[0162] The cancer amendable for treatment according to the present invention includes, but not limited to: carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non- Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post- transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0163] According to some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The cancerous conditions amendable for treatment of the invention include metastatic cancers.

[0164] According to other embodiments, the cancer is a solid cancer.

[0165] The pharmaceutical composition according to the present invention may be administered as a stand-alone treatment or in combination with a treatment with any other agent. According to a specific embodiment, constructs according to the present invention are administered to a subject in need thereof as part of a treatment regimen in combination with at least one anti-cancerous agent. The pharmaceutical composition according to the present invention may be administered in combination with the anti- cancerous agent or separately.

[0166] The pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition.

[0167] According to a specific embodiment, the anti-neoplastic composition comprises at least one chemotherapeutic agent.

[0168] The term "anti-neoplastic composition" refers to a composition useful in treating cancer comprising at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function or metastasis, and/or causing destruction of tumor cells. Therapeutic agents suitable in an anti-neoplastic composition for treating cancer include, but not limited to, chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons, and antagonistic agents targeting cytokines, cytokine receptors or antigens associated with tumor cells. [0169] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN<R>™ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (e.g., Agnew, Chem Intl. Ed. Engl. 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN<R>™ doxorubicin (including morpholino -doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fiuorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK<R>™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL<R>™ paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin- engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE<R>™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR<R>™ gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE<R>™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difiuorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0170] Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX<R>™ tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE<R>™ megestrol acetate, AROMASIN<R>™ exemestane, formestanie, fadrozole, RIVISOR<R>™ vorozole, FEMARA<R>™ letrozole, and ARIMIDEX<R>™ anastrozole; and anti-andro gens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME<R>™ ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy DNA-based vaccines, for example, ALLOVECTIN<R>™ vaccine, LEUVECTIN<R>™ vaccine, and VAXID<R>™ vaccine; PROLEUKIN<R>™ rIL-2; LURTOTECAN<R>™ topoisomerase 1 inhibitor; ABARELIX<R>™ rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0171] According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to said subject a pharmaceutical composition of the present invention. According to one embodiment, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a construct of the present invention. According to some embodiments, the pharmaceutical composition is administered as part of a treatment regimen together with at least one anti-cancer agent. The term "therapeutically effective amount" is an amount of a drug, compound, construct etc. that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.

[0172] According to another aspect, the present invention provides a peptide that binds specifically to human eukaryotic Elongation Factor 2 (eEF2), an analog or fragment thereof. According to one embodiment, the present invention provides a peptide that binds specifically to human eEF2. According to some embodiments, the peptide is a toxin. According some embodiments, the peptide consists of 5 to 30 amino acids. According to other embodiments, each peptide consists of 6 to 25 amino acids. According to yet other embodiments, each peptide consists of 7 to 20 amino acids. According to some embodiments, each peptide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Each possibility represents a separate embodiment of the invention.

[0173] According to some embodiments, the peptide that binds to human eEF2 is a peptide having SEQ ID NO: 3. According to certain embodiments the present invention provides an analog of SEQ ID NO:3. According to a further embodiment, the present invention provides a the fragment of the peptide or of the analog. According to one embodiment, the peptide is a peptide having SEQ ID NO: 3. According to another embodiment, the peptide is a peptide of SEQ ID NO: 3. According to some embodiments, the peptide is a cyclic peptide.

[0174] According to some embodiments, the analog has a sequence identity of at least 70%, at least 80%, or at least 90% to SEQ ID NO: 3. According to some embodiments, the analog has at least 70%, at least 75%, at least 80%, at least 85, at least 90% or at least 95% sequence identity to SEQ ID NO: 3. According to other embodiments, the analog has about 70% to 95%, 75% to 90%, or 80% to 85% sequence identity to SEQ ID NO: 3. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 3. According to some embodiments, the conservative analog of SEQ ID NO: 3 has 1 , 2, 3, 4 or 5 conservative substitutions in SEQ ID NO: 3. According to some embodiments, the analog is a cyclopeptide.

[0175] According to one embodiment, the fragment consists of 6 to 11 , 7 to 10 or 8 to 9 consecutive amino acids of SEQ ID NO: 3 or analog thereof.

[0176] According to some embodiments, the peptide comprising or consisting of SEQ ID NO: 3 enhances the activity of human eEF2. According to one embodiment, the peptide is an agonist of eEF2. According to another embodiment, the analog of SEQ ID NO: 3 or the fragment of the peptide or the analog enhances the activity of eEF2.

[0177] According to one embodiment, the peptide comprising or consisting of SEQ ID NO: 3, analog thereof or the fragment of the peptide or said analog is a toxin. In one embodiment, the peptide is for use in inducing cell death in target cells. According to some embodiments, the cells are cancer cells. According to one embodiment, the peptide comprising SEQ ID NO:3 is for use in inducing cell death in target cells. According to another embodiment, the peptide consisting of SEQ ID NO:3 is for use in inducing cell death in target cells. According to a further embodiment, the analog of a peptide comprising or consisting of SEQ ID NO: 3 is for use in inducing cell death in target cells.

[0178] The terms "induce cell death" and "promote cell death" are used herein interchangeably and mean that the of the present invention (i.e. the peptide, the analog or the fragment) can directly inducing cell death to cells, where cell death includes apoptosis and necrosis. The cell death may be caused due to interaction of the compound of the present invention with molecules molecule expressed on the cell surface or with molecules located within the cell such as molecule located in the cytosol, bound to the inner side of the cell membrane, located in the organelles or present on the membrane of the organelles, either inner or outer part of it. [0179] The term "cell death" as used herein encompasses both destruction and damage or impairment of cells. The term "cell death" encompasses cell ablation.

[0180] According to some embodiments, the peptide that binds to human eEF2 is a peptide having SEQ ID NO: 4. According to certain embodiments, the present invention provides an analog of SEQ ID NO: 4. According to a further embodiment, the present invention provides a fragment of the peptide or of the analog. According to one embodiment, the peptide is a peptide having SEQ ID NO: 4. According to another embodiment, the peptide is a peptide of SEQ ID NO: 4. According to some embodiments, the peptide is a cyclic peptide.

[0181] According to some embodiments, the analog has a sequence identity of at least 70%, at least 80%, or at least 90% to SEQ ID NO: 4. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 4. According to other embodiments, the analog has 70% to 95%, 75% to 90%, or 80% to 85% identity to SEQ ID NO: 4. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 4. According to some embodiments, the conservative analog of SEQ ID NO: 4 has 1, 2, 3, 4 or 5 conservative substitutions in SEQ ID NO: 4. According to some embodiments, the analog is a cyclic peptide.

[0182] According to one embodiment, the fragment consists of 6 to 11 , 7 to 10 or 8 to 9 consecutive amino acids of SEQ ID NO: 4 or analog thereof.

[0183] According to some embodiments, the peptide comprising or consisting of SEQ ID NO: 4 enhances the activity of human eEF2. According to one embodiment, the peptide is an agonist of eEF2. According to another embodiment, the analog of SEQ ID NO: 4 or the fragment of the peptide or the analog enhances the activity of eEF2.

[0184] According to one embodiment, the peptide comprising or consisting of SEQ ID NO: 4, analog thereof or the fragment of the peptide or said analog is a toxin. In one embodiment, the peptide is for use in inducing cell death in target cells. According to some embodiments, the cells are cancer cells. According to one embodiment, the peptide comprising SEQ ID NO: 4 is for use in inducing cell death in target cells. According to another embodiment, the peptide consisting of SEQ ID NO: 4 is for use in inducing cell death in target cells. According to a further embodiment, the analog of a peptide comprising or consisting of SEQ ID NO: 4 is for use in inducing cell death in target cells.

[0185] According to another aspect, the present invention provides a conjugate of the peptide that binds specifically to human eEF2. [0186] According to one embodiment, the present invention provides a conjugate of the peptide selected from a peptide having or consisting of SEQ ID NO: 3, analog thereof or fragment thereof. According to one embodiment, the present invention provides a conjugate of the cyclopepide having or consisting of SEQ ID NO: 3.

[0187] According to one embodiment, the present invention provides a conjugate of the peptide selected from a peptide having or consisting of SEQ ID NO: 4, analog thereof or fragment thereof. According to one embodiment, the present invention provides a conjugate of the cyclopepide having or consisting of SEQ ID NO: 4.

[0188] The term "conjugate" refers to any substance formed from the joining together or binding of two or more molecules. In particular, the term conjugate encompasses a compound formed from binding of two or more peptides of any one of the above embodiments or a compound comprising said peptide bound to another molecule. According to some embodiments, the peptide, analog or fragment of the present invention is conjugated with a carrier protein or moiety which improves the peptide's antigenicity, solubility, stability or permeability. A fusion protein comprising at least one peptide according to the invention is also within this scope.

[0189] Thus, according to some embodiments, the conjugate comprises at least two copies of the peptides comprising or consisting of SEQ ID NO: 3, analog or fragment thereof covalently bound.

[0190] According to another embodiment, the conjugate comprises at least one peptide comprising or consisting of SEQ ID NO: 3, analog or fragment thereof and another molecule. According to some embodiments, said molecule can be any molecule. According to one embodiment, the molecule is selected from an active agent, an extracellular tumor antigen targeting molecule, a carrier, a toxin, a permeability- enhancing moiety and an anti-cancer agent.

[0191] According to some embodiments, the conjugate comprises at least two copies of the peptide comprising or consisting of SEQ ID NO: 4, analog or fragment thereof covalently bound.

[0192] According to another embodiment, the conjugate comprises at least one peptide comprising or consisting of SEQ ID NO: 4, analog or fragment thereof and another molecule. According to some embodiments, said molecule can be any molecule. According to one embodiment, the molecule is selected from an active agent, an extracellular tumor antigen targeting molecule, a carrier, a toxin, a permeability- enhancing moiety and an anti-cancer agent. [0193] The extracellular tumor antigen targeting molecule, a carrier, a toxin, an anticancer agent are as defined according to the present invention. The term "active agent" refers to an agent that has biological activity, pharmacologic effects and/or therapeutic utility.

[0194] According to one embodiment, the extracellular tumor antigen is selected from EGFR, PD-L1, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1 , PD-L2, PDGFR, and VEGFR.

[0195] According to another embodiment, the toxin is selected from the group consisting of a toxin binding to a eukaryotic elongation factor 2, BIM-BH3 toxin having the amino acid sequence set forth in SEQ ID NO: 5, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin,

ProTx-I ProTx-II, Conus californicus toxin, snake-venom toxin, and cyanotoxin.

[0196] According to yet another embodiment, the carrier may be a scaffold carrier such as PEG carrier or peptidic carrier.

[0197] According to some embodiments, the conjugate of the present invention is for use in inducing cell death in target cells.

[0198] According to another aspect, the present invention provides a peptide comprising the amino acids sequence set forth in SEQ ID NO: 1. According to one embodiment, the present invention provides an analog of the peptide having SEQ ID NO: 1. According to a further embodiment, the present invention provides a fragment of said peptide or said analog. According to one embodiment, the peptide consists of SEQ ID NO: 1.

[0199] According some embodiments, the peptide consists of 5 to 30 amino acids. According to other embodiments, each peptide consists of 6 to 25 amino acids. According to yet other embodiments, each peptide consists of 7 to 20 amino acids. According to some embodiments, each peptide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Each possibility represents a separate embodiment of the invention.

[0200] According to some embodiments, the peptide having or consisting of SEQ ID NO: l, the analog or the fragment thereof binds specifically to a human Epidermal Growth Factor Receptor (EGFR). According to one embodiment, the peptide, analog of the fragment is an antagonist of EGFR. According to some embodiments, the peptide is a cyclopeptide. [0201] According to some embodiments, the analog of SEQ ID NO: 1 has a sequence identity of at least 70%, at least 80%, or at least 90% to SEQ ID NO: 1. According to other embodiments, the analog has 70% to 95%, 75% to 90%, or 80% to 85% sequence identity to SEQ ID NO: 1. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 1. According to some embodiments, the conservative analog of SEQ ID NO: 1 has 1 , 2, 3, 4 or 5 conservative substitutions. According to some embodiments, the analog is a cyclopeptide.

[0202] According to one embodiment, the fragment consists of 6 to 11 , 7 to 10 or 8 to 9 consecutive amino acids of SEQ ID NO: 1 or of analog thereof.

[0203] According to some embodiments, the peptide comprising or consisting of SEQ ID NO:l , analog or fragment thereof is a cancer cells targeting peptide. Thus, in one embodiment, the peptide comprising or consisting of SEQ ID NO: 1 , analog or fragment thereof is for use in cancer cell targeting.

[0204] According to one embodiment, the present invention provides a conjugate of the peptide selected from a peptide having or consisting of SEQ ID NO: 1 , analog thereof or fragment thereof. According to one embodiment, the present invention provides a conjugate of the cyclopepide having or consisting of SEQ ID NO: 1.

[0205] According to some embodiments, the conjugate comprises at least two copies of the peptide comprising or consisting of SEQ ID NO: 1, analog or fragment thereof covalently bound. According to another embodiment, the conjugate comprises at least one peptide comprising or consisting of SEQ ID NO: 1 , analog or fragment thereof and another molecule. According to some embodiments, said molecule can be any molecule. According to one embodiment, the molecule is selected from an active agent, an extracellular tumor antigen targeting molecule, a carrier, a permeability-enhancing moiety, a toxin, an anti-cancer agent and a combination thereof.

[0206] The terms extracellular tumor antigen targeting molecule, a carrier, a toxin, an anti-cancer agent are as defined in the present invention.

[0207] According to one embodiment, the extracellular tumor antigen is selected from EGFR, PD-L1, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1, OGFR, PD-1 , PD-L2, PDGFR, and VEGFR.

[0208] According to another embodiment, the toxin is selected from the group consisting of a toxin binding to a eukaryotic elongation factor 2, BIM-BH3 toxin of SEQ ID NO: 5, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake- venom toxin, and cyanotoxin.

[0209] According to yet another embodiment, the carrier may be a scaffold carrier such as PEG carrier or peptidic carrier.

[0210] According to another aspect, the present invention provides a peptide comprising the amino acids sequence set forth in SEQ ID NO: 2. According to one embodiment, the present invention provides an analog of the peptide having SEQ ID NO:2. According to a further embodiment, the present invention provides a fragment of said peptide or said analog. According to one embodiment, the peptide consists of SEQ ID NO: 2.

[0211] According some embodiments, the peptide consists of 5 to 30 amino acids. According to other embodiments, each peptide consists of 6 to 25 amino acids. According to yet other embodiments, each peptide consists of 7 to 20 amino acids. According to some embodiments, each peptide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Each possibility represents a separate embodiment of the invention.

[0212] According to some embodiments, the peptide having or consisting of SEQ ID NO:2, the analog or the fragment thereof binds specifically to a human Programmed death-ligand 1 (PD-L1).

[0213] According to any one of the above embodiments, the peptide, analog of the fragment is an antagonist of PD-L1.

[0214] According to some embodiments, the peptide is a cyclopeptide.

[0215] According to some embodiments, the analog of SEQ ID NO: 2 has a sequence identity of at least 70%, at least 80%, or at least 90% to SEQ ID NO: 2. According to other embodiments, the analog has 70% to 95%, 75% to 90%, or 80% to 85% identity to SEQ ID NO: 2. According to some embodiments, the analog is a conservative analog of SEQ ID NO: 2. According to some embodiments, the conservative analog of SEQ ID NO: 2 has 1 , 2, 3, 4 or 5 conservative substitutions. According to some embodiments, the analog is a cyclopeptide.

[0216] According to one embodiment, the fragment consists of 6 to 11 , 7 to 10 or 8 to 9 consecutive amino acids of SEQ ID NO: 2 or of an analog thereof.

[0217] According to some embodiments, the peptide comprising or consisting of SEQ ID NO:2, analog or fragment thereof is a cancer cells targeting peptide. Thus, in one embodiment, the peptide comprising or consisting of SEQ ID NO:2, analog or fragment thereof is for use in cancer cell targeting.

[0218] According to one embodiment, the present invention provides a conjugate of the peptide selected from a peptide having or consisting of SEQ ID NO: 2, analog thereof or fragment thereof. According to one embodiment, the present invention provides a conjugate of the cyclopepide having or consisting of SEQ ID NO: 2.

[0219] Thus, according to some embodiments, the conjugate comprises at least two copies of the peptide comprising or consisting of SEQ ID NO: 2, analog or fragment thereof covalently bound. According to another embodiment, the conjugate comprises at least one peptide comprising or consisting of SEQ ID NO: 2, analog or fragment thereof and another molecule. According to some embodiments, said molecule can be any molecule. According to one embodiment, the molecule is selected from an active agent, an extracellular tumor antigen targeting molecule, a carrier, a toxin, an anti-cancer agent, a permeability-enhancing moiety and a combination thereof.

[0220] The extracellular tumor antigen targeting molecule, a carrier, a toxin, an anticancer agent are as defined in the present invention.

[0221] According to one embodiment, the extracellular tumor antigen is selected from EGFR, PD-L1, HER2, androgen receptor, benzodiazepine receptor, Cadherin, CXCR4, CTLA- 4, CD2, CD19, endothelin receptor, ERBB4, FGFR, folate receptor, HER4, HGFR, Mucin 1 , OGFR, PD-1 , PD-L2, PDGFR, and VEGFR.

[0222] According to another embodiment, the toxin is selected from the group consisting of a toxin binding to a eukaryotic elongation factor 2, BIM-BH3 toxin having the amino acid sequence set forth in SEQ ID NO: 5, Diphtheria toxin, Pseudomonas exotoxin, Anthrax toxin, botulinum toxin, Ricin, PAP, Saporin, Gelonin, Momordin, ProTx-I ProTx-II, Conus californicus toxin, snake-venom toxin, cyanotoxin, and any combination thereof.

[0223] According to yet another embodiment, the carrier may be a scaffold carrier such as PEG carrier of peptidic carrier.

[0224] According to another aspect, the present invention provides a composition comprising the peptide of the present invention, or the conjugate of the present invention. According to one embodiment, the composition is a pharmaceutical composition. Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising the peptide of the present invention, or the conjugate of the present invention. [0225] According to one embodiment, the pharmaceutical composition comprises a peptide comprising or consisting of SEQ ID NO: 1 according to any one of the above embodiments. According to another embodiment, the pharmaceutical composition comprises the analog of SEQ ID NO: 1 or a fragment of said peptide or said analog. According to some embodiments, the pharmaceutical composition comprises a plurality of said peptides, analogs or fragments. According to yet another embodiment, the pharmaceutical composition comprises one or more conjugates of the peptide comprising or consisting of SEQ ID NO:l, analog or fragment thereof according to any one of the above embodiments.

[0226] According to some embodiments, the pharmaceutical composition comprises a peptide comprising or consisting of SEQ ID NO: 2 according to any one of the above embodiments. According to another embodiment, the pharmaceutical composition comprises the analog of SEQ ID NO: 2 or a fragment of said peptide or said analog. According to some embodiments, the pharmaceutical composition comprises a plurality of said peptides, analogs or fragments. According to yet another embodiment, the pharmaceutical composition comprises one or more conjugates of the peptide comprising or consisting of SEQ ID NO:2, analog or fragment thereof according to any one of the above embodiments.

[0227] According to certain embodiments, the pharmaceutical composition comprises a peptide comprising or consisting of SEQ ID NO: 3 according to any one of the above embodiments. According to another embodiment, the pharmaceutical composition comprises the analog of SEQ ID NO: 3 or a fragment of said peptide or said analog.

According to some embodiments, the pharmaceutical composition comprises a plurality of said peptides, analogs or fragments. According to yet another embodiment, the pharmaceutical composition comprises one or more conjugates of the peptide comprising or consisting of SEQ ID NO:3, analog or fragment thereof according to any one of the above embodiments.

[0228] According to another embodiment, the pharmaceutical composition comprises a peptide comprising or consisting of SEQ ID NO: 4 according to any one of the above embodiments. According to another embodiment, the pharmaceutical composition comprises the analog of SEQ ID NO: 4 or a fragment of said peptide or said analog. According to some embodiments, the pharmaceutical composition comprises a plurality of said peptides, analogs or fragments. According to yet another embodiment, the pharmaceutical composition comprises one or more conjugates of the peptide comprising or consisting of SEQ ID NO:4, analog or fragment thereof according to any one of the above embodiments.

[0229] All definitions and embodiments of other aspects of the present invention related to said peptides and conjugates are encompassed by this aspect as well.

[0230] According to some embodiments, the pharmaceutical composition is for treating a cell proliferative disease or disorder. According to some embodiments, cell proliferative disease or disorder is cancer. According to one embodiment, the pharmaceutical composition comprises a peptide selected from a peptide comprising or consisting of amino acid sequence selected from SEQ ID NO: 1 , 2, 3 and 4, analog thereof or fragment thereof, as defined in any one of the embodiments of the present invention. Thus, in certain embodiment, the present invention provides a pharmaceutical composition comprising a peptide comprising or consisting of SEQ ID NO: 1 for use in treating cancer. According to a further embodiment, the present invention provides a pharmaceutical composition comprising a peptide comprising or consisting of SEQ ID NO: 2 for use in treating cancer. According to yet another embodiment, the present invention provides a pharmaceutical composition comprising a peptide comprising or consisting of SEQ ID NO: 3 for use in treating cancer. According to certain embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising or consisting of SEQ ID NO: 4 for use in treating cancer. According to another embodiment, the pharmaceutical composition comprises one or more conjugates of said peptides as defined in any one of the embodiments of the present invention.

[0231] According to another aspect, the present invention provides a method of treating a proliferative disease or disorder in a subject in need thereof comprising administering a therapeutically effective amount of the peptides or conjugates of the present invention. According to one embodiment, the method comprises administering a pharmaceutical composition comprising the peptides or conjugates of the present invention. According to one embodiments, the peptide is selected from the group consisting of a peptide comprising or consisting of SEQ ID NO: 1 , a peptide comprising or consisting of SEQ ID NO: 2, a peptide comprising or consisting of SEQ ID NO: 3, a peptide comprising or consisting of SEQ ID NO: 4, analogs thereof, and fragments of said peptides. According to one embodiment, the conjugate is a conjugated of said peptides. According to one embodiment, the peptide, analog or fragment is cyclic. [0232] According to another aspect, the present invention provides an isolated polynucleotide comprising a sequence encoding the peptide selected from a peptide comprising or consisting of amino acid sequence selected from SEQ ID NO: 1 , 2, 3 and 4. According to some embodiment, the polynucleotide comprises a sequence encoding an analog of a peptide selected from a peptide comprising or consisting of amino acid sequence selected from SEQ ID NO: 1, 2, 3 and 4, or fragment thereof, as defined in any one of the embodiments of the present invention.

[0233] According to some embodiments, the polynucleotide comprises a sequence encoding the peptide comprising or consisting of SEQ ID NO: 1 , analog thereof or fragment thereof. According to one embodiment, the polynucleotide comprises the sequence encoding the peptide having SEQ ID NO: 1. According to another embodiment, the polynucleotide comprises the sequence encoding the peptide of SEQ ID NO: 1.

[0234] According to certain embodiments, the polynucleotide comprises a sequence encoding the peptide comprising or consisting of SEQ ID NO: 2, analog thereof or fragment thereof. According to one embodiment, the polynucleotide comprises the sequence encoding the peptide having SEQ ID NO: 2. According to another embodiment, the polynucleotide comprises the sequence encoding the peptide of SEQ ID NO: 2.

[0235] According to another embodiment, the polynucleotide comprises a sequence encoding the peptide comprising or consisting of SEQ ID NO: 3, analog thereof or fragment thereof. According to one embodiment, the polynucleotide comprises the sequence encoding the peptide having SEQ ID NO: 3. According to another embodiment, the polynucleotide comprises the sequence encoding the peptide of SEQ ID NO: 3.

[0236] According to yet another embodiment, the polynucleotide comprises a sequence encoding the peptide comprising or consisting of SEQ ID NO: 4, analog thereof or fragment thereof. According to one embodiment, the polynucleotide comprises the sequence encoding the peptide having SEQ ID NO: 4. According to another embodiment, the polynucleotide comprises the sequence encoding the peptide of SEQ ID NO: 4

[0237] According to one embodiment, the present invention provides a polynucleotide comprising a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1 , (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 3. According to another embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 4. According to a further embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2, (iii) SEQ ID NO: 3 and SEQ ID NO: 4.

[0238] According to another aspect, the present invention provides a nucleic acid construct, comprising the polynucleotide according to any one of the above embodiments. According to one embodiment, the polynucleotide is operably linked to a promoter. According to one embodiment, the nucleic acid construct comprises a polynucleotide comprising a sequence encoding the peptide selected from a peptide comprising or consisting of amino acid sequence selected from SEQ ID NO: 1 , 2, 3 and 4, analog thereof or fragment thereof, as defined in any one of the embodiments of the present invention. According to another embodiment, the nucleic acid construct comprises a polynucleotide comprising a sequence encoding the comprising a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 3. According to another embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1 , (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 4. According to a further embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1 , (ii) SEQ ID NO: 2, (iii) SEQ ID NO: 3 and SEQ ID NO: 4.

[0239] The term "nucleic acid construct", as used herein, refers to an artificially constructed segment of nucleic acid. It can be an isolated or integrated in another nucleic acid molecule.

[0240] As used herein, the term "operably linked", "operably encodes", and "operably associated" are used herein interchangeably and refer to the functional linkage between a promoter and nucleic acid sequence, wherein the promoter initiates transcription of RNA corresponding to the DNA sequence.

[0241] The term "promoter" is a regulatory sequence that initiates transcription of a downstream nucleic acid. The term "promoter" refers to a DNA sequence within a larger DNA sequence defining a site to which RNA polymerase may bind and initiate transcription. A promoter may include optional distal enhancer or repressor elements. The promoter may be either homologous, i.e., occurring naturally to direct the expression of the desired nucleic acid, or heterologous, i.e., occurring naturally to direct the expression of a nucleic acid derived from a gene other than the desired nucleic acid. A promoter may be constitutive or inducible. A constitutive promoter is a promoter that is active under most environmental and developmental conditions. An inducible promoter is a promoter that is active under environmental or developmental regulation, e.g., upregulation in response to xylose availability.

[0242] According to another aspect, the present invention provides a vector comprising the polynucleotide sequence or the nucleic acid construct of the present invention. Thus, in one embodiment, the present invention provides a vector comprising the polynucleotide comprising a sequence encoding the peptide selected from a peptide comprising or consisting of amino acid sequence selected from SEQ ID NO: 1 , 2, 3 and 4, analog thereof or fragment thereof, as defined in any one of the embodiments of the present invention. According to another embodiment, the vector comprises the polynucleotide comprising a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1 , (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 3. According to another embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 4. According to a further embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2, (iii) SEQ ID NO: 3 and SEQ ID NO: 4.

[0243] The terms "vector" and "expression vector" are used herein interchangeably and refer to any non-viral vector such as plasmid, cosmid, artificial chromosome (bacterial or yeast), or viral vector such as virus, retrovirus, bacteriophage, or phage, binary vector in double or single stranded linear or circular form, or nucleic acid, sequence which is able to transform host cells and optionally capable of replicating in a host cell. The vector may contain an optional marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. According to one embodiment, the vector is a plasmid. According to another embodiment, the vector is a phage or bacteriophage.

[0244] The term "plasmid" refers to circular, optionally double-stranded DNA capable of inserting a foreign DNA fragment to a cell and optionally capable of autonomous replication in a given cell. Plasmids usually contain further sequences in addition to the ones, which should be expressed, like marker genes for their specific selection and in some cases sequences for their episomal replication in a target cell. In certain embodiments, the plasmid is designed for amplification and expression in bacteria. Plasmids can be engineered by standard molecular biology techniques.

[0245] According to another aspect, the present invention provides a cell comprising the polynucleotide comprising a sequence encoding the peptide selected from a peptide comprising or consisting of amino acid sequence selected from SEQ ID NO: 1 , 2, 3 and 4, analog thereof or fragment thereof, as defined in any one of the embodiments of the present invention, the nucleic acid construct of the present invention. According to another embodiment, the present invention provides a cell comprising the polynucleotide comprising a sequence encoding the polypeptide comprising at least one copy of (i) SEQ ID NO: 1 , (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 3. According to another embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2 and (iii) SEQ ID NO: 4. According to a further embodiment, the polynucleotide comprises a sequence encoding a polypeptide comprising at least one copy of (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2, (iii) SEQ ID NO: 3 and SEQ ID NO: 4.

[0246] The terms "comprising", "comprise(s)" "include(s)," "having," "has, " "contain(s)," as used in this specification means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. The terms "have", "has", having" and "comprising" may also encompass the meaning of "consisting" and "consisting essentially of, and may be substituted by these terms. The term "consisting of excludes any component, step or procedure not specifically delineated or listed. The term "consisting essentially of means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0247] As used herein, the term "about", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10%, or +/-5%, +/-1%, or even +/-0.1 % from the specified value.

[0248] The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the appended claims. EXAMPLES

Example 1. Library of Constructs

[0249] A library of constructs comprising a branched PEG, a toxin peptide and two target-binding peptides. Each construct comprises a branched PEG with eight connecting arms, each having an NHS (N-Hydroxysuccinimide) terminus to which an amino moiety of a peptide is connected. To each scaffold eight peptides are connected: six copies of a peptide toxin and 1 copy of each of two target-binding peptides.

Different combinations of peptide toxins and target-binding peptides are included in the different constructs of the library (see Table 1).

Table 1. Examples of constructs

Image available on "Original document"


[0250] One exemplary arrangement is as following the toxins are 1 - Nodularin, 2 - ProTx-I, 3 - Viperistatin fragment and the binding peptides are directed against the following targets: A- androgen receptor, B - ERBB4, and C- CXCR4.

Another exemplary arrangement is Toxin 1 - cyclotoxin of SEQ ID NO: 3, Toxin 2 - cyclo-toxin of SEQ ID NO: 4; Toxin 3 - combination of cyclotoxins of SEQ ID NO: 3 and 4; the peptides are Peptide A - cyclic peptide SEQ ID NO: 1 ; Peptide B - cyclic peptide SEQ ID NO: 3; Peptide C is directed to bind androgen receptor, B - ERBB4, or C- CXCR4. [0251] The constructs are synthesized using methods known in the art, including Fmoc- solid phase peptide synthesis, purified using HPLC and tested in in-vitro and in- vivo for a specific activity, such as anti-proliferative activity using assays and animal models well known in the art.

Example 2. Preparation of cyclotoxins Toxl and Tox2

[0252] Using the technique described in WO 2007/010525, cyclopeptides (referred as toxins or cyclotoxins Toxl and Tox2) binding to human eukaryotic elongation factor 2 (eEF2) were generated and tested. The sequences of the cyclic peptides denoted as Toxl (consisting of SEQ ID NO: 3) and Tox2 (consisting of SEQ ID NO: 4) are provided in Table 2.

Table 2. Two toxic peptides.


Example 3. Binding of Toxl and Tox2 to eEF2

[0253] Binding of Tox 1 and Tox2 to eEF2 was tested by ELISA using eEF2 or BSA as ligands.

[0254] Experimental part

[0255] 0.25 μg of target proteins, eEF2 (Human; Yeast derived) or BSA (negative control) were applied to several wells of maxisorp plate (NUNC) in 50μ1 PBS and incubated over night at 4°C. The solutions were removed, and each well was supplemented with 280μ1 blocking solution (BSA 2mg/ml). The plate was incubated lhr at 25°C.

[0256] To 1.5ml tubes ΙΟΟμΙ of blocking solution + 10<9>pfu(plaque forming units) of M13 phages that express the following peptides: eEF2-binding : RB, LBR1, TB2 (Toxl), Y02, GW (Tox2), DRB, PY, BW were added. The plate was incubated lhr at 25°C. The solution were discarded and the wells were washed 7 times with 280μ1 washing solution (Tween 20 0.05%).

[0257] Each well was supplemented with 50μ1 of HRP/Anti-M13 Monoclonal Conjugate (GE Healthcare) diluted 1 :5000. The plate was incubated lhr at 25°C. The solutions were discarded and the wells were washed 7 times with 280μ1 washing solution (Tween 20 0.05%). Each well was supplemented with 50μ1 of TMB (T0440 ; Sigma). [0258] The plate was photographed using a scanner after incubation time of 1.5min and 30min. It can be seen from Fig. 2 that Toxl (denoted as TB2 in the figures) had the strongest effect. Example 4. Toxl and Tox2 activate eEF2

[0259] The effect of Toxl and Tox2 was tested in the in vitro transcription/translation system using HeLa Lysate system 1-Step Human Coupled IVT Kit-DNA(ThermoFisher Scientific). The following peptides were tested: GW (Toxl), DRB RB,TB2 (Tox2), and BW. In addition, a non eEF2 -binding control peptide, GR, was also tested.

[0260] The IVT Kit components were mixed, and one portion was taken out to serve as a negative control. The rest of the mix was supplemented with pCFE-GFP DNA. This DNA, when transcribed and translated gives a fluorescence protein, GFP. The extent of fluorescence gives a measure of the extent of protein synthesis.

[0261] The mix was split into 9ml aliquots. Each aliquot was supplemented with 1ml of one concentration of a specific peptide. A positive control was supplemented with 1ml of PBS. The reaction mixtures were incubated 4hr at 30°C.

[0262] 40ml of PBS were added to each reaction mixtures. The mixtures were transferred to a 96 well black ELISA plate, and the fluorescence was measured at ex/em 482/512nm.

[0263] It can be seen from the results (see Fig. 3), most of the peptides gave higher fluorescence than the positive control (that contained no peptide ; orange dot), and more than the non eEF2 -binding control peptide at concentration of 5μΜ. That means that they enhanced protein synthesis, when TB2 and GW provided the highest effect. Example 5. Preparation of multi-armed PEG complex loaded with 2 targeting peptides and 2 toxins

[0264] A construct of a branched PEG molecule covalently coupled with two different cancer-targeting moieties and two different toxin moieties was designed and synthesized (the schematic representation of the scaffold is shown in Fig. 1). The targeting moieties included in this example construct were the cyclic peptides E13.3 (consisting of SEQ ID NO: l) and PD-L1-GR (consisting of SEQ ID NO:2), and the toxin moieties were the cyclic peptides Toxl (consisting of SEQ ID NO:3), and Tox2 (consisting of SEQ ID NO:4). [0265] The preparation method comprised two steps. At the first step a branched PEG containing eight arms was produced in which seven arms were coupled with targeting/toxin moieties (protected peptides) and one with a Lysine residue protected with FMOC (Fmoc-Lys). At the second step eight of the peptide/toxin-PEG molecules produced in step 1 were coupled to another branched PEG molecule of eight arms to obtain a construct of multi-branched PEG coupled with 56 toxin/targeting moieties, of which 42 moieties are toxin peptides (21 Toxl and 21 Tox2), and 14 are targeting peptides (7 copies of EGRF targeting peptide E13.3 and 7 copies of PD-L1 targeting peptide PD-L1-GR).

[0266] In more details:

[0267] Step 1 - preparation of branched PEG coupled with one type of targeting or toxin moiety

[0268] 2.4μΓηο1ε of a targeting peptide or 7.3μΓηο1ε of toxin peptide were dissolved in DMSO.

[0269] All peptides have only one primary amine, except for E13.3, which has 3, of which one is protected with dde, and the N-terminal is blocked with acetate residue.

[0270] 5.9mg Fmoc-Lys-OH (Novabiochem (Merck) Cat. Num. 852023; MW=368.43) was dissolved in 150 μΐ of HC1 0.1 M, followed by addition of 650μ1 of DMSO to reach a concentration of 20 mM.

[0271] 33.4mg of 8-arm star PEG-NHS (Mw lOKDa, Creative Biotechnologies) were dissolved in 167μ1 of dioxane to reach a concentration of 20 mM.

[0272] Each of the targeting peptides solutions were mixed with 17μ1 of Fmoc-Lys-OH solution and 17μ1 of PEG solution.

[0273] Each of the toxin peptides solutions were mixed with 52μ1 of Fmoc-Lys-OH solution and 52μ1 of PEG solution. Each mix was supplemented with TEA (trimethylamine) to 5%. Each solution was incubated for 15.5 hours at room temperature on a Rotamix at 30 rpm to obtain a clear solution of 8 armed PEG coupled with 7 molecules of a specific targeting/toxin moiety and one arm containing a primary amine(The Fmoc protection is removed in this process to give one free primary amine on each PEG molecule).

[0274] The branched PEG-peptide molecules are denoted PEG-E13.3, PEG-PD-L1-GR, PEG-Tox 1 and PEG-Tox 2.

[0275] Step 2 - construction of multi-branched PEG construct coupled to 56 targeting/toxin moieties. [0276] The branched PEG-peptide solutions: PEG-E13.3, PEG-PD-L1-GR, PEG-Toxl and PEG-Tox2 were mixed together with 20mM PEG-NHS solution in a stoichiometric molar ratio of PEG-NHS:PEG-E13.3:PEG-PD-Ll-GR:PEG-Toxl :PEG-Tox2 of 1 :1 :1 :3:3, and incubated for 2 hours at room temperature on a Rotamix at 30 rpm, followed by slow addition of 80% hydrazine to a final concentration of 5%. Hydrazine was used to remove the dde protecting group from the El 3.3 moiety. The mixture was incubated for 2 hours at room temperature on a Rotamix at 30 rpm. The resultant construct is a multi-branched PEG coupled with 56 targeting/toxin moieties: 7 copies of El 3.3 peptide, 7 copies of PD-L1-GR peptide, 21 copies of Toxl and 21 copies of Tox 2. At the end of the reaction, PBS was added with gentle mixing.

[0277] Step 3 - Ultrafiltration

[0278] The samples were ultrafiltrated with two additions of 20ml PBS using Vivaspin 20 concentrator (30 K MWCO PES) to a concentration of -206 μΜ of loaded multi- armed PEG denoted as PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2, and the buffer was substituted to PBS.

[0279] In a similar way, additional multi-branched PEGs carrying alternative toxins or peptides (such as BIM) were produces. Examples of such multi-armed PEG is PEG- E13.3-PD-L1-GR-BIM, in which the toxins Tox 1 and Tox 2 were substituted by BIM. Example 6. Toxicity of a construct comprising E13.3, Toxl and Tox2

[0280] A construct comprising a multi-arm-PEG scaffold bound to E13.3 targeting peptide having the sequence SEQ ID NO: 1 (CHPGDKQEDPNCLQADK) and a toxin selected from BIMBH3 (referred also as BIM and having the sequence SEQ ID NO: 5 MRPEIWIAQELRRIGDEFNA) or a combination of Toxl and Tox2 was generated. The scaffolds were prepared as described in Example 5 and is denoted as PEG-E13.3- Toxl-Tox2 and PEG-13.3-BIM, accordingly

[0281] Cells Culture and Seeding:

[0282] A431 cells (human squamous carcinoma express about 100,000 copies of EGFR on each cell) and MCF-7 cells ( breast cancer cell expressing about 3,000 copies of EGFR on each cell) were thawed and cultivated to achieve exponentially growing cultures. Cells were collected, counted and seeded at the density of 7,000 cells/well and 5,000 cells/well, respectively, in a 96 well tissue culture plate.

[0283] The plates were incubated until the next day at the following conditions: 37+1 °C, humidified, and 5+0.5% C02/air, to enable cells adherence to the wells. [0284] Treatment:

[0285] The cell viability of A549 cell was tested using Alamar Blue viability assay. At the next day following the seeding, Growth Media was replaced with 200μ1 Assay Media that contained 2% FBS and Test Items at different concentrations of the construct (1, 3 and 8 μΜ), or Vehicle Control (PBS; concentration-0). Plates were incubated at 37+1 °C, humidified 5+0.5% CCVair. After 48 hours of incubation, images of cells treatments were taken on microscope (see Figs. 4-7).

[0286] Several concussions can be made from these experiments. First, it can be seen on the figures that the typical cells aggregates characterizing A431 and MCF-7 disappeared when a construct comprising PEG-E13.3 and any one of the toxin was added (Figs. 5-6). Moreover the phenomena was dose dependent. However, when the construct lacked El 3.3 peptide (Fig. 7), increasing the concentration of the toxin did not increase the ratio of dead cells significantly and actually was not different from the control. This result clearly indicate that E13.3 targeted the construct to the cell.

[0287] Second, the proportion of dead cells increased with increasing the concentration of the toxins (for both, BIM and combination of Toxl and Tox2), indicating for dose dependent effect. Moreover, comparing the images obtained for BIM and a combination of Toxl and Tox2, it can be seen that the combination was more potent causing to more severe cell death. As expected MCF-7 cells, expressing less EGFR were less sensitive than A431 cells.

[0288] Concluding all said above it is clear that a construct comprising a toxin such as Toxl, Tox2 or a combination thereof and targeting peptides, wherein at least one of them is E13.3 are potent in targeting and treating cancer. Example 7. Cytotoxicity of the constructs as tested on A431 cells

[0289] In condition similar to those of Examples 5 and 6, PEG-PD-L1-GR-BIM, PEG- E13.3-BIM and PEG-E13.3-PD-L1-GR-BIM constructs were prepared and tested for cytotoxicity using A431 cells and Alamar Blue Blue viability assay varying the concentration of the construct from 10 nM to 1 μΜ. After 48 hours of incubation, images of cells treatments were taken on microscope.

[0290] The results are presented in Fig. 8. It can be seen that the construct PEG- PD- Ll-GR-BIM and PEG-E13.3-BIM had limited ability of killing A431 cells at 1 μΜ concentration. The combination of E13.3 and PD-L1-GR targeting peptides on the other hand provided killing or more than 60% of the cells. Actually the cytotoxic effect of the construct comprising both targeting peptides was higher than the additive effect of the two constructs comprising one of two these peptides. This clearly indicates for the synergistic cytotoxic effect that the construct comprising two targeting peptides and a combination of Toxl and Tox2 has.

Example 8. Effect of PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2, and PEG-E13.3-(PD-L1- GR)-BIM constructs on the growth and viability of A549 cell line

[0291] Materials and methods

[0292] The test items PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2, and PEG-E13.3-(PD-L1- GR)-BIM were prepared as described in Example 5 and were used at concentration of 10μΜ. Phosphate Buffered Saline (PBS) is used as a control.

[0293] A-549 cells (human lung tumor cells) were thawed and cultivated to achieve exponentially growing cultures. Cells were collected, counted and seeded in a 96 well tissue culture plate at the following densities: A-549: 5,000cells/well.

[0294] The plate was incubated until the next day at 37+1 °C, humidified, 5+0.5% CCVair, to enable cells adherence to the wells.

[0295] Treatment

[0296] At the next day after the seeding, Growth Media were replaced with Test Items

Solutions prepared in Assay Medium (2%f FBS). Test Items Solutions are applied carefully (onto the sides of the well, not directly onto the cells) in volume of 200μ1 ε11 to achieve the final concentrations as following: PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2: 3 or 10 μΜ and PEG-E13.3-(PD-L1-GR)-BIM - 10 μΜ.

[0297] The plate was incubated at 37+1 °C, humidified 5+0.5% C02/air.

[0298] After 48 hours of incubation, representative images of cells treatments were taken on microscope. The results are presented on Fig. 9

[0299] Results

[0300] As can be clearly seen from Fig. 9 PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2 was effective in killing A549 cell both in concentration of 3 and 10 μΜ. Interestingly, PEG- E13.3-(PD-Ll-GR)-Toxl-Tox2 at the concentration of 3 μΜ it was much more efficient than 10 μΜ PEG-E13.3-(PD-L1-GR)-BIM construct comprising well known BIM toxin. Example 9. Acute IV toxicity of PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2 in mice

[0301] PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2 was prepared as described in Example 5 and injected intravenously to 3 Female Hsd:ICR (CD-I ®) mice, 7 weeks old using 4 ml/kg dose according to the regiments described in Table 3.

Table 3. Administration schedule

Image available on "Original document"


[0302] The weight and individual clinical signs were observed for 20 days. No significant abnormalities were seen neither in weight nor in the tested clinical signs. The animals were euthanized on day 20 and individual gross necropsy was performed. No abnormality was detected during the examination. Results of this example clearly indicate that PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2 construct is perfectly safe in vivo.

Example 10. Evaluation of antitumor effect of PEG-E13.3-(PD-Ll-GR)-Toxl-Tox2 in vivo

[0303] Material and methods

[0304] Animals: 18 athymic nude female mice 6-7 weeks old divided into 3 groups (1 control group and 2 test items groups) are allowed to accumulate for at least 5 days. Following acumulation, A431 tumor cells are subcutaneously injected to right flan region of each mouse, the day of injection is denoted as Day 0.

[0305] The following parameters are monitored: weight (twice a week), tumor size (measured with digital caliper and the tumor volume is calculated as width<2>xlength/2.

[0306] When the tumor reaches the size of 100-150 mm3, mice are subbbjected to 3 IV injections of test items during the first week. Animals are observed from additional 3 weeks.

[0307] Following observation period, mice are euthanized, the tumor is excised, measured and fixed in 4% formaldehyde solution for further analysis. Example 11: Antagonist of EGF receptor: screening, purification

[0308] Screening

[0309] Using the technique described in WO 2007/010525, a series of new peptides (cyclopeptides) binding to human Epidermal Growth Factor Receptor (EGFR) were generated and tested. After identification of several potential peptide, a few further cycles of optimization were performed. One of the peptides, denoted as El 3.3 and having the sequence of CHPGDKQEDPNCLQADK (SEQ ID NO: 1) showed high affinity to the receptor at its binding site.

[0310] Expression and purification

[0311] Bacteria comprising plasmids for expression of the identified peptides E7.1 , E10.2, E10.3, E13.3, E15.1.3-T, E14.1.1 , E14.1.4, E23I3, E23I5 and A4.3.12-T were started with 2.5 μΐ of cells comprising the plasmid of a relevant peptide in 5 ml 2YT medium with ampicillin and grown at 38°C over night at 350 rpm. 2 ml of each starter were grown in 50 ml 2YT at 37°C; the expression was induces with IPTG, 0.43 mM at OD 1.5-2.5 for 3 hours following which the cells were centrifuged and kept at -20°C.

[0312] The cells were lysed with lysozyme in the presence of DNases I and B-Per (Bacterial Protein Extraction Reagent), and the peptides were purified by affinity chromatography using Ni-NTA beads in a batch mode. Shortly, the peptides were loaded on Ni-NTA beads in the presence of 20 mM Imidazole, washed with PBS and eluted with 250 mM Imidazole. The buffer was exchange using PALL Life Sceince, Nanosep Centrifugal devices, 3K gray. The quantity of the peptides was tested by Coomassie Plus Protein Assay (see Fig. 10).

Example 12. The effect of the peptides on EGFR

[0313] The effect of several peptides on the phosphorylation levels of human EGFR in human epidermoid carcinoma cell line A431 was assessed by ELISA test. Briefly, exponentially growing A431 cell culture were detaches from the flask with 0.25% trypsin/EDTA solution, and 200 μΐ of cell suspension were transferred to 96-well plates at the concentration of 2xl0<5>cells/ml and grown for about 3 days. Following medium exchange, 50 μΐ EGF dilutions and EGF +peptides (50 ng/ml and 0.2 mg/ml, respectively) were added. The plate was incubated for 7.5 min at 37 °C. EGF- containing medium was removed and the cells were fixed by 150 ul of fixing solution and incubated for 20 min. at room temperature, following which the plate was washed 2 times with triton washing solution. The level of phosphorylates was assessed by incubation with phospho-EGFR (Tyrl045) antibody as a primary antibody and Anti- rabbit IgG as a secondary antibody. The ability of different peptides to inhibit auto- phosphorylation of EGFR is presented on Figure 2. The normalized percent of inhibition (of the EGFR auto-phosphorylation is presented in Fig. 11 and Table 4. The normalized percent of inhibition is calculated as the fluorescence signal of the test item divided by the fluorescence signal of the control that contains no test item, with the same concentration of EGF.

Table 4. Normalized Percent of EGFR autophosphorylation inhibition
Image available on "Original document"

[0314] As it can be clearly seen from the Fig. 11 and Table 4, E13.3 has the higher inhibitory activity among the peptides, having calculated IC50of 2 μΜ.

Example 13. Stability of E13.3 in bovine serum

[0315] The stability of the selected peptides in bovine serum was assessed by measurement of the inhibitory activity of the peptides after incubation of the peptides with bovine serum. The inhibitory activity was measured as described in Example 12. The inhibitory activity of the peptide was assessed following incubation of the peptides with bovine serum at 37°C for different periods of time. EGF concentration in the samples was 50 ng/ml. The results are presented in Figs. 12. It can be clearly seen that all peptides have similar stability in the bovine serum with t0.5 of about 1.5 hours.

Example 14. Inhibition efficacy of the peptides is dose dependent

[0316] Efficacy of different concentration of the selected peptide was assessed by ELISA in a similar was as in Example 12. The results are presented in Figs. 13. The IC50 of all peptides was about 0.5-1 μΜ.

Example 15. Preparation of E13.3 bound to 8-arm PEG

[0317] 9.8 mg of E13.3(fmoc)Lys was dissolved in water to the final concentration of 20 mg/ml. 11.3 mg of 8 arm PEG Succinimidyl Carboxymethyl Ester, MW 73,000 (JENKEM TECHNOLOGY USA INC) with 565 μΐ dioxane was heated at 37°C to a complete dissolution. El 3.3 and PEG solution were mixed in the presence of 50 μΐ TEA and incubated overnight at room temperature. To the obtained solution, 50 μΐ piperidine was added and incubated for 0.5 at room temperature. To the solution, 1 ml of ethyl acetate was added to obtain a suspension which was than centrifuged and the upper phase was removed. These steps of washing with ethyl acetate were repeated 4-5 times. Finally, the upper phase was removed completely and the remained pellet was dissolved in 200 μΐ PBS. The buffer was further exchanged to PBS using Vivaspin 20ml Concentrator to eliminate any traces of ethyl acetate.

Example 16. Stability of E13.3 in mice

[0318] The stability of the fluorescently marked peptide E13.3 alone or bound to 8- armed PEG was evaluated in vivo by injecting the compounds to the tail vein of mice. The blood of the animals was analyzed for the presence of the peptide (fluorescence) at different time intervals. It can be clearly seen from the result presented in Fig. 14 that to.5 of the free peptide is much shorter (about several minutes) than that of the peptides bound to PEG (t0.5 of about 3.5 hours).

Example 17. The effect of E13.3 on the viability of different cancer cell lines

[0319] The anti-cancer activity of E13.3 was assessed using several cancer cell lines (A549 - human lung carcinoma cell line and FaDu - human pharyngeal carcinoma cell line). The cell cultures were incubated in the presence or absence of El 3.3 (at different concentrations) and tested for viability using alamarBlue reagent. The results are presented on Fig. 15. It can be seen, that El 3.3 bound to PEG could successfully reduce the viability of the cancer cells in all tested concentrations.

Example 18. Accumulation of fluorescent PEG-E13.3 in cancer tumors

[0320] El 3.3 -PEG was labeled with Flourescein and injected IV to Xenograft mice bearing subcutaneous NCI-H1650 tumor (lung cancer). Following anesthesia, kidney, liver and tumor were collected at specific time points and the fluorescence was measured. The results are presented in Figs. 16 and 17.

[0321] As it can be seen from Fig. 16, there was a fast increase in the fluorescence in kidney and liver with a typical elimination curve afterwards. Contrary to that, the fluorescence was accumulated in the cancer cells indicating that E13.3 effectively binds, enters and accumulated in the cancer cells. Results shown on Fig. 17 further support that most of the cancer cells interact with E13.3-PEG and internalize the fluorescent peptide. Example 19: PD-Ll binding proteins

[0322] Screening

[0323] Using the technique described in WO 2007/010525, a series of new peptides (cyclopeptides) binding to binding to a human PD-Ll were generated and tested. After identification of several potential peptides, a few further cycles of optimization were performed. One of the peptides, denoted as PD-Ll-GR and having the sequence of CysGluGlyLeuProAlaAspTrpAlaAlaAlaCys (SEQ ID NO: 2) showed high affinity to the receptor at its binding site. Example 20. Preparation of PD-Ll-GR peptide bound to multi-armed PEG construct

[0324] Multi-armed PEG constructs comprising (i) PD-Ll-GR cyclic peptide and BIM- BH3 (denoted as PEG-(PD-Ll-GR)-BIM), (ii) E13.3 targeting peptide (SEQ ID NO:l) and BIM-BH3 toxin (denoted as PEG-E13.3-BIM), and (iii) E13.3, PD-Ll-GR and BIM-BH3 toxin (denoted as PEG-E13.3-(PD-L1-GR)-BIM) were prepared as described in Example 5.

[0325] The constructs were used as Test Items in cell proliferation assay in concentration of ΙμΜ. PBS was used as a control. For the assay, A549 cells (human lung carcinoma cell line) were thawed and cultivate to achieve exponentially growing. The cells were collected, counted and seeded at the density of 7,000 cells/well in a 96 well tissue culture plate. The plate was incubated until the next day at 37+1 °C, humidified, 5+0.5% C02/air, to enable cells adherence to the wells. At the next day, Growth Media are replaced with Test Items Solutions prepared in Assay Medium (2%f FBS). Test Items Solutions are applied carefully in volume of 200μ1Λνε11 to achieve the final concentrations of the Test Items of ΙμΜ. After 48 hours of incubation, representative images of cells treatments were taken on microscope and are presented in Fig. 18.

[0326] It can be seen from Fig. 18, the construct comprising PEG-E13.3-(PD-L1-GR)- BIM was the only construct to inhibit cell proliferation at a concentration of ΙμΜ. This indicates that the complex comprising a combination of E13.3 and PD-Ll-GR peptides has significantly higher cytotoxicity than the combined cytotoxicity of the constructs comprising only one of the peptides. [0327] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.



US10160967
METHODS AND COMPOSITIONS FOR IDENTIFYING A PEPTIDE HAVING AN INTERMOLECULAR INTERACTION WITH A TARGET OF INTEREST

[ PDF ]

Inventor: MORAD ILAN /ITZHAKI HANAN

This invention provides, in one embodiment, a recombinant virus or a recombinant virus library wherein each virus comprises a protein involved in viral attachment or infection, a polypeptide which differs by at least a single amino acid from another peptide or polypeptide in the library, and a modified cleavage site that is proximal to the peptide and the protein, wherein the cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for it as compared to a non-modified cleavage site. The invention further provides a target of interest complex comprising a protease, a target of interest involved in an intermolecular interaction, and a flexible linker that attaches the protease and target of interest. The invention further provides uses thereof, including a method for identifying a peptide which has an intermolecular interaction with a target of interest or identifying the agonistic or antagonistic feature of a peptide that has an intermolecular reaction with a target of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 11/989,203 filed on Jan. 22, 2008, which is the 371 filing of International application no. PCT/IL2006/000815 filed on Jul. 12, 2006, which claims the benefit of U.S. provisional application No. 60/701,092 filed on Jul. 21, 2005, the entire content of each of which is incorporated herein by reference thereto.

FIELD OF THE INVENTION

[0002] This invention is directed to a method of identifying a polypeptide having an intermolecular interaction with a target of interest and functional features thereof.

BACKGROUND OF THE INVENTION

[0003] Phage display has become a powerful method for screening populations of peptide or polypeptides, mutated proteins, and cDNAs for members that have affinity to target molecules of interest. It is possible to generate many different recombinants from which one or more clones can be selected with affinity to antigens, antibodies, cell surface receptors, protein chaperones, DNA, metal ions, etc. Screening libraries are versatile because the displayed elements are expressed on the surface of the virus as capsid-fusion proteins. The most important consequence of this arrangement is that there is a physical linkage between phenotype and genotype. There are several other advantages as well: 1) virus particles which have been isolated from libraries by affinity selection can be regenerated by simple bacterial infection and 2) the primary structure of the displayed binding peptide or protein can be easily deduced by DNA sequencing of the cloned segment in the viral genome.

[0004] Synthetic oligonucleotides that are fixed in length, but with multiple unspecified codons can be cloned into genes III, VI, or VIII of bacteriophage M1 3 where they are expressed on the capsid fusion protein. The libraries, often referred to as random peptide libraries, can be screened for binding to target molecules of interest.

[0005] Most vital cellular processes are regulated by the transmission of signals wherein such signal transduction is likely mediated by protein-protein interactions involving modular domains within the signaling proteins.

[0006] Methods for isolating partner proteins involved in protein-protein interactions have generally focused on finding a ligand to a characterized protein. Such approaches have included using anti-idiotypic antibodies that mimic the known protein to screen cDNA expression libraries for a binding ligand (Jerne, 1974, Ann. Immunol. (Inst. Pasteur) 125c:373-389; Sudol, 1994, Oncogene 9:2145-2152). Skolnick et al. (1991, Cell 65:83-90) isolated a binding partner for PI3-kinase by screening a cDNA expression library with the <32>P-labeled tyrosine phosphorylated carboxyl terminus of the epidermal growth factor receptor (EGFR). While current methods provide for the identification and isolation of a peptides having an intermolecular interaction with a target of interest, functional analysis of the screening steps is currently lacking.

SUMMARY OF THE INVENTION

[0007] This invention provides, in one embodiment, a recombinant virus comprising a protein or protein fragment, comprising segments which are involved in viral attachment, infection or a combination thereof, a peptide or polypeptide involved in an intermolecular interaction, and a modified cleavage site that is proximal to said peptide and to said segments of said protein, wherein said cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for it.

[0008] In another embodiment, this invention provides a recombinant virus library wherein each virus comprises a protein or protein fragment, comprising segments which are involved in viral attachment, infection or a combination thereof, a peptide or polypeptide which differs by at least a single amino acid from another peptide or polypeptide in said library, and a modified cleavage site that is proximal to said peptide and to said segments of said protein, wherein said cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for said cleavage site as compared to a non-modified cleavage site.

[0009] In another embodiment, the invention provides a target of interest complex comprising a protease, a target of interest involved in an intermolecular interaction, and a flexible linker that attaches the protease and target of interest.

[0010] In another embodiment, this invention provides a method of identifying a peptide or polypeptide having an intermolecular interaction with a target of interest comprising the steps of: (a) contacting a recombinant virus library as described hereinabove with a plurality of complexes as described hereinabove; (b) contacting said library with a plurality of cells; (c) isolating viruses that did not infect said cells; (d) providing infectious clones of isolated viruses by amplifying and expressing the genomes of said isolated viruses; (e) repeating the above steps; and (f) identifying peptides expressed by said viruses whereby intermolecular interactions between said target of interest and a peptide expressed by said recombinant virus result in said protease being in close proximity to said cleavage site, resulting in cleavage of said protein or protein fragment, comprising segments which are involved in viral attachment to, infection of, or a combination thereof of cells, and prevention of entry of a virus that had comprised a peptide involved in intermolecular interactions into said cells.

[0011] In another embodiment, this invention provides a kit for identifying a peptide or polypeptide involved in an intermolecular interaction with a target of interest comprising a recombinant virus peptide library as described hereinabove, a target of interest complex as described hereinabove, and cells that are susceptible to viral attachment, infection or a combination thereof.

[0012] In another embodiment, this invention provides a method for identifying an agonistic or antagonistic feature of a peptide or polypeptide that has an inter molecular interaction with a receptor of interest comprising the steps of: (a) contacting the recombinant virus library as described hereinabove with a plurality of complexes as described hereinabove, wherein the target of interest is a receptor; (b) contacting said recombinant virus library of step (a) with cells; (c) isolating viruses in (b) which have not infected said cells; (d) providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses; (e) contacting infectious clones of viruses of step (c) with the receptor of interest, wherein said receptor is not attached to a protease; (f) contacting the viruses of step (e) with a protein attached to a protease via a flexible linker, wherein said protein is involved in the downstream signal transduction pathway of said receptor of interest; (g) contacting the viruses of step (f) with cells; (h) separating viruses in (g) that have not infected said cells from viruses which have infected cells; (i) providing infectious clones of second isolated viruses of step (h) by amplifying and expressing the genomes of said second isolated viruses; (j) repeating steps (a)-(i); and (k) identifying peptides expressed by the viruses in step (h), whereby viruses which have not infected said cells in (h) express a peptide which has agonistic activity for said receptor of interest, and viruses which have infected said cells in (h) express a peptide which has antagonistic activity for said receptor.

[0013] In another embodiment, this invention provides a method for identifying a peptide or polypeptide that inhibits an enzyme of interest comprising the steps of: (a) contacting the recombinant virus library as described hereinabove with a plurality of complexes as described hereinabove, wherein the target of interest is an enzyme; (b) contacting said recombinant virus library of step (a) with cells; (c) isolating viruses in (b) which have not infected said cells; (d) providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses; (e) contacting infectious clones of viruses of step (c) with the enzyme of interest, wherein said enzyme is not attached to a protease; (f) contacting the viruses of step (e) with a substrate of the enzyme attached to a protease via a flexible linker; (g) contacting the viruses of step (f) with cells; (h) separating viruses in (g) which have not infected said cells from viruses which have infected cells; (i) providing infectious clones of second isolated viruses of step (i) by amplifying and expressing the genomes of said second isolated viruses; (j) repeating steps (a)-(i); and (k) identifying peptides expressed by the viruses in step (h), whereby viruses which have not infected said cells in (h) express a peptide which does not affect said enzyme of interest, and viruses which have infected said cells in (h) express a peptide which inhibits said enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is an illustration of the conversion, in one embodiment, of an infective (A) to a non-infective (B) via cleavage of the N1 and N2 domains from the CT domain of the phage minor coat gene 3 (pIII) protein of the M13 bacteriophage, which is attached to the bacteriophage capsid.

[0015] FIG. 2 is an illustration in one embodiment of a genetically engineered infective M13 bacteriophage termed SoIP. SoIP comprises the pIII domains of wild type bacteriophage, but with an insertion of a modified protease site and a peptide between the N2 and CT domains of each pIII protein. Cleavage of the N1 and N2 domains of pIII occurs in SoIP when a target protein (Target) which is fused to a protease (Protease) binds to the peptide expressed on the pIII protein (Peptide). The resultant proximity of Protease to the modified protease site results in proteolysis of N1, N2, the peptide, and the modified protease site from the CT domain of pIII and the capsid.

[0016] FIG. 3 is an illustration of a preferred embodiment of a SoIP, specifying the identity of the modified protease site. SoIP comprises the pIII domains of wild type bacteriophage, but with an insertion of a modified ubiquitin sequence (mUb) and one half of a leucine zipper peptide (LeuZip2) between the N2 and CT domains of each pIII protein. Cleavage of the N1 and N2 domains of pIII occurs in SoIP when the second half of a leucine zipper peptide (LeuZip1), which is fused to a UBP protease, binds to LeuZip2 expressed on the pIII protein. The resultant proximity of UBP to mUb results in proteolysis of N1, N2, LeuZip2, and mUb from the CT domain of pIII and the capsid.

[0017] FIG. 4 is an illustration in one embodiment of the method of incubating infective and non-infective SoIP with bacteria. Only infective SoIP are able to enter the bacterial cell. After incubation, bacterial cells containing infecting SoIP are removed, and non-infective SoIP remain in the medium.

[0018] FIGS. 5A-D are an illustration in one embodiment of the method of identifying proteins that interact with a protein of interest. A recombinant virus library, in which each recombinant virus expresses a peptide unique from those expressed by other recombinant viruses in the library, is incubated with a fusion protein comprising a known target peptide or polypeptide fused to a protease via a flexible linker. Library peptides with an affinity for the target will be cleaved. The library is then incubated with cells. Recombinant viruses with cleaved pIII proteins will be unable to infect cells.

[0019] FIGS. 6A-6D are an illustration of an embodiment of the present invention in which the invention can distinguish between functional and non-functional peptides or polypeptides using the SoIP assay. Functional proteins may be further categorized as antagonist, agonist, and/or inhibitor. Recombinant viruses that comprise a peptide from the peptide library (referred to as “Peptide”) that demonstrates binding to a target protein of interest are incubated with the target (6A). Next, a fusion protein comprising a protease fused to a known target's ligand is added to the incubation mixture comprising the recombinant virus (6B). The ligand can be a downstream element, in case the target is a receptor, or an enzyme, that works on the target. The peptide can either facilitate the binding of the target and its ligand, or not. In case the peptide facilitates binding of the target and its ligand, the protease and the modified protease site are brought into close proximity. This will lead to the cleavage of the pIII protein, which will render the recombinant virus non-infective (6C). If the peptide inhibits the binding of the target and its ligand, the pIII protein will not be cleaved and the recombinant virus will remain infective (6D).

DETAILED DESCRIPTION OF THE INVENTION

[0020] This invention provides a recombinant virus and a recombinant virus library and its use in identifying interacting polypeptides, which may find application in diagnostics and/or therapeutics.

Recombinant Virus

[0021] In one embodiment, a recombinant virus of this invention comprises: a protein or protein fragment, comprising segments which are involved in viral attachment to, infection of or a combination thereof of a host cell; a peptide or polypeptide involved in an intermolecular interaction; and a modified cleavage site that is proximal to said peptide and to said segments of said protein, wherein the cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for said cleavage site, as compared to a non-modified cleavage site.

[0022] In one embodiment, viruses of the present invention may infect mammalian, insect, plant, bacteria cells or a combination thereof. In one embodiment, a virus of the present invention may be a bacteriophage. In another embodiment, a virus of the present invention may be a filamentous bacteriophage. In another embodiment, a virus of the present invention may be M13. In another embodiment, it may be lambda phage, fd, f1, or T4. In another embodiment, a virus of the present invention may be Mycobacteria phage. In one embodiment, it might be Mycobacteria phage D29, Mycobacteriophage Bethlehem, Mycobacteriophage U2, Mycobacterium phage L5, or Mycobacterium phage TM4.

[0023] Bacteriophage (phage) are viruses that have a specific affinity for and infect bacteria. Phages consist of a protein coat or capsid enclosing the genetic material (DNA or RNA) that is injected into a bacterium upon infection. Phages may be lytic or temperate. Lytic phages lyse, or break apart, the host cell, while temperate phages integrate their DNA into that of the host (lysogeny). In the case of lytic phages, all synthesis of host DNA, RNA and proteins ceases, and the phage genome is used to direct the synthesis of phage nucleic acids and proteins using the host's transcriptional and translational apparatus. These phage components then self assemble to form new phage particles. The synthesis of a phage lysozyme leads to rupture of the bacterial cell wall releasing typically 100-200 phage progeny. The temperate phages, such as lambda, may also show this lytic cycle when they infect a cell, but more frequently they induce lysogeny. Examples of bacteriophages that attack Escherichia coli are lambda phage and the T even phages, T2, T4 and T6.

[0024] In another embodiment, viruses can include any desired virus, as will be recognized by those of skill in the art, including but not limited to Adenoviruses, Herpesviruses, Poxviruses, Parvoviruses, Reoviruses, Birnaviruses, Picornaviruses, Togaviruses, Orthomyxoviruses, Rhabdoviruses, Retroviruses, Hepadnaviruses, Enterovirus, Cardiovirus, Rhinovirus, Apthovirus, Hepatovirus, etc. In one embodiment, viruses are lysogenic, while in another embodiment, viruses are temperate.

[0025] In one embodiment, the term “recombinant virus” refers to viruses that are engineered to express foreign or modified protein or proteins, which are not natively expressed in that virus. At least one protein or protein fragment is expressed by the recombinant virus.

[0026] In another embodiment, the virus can be modified in any of various ways known in the art, such as to introduce temperature sensitivity, to introduce a reporter gene, to be rendered replication-deficient, or to eliminate other viral genes. Methods of such modifications are standard in the art. In one embodiment, the virus is modified to express the peptide on the surface of the virus, as by engineering the virus genome to encode a fusion protein for a coat protein and the peptide. In one embodiment, the present invention further provides a recombinant virus comprising a selectable marker or label such that the label can be directly detected. The selectable marker can be any selectable marker, which is known in the art, such as antibiotic resistance protein, for example, without being limited, ampicillin. The selectable marker can be also used for deletion mutants counter selection, when there is a need to select cells that are infected by virus that did not lose essential genes.

[0027] In one embodiment, the term “protein or protein fragment” refers to a molecule comprised of amino acid residues joined by peptide (i.e., amide) bonds and includes peptides, polypeptides, and proteins. In another embodiment, “protein or protein fragment” refers to a protein or protein fragment comprising segments which are involved in viral attachment, infection, or a combination thereof. In one embodiment, the term “segment” refers to a sequence of amino acids of any length with functional or structural homology to proteins known to mediate viral attachment, infection, or a combination thereof. In one embodiment, a “tail” refers to a protein involved in viral attachment, infection, or a combination thereof.

[0028] In one embodiment, a segment of said protein or protein fragment mediates attachment. In one embodiment, the term “attachment” refers to a physical connection between a virus and a host cell. In the process of viral infection, a virus attaches to a host cell in order to enter or penetrate it. Many viruses comprise a protein expressed on its surface, sometimes referred to as an “antireceptor” which binds to a constituent of the cell surface of a potential host cell, sometimes referred to as a “receptor.” In one embodiment, the proteins or protein segments involved in viral attachment comprise the pIII protein. In another embodiment, the protein or protein segments comprise the PVIII protein. In one embodiment, the proteins or protein segments involved in viral attachment comprise the N2 domain of the pIII protein (a.k.a. Coat Protein A) of filamentous bacteriophage (M13, fd, f1, I2-2, If1, Ike, Pf1), the A protein of Bacteriophage fr, GA, or MS2, the Gp38 protein (a.k.a. receptor recognizing protein) of Bacteriophage AR1, K3, M1, Ox2, or T2, the J protein of Bacteriophage lambda, φx174, α3, or G4, and others, known to one skilled in the art. In another embodiment, the proteins or protein segments involved in viral attachment comprise hemagglutinin of influenza virus, gp120 envelope glycoprotein of HIV or segments thereof, and others, known to one skilled in the art.

[0029] In one embodiment, a segment of said protein or protein fragment mediates infection. In one embodiment, the term “infection”, “entry,” “fusion” and “penetration” refer to fusion of a virus with the membrane of a host cell to allow the passage of genetic material or a complete virus into the host cell. Some viruses comprise a protein or a protein segment that is involved in cell entry. In one embodiment, the proteins or protein segments involved in infection comprise the pIII protein. In another embodiment, the protein or protein segments comprise the PVIII protein. In one embodiment, a protein or a protein segment that is involved in infection is the N1 domain of the pIII protein of filamentous bacteriophage. In another embodiment, a protein or a protein segment that is involved in infection is the gp41 of HIV-1, while in another embodiment, it's the V3 domain or other segments of the gp120 of HIV-1.

[0030] In one embodiment, the protein or protein fragment comprising segments involved in viral attachment, infection or a combination thereof are structural or functional homologs of those mentioned hereinabove. In another embodiment, the segment involved in viral attachment, infection or a combination thereof are synthetically constructed using methods well known in the art.

[0031] In one embodiment, two protein fragments of a single protein mediate mediate attachment and infection. In another embodiment, a single protein or protein fragment mediates both attachment and infection. In another embodiment, two protein fragments of two distinct proteins mediate attachment and infection. In another embodiment, two protein fragments of two distinct proteins mediate attachment and infection. In another embodiment, two distinct proteins mediate attachment and infection.

Non-Endogenous Peptides

[0032] In one embodiment, each recombinant virus of the present invention comprises a peptide involved in at least one intermolecular interaction. In one embodiment, the term “polypeptide” or “peptide” refers to a molecule comprised of amino acid residues joined by peptide (i.e., amide) bonds and includes peptides, polypeptides, and proteins. Hence, in one embodiment, the polypeptides of this invention may have single or multiple chains of covalently linked amino acids and may further contain intrachain or interchain linkages comprised of disulfide bonds. In one embodiment, some polypeptides may also form a subunit of a multiunit macromolecular complex. In one embodiment, the polypeptides can be expected to possess conformational preferences and to exhibit a three-dimensional structure. Both the conformational preferences and the three-dimensional structure will usually be defined by the polypeptide's primary (i.e., amino acid) sequence and/or the presence (or absence) of disulfide bonds or other covalent or non-covalent intrachain or interchain interactions. In another embodiment, the polypeptides do not possess conformational preferences or exhibit a three-dimensional structure.

[0033] In one embodiment, the terms “peptides”, “polypeptides”, “plurality of peptides” or “plurality of polypeptides” can be used interchangeably and refer to more than one peptide or polypeptide.

[0034] The polypeptide of the present invention can be of any size. As can be expected, the polypeptides can exhibit a wide variety of molecular weights, some exceeding 150 to 200 kilodaltons (kD). Typically, the polypeptides may have a molecular weight ranging from about 5,000 to about 100,000 daltons. Still others may fall in a narrower range, for example, about 10,000 to about 75,000 daltons, or about 20,000 to about 50,000 daltons. In an alternative embodiment, the polypeptides of the present invention may be 1-250 amino acid residues long. In another embodiment, the polypeptides of the present invention may be 10-200 amino acid residues long. In an alternative embodiment, the polypeptides of the present invention may be 50-100 amino acid residues long. In an alternative embodiment, the polypeptides of the present invention may be 1-250 amino acid residues long. In an alternative embodiment, the polypeptides of the present invention may be 1-250 amino acid residues long.

[0035] The peptides or polypeptides, or the DNA sequences encoding same, may be obtained from a variety of natural or unnatural sources, such as a prokaryotic or a eukaryotic cell. In one embodiment, the source cell may be wild type, recombinant, or mutant. In another embodiment, the plurality of peptides or polypeptides may be endogenous to microorganisms, such as bacteria, yeast, or fungi, to a virus, to an animal (including mammals, invertebrates, reptiles, birds, and insects) or to a plant cell.

[0036] In another embodiment, the peptides or polypeptides may be obtained from more specific sources, such as the surface coat of a virion particle, a particular cell lysate, a tissue extract, or they may be restricted to those polypeptides that are expressed on the surface of a cell membrane.

[0037] In another embodiment, the peptide or polypeptide is derived from a particular cell or tissue type, developmental stage or disease condition or stage. In one embodiment, the disease condition or stage is cancer, in another embodiment, it's an infection, in another embodiment, it's an HIV infection, in another embodiment, it's a developmental disorder, while in another embodiment, it's a metabolic disorder.

[0038] In one embodiment, the peptide or polypeptide is obtained from a peptide or polypeptide library as is described hereinbelow. In another embodiment, it's created synthetically and inserted into a vector as is well known in the art. In one embodiment, the term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.

[0039] In one embodiment, the peptide or polypeptide is expressed on the external surface of the virus. In another embodiment, the peptide or polypeptide is not the most terminal peptide on a protein or recombinant protein expressed on the external surface of the virus. In one embodiment, “terminal” refers to the absolute end of a protein or polypeptide. In one embodiment, the peptide or polypeptide is not at the 3′ end of a protein or recombinant protein expressed on the external surface of the virus, while in another embodiment, it is not at 5′ end of a protein or recombinant protein. In one embodiment, the peptide or polypeptide is not among the last 50 amino acids of a protein or recombinant protein. In another embodiment, the peptide or polypeptide is not among the last 100 amino acids of a protein or recombinant protein. In another embodiment, the peptide or polypeptide is not among the last 500 amino acids of a protein or recombinant protein.

[0040] In another embodiment, the peptide or polypeptide is connected to the proximal segments of the protein or protein fragment involved in viral attachment, infection, or a combination thereof via a flexible linker. In one embodiment, the linker is G1 or G2 of the protein pIII of M13. In another embodiment, the flexible linker is a (Gly-Gly-Gly-Ser-)n (SEQ ID No. 1) synthetic linker. In another embodiment, any other flexible linker could be used.

[0041] In another embodiment, the peptides or polypeptides are agonists. In another embodiment, the peptides or polypeptides are antagonists. In another embodiment, the peptides or polypeptides are antigens. In another embodiment, the peptides or polypeptides are enzymes. In another embodiment, the peptides or polypeptides are activators of enzymes or other substrates. In another embodiment, the peptides or polypeptides are inhibitors of enzymes or other substrates. In another embodiment, the peptides or polypeptides are hormones. In another embodiment, the peptides or polypeptides are regulatory proteins. Regulatory proteins command the numerous interactions that govern the expression and replication of genes, the performance of enzymes, the interplay between cells and their environment, and many other manifestations. In another embodiment, the peptides or polypeptides are cytoskeletal proteins. Cytoskeletal proteins form a flexible framework for the cell, provide attachment points for organelles and formed bodies, and make communication between parts of the cell possible. In another embodiment, the peptides or polypeptides are toxins. In another embodiment, the peptides or polypeptides are functional fragments of agonists, antagonists, antigens, enzymes, enzyme activators, enzyme inhibitors, enzyme substrates, hormones, regulatory proteins, cytoskeletal proteins, or toxins. “Functional fragments” are meant to indicate a portion of the peptide or polypeptide which is capable of performing one or more of the functions of the peptide or polypeptide, even in the absence of the remainder of the peptide or polypeptide. In one embodiment, the functional fragment is sufficient to mediate an intermolecular interaction with a target of interest.

[0042] In an alternative embodiment, the peptide binds DNA or RNA or a fragment thereof. In one embodiment, the DNA or RNA binding peptide may be any of the many known in the art including, but not limited to: Zinc finger proteins such as Beta-beta-alpha zinc finger proteins, Nuclear receptor proteins, Loop-sheet-helix type protein, and GAL4 type protein; the Helix-turn-helix proteins such as Cro and repressor proteins, Lad purine repressor proteins (PurR), Fold restriction endonuclease (DNA-recognition region), Gamma-delta recombinase protein (C-terminal domain), Hin recombinase protein, Trp repressor protein, Diptheria tox repressor, Catabolite gene activator proteins (CAP), Homeodomain proteins, RAP1 protein, Prd paired protein, Tc3 transposase protein, TFIIB family, Interferon regulatory factor, Transcription factor family, and ETS domain family bacteriophage; and the Leucine zipper proteins such as Basic zipper proteins and Zipper-type proteins (helix-loop-helix). In another embodiment, the DNA or RNA binding peptide may be other alpha-helix proteins such as Cre recombinase family, Papillomavirus-1 E2 protein, Histone family, Ebna1 nuclear protein family, Skn-1 transcription factor, High mobility group family, and MADS box family; Beta-sheet proteins such as TATA Box-Binding Proteins; Beta-hairpin/ribbon proteins such as Met repressor protein, Tus replication terminator protein, Integration host factor protein, Hyperthermophile DNA binding protein, Arc repressor, Transcription factor T domain; and other protein families such as Rel homology region proteins and Stat family. In another embodiment, the DNA or RNA binding peptide may be enzymes such as Methyl transferase proteins, PvuII Endonuclease protein, Endonuclease V protein, EcoRV Endonuclease family, BamHI Endonuclease family, EcoRI endonuclease family, DNA mismatch endonuclease, DNA polymerase I protein, DNA polymerase T7, Dnase I proteins, DNA polymerase beta proteins, Uraci-DNA glycosylase, Methyladenine-DNA glycosylase, Homing endonuclease, and Topoisomerase I or viral proteins such as HIV reverse transcriptase.

[0043] In another embodiment, the peptide or polypeptide is a transcriptional or translational activator or a fragment thereof. In another embodiment, the peptide or polypeptide is a transcriptional or translational repressor or a fragment thereof. In another embodiment, the peptide or polypeptide is a receptor or a fragment thereof. In another embodiment, the peptide or polypeptide is an organic molecule, inorganic compound, or organometallic compound or a fragment thereof.

[0044] In one embodiment, the peptide or polypeptide may represent a cognate peptide of any of the peptides or polypeptides described hereinabove. A “cognate” peptide is any peptide that interacts and/or binds to another molecule.

[0045] The peptide or polypeptide identified by this invention can be in another embodiment, a potential drug candidate or a “lead” compound.

[0046] Compounds that pass an initial in vitro screening test, such as the methods of the present invention, are known as “lead” compounds. These lead compounds are then put through further testing, including, eventually, in vivo testing in animals and humans, from which the promise shown by the lead compounds in the original in vitro tests is either, confirmed or refuted. See Remington's Pharmaceutical Sciences, 1990, A. R. Gennaro, ed., Chapter 8, pages 60-62, Mack Publishing Co., Easton, Pa.; Ecker and Crooke, 1995, Bio/Technology 13:351-360.

Intermolecular Interaction

[0047] In one embodiment, the term “intermolecular interaction” refers to an expressed peptide or polypeptide of a virus of the present invention which has the capacity to bind to a target of interest. In one embodiment, the interaction is of high affinity, while in another embodiment, it is of low affinity. In one embodiment, the interaction is mediated by a covalent bond, an ionic bond, a hydrogen bond, Van der Waal's force (a.k.a. weak London dispersion forces), dipole-dipole forces, metallic bonding or any attractive force which provides a physical proximity of between, in one embodiment, 1-5, in another embodiment, 1-20 angstroms.

[0048] In another embodiment, the peptide may perform a particular function with a target of interest. In one embodiment, the intermolecular interaction may give rise to a biological, chemical, or physiological consequence. In one embodiment, the consequence may be reversible, while in another embodiment, it may be irreversible. In one embodiment, the intermolecular interaction induces a conformational change, a transformation into a different chemical state of the functional domain or of molecules acted upon by the functional domain, the transduction of an intracellular or intercellular signal, the regulation of gene or protein expression, the regulation of cell growth or death, the activation or inhibition of an immune response, or any combination thereof.

[0049] In one embodiment, the term “intermolecular interaction” refers to an affinity between a peptide and a target of interest, i.e. the tendency of a peptide to attach to a specific protein target. The affinity binding is a measure of the intrinsic binding strength of the ligand binding reaction. The intrinsic attractiveness of the binder for the ligand is typically expressed as the equilibrium association constant (Ka) of the reaction. The equilibrium constant Ka=[Ligand-Binder]/[Ligand][Binder], where [ ] represents the molar concentration of the material at equilibrium. The Ka describing the affinity between the peptide and the target of interest can be 10<−4>, 10<−5>, 10<−6>, 10<−7>, 10<−8>, 10<−9>, 10<−10>, 10<−11>, 10<−12>, 10<−13>, 10<−14>, 10<−15 >M or lower.

[0050] In one embodiment, the intermolecular interaction may be between a receptor and a ligand, a receptor and a downstream signal transduction molecule, a receptor and a hormone, a DNA binding protein and a DNA oligonucleotide, an RNA binding protein and an RNA oligonucleotide, an enzyme and a substrate, a toxin and a receptor, a protease and a cleavage site oligonucleotide, an antigen and an antibody, two cell adhesion molecules, or two cytoskeletal proteins. In another embodiment, the intermolecular interaction may refer to an association of any of the above proteins with a protein that enhances binding or activity of the protein or with a protein that inhibits bind binding or activity of the protein. In another embodiment, the intermolecular interaction may involve proteins involved in the regulation of cellular events such as signal transduction, the cell cycle, protein trafficking, targeted proteolysis, cytoskeletal organization and gene expression.

[0051] In one embodiment, the environmental conditions such as salt concentration, pH, hydrophobicity, temperature and pressure may affect the nature of an intermolecular interaction, and those factors can be adjusted to the interaction of interest by a person skilled in the art.

Modified Cleavage Site

[0052] In one embodiment, the viruses of the present invention further comprise modified cleavage sites. In one embodiment, the term “modified cleavage site” refers to a cleavage site that has a reduced binding affinity to a compound mediating its cleavage. In one embodiment, a reduced binding affinity is a relative determination where binding affinity of the modified cleavage site is lower than that of the natural cleavage site. In one embodiment, the capacity of the modified cleavage site to be cleaved is similar to that of the natural cleavage site. Cleavage may be mediated by proteases or other compounds as will be described hereinbelow.

[0053] In one embodiment, the term “reduced binding affinity,” “significantly reduced binding affinity” or “reduced affinity”, means that the binding affinity of the compound mediating cleavage to the cleavage site is as low as possible using the methods existing in the art, such as ELISA, Gel Shift, Plasmon Resonance (BioCore) etc. In one embodiment, the reduced binding affinity is reflected by changes in off-rate, on-rate, free energy, interatomic distances, binding entropy or binding enthalpy.

[0054] In one embodiment, the modified cleavage site of the present invention is proximal to the peptide and protein segment portions of the invention. “Proximal” refers, in one embodiment, to a distance of between 1 and 1000 nucleotides between the modified cleavage site and a peptide or between the modified cleavage site and a protein involved in attachment or infection. In one embodiment, the modified cleavage site and a peptide are inserted in any order between the domain of a protein involved in attachment or infection that anchors the protein to the phage coat and the domain of said protein that mediates attachment. In another embodiment, the modified cleavage site and a peptide are inserted in any order between a domain of a protein that mediates attachment and a domain that mediates infection.

[0055] In one embodiment, the present invention also provides an oligonucleotide vector of a recombinant virus of the present invention as described hereinabove. In another embodiment, the present invention provides the use of an oligonucleotide vector of the present invention to prepare a recombinant virus.

[0056] In one embodiment, a recombinant virus is created by constructing a recombinant vector using standard recombinant techniques (see, for example, Maniatis, et al., Molecular Cloning, A laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al., 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996), both incorporated herein by reference) as is well know by one of skill in the art. In one embodiment, cells may be transfected with the vector, allowed to multiply, and DNA isolated therefrom. In another embodiment, viruses may be transduced with the vector.

[0057] In one embodiment, a recombinant virus of the present invention may be used in a method to identify a peptide or polypeptide that has an intermolecular interaction with a target of interest as described hereinbelow. In another embodiment, the recombinant virus may be used as part of a kit for identifying a peptide or polypeptide that has an intermolecular interaction with a target of interest, as described hereinbelow. In another embodiment, the recombinant virus may be used in a method to identify an agonistic feature, an antagonistic feature of a peptide or polypeptide that has an intermolecular interaction with a receptor of interest, as described hereinbelow. In another embodiment, the recombinant virus may be used in a method to identify a peptide or polypeptide that inhibits an enzyme of interest, as described hereinbelow. In another embodiment, the recombinant virus may be used in a method to identify a peptide or polypeptide that has a functional feature with a receptor of interest, as described hereinbelow.

Recombinant Virus Library

[0058] In one embodiment, a recombinant virus library of the present invention comprises a plurality of viruses as described hereinabove. In one embodiment, each virus of the virus library comprises a peptide that differs by at least one amino acid from a peptide expressed by another virus of the virus library. In one embodiment, the viruses of the present invention comprise peptides involved in an intermolecular interaction. The peptides can be in one embodiment, conveniently selected from any peptide library. In one embodiment, they are selected from random peptide libraries, in another embodiment, combinatorial peptide libraries, while in another embodiment, simulated molecular evolution peptide libraries.

[0059] In one embodiment, a random peptide library may generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all amino acids. In another embodiment, a bias is introduced into the library. For example, a bias that a lysine occurs every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine may be specified. Clearly, many types of biases can be contemplated, and this invention is not restricted to any particular bias.

[0060] In another embodiment, the peptide or polypeptide library is created as is well known in the art. For example, in one embodiment, a sample of genomic DNA is mechanically sheared or partly digested by restriction enzymes to form large fragments. In one embodiment, this population of overlapping DNA fragments may then be separated by gel electrophoresis to isolate a set of a particular length (15 kb for example). In one embodiment, synthetic linkers may be attached to the ends of these fragments, in one embodiment, cohesive ends may be formed, while in another embodiment, blunt ends may be formed.

[0061] In one embodiment, the fragments are then inserted into a vector. In one embodiment, the recombinant vector represents the virus of this invention. In one embodiment, the vector is a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome. In one embodiment, the vector is derived from a bacteriophage, which infects E. coli. In one embodiment, the vector is derived from a lambda phage, M13, fd, fl, or T4. Cells may be transfected with the vector, and DNA isolated therefrom. In one embodiment, cells are propagated so the library can be used repeatedly.

[0062] As described above, the virus library may utilize, in one embodiment, a recombinant plasmids or cosmids. In one embodiment, the term “plasmid” is meant to refer to an autonomously replicating extrachromosomal DNA molecule or other genetic particle, often, but not always, comprised of circular double-stranded DNA. Plasmids may become incorporated into the genome of the host or may remain independent. In one embodiment, the term “cosmid” is meant to refer to a hybrid plasmid that contains cos sites at each end. Cos sites are recognized during head filling of lambda phages. Cosmids are useful for cloning large segments of foreign DNA (up to 50 kb).

[0063] In another embodiment, the library comprises a plurality of polypeptides from a polypeptide expression library. The polypeptide expression library may be obtained, in one embodiment, from cDNA, fragmented genomic DNA, etc. In one embodiment, the library is a cDNA library comprising total poly A+RNA of a given organism. In one embodiment, the polypeptide is labeled using a label as described hereinbelow.

[0064] In one embodiment, the recombinant virus library may be used in a method to identify a peptide or polypeptide that has an intermolecular interaction with a target of interest as described hereinbelow. In another embodiment, the library may be used as part of a kit for identifying a peptide or polypeptide that has an intermolecular interaction with a target of interest, as described hereinbelow. In another embodiment, the library may be used in a method to identify an agonistic feature, an antagonistic feature of a peptide or polypeptide that has an intermolecular interaction with a receptor of interest, as described hereinbelow. In another embodiment, the library may be used in a method to identify a peptide or polypeptide that inhibits an enzyme of interest, as described hereinbelow.

Host Cells

[0065] In one embodiment, the invention comprises a cell comprising a recombinant virus of the present invention as described hereinabove. In another embodiment, the invention comprises a plurality of cells comprising a recombinant virus library of the present invention as described hereinabove. “A plurality of cells” is meant to indicate more than one cell. Cell types may include but are not limited to mammalian, insect, plant, or bacterial cells.

[0066] In one embodiment, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In one embodiment, all of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In one embodiment, host cells will have been engineered to express a screenable or selectable marker which is activated by the transcription factor that is part of a fusion protein.

[0067] In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. When host cells are “transfected”, “transformed”, or “transduced” with nucleic acid molecules, they are referred to as “engineered” or “recombinant” cells or host cells, e.g., a cell into which an non-endogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally-occurring cells which do not contain a recombinantly introduced nucleic acid. In one embodiment, “non-endogenous” refer to a molecule that it is foreign to the cell into which it is being introduced. In one embodiment, the non-endogenous molecule is DNA. In another embodiment, non-endogenous refers to a nucleic acid sequence that is homologous to a sequence in the cell but is in a position within the host cell nucleic acid in which the sequence is ordinarily not found.

[0068] Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Cell types available for vector replication and/or expression include, but are not limited to, bacteria, such as E. coli (e.g., E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), DHSα, JM109, and KC8, bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas species, as well as a number of commercially available bacterial hosts such as SURER Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE, La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses. In one embodiment, E. Coli may be used as a host cell. Useful strain of E. Coli may be determined using Genbank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) by one skilled in the art.

[0069] Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC 12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

[0070] In another embodiment, the present invention also provides a plurality of oligonucleotide vectors of the recombinant virus library of the present invention as described hereinabove. In another embodiment, the present invention provides the use of oligonucleotide vectors of the present invention to prepare a recombinant virus library.

[0071] In one embodiment, a recombinant virus library of the present invention may be used in a method to identify a peptide or polypeptide that has an intermolecular interaction with a target of interest as described hereinbelow. In another embodiment, the recombinant virus library may be used as part of a kit for identifying a peptide or polypeptide that has an intermolecular interaction with a target of interest, as described hereinbelow. In another embodiment, the recombinant virus library may be used in a method to identify an agonistic feature, an antagonistic feature of a peptide or polypeptide that has an intermolecular interaction with a receptor of interest, as described hereinbelow. In another embodiment, the recombinant virus library may be used in a method to identify a peptide or polypeptide that inhibits an enzyme of interest, as described hereinbelow. In another embodiment, the recombinant virus library may be used in a method to identify a peptide or polypeptide that has a functional feature with a receptor of interest, as described hereinbelow.

Target of Interest Complex

[0072] In another embodiment, the invention provides a protein-protein or protein-non-protein complex (referred to herein as “Target of interest (TOI) complex”) comprising a protease or functional domain thereof, a target of interest involved in an intermolecular interaction, and a flexible linker that attaches the protease and target of interest.

Protease

[0073] In one embodiment, the cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for said site, as compared to a non-modified cleavage site. In one embodiment, a compound that cleaves DNA is a protease or other enzyme. “Protease” refers to the enzymes included under E.C.3.4. (Cf. Kirk-Othmer, Encyclopedia of Chemical Technology, 3rd edition, volume 9, pages 173-223, J. Wiley 1980; E. Pfleiderer and R. Reiner in H. J. Rehm & G. Reed, Biotechnology, volume 6b, pages 729-742, VCH 1988; K. Aunstrup in Industrial Aspects of Biochemistry, B. Spencer, editor, volume 30(I), pages 23-46, North Holland 1974). Proteases are well characterized enzymes that cleave other proteins at a particular site. One family, the Ser/Thr proteases, cleave at serine and threonine residues. Other proteases include cysteine or thiol proteases, aspartic proteases, metalloproteinases, aminopeptidases, di & tripeptidases, carboxypeptidases, and peptidyl peptidases. The choice of these is left to the skilled artisan and certainly need not be limited to the molecules described herein. It is well known that enzymes have catalytic domains and these can be used in place of full length proteases. Such are encompassed by the invention as well. One preferred embodiment is the ubiquitin binding protein-1 protease, or an active portion thereof. Other specific cleavage sites for proteases may also be used, as will be clear to the skilled artisan.

[0074] The proteases of the invention may be, for example, specific ubiquitin protease UBP1, aspartyl protease, herpes protease, herpes simplex 1 protease, retroviral protease, cysteine protease, matrix metalloproteinase, interstitial collagenase (MMP-1), gelatinase A (MMP-2) and gelatinase B (MMP-9); serine proteases such as plasminogen activator (PA) and the like. However, the methods of the present invention are not limited to these particular proteases. Endogenous proteolytic enzymes provide a variety of useful functions, including the degradation of invading organisms, antigen-antibody complexes, and certain tissue proteins that are no longer necessary. The serine proteases comprise a large family of enzymes that use an activated serine residue in the substrate-binding site to catalytically hydrolyze peptide bonds the aspartate-specific cysteine proteases (ASCPs).

[0075] In one embodiment, any member of a particular family of proteases may be used in place of the protease specifically described. Further, any member of a particular family of proteases may be used on a particular modified cleavage site.

[0076] In one embodiment, there is provided a recombinant virus library in which at least one virus comprises a peptide and a modified cleavage site located proximally to a protein or protein fragment, comprising segments which are involved in viral attachment, infection, or a combination thereof and a protein or protein fragment, comprising segments that serve as an anchor. In another embodiment, there is provided a recombinant virus library in which at least one virus comprises a peptide and a modified cleavage site located between a protein or protein fragment, comprising segments which are involved in viral attachment, infection, or a combination thereof and a protein or protein fragment, comprising segments that serve as an anchor. An “anchoring protein or fragment” refers to a protein or fragment that connects another protein or fragment to a larger body such as a cell wall or cell membrane.

Linkers

[0077] In one embodiment, all components of the TOI complex are separated by long flexible linkers or spacers to enable a good degree of freedom that would allow the components of the system to interact with each other. In one embodiment, the linkers allow structural folding, while in another embodiment, they allow hydrogen bonding.

[0078] The linkers can be, without limitation, leucine zipper, SH2 domains, PDZ domains, antibody domains, and the like. Any other flexible linker could be used as well. In one embodiment, the flexible linker is at least 3 amino acids in length. In another embodiment the flexible linker is at least 5 amino acids in length. In another embodiment the flexible linker is at least 7 amino acids in length. In another embodiment the flexible linker is at least 9 amino acids in length. In another embodiment the flexible linker is at least 13 amino acids in length. In another embodiment the flexible linker is at least 15 amino acids in length. In another embodiment the flexible linker is at least 18 amino acids in length. In another embodiment the flexible linker is between 1 and 20 amino acids in length. In another embodiment the flexible linker is between 3 and 15 amino acids in length. In another embodiment the flexible linker is between 3 and 10 amino acids in length. In another embodiment the flexible linker is between 5 and 10 amino acids in length. In another embodiment the flexible linker is between 10 and 15 amino acids in length. In another embodiment the flexible linker is between 15 and 20 amino acids in length. In another embodiment, the target of interest is bound directly to a protease without a linker.

Target of Interest

[0079] In one embodiment, the TOI complex of the present invention comprises a target of interest. The target of interest is in one embodiment, a receptor. In one embodiment, the term “receptor” refers to receptors that bind peptide messengers with high affinity and regulate intracellular signals which influence the behavior of cells. In another embodiment, the target of interest is an agonist. In another embodiment, the target of interest is an antagonist. In another embodiment, the target of interest is an antibody. In another embodiment, the target of interest is an antigen. In another embodiment, the target of interest is an enzyme. In another embodiment, the target of interest is an enzyme-activating substrate. In another embodiment, the target of interest is an inhibitor. In another embodiment, the target of interest is a hormone. In another embodiment, the target of interest is a cytoskeletal protein. In another embodiment, the target of interest is a toxin. In another embodiment, the target of interest is synthetic or physiological polymer. In another embodiment, the target of interest is a small organic molecule. In another embodiment, the target of interest is a DNA sequence. In another embodiment, the target of interest is a RNA sequence. In another embodiment, the target of interest is an oligonucleotide. In another embodiment, the target of interest is a transcriptional activator or repressor. In another embodiment, the target of interest is a translational activator or repressor. In another embodiment, the target of interest is a functional fragment of an agonist, antagonist, receptor, antibody, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, synthetic or physiological polymer, small organic molecule, DNA, RNA, oligonucleotide, transcriptional activator or repressor, translational activator or repressor.

[0080] In another embodiment, the target of interest is a carrier. In another embodiment, the target of interest is an information protein. Information proteins are proteins involved in DNA replication, repair, recombination and transcription. In another embodiment, the target of interest is a structural protein. Structural proteins are fibrous proteins, including keratins, actin, myosin, and collagens involved building components inside the cell and around it. In another embodiment, the target of interest is a regulatory protein. Regulatory proteins command the numerous interactions that govern the expression and replication of genes, the performance of enzymes, the interplay between cells and their environment, and many other manifestations. In another embodiment, the target of interest is a DNA or RNA binding peptide.

[0081] In one embodiment, the target materials may be organic macromolecules, such as polypeptides, lipids, polynucleic acids, and polysaccharides, but are not so limited. Almost any molecule that is stable in aqueous solvent may be used as a target. The following list of possible targets is given as illustration and not as limitation. The categories are not strictly mutually exclusive. The omission of any category is not to be construed to imply that said category is unsuitable as a target. Merck Index refers to the Eleventh Edition.

[0082] In one embodiment, the target of interest may be a peptide such as human β endorphin (Merck Index 3528), dynorphin (MI 3458), Substance P (MI 8834), Porcine somatostatin (MI 8671), human atrial natriuretic factor (MI 887), human calcitonin, or glucagons. In another embodiment, the target of interest may be a hormone such as human TNF (MI-9411), Interleukin-1 (MI 4895), Interferon-.lambda. (MI 4894), Thyrotropin (MI 9709), Interferon-α (MI 4892), or Insulin (MI 4887, p. 789). In another embodiment, the target of interest may be an enzyme such as human neutrophil elastase, Human thrombin, human Cathepsin G, human tryptase, human chymase, human blood clotting Factor Xa, any retro-viral Poi protease, any retro-viral Gag protease, dihydrofolate reductase, Pseudomonas putida cytochrome P450CAM, human pyruvate kinase, E. Coli pyruvate kinase, jack bean urease, or aspartate transcarbamylase (E. coli), ras protein, or any protein-tyrosine kinase. In another embodiment, the target of interest may be an inhibitor such as aprotinin (MI 784), human el-anti-trypsin, or phage .lambda. cI (inhibits DNA transcription). In another embodiment, the target of interest may be a receptor, such as TNF receptor, IgE receptor, LamB, CD4, or IL-1 receptor. In another embodiment, the target of interest may be a toxin such as ricin (also an enzyme), a Conotoxin GI, mellitin, Bordetella pertussis adenylate cyclase (also an enzyme), or Pseudomonas aeruginosa hemolysin. In another embodiment, the target of interest may be another protein such as horse heart myoglobin, human sickle-cell haemoglobin, human deoxy haemoglobin, human CO haemoglobin, human low-density lipoprotein (a lipoprotein), human IgG (combining site removed or blocked) (a glycoprotein), influenza haemagglutinin, phage .lambda. capsid, fibrinogen, HIV-1 gp120, Neisseria gonorrhoeae pilin, fibril or flagellar protein from spirochaete bacterial species such as those that cause syphilis, Lyme disease, or relapsing fever, or pro-enzymes such as prothrombin or trypsinogen. In another embodiment, the target of interest may be an insoluble protein such as silk, human elastin, keratin, collagen, or fibrin. In another embodiment, the target of interest may be a nucleic acid such as DNA, RNA, yeast Phe tRNA, ribosomal RNA, or a segment of mRNA. In another embodiment, the target of interest may be an organic monomer (not peptide, protein, or nucleic acid) such as cholesterol, aspartame, bilirubin, morphine, codeine, heroine, dichlorodiphenyltrichlorethane (DDT), prostaglandin PGE2, actinomycin, 2,2,3 trimethyldecane, Buckminsterfullerene, or cortavazol (MI 2536, p. 397). In another embodiment, the target of interest may be an organic polymer such as cellulose or chitin. In another embodiment, the target of interest may be 0-antigen of Salmonella enteritidis (a lipopolysaccharide). In another embodiment, the target of interest may be an inorganic compound such as asbestos, zeolites, hydroxylapatite, 111 face of crystalline silicon, paulingite, U (IV) (uranium ions), or Au(III) (gold ions). In another embodiment, the target of interest may be an organometallic compound such as iron(III) haem, cobalt haem, cobalamine, or (isopropylamino)6 Cr(III).

[0083] In one embodiment, the peptide or polypeptide may represent a cognate peptide of any of the peptides or polypeptides described herein. In another embodiment, said target of interest has one or more intermolecular interactions with a cognate ligand, antigen, enzyme substrate, enzyme, regulatory protein, or cytoskeletal protein expressed on the surface of a recombinant virus. An “enzyme substrate” is a substrate on which an enzyme acts to catalyze a reaction. In another embodiment, said target of interest has one or more intermolecular interactions with an agonist, antagonist, antigen, enzyme activator, enzyme inhibitor, hormone, regulatory protein, toxin, or a functional fragment thereof.

[0084] In one embodiment, the target of interest is a peptide, which has approximately 6 to 60 amino acid residues. In another embodiment, a peptide target of interest has approximately 20-100 amino acids, or in another embodiment 20-50 amino acids. In the case of a bile acid receptor, for example, the target of interest may be a bile acid, such as cholic acid or cholesterol, and may have a molecular weight of about 300 to about 600 kDa. If the functional domain relates to transcriptional control, the target of interest may be a portion of a transcriptional factor, which may bind to a region of a gene of interest or to an RNA polymerase. The target of interest may even be a nucleoside analog, such as cordycepin or the triphosphate thereof, capable of inhibiting RNA biosynthesis. The target of interest may also be the carbohydrate portion of a glycoprotein, which may have a selective affinity for the asialoglycoprotein receptor, or the repeating glucan unit that exhibits a selective affinity for cellulose binding domain or the active site of heparinase.

[0085] In one embodiment, the target of interest may refer to a functional domain of a target of interest. In another embodiment, the functional domain may include a ligand binding domain, an activation domain, or any other domain of a target of interest.

[0086] In one embodiment, the target of interest is labeled. In another embodiment, the TOT complex additionally comprises a tag that allows affinity purification. In another embodiment, the tag is a His-Tag.

[0087] As described hereinabove, vectors include plasmids, cosmids, viruses (bacteriophage, mammalian viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis, et al., Molecular Cloning, A laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al., 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996), both incorporated herein by reference).

[0088] The term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleotide sequences that serve other functions as well and are described infra.

[0089] In one embodiment, the TOI complex of the present invention is produced by methods known in the art. In one embodiment, the TOI complex is produced by in vitro translation. In certain embodiments, a plasmid vector is contemplated for use in cloning and gene transfer. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides an easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.

[0090] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM™-11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.

[0091] Bacterial host cells, for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.

[0092] In another embodiment, prokaryotic vectors can be used to transform eukaryotic host cells. However, it may be desirable to select vectors that have been modified for the specific purpose of expressing proteins in eukaryotic host cells. Expression systems hare been designed for regulated and/or high level expression in such cells. For example, the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM from CLONTECH®.

[0093] Other examples of expression systems include STRATAGENE®'S COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0094] The construct may contain additional 5′ and/or 3′ elements, such as promoters, enhancers, poly A sequences, and so forth. The elements may be derived from the host cell, i.e., homologous to the host, or they may be derived from distinct source, i.e., heterologous. It is to be understood that various elements of the vector may be manipulated such as, for example, promoters, enhancers, high copy or low copy, etc as will be understood by one skilled in the art. Any manipulation of the vector is considered to be part of the present invention.

[0095] Vectors can include a multiple cloning site (MCS), donor and/or acceptor splicing sites, termination signals, polyadenylation sites, etc., as are well known to those of skill in the art of recombinant technology.

[0096] In order to propagate a vector in a host cell, it may contain one or more origins of replication (often termed “ori”) sites′, which are specific nucleotide sequences at which replication is initiated. Alternatively, an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

Transformation Methodology

[0097] Suitable methods for nucleic acid delivery for use with the current invention are believed to include virtually any method by which a nucleic acid molecule (e.g., DNA) can be introduced into a cell as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, including microinjection; by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor-mediated transfection; by PEG-mediated transformation of protoplasts; by desiccation/inhibition-mediated DNA uptake, and any combination of such methods, as are well known to those skilled in the art.

[0098] In one embodiment, TOI complexes comprising non-protein components, once created by any method known in the art, are tested for binding specificity using any of the methods known in the art including but not limited to electrophoretic mobility shift assay (EMSA, Gel Shift Assay, or Band Shift Assay), footprinting, and methylation interference with a known ligand of the target of interest as a probe. In another embodiment, TOI complexes comprising protein components, once created by any method known in the art, are tested for binding specificity using any of the methods known in the art including but not limited to co-immunoprecipitation, yeast two-hybrid, density gradient centrifugation, GFP tagging (fluorescence resonance energy transfer (FRET)), protein affinity chromatography, protein arrays, surface plasmon resonance (SPR) and GST pulldown assay with a known ligand of the target of interest as a probe. These same methods will be used to confirm the interaction of the target of interest and a peptide expressed by a recombinant virus.

[0099] In another embodiment, the present invention provides the use of any of the peptides, polypeptides, proteins, protein fragments, and TOI complexes of the present invention in executing the methods of this invention. In another embodiment, the TOI complexes are

[0100] In one embodiment, a TOI complex of the present invention may be used in a method to identify a peptide or polypeptide that has an intermolecular interaction with a target of interest as described hereinbelow. In another embodiment, the TOI complex may be used as part of a kit for identifying a peptide or polypeptide that has an intermolecular interaction with a target of interest, as described hereinbelow. In another embodiment, the TOI complex may be used in a method to identify an agonistic feature, an antagonistic feature of a peptide or polypeptide that has an intermolecular interaction with a receptor of interest, as described hereinbelow. In another embodiment, the TOI complex may be used in a method to identify a peptide or polypeptide that inhibits an enzyme of interest, as described hereinbelow. In another embodiment, the TOI complex may be used in a method to identify a peptide or polypeptide that has a functional feature with a receptor of interest, as described hereinbelow.

Method of Identifying a Peptide with an Intermolecular Interaction

[0101] In another embodiment, this invention provides a method of identifying a peptide or polypeptide having an intermolecular interaction with a target of interest by employing a recombinant virus or recombinant virus library of the present invention with a TOI complex also of the present invention or any embodiment thereof. The method comprises the steps of: contacting a recombinant virus library as exemplified hereinabove with a TOI complex as exemplified hereinabove, contacting said library with a plurality of cells, isolating viruses that did not infect said cells, providing infectious clones of isolated viruses by amplifying and expressing the genomes of said isolated viruses, repeating the above steps, and identifying peptides expressed by said viruses whereby intermolecular interactions between said target of interest and a peptide expressed by said recombinant virus result in said protease being in close proximity to said cleavage site, resulting in cleavage of said protein or protein fragment, comprising segments which are involved in viral attachment to, infection of, or a combination thereof of cells, and prevention of entry of a virus comprising a peptide involved in intermolecular interactions into said cells.

[0102] In one embodiment, the method is predicated on the fact that viruses are inhibited or prevented from entry into a host cell as a result of the cleavage of their pIII protein as depicted in FIGS. 1 and 4. In one embodiment, if a protease were to cleave the pIII proteins of a bacteriophage at a site between the N2 and CT domains of pIII, the M13 bacteriophage would not be infective. In another embodiment, if a protease were to cleave the pIII proteins of a bacteriophage at a site between the N1 and N2 domains of pIII, the M13 bacteriophage would not be infective. In one embodiment, the pIII protein cleavage is mediated by a protease that was brought into proximity of its cleavage site by the interaction of a target of interest proximal to the protease with a peptide proximal to the modified cleavage site on the pIII protein of the virus as depicted in FIG. 2.

[0103] The steps are repeated for any number of cycles deemed sufficient to enrich the fraction for recombinant viruses that harbor peptides that bind to a target of interest and until there is a negligible number of background clones. In one embodiment, background clones refers to clones which harbor peptides that do not bind to a target of interest. In one embodiment, the number of cycles ranges between 3 and 50.

[0104] In one embodiment, the identified peptide that has an intermolecular interaction with the target of interest is isolated. In one embodiment, the isolated viruses which have been prevented from entering into cells are found in the medium. The step of the isolation can be performed using methods that are well known in the art. In one embodiment the separation is performed by filtration. In another embodiment the separation is performed by spinning down, for example in a centrifuge. In another embodiment, the separation can be conducted by selectively providing conditions for the maintenance of the infected cells or, alternatively, conditions for the maintenance of the non-infective viruses.

[0105] In another embodiment, the isolated viruses which have not infected cells are attached to the surface of the cells, but have not entered said cells. In one embodiment, the present invention further provides a recombinant virus, including a bacteriophage, comprising a label such that the label can be directly detected. The recombinant virus can express on its surface a peptide that has an intermolecular interaction with a selected protein. By “directly detected” is meant that the recombinant virus be labeled in advance and still comprise a peptide that binds its target upon addition of the target. Furthermore, by “label” is meant a means for visualization, such as a recognition site for direct phosphorylation, biotinylation, chemical linkages, etc. engineered into the recombinant virus, or such as a directly visualized label requiring no chemical reaction to detect, e.g., the recombinant virus expresses a fluorescent protein or is labeled by a radioactive moiety. Recombinant virus can be modified to include a label in advance, allowed to interact with the fusion protein of the present invention, be exposed to cells, and the cells separated from free recombinant virus in solution. Recombinant virus can then be visualized to detect the presence and localization of the recombinant virus, whether in the solution or in the cell fraction. Visualization may or may not require a chemical reaction. An example of a fluorescent protein is the green fluorescent protein (GFP) originally isolated from the jellyfish Aequorea Victoria. Another example of a fluorescent protein is the green fluorescent protein originally isolated from Renilla reniforms, which demonstrates a single absorption peak at 498 nm and an emission peak at 509 nm. (Cubitt, et el. (1995) TUBS 20: 448-455).

[0106] The amino acid sequence of the identified peptide can be determined directly by conventional means of amino acid sequencing, or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently by use of standard DNA sequencing methods. The primary amino acid sequence can then be deduced from the corresponding DNA sequence.

[0107] If the amino acid sequence is to be determined from the polypeptide itself, one may use micro sequencing techniques. The sequencing technique may include mass spectroscopy.

[0108] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and) (BLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17): 3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0109] In the method of the present invention, the recombinant virus library is contacted with a plurality of cells. The step of “contacting” or “introducing” refers hereinafter to bringing a solution comprising at least one element, (e.g., the recombinant virus) in contact with another element (e.g., the cells or a composition which comprise the cells).

[0110] In one embodiment, the recombinant virus library of the method of identifying a peptide or polypeptide with an intermolecular interaction with a target of interest is produced in a phage. In another embodiment, the recombinant library is produced in M13 bacteriophage.

[0111] In another embodiment, the recombinant viruses comprise peptides with mutations that abrogate binding to target of interest.

[0112] In one embodiment, the term “mutation” refers to an insertion, deletion, or substitution of one or more natural or wild type nucleic acids for alternate nucleic acids. In one embodiment, the term “abrogate” means to abolish, do away with, or annul.

Epitope Mapping

[0113] In another embodiment, the peptide identified by this invention can serve for epitope mapping of the target of interest. “Epitope mapping” refers to methods used for studying the interactions of antibodies with specific regions of protein antigens.

[0114] In one embodiment, the polypeptide is labeled. In another embodiment, the target of interest is labeled. A wide range of labels can be used, including but not limited to conjugating the target to biotin by conventional means. Alternatively, the label may comprise a fluorogen, an enzyme, an epitope, a chromogen, or a radionuclide. The detection means employed to detect the label will depend on the nature of the label and are known in the art, e.g., film to detect a radionuclide; an enzyme substrate that gives rise to a detectable signal to detect the presence of an enzyme; antibody to detect the presence of an epitope, etc.

Solid Support

[0115] In one embodiment, the target is not bound to a solid support and is added to the medium by, for example, contacting the solution of the recombinant virus with a solution that comprises the target of interest. In another embodiment, the target of interest of this invention may be bound to a solid support.

Kit

[0116] In another embodiment, this invention provides a kit for identifying a peptide or polypeptide involved in an intermolecular interaction with a target of interest comprising a recombinant virus peptide library of the present invention as exemplified hereinabove, a TOI complex of the present invention as exemplified hereinabove, and cells that are susceptible to viral attachment, infection or a combination thereof. In one embodiment, the kit of the present invention comprises a virus of the present invention, a library of the present invention, a TOI complex of the present invention or any combination thereof.

[0117] Any of the compositions described herein may be comprised in a kit. The kits will thus comprise, in suitable container means for the vectors or cells of the present invention, and any additional agents that can be used in accordance with the present invention. In one embodiment, any container of the kit will additionally comprise a preservative, which, in one embodiment, will increase the “shelf life” of the kit component or components to which it is added.

[0118] The kits may comprise suitably aliquoted compositions of the present invention. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.

[0119] The kits of the present invention also will typically include a means for containing reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0120] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0121] The kit optionally further comprises a means for detecting the presence of an interaction between a polypeptide and a target of interest or the absence of an interaction thereof.

[0122] In one embodiment, the recombinant virus library of the kit for identifying a peptide or polypeptide with an intermolecular interaction with a target of interest is produced in a phage. In another embodiment, the recombinant library is produced in M13 bacteriophage. In another embodiment, the recombinant viruses of the kit comprise peptides with mutations that abrogate binding to target of interest. In another embodiment, the peptide identified by said kit can be used for epitope mapping of the target of interest. In another embodiment, the target of interest of the kit is not bound to a solid support.

Identifying an Agonist or Antagonist

[0123] It should be likewise be apparent that the function of polypeptides can be identified, i.e. this invention enables, in one embodiment, to determine whether the peptide is an agonist or an antagonist to a receptor or if it is an inhibitor of an enzyme. In one embodiment, this invention provides a method of identifying a peptide or polypeptide that has an agonistic or an antagonistic effect on the target of interest of the invention by employing a recombinant virus or recombinant virus library of the present invention with a TOI complex also of the present invention or any embodiment thereof.

[0124] It should likewise be apparent that a wide range of peptides or polypeptides that have an agonistic or an antagonistic effect on the target of interest of the invention, can be identified by the process of the invention, which comprises: (a) contacting the recombinant virus library as described hereinabove with a plurality of complexes as described hereinabove wherein the target of interest is a receptor; (b) contacting said recombinant virus library of step (a) with cells; (c) isolating viruses in (b) which have not infected said cells; (d) providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses; (e) contacting infectious clones of viruses of step (c) with the receptor of interest, wherein said receptor is not attached to a protease; (f) contacting the viruses of step (e) with a protein attached to a protease via a flexible linker, wherein said protein is involved in the downstream signal transduction pathway of said receptor of interest; (g) contacting the viruses of step (f) with cells; (h) separating viruses in (g) that have not infected said cells from viruses which have infected cells; (i) providing infectious clones of second isolated viruses of step (h) by amplifying and expressing the genomes of said second isolated viruses; (j) repeating steps (a)-(i); and (k) identifying peptides expressed by the viruses in step (h), whereby viruses which have not infected said cells in (h) express a peptide which has agonistic activity for said receptor of interest, and viruses which have infected said cells in (h) express a peptide which has antagonistic activity for said receptor.

[0125] Knowing that a polypeptide exhibits a selective affinity to a target of interest, one may attempt to identify a drug that can exert an effect on the polypeptide-target of interest interaction, e.g., either as an agonist or as an antagonist of the interaction. With this assay, one can screen a collection of candidate “drugs” for the one exhibiting the most desired characteristic, e.g., the most efficacious in reverting the required vital process or function and whether it is an agonist or an antagonist to the target of interest.

[0126] In one embodiment, the term “agonist” refers to an endogenous substance or drug that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.)

[0127] In one embodiment, the term “antagonist” refers to an endogenous substance or drug that blocks or nullifies an action of another endogenous substance or drug, such as a drug that binds to a receptor without initiating a physiological or a pharmacological response characteristic of that receptor.

[0128] In order to isolate a peptide which has an antagonistic feature, the medium further comprises a ligand, which naturally binds the receptor of interest, and other components, may be added if required for the activation of the receptor, for example without limitation calcium, sodium, and magnesium ions, ATP, EDTA, DTT, etc.

[0129] In one embodiment, the receptor of interest may be any receptor from the following families, including subtypes thereof: 5-Hydroxytryptamine, Acetylcholine (muscarinic), Adenosine, Adrenoceptors, Anaphylatoxin, Angiotensin, Apelin, Bombesin, Bradykinin, Cannabinoid, Chemokine, Cholecystokinin, Dopamine, Endothelin, Free fatty acid, G protein-coupled bile acid, Galanin, Motilin, Ghrelin, Glycoprotein hormone, GnRH, Histamine, KiSS1-derived peptide, Leukotriene and lipoxin, Lysophospholipid, Melanin-concentrating hormone, Melanocortin, Melatonin, Neuromedin U, Neuropeptide FF/neuropeptide AF, Neuropeptide S, Neuropeptide W/neuropeptide B, Neuropeptide Y, Neurotensin, N-Formylpeptide family, Nicotinic acid, Opioid, Opsin-like, Orexin (hypocretin), P2Y, Peptide P518, Platelet-activating factor, Prokineticin, Prolactin-releasing peptide, Prostanoid, Protease-activated, Relaxin, Somatostatin, SPC/LPC, Tachykinin, Trace Amine, TRH, Urotensin, Vasopressin/oxytocin, OrphanA1, OrphanA2, OrphanA3, OrphanA4, OrphanA6, OrphanA7, OrphanA9, OrphanA12, OrphanA13, OrphanA14, OrphanA15, OrphanLGR, OrphanSREB, Orphan, Orphan (chemokine receptor-like), Orphan (Mas-related), Orphan (melatonin-like), Orphan (P2Y-like), Orphan (trace amine-like), or Other orphan genes. In another embodiment, the receptor of interest may be any receptor from the following families, including subtypes thereof: Calcitonin receptor family, CRF receptor family, CRF receptor family, Glucagon receptor family, PTH receptor family, VIP/PACAP, LNB7TM, LNB7TM:Brain specific angiogenesis inhibitor, LNB7TM:Proto-cadherin, LNB7TM:EGF, mucin-like receptor, LNB7TM, or LNB7TM:Latrophilin substrate. In another embodiment, the receptor of interest may be any receptor from the following families, including subtypes thereof: GABAB, Metabotropic glutamate, Calcium sensor, GPRC5, or Unclassified.

[0130] In another embodiment, the receptor of interest may be TNF receptor, IgE receptor, LamB, CD4, or IL-1 receptor, any of the serotonergic receptors, cholinergic receptors, rhodopsin receptor, thyroid-stimulating hormone receptor, follicle-stimulating hormone receptor, any of the odorant receptors, and parathyroid hormone receptor. In another embodiment, the receptor of interest may be a nuclear receptor such as an estrogen receptor, progesterone receptor, androgen receptor, thyroid receptor, Vitamin D receptor, glucocorticoid receptor, mineralocorticoid receptor, peroxisome proliferator-activated receptor, insulin receptor, gonadotrophin-releasing hormone receptor, or cathepsin D receptor.

[0131] In another embodiment, the protein involved in the downstream signal transduction pathway of the receptor of interest is a guanine nucleotide binding protein (G protein) or any subunit thereof, a small GTPase, a cyclic nucleotide such as cyclic AMP or cyclic GMP, calcium, phosphoinositide derivatives such as phosphatidylinositol-triphosphate (PIP3), Diacylglycerol (DAG) or inositol-triphosphate (IP3), or various protein kinases or phosphatases. In another embodiment, the protein involved in the downstream signal transduction pathway of the receptor of interest is nitric oxide, carbon oxide, ceramide, or lysophosphatic acid.

Identifying an Enzyme Inhibitor

[0132] In another embodiment, this invention provides a method for identifying a peptide or polypeptide that inhibits an enzyme of interest by employing a recombinant virus or recombinant virus library of the present invention with a TOI complex also of the present invention or any embodiment thereof. In another embodiment, this invention provides a method for identifying a peptide or polypeptide that inhibits an enzyme of interest comprising the steps of: (a) contacting the recombinant virus library as described hereinabove with a plurality of complexes as described hereinabove wherein the target of interest is an enzyme; (b) contacting said recombinant virus library of step (a) with cells; (c) isolating viruses in (b) which have not infected said cells; (d) providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses; (e) contacting infectious clones of viruses of step (c) with the enzyme of interest, wherein said enzyme is not attached to a protease; (f) contacting the viruses of step (e) with a substrate of the enzyme attached to a protease via a flexible linker; (g) contacting the viruses of step (f) with cells; (h) separating viruses in (g) which have not infected said cells from viruses which have infected cells; (i) providing infectious clones of second isolated viruses of step (i) by amplifying and expressing the genomes of said second isolated viruses; (j) repeating steps (a)-(i); and (k) identifying peptides expressed by the viruses in step (h), whereby viruses which have not infected said cells in (h) express a peptide which does not affect said enzyme of interest, and viruses which have infected said cells in (h) express a peptide which inhibits said enzyme.

Identifying a Peptide with a Functional Feature

[0133] In another embodiment, this invention provides a method of identifying a peptide or polypeptide which has a functional feature with a receptor of interest by employing a recombinant virus or recombinant virus library of the present invention with a TOI complex also of the present invention or any embodiment thereof. In another embodiment, this invention provides a method of identifying a peptide or polypeptide which has a functional feature with a receptor of interest comprising the steps of: (a) contacting the recombinant virus library as described hereinabove with a plurality of complexes as described hereinabove wherein the target of interest is a receptor; (b) contacting said recombinant virus library of step (a) with cells; (c) isolating viruses in (b) which have not infected said cells; (d) providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses; (e) contacting infectious clones of viruses of step (c) with the receptor of interest, wherein said receptor is not attached to a protease; (f) contacting the viruses of step (e) with a ligand of the receptor attached to a protease via a flexible linker; (g) contacting the viruses of step (f) with cells; (h) separating viruses in (g) which have not infected said cells from viruses which have infected cells; (i) providing infectious clones of second isolated viruses of step (i) by amplifying and expressing the genomes of said second isolated viruses; (j) repeating steps (a)-(i); and (k) identifying peptides expressed by the viruses in step (h), whereby viruses which have not infected said cells in (h) express a peptide which does not have a functional feature, and viruses which have infected said cells in (h) express a peptide which has a functional feature with regards to said receptor of interest.

[0134] In one embodiment, the recombinant virus library of the method for identifying a peptide or polypeptide with an agonistic, antagonistic, enzyme inhibitory, or functional effect with respect to a target of interest is produced in a phage. In another embodiment, the recombinant library is produced in M13 bacteriophage. In another embodiment, the recombinant viruses of the methods comprise peptides with mutations that abrogate binding to target of interest. In another embodiment, the receptor or enzyme of interest is not bound to a solid support. In another embodiment, the isolated viruses which have not infected cells, either after the step of introducing the receptor or enzyme of interest attached to a protease or after the step of introducing the protein (downstream signal transducer or enzyme substrate) attached to a protease or both, are attached to the surface of the cells, but have not entered said cells.

[0135] This invention is also directed to a composition which can be useful in the screening of drug candidates. In one embodiment of the invention, this invention provides a composition comprising: a recombinant virus peptide library made in accordance with the embodiments of the present invention and a target of interest connected to a protease.

[0136] With this assay, one can screen a collection of peptides for the one that exhibits the most desired characteristic. For example, the peptide that is most efficacious in activating the target of interest may be selected. Alternatively, the peptide that is most efficacious in blocking, inhibiting or competing with a ligand, which binds and activates the target of interest, may be selected.

[0137] In one embodiment, an advantage to the present methodology may comprise functional selection, which is an inherent part of the assay, saving cumbersome and time-consuming screenings at later stages. In another embodiment, sporadic deletion mutants that occur in the selection process using conventional techniques are avoided in the technology of the present invention. In another embodiment, non-specific binding problems resulting from tagging targets to a solid matrix are avoided, because a solid support is not necessary for the technology. In another embodiment, transient interactions, such as enzymes with their substrates can be detected by this technology. Finally, in another embodiment, this invention allows superior detection of proteins with either low- or high-affinity binding to a target of interest.

[0138] It should be likewise be apparent that the function of the polypeptides can be identified, i.e. this invention enables, in one embodiment, to determine whether the polypeptide can block the action of a toxin or whether it has an enhancing or an inhibiting effect on other processes such as cellular, biochemical, or physiological processes, such as cellular signal transduction, transcriptional regulation, translational regulation, cell adhesion, migration or transport, cytokine secretion and other aspects of the immune response, and the like.

[0139] In one embodiment, the peptides identified by the present invention may be used for diagnosis of a disease or for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Accordingly, methods for treatment include the use of the identified protein or fragments thereof. In another embodiment, the peptides identified by the present invention may be used to identify a person who is vulnerable to or has a proliferative disorder, which in one embodiment, is a cancer. In another embodiment, may be used to identify a person who is vulnerable to or has an infection, in another embodiment, an HIV infection, in another embodiment, a developmental disorder, while in another embodiment, a metabolic disorder. In another embodiment, peptides identified by the present invention may be used to treat a person with cancer. In another embodiment, peptides identified by the present invention may be used to treat a person with a proliferative disorder, an infection, an HIV infection, a developmental disorder, or a metabolic disorder. In another embodiment, it may be used to suppress, inhibit, or prevent any of the disorders mentioned hereinabove.

SoAP Assay Containing a Sterically Inhibiting Group

[0140] In another embodiment, this invention provides a second recombinant virus comprising: at least one protein or protein fragment, comprising segments which are involved in a vital process; a peptide or polypeptide which differs by at least one amino acid from another peptide or polypeptide in said library; a modified cleavage site proximal to said peptide and to said protein; and a sterically inhibiting group, wherein said sterically inhibiting group inhibits the function of said protein essential for a vital process.

[0141] The invention also provides a second library comprising such recombinant viruses.

[0142] In one embodiment, this invention provides a replicable genetic package peptide library, such as a second recombinant virus library, for identifying potential new drug candidates and lead compounds. In another embodiment, the invention provides methods for identifying the agonistic or antagonistic features of a peptide identified as having an intermolecular interaction with a target of interest. In another embodiment, this invention provides compositions, which can serve in an assay kit, for screening peptides for their ability to bind to a target of interest. In another embodiment, this invention provides compositions for screening peptides for their agonistic or antagonistic features.

[0143] In one embodiment, the sterically inhibiting group comprises a selectable marker. The selectable marker can be any selectable marker, which is known in the art, such as antibiotic resistance protein, for example, without being limited, ampicillin. The selectable marker can be also used for deletion mutants counter selection, when there is a need to select cells that are infected by virus that did not lose essential genes. In another embodiment, the protein essential for vital cellular process is a protein required for survival, infectivity or propagation. In another embodiment, the modified cleavage site is inserted between said peptide and said at least one protein essential for infectivity or propagation.

[0144] In one embodiment, the present invention provides a replicable genetic package peptide library wherein each replicable genetic package comprises at least one protein or protein fragment, which is involved in a vital process; a peptide or polypeptide, which differs by at least one amino acid from another peptide or polypeptide in said library; a modified cleavage site proximal to said peptide and to said protein; and a sterically inhibiting group, wherein said sterically inhibiting group inhibits the function of said protein essential for a vital process.

[0145] The invention also provides a library of such replicable genetic packages.

[0146] In one embodiment, the selectable marker is a part of the sterically inhibiting group. In another embodiment, the selectable marker is an antibiotic resistance gene. In another embodiment, the protein essential for vital cellular process is a protein required for survival, infectivity or propagation. In another embodiment, the modified cleavage site is inserted between said peptide and said at least one protein essential for infectivity or propagation.

[0147] In another embodiment, the genetic package is for example, without limitation, eukaryotic cell, bacteria, a virus, or a phage.

[0148] In one embodiment, the present invention provides a method of identifying a peptide having intermolecular interaction with a target of interest comprising the steps of: (a) introducing a target of interest attached to a protease to said second recombinant virus library of claim 1; (b) contacting said second recombinant virus library with cells; and (c) detecting viruses that are propagating, wherein if a cell comprises a recombinant virus which contain a peptide that has affinity to the target of interest, the sterically inhibiting group will be cleaved off and the virus will propagate.

[0149] In one embodiment, said peptide is an agonist, an antagonist, an antigen, an enzyme activating substrate, an inhibitor, a DNA or RNA binding peptide, transcription or translation activator or repressor. In another embodiment, the target of interest is a receptor, an antibody, a carrier, an information protein, a hormone, a regulatory protein, a structural protein, a toxin, an enzyme, DNA, RNA, oligonucleotide, synthetic or physiological polymer or a small organic molecule. In another embodiment, said peptide is used for epitope mapping of the target of interest. In another embodiment, the recombinant virus library is in M13 bacteriophage. In another embodiment, the protein essential for infectivity or propagation is pIII. In another embodiment, the target of interest is not bound to a solid support.

[0150] In one embodiment, there is provided a method of identifying a peptide having an intermolecular interaction with a target of interest comprising the steps of: preparing a second recombinant virus library according to an embodiment of the invention, wherein at least portion of the second recombinant virus is inactivated by the sterically inhibiting group attached to a protein essential for a vital process, and the inactivation can be reverted upon the removal of the sterically inhibiting group; introducing a target of interest, which is attached to a protease to said second recombinant virus library; the cell culture will be enriched with cells infected by a second recombinant virus which comprises a peptide that has affinity to the target of interest, due to the removal of the sterically inhibiting group, and the second virus will propagate.

[0151] The method is based on the concept that only when the target of interest interacts with a peptide, the susceptibility of the modified protease site to proteolysis is reverted, and the sterically inhibiting group is removed and enables the protein which is essential to a vital process to function. It should be noted in this respect that the level of the proteolytic activity is dependent on the affinity between the target protein and the peptide of the second library which is attached to the modified protease site. The methods of the invention are not limited to one cycle. Rather, in one embodiment, the method is performed with one cycle. In another embodiment, the method is performed with two cycles. In another embodiment, the method is performed with three cycles. In another embodiment, the method is performed with four cycles. In another embodiment, the method is performed with five cycles. In another embodiment, the method is performed with seven cycles. In another embodiment, the method is performed with nine cycles.

[0152] In one embodiment, the present invention provides a method of identifying a peptide or polypeptide, which has intermolecular interaction with a target of interest comprising the steps of: introducing a target of interest contacted to a protease to said replicable genetic package of claim 6; and detecting the replicable genetic package which propagates or survives, wherein only if the replicable genetic package contains a peptide that has affinity to the target of interest, the sterically inhibiting group will be cleaved off and the replicable genetic package will be able to propagate or survive.

[0153] In one embodiment, said peptide is an agonist, an antagonist, an antigen, an enzyme activating substrate, an inhibitor, a DNA or RNA binding peptide, transcription or translation activator or repressor. In another embodiment, the target of interest is a receptor, an antibody, a carrier, an information protein, a hormone, a regulatory protein, a structural protein, a toxin or an enzyme, a DNA, an RNA, an oligonucleotide, a synthetic polymer or a small organic molecule. In another embodiment, said replicable genetic package is a eukaryotic cell, bacteria, a virus, or a phage. In another embodiment, the target of interest is not bound to a solid support.

[0154] In one embodiment, the present invention provides a composition for identifying a peptide that has intermolecular interaction with a target of interest comprising: the second recombinant virus peptide library described above; and a target of interest connected to a protease.

[0155] In one embodiment, the present invention provides a composition for identifying a peptide that has intermolecular interaction with a target of interest comprising: a replicable genetic package peptide library as described hereinabove and a target of interest connected to a protease.

[0156] In one embodiment, the present invention provides a peptide or polypeptide, which has an intermolecular interaction with a target of interest, isolated according to any of the methods described herein.

[0157] It should likewise be apparent that a wide range of polypeptides that have a function i.e. have an agonistic or an antagonistic effect on the target of interest of the invention, can be identified by the process of the invention. In one embodiment, the present invention provides a method of identifying an agonistic or antagonistic feature of a peptide, which has intermolecular interaction with a receptor of interest comprising the steps of: (a) contacting a receptor of interest connected to a protease, to said second recombinant virus library as described hereinabove; (b) contacting said second recombinant virus library with cells; (c) detecting the propagated viruses, wherein if a cell comprises a recombinant virus which comprises a peptide that has affinity to the to the target of interest the sterically inhibiting group will be cleaved off and the virus will propagate; (d) contacting the propagated viruses of step c with the receptor of interest; (e) contacting the viruses of step d with a protein attached to protease, wherein said protein is downstream in the signal transduction pathway of the receptor of interest; (f) contacting the viruses of step e with cells; and (g) separating the cells obtained in step f, so as to obtain infected cells and non infecting viruses, wherein if a virus infects a cell, it contains a peptide which has an agonistic feature and if a virus is a non infecting virus it contains a peptide which has an antagonistic feature, thereby identifying an agonistic or antagonistic feature of a peptide, which has intermolecular interaction with a receptor of interest.

[0158] In one embodiment, the present invention provides a method of identifying peptide which has a functional feature with a receptor of interest comprising the steps of: (a) contacting a receptor of interest connected to a protease, to the second recombinant virus peptide library as described hereinabove; (b) contacting said second recombinant virus library with cells; (c) detecting the propagated viruses, wherein if a cell comprises a second recombinant virus which comprises a peptide that has affinity to the target of interest, the sterically inhibiting group will cleave off enabling the virus propagation; (d) contacting the propagated viruses of step c with said receptor of interest; (e) contacting the viruses of step d with a ligand to the receptor of interest attached to the protease; (f) contacting the viruses of step e with host cells; and (g) separating the infected cells and the non infecting viruses obtain in step f, so as to obtain infected cells and non infecting viruses, wherein if a virus infects a cell, it contains a peptide which does not have functional feature and if the virus is a non infecting virus it contains a peptide which has a functional feature, thereby identifying a peptide which has a functional feature with a receptor of interest.

[0159] In one embodiment, the term “functional feature” refers hereinbelow to any function that the peptide might produce on the receptor of interest, namely agonistic reaction or antagonistic reaction.

[0160] In one embodiment, the present invention provides a method of identifying peptide that inhibits an enzyme of interest comprising the steps of: (a) contacting an enzyme of interest connected to a protease, to the second recombinant virus peptide library as described hereinabove; (b) contacting said second recombinant virus library with cells; (c) detecting the propagated viruses, wherein if a cell comprises a second recombinant virus, which comprises a peptide that has affinity to the enzyme of interest, the sterically inhibiting group will cleave off enabling the virus propagation; (d) contacting the viruses of step c with said enzyme of interest; (e) contacting the viruses of step d with a substrate of the enzyme, wherein said substrate is attached to a protease; (f) contacting the viruses of claim e with cells; and (g) separating said cells by spinning down or filtration, so as to obtain infected cells in the pellet or and non infecting viruses which contain a peptide which inhibits an enzyme inhibiting peptide in the supernatant; thereby identifying peptide which inhibits an enzyme of interest.

[0161] In one embodiment, the present invention provides a method of identifying a peptide that activates an enzyme of interest comprising the steps of: (a) contacting an enzyme of interest connected to a protease, to the second recombinant virus peptide library as described hereinabove; (b) contacting said second recombinant virus library with cells; (c) detecting the propagated viruses, wherein if a cell comprises a second recombinant virus, which comprises a peptide that has affinity to the enzyme of interest, the sterically inhibiting group will cleave off enabling the virus propagation; (d) contacting the viruses propagated in step c with said enzyme of interest; (e) contacting the viruses of step d with a substrate of the enzyme, wherein said substrate is attached to a protease; (f) contacting the viruses of claim e with cells; (g) detecting the propagated viruses, wherein if a cell comprises a recombinant virus, the virus comprises a peptide which activates the enzyme of interest, the sterically inhibiting group will cleave off enabling the virus propagation; and (h) separating the non infecting viruses from infected cells, thereby obtaining enriched population of peptides which activates an enzyme of interest.

[0162] The methods of identification the function of the identified peptide, its agonistic or antagonistic property, or whether it activates or inhibits an enzyme are not limited to second recombinant virus libraries. The methods may be applied in any replicable genetic packages. In one embodiment, the present invention provides a method of identifying an agonistic or antagonistic feature of a peptide, which has intermolecular interaction with a receptor of interest comprising the steps of: (a) contacting a receptor of interest connected to a protease, to said replicable genetic package as described hereinabove; (b) detecting the replicable genetic package which propagate or survive, wherein if a replicable genetic package comprises peptide that has affinity to the to the target of interest, the sterically inhibiting group will be cleave off and the replicable genetic package will propagate or survival; (c) contacting the replicable genetic package of step b with the receptor of interest; (d) contacting the replicable genetic package of step d with a protein attached to protease, wherein said protein is downstream in the signal transduction pathway of the receptor of interest; and (e) separating the replicable genetic package obtained in step d, wherein if a replicable genetic package propagates or survives, it contains a peptide which has an agonistic feature.

[0163] In one embodiment, the present invention provides a method of identifying peptide which has a functional feature with a receptor of interest comprising the steps of: (a) contacting a receptor of interest connected to a protease, to the replicable genetic package as described hereinabove; (b) detecting the replicable genetic package which propagate or survive, wherein if a replicable genetic package comprises a peptide that has affinity to the target of interest the sterically inhibiting group will cleaved off enabling the replicable genetic package propagation or survive; (c) contacting the replicable genetic package of step b with a ligand to the receptor of interest attached to the protease; and (d) separating the replicable genetic package which survive or propagate, thereby identifying a peptide which has a functional feature with a receptor of interest.

[0164] In one embodiment, the present invention provides a method of identifying a peptide that activates an enzyme of interest comprising the steps of: (a) contacting an enzyme of interest connected to a protease, to the replicable genetic package as described hereinabove; (b) detecting the replicable genetic package which survives or propagates, wherein if a replicable genetic package comprises a peptide that has affinity to the enzyme of interest, the sterically inhibiting group was cleave off enabling the virus propagation; (c) contacting the replicable genetic package of step b with said enzyme of interest; (d) contacting the replicable genetic package of step c with a substrate of the enzyme, wherein said substrate is attached to a protease; and (e) detecting the replicable genetic package which survives or propagates, wherein if said replicable genetic package comprises a peptide that activates the enzyme of interest, the inhibiting group is cleaved off enabling the replicable genetic package propagation; thereby identifying a peptide which activates an enzyme of interest.

[0165] The concept of this embodiment of the invention is to attach a sterically inhibiting group, which comprises a modified protease cleavage site and a peptide library to a protein which is essential to any vital process of any organism so as to inhibit a function of the essential protein. The modified organism is then contacted with a target of interest attached to a protease. Only when the target of interest has affinity to a peptide of the peptide library, the sterically inhibiting group will be cleaved off and the function of the protein, which is essential to a vital process, will be reverted.

[0166] In one embodiment, the peptide and modified protease site, which are attached to the protein that is essential to a vital process, form a sterically inhibiting group.

[0167] By the word “sterically inhibiting group” or “steric hindrance” it is meant interference with a feasible chemical reaction. Here it means physical inhibition of the protein function by attaching a chemical group to a protein, which protein is essential for a vital process.

[0168] The virus library may utilize a number of expression vehicles known to those of ordinary skill, including but not limited to, recombinant bacteriophage, lambda phage, M13, a recombinant plasmid or cosmid, and the like. If the virus used is M13, the protein, which is essential for propagation and infectivity, may be without limitation, pIII or pVIII and the like. Accordingly, the M13 cannot be infective when a sterically inhibiting group is attached for example, to the N terminus of pIII and would be activated only upon the removal of the sterically inhibiting group by, for example, a suitable protease.

[0169] A prophetic example which demonstrates the concept of the invention is the use of auxotrophic bacteria. An auxotrophic bacterium is a mutant bacterium that requires nutrients that wild type bacteria are able to synthesize themselves. For example, an arginine mutant requires a nutritional supplement of arginine in order to grow, because it lacks the enzyme responsible for arginine production. Any of the methods of the present invention may use auxotrophic bacteria, in which the missing enzyme is expressed in a plasmid, fused to a sterically inhibiting group that comprises peptide library, a modified protease site, and optionally an antibiotic resistance protein (for the deletion mutants counter selection), wherein the sterically inhibiting group inhibits the activity of the enzyme. These bacteria can grow only on rich media. Growing the bacteria on minimal medium will select for the bacterial cells expressing peptide that interact with the target of interest, because the sterically inhibiting group in these cells will be preferably cleaved off, and the enzyme will revert its full activity. It should be noted that the rate of the bacteria growth will be dependent on the interaction of the target with the peptide.

[0170] In another embodiment, the method can be performed with cells that can grow in a medium containing a specific antibiotic and express the antibiotic resistance gene fused to the sterically inhibiting group. The antibiotic resistance gene will be activated only when the sterically inhibiting group will be removed and the cells will be able than to propagate. The cells may be further containing an antibiotic resistance gene for the deletion mutants counter selection.

EXAMPLES

[0171] M13, as well as fd and fl, is a filamentous Ff bacteriophage. The phage minor coat gene 3 protein (pIII) of M13 bacteriophage (FIG. 1A) allows the phage to infect bacteria by interacting with the bacterial F pilus and later with the integral membrane protein ToIA. Specifically, the CT domain of pIII anchors pIII to the phage coat, while the N2 domain interacts with bacterial F pilus and the N1 domain forms a complex with the C-terminal of ToIA at later stages of infection.

[0172] One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis, et al., Molecular Cloning, A laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et al., 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996), both incorporated herein by reference).

Example 1
Development of a Recombinant Virus Library with a Modified Protease Site

[0173] M13 is only active (i.e. able to infect) when the N2 and N1 domains are tightly linked to the CT domain, which is linked to the capsid of the phage. In addition, a phage remains active if any one of its 3-5 pIII units is functional. Thus, insertion of a protease cleavage site between the CT and the N2 domains of pIII would render the phage inactive (FIG. 1B) upon incubation with a suitable amount and type of protease.

[0174] In order to develop a system in which interaction of heterologous proteins may be assessed, a protease and protease cleavage site pair is chosen, wherein cleavage will only occur if the protease and the protease cleavage site are brought into close proximity by another group (FIG. 2). Thus, an appropriate modified protease cleavage site of the pair should have minimal affinity for its protease but at the same time, should sustain high proteolytic activity when it comes into contact with its protease. Thus, if the protease described above is fused to a target protein and the protease cleavage site in the pIII protein is cross-linked to a target protein ligand, the specific proteolytic activity of the protease will be activated by bringing the protease and protease cleavage site into close proximity (FIG. 2). A higher affinity of the target protein to the target protein ligand would be expected to increase the proteolytic activity.

[0175] Each of the recombinant proteins in the system, such as the protease-target protein and the modified cleavage site-target protein ligand, are separated by long flexible linkers to enable a good degree of rotational freedom, which in turn allows interaction between the components of the system. The linkers chosen may include a leucine zipper. Any other flexible linker could be used as well.

[0176] The 76 amino acid-long ubiquitin proteolytic site (Genbank X01474; PID: g4741) and one of ubiquitin's specific proteases, UBP1 (Genbank M63484; PID: g173126) are identified as suitable candidates for modified cleavage site and protease, respectively (FIG. 3). UBP1 specifically cleaves ubiquitin after its C-terminal glycine. A modified ubiquitin structure that is bound by UBP1 with very low or no affinity but has a preserved UBP1 cleavage site is generated using a mutagenesis library of ubiquitin generated by PCR.

Example 2
Testing of a Recombinant Virus Library with a Modified Ubiquitin Site

[0177] The two parts of a leucine-zipper are used to evaluate the functionality of the modified ubiquitin system in the present invention (FIG. 3). The protease cleavage site is cross-linked to one part of the leucine zipper, and the protease is engineered as a fusion protein with another part of the leucine zipper. When combined, the two parts of the leucine zipper interact, resulting in the association of the LTBP1 with modified ubiquitin. Any other two interacting molecules could be used, but they should preferably be small and have a strong affinity for one another.

[0178] Engineered and wild type bacteriophages are incubated with and without the target ligand fusion protein. After incubation of the phages with the fusion protein, bacteria are added to the medium. After incubation of the phages with bacteria, bacteria are removed from the medium to separate bacteriophages in the medium from those that invaded the bacterial host cell (FIG. 4). Bacteria are then washed and resuspended in water. The genomes of the inactive phages that are not able to infect the bacterial cells are rescued from the filtrate by PCR. Alternatively, the genomes of the active phages from the bacterial cells are rescued by PCR.

[0179] Wild type bacteriophages and engineered bacteriophages that were not exposed to the target protein ligand are found in high levels in the host bacteria. However, bacteriophages engineered with the leucine zipper construct cross-linked to the modified ubiquitin described above are found in high levels in the incubation medium, indicating that the pIII protein was cleaved and the phage thus rendered non-infective. Thus, in the engineered bacteriophage system described hereinabove, the inability of a phage to invade a host cell indicates the presence of a protein-protein interaction.

Example 3
SoIP Assay to Identify Peptide that Interacts with a Target of Interest

[0180] Next, a library of M13 bacteriophages is created which contain an insertion of the modified protease cleavage site (ubiquitin) and a random peptide between the CT and N2 domains of pIII (FIG. 2, 5A). The target protein is fused to one half of a leucine zipper, and a protease (UBP1) is fused to a second half of a leucine zipper. The target protein and UBP1 are then cross-linked by the leucine zipper (FIG. 2, 5A), creating a complex comprising the target of interest. After incubation of the M13 library with the target fusion protein (FIG. 5A-B), bacteria are added to the medium (FIG. 5C). After an appropriate incubation (FIG. 5D), bacteria are separated from the medium, and the genomes of the active and inactive phages are separated, recovered, and sequenced as described hereinabove.

[0181] Bacteriophages that successfully invade the bacterial host cell are expected have at least one intact pIII protein, indicating that the target protein and target protein ligand did not have a high affinity (FIG. 4). Bacteriophage in the medium are expected to be missing all of their pIII proteins, indicating that the target protein and target protein ligand displayed high affinity binding (FIG. 4). In this way, novel targets of a characterized protein can be isolated from libraries.

Example 4
SoIP Assay to Identify a Functional Interaction Between Peptide and Target of Interest

[0182] The positive or negative functionality of novel targets of a characterized protein may be characterized using the method described herein.

[0183] In the case where the target of interest is a receptor, the first step is to perform the technique described Example 3 to screen a peptide library for a peptide that interacts with a receptor protein whose downstream signaling molecule is characterized. Then, clones of cells that were non-infective in Example 3 are incubated with a receptor protein that is not fused to a protease (UBP1). Next, the protease (UBP1) is engineered as a fusion protein with a protein known to be involved in the downstream signaling of the target (FIG. 6B). After incubation of the recombinant virus library with the known signaling molecule fusion protein, bacteria are added to the medium. After an appropriate incubation, bacteria are separated from the medium, and the genomes of the active and inactive phages are separated and recovered as described hereinabove.

[0184] If the peptide expressed by the recombinant bacteriophage is an antagonist of the receptor of interest, then it will bind the receptor of interest and prevent an interaction of the receptor with its downstream protein. Thus, there will be no opportunity for interaction between the protease and modified cleavage site, because the downstream protein, which is linked to a protease, will not interact with the receptor of interest and the peptide, which are in close proximity to the modified cleavage site. Thus, the pIII protein of the bacteriophage will remain intact, along with the ability of the phage to infect (FIG. 6D). Thus, bacteriophage localized in cells express peptides that are antagonistic with respect to the receptor of interest.

[0185] If the peptide expressed by the recombinant bacteriophage is an agonist of the receptor of interest, then it will bind the receptor of interest and lead to an interaction of the activated receptor with its downstream protein. The proximity of the downstream protein, which is linked to a protease, to the receptor of interest and the peptide, which are in close proximity to the modified cleavage site, will lead to the cleavage of the cleavage site by the protease, the loss of the pIII protein, and the loss of ability to infect (FIG. 6C). Thus, bacteriophage in the medium express peptides that are agonistic with respect to the receptor of interest.

[0186] A second illustration of the assay's ability to exhibit functionality of a peptide is the case where the target of interest is an enzyme. The first step is to perform the technique described Example 3 to screen a peptide library for a peptide or peptides that interact with an enzyme whose substrate is characterized. Then, clones of cells that were non-infective in Example 3 are incubated with an enzyme that is not fused to a protease (UBP1). Next, the protease (UBP1) is engineered as a fusion protein with the enzyme substrate (FIG. 6B). After incubation of the recombinant virus library with the known enzyme substrate fusion protein, bacteria are added to the medium. After an appropriate incubation, bacteria are separated from the medium, and the genomes of the active and inactive phages are separated and recovered as described hereinabove.

[0187] If the peptide expressed by the recombinant bacteriophage is an inhibitor of the enzyme of interest, then it will bind the enzyme of interest and prevent an interaction of the enzyme with its substrate. Thus, there will be no opportunity for interaction between the protease and modified cleavage site, because the enzyme substrate, which is linked to a protease, will not interact with the enzyme of interest and the peptide, which are in close proximity to the modified cleavage site. Thus, the pIII protein of the bacteriophage will remain intact, along with the ability of the phage to infect (FIG. 6D). Thus, bacteriophage localized in cells express peptides that are inhibitors of the enzyme of interest.

[0188] If the peptide expressed by the recombinant bacteriophage is an activator of the enzyme of interest, then it will bind the enzyme of interest and lead to an interaction of the activated enzyme with its substrate. The proximity of the substrate, which is linked to a protease, to the enzyme of interest and the peptide, which are in close proximity to the modified cleavage site, will lead to the cleavage of the cleavage site by the protease, the loss of the pIII protein, and the loss of ability to infect (FIG. 6C). Thus, bacteriophage in the medium express peptides that are activators of the enzyme of interest. In this way, the way in which the novel peptide affects characterized target-ligand interactions may be assessed.