WO2012046238A2 - Erythropoietin receptor antagonists - Google Patents

Abstract

The present invention provides erythropoietin derived peptides which bind the erythropoietin receptor and inhibit EPO-induced cell proliferation. The invention further provides pharmaceutical compositions comprising same, and uses thereof in the treatment of primary and secondary erythrocytosis as well as for inhibiting undesirable cell proliferation induced by erythropoietin receptor in patients treated with erythropoietin.

Description

ERYTHROPOIETIN RECEPTOR ANTAGONISTS

FIELD OF THE INVENTION

The present invention relates to erythropoietin derived peptide antagonists of the erythropoietin receptor, to pharmaceutical compositions comprising same, and uses thereof including, inter alia, the treatment of primary and secondary erythrocytosis, as well as for inhibiting undesirable proliferation, migration and/or survival of cancer cells expressing erythropoietin receptor.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a 34 kDa cytokine required for the survival, proliferation, and differentiation of committed erythroid progenitor cells (Sasaki et al. (2000) Biosci. Biotechnol. Biochem, 64: 1775-93). EPO is produced mainly by the kidney and from there reaches its destination through the blood circulation. EPO functionality is mediated via erythropoietin receptor (EPO-R), a 62 kDa protein that belongs to the cytokine receptor superfamily (Youssoufian, H. et al., (1993) Blood, 81 :2223-36). EPO-R has no intrinsic kinase activity and therefore relies on the tyrosine kinase, Janus kinase 2 (JAK2) to initiate downstream signaling. EPO binding to the receptor induces a conformational change in the EPO-R homodimer which initiates the activation of JAK2 (Wojchowski, D.M. et al., 1999, Exp. Cell Res., 253:143-56). Activated JAK2 phosphorylates the EPO-R at multiple cytoplasmic tyrosine residues, which results in the recruitment to the receptor of SH2-containing proteins such as, the Signal Transducer and Activator of Transcription 5 (STAT5) (Richmond, T. D. et al., 2005, Trends Cell Biol. 15:146-55), MAPK (Chen, J. et al., 2004, Exp. Cell Res. 298: 155-66), and regulatory subunit of phosphoinositide 3- Kinase (PI3K) (Zhao, W. et al, 2006, Blood 107:907-15). Enhancement of EPO-R signaling via administration of Recombinant Human EPO (rHuEPO) is commonly used as treatment for various diseases and clinical conditions, including treatment of patients suffering from anemia. EPO is indicated for therapy of e.g., (a) anemia associated with chronic renal failure, (b) anemia related to zidovudine therapy in HIV-infected patients, and (c) anemia in cancer patients undergoing chemotherapy. In addition, EPO has been used to reduce the need for allogeneic blood transfusion in patients undergoing surgery, and for pruritus associated with renal failure. The wide usage of rHuEPO is reflected in its global annual sales which exceeds $13 billion.

In contrast to patients in need for treatment with rHuEPO, certain patients may benefit from inhibition of EPO-R activity. These include patients suffering from increased red cell mass (erythrocytosis) that stems from a wide range of etiologies. Erythrocytosis can be primary or secondary to other diseases. Primary erythrocytosis is a rare disease encompassing mutations in EPO-R which enhance its activity. In most cases the mutations in the EPO-R gene result in truncations in the EPO-R intracellular domain (Van Maerken, T. et al., 2004, J Pediatr. Hematol. Oncol. 26, 407-16). These truncated EPO-Rs lack the intracellular negative regulatory domain of the receptor and therefore exhibit prolonged activation and mediate enhanced cell proliferation (Arcasoy, M. O. et al., (1999) Exp. Hematol. 27:63-74). Secondary erythrocytosis is either caused by defects in the oxygen sensing pathway (mutation in VHL, PHD2 or HIF-2a) (Gordeuk, V. R. et al., 2005, Haematologica 90:109-16) or can be acquired. There is long list of diseases and conditions that lead to acquired secondary erythrocytosis, these include hypoxia related diseases (e.g, chronic lung disease, right-to-left cardiopulmonary vascular shunts, carbon monoxide poisoning, smoker's erythrocytosis, hypoventilation syndromes including obstructive sleep apnea, high-altitude) (McMullin M. F., 2008, Int. J. Lab. Hematol. 30:447-59), or diseases resulting with pathologic production of EPO, these include several types of cancer (e.g., cerebellar hemangioblastoma, meningioma, parathyroid carcinoma/adenomas, hepatocellular carcinoma, renal cell cancer, pheochromocytoma, and uterine leiomyomas) (Patnaik M. M. and Tefferi A., 2009, Leukemia 23, 834-44).

Recently, EPO-R was identified in non-erythroid cells in the heart, lung, brain and the immune system as well as in several cancer types (Arcasoy M. O., 2008, Br. J. Haematol 141 : 14-31). EPO-R expression on cancer cells was indicated to functionally induce migration and proliferation, and to increase survival of cancer cells (Richmond, T. D. et al., 2005, Trends Cell Biol 15:146-55). Consequently, this raised the concern that treatment of cancer patients having chemotherapy-associated anemia or radiation-associated anemia with EPO would stimulate neoplastic cells proliferation and spread. It would be thus beneficial to combine to EPO treatment an EPO-R blocking agent that would be targeted to the malignant cells. Such combination could provide medical solution to chemotherapy-associated anemia or radiation-associated anemia, while preventing the undesirable activation of the tumor cells by EPO.

Taken together, there remains an unmet medical need for developing effective agents and protocols for the inhibition of EPO-induced activity. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention is directed to erythropoietin-derived peptides capable of inhibiting EPO induced EPO receptor (EPO-R) activation. Specifically, the invention provides short peptides derived from highly conserved helical regions of EPO, homologues and fragments thereof.

According to some aspects the inhibitory peptides are capable of binding to EPO-R and interfere with its binding to EPO. The invention is further directed to the use of these peptides for the treatment of primary and secondary erythrocytosis, as well as to their utility as inhibitors or modulators of undesirable cancer cell proliferation, migration and survival in chemotherapy-associated or radiation- associated anemia of cancer patients treated with EPO.

It is now disclosed for the first time that a 17-mer EPO-derived peptide antagonist, corresponding to residues 121-137 of mouse EPO, comprising the sequence set forth in SEQ ID NO: 25 was capable of inhibiting EPO-induced EPO-R phosphorylation and the activation of downstream effectors (figure 6). Consequently, the proliferation and survival of Ba/F3 cells (a mouse pro-B cell line) stably transfected with EPO-R and the migration of MDA-MB-435 (a human breast cancer cell line) were inhibited by the EPO-derived peptide antagonist (figures 7-9). Further, in vivo administration of EPO-derived peptide antagonist resulted with reduced mean corpuscular volume (MCV), and elevated EPO-R mRNA expression in bone marrow of mice, indicating that the antagonist mediated anemia (figures 10-11). Thus, EPO- derived peptide antagonist may be used as inhibitor or modulator of primary and secondary erythrocytosis and as inhibitor or modulator of undesirable cancer cell progression in cancer patients treated with EPO.

In one aspect, the present invention provides synthetic or recombinant peptides comprising 9-30 amino acids. Other embodiments are directed to peptides comprising 12-30 amino acids. According to a specific embodiment, the peptides comprise at least 9 contiguous amino acids derived from general formula I:

Xi -X2-X3-X4-X5-X6- 7-X8-X9-X1 o"Xi 1 -X12

wherein X] and X₇, each is independently selected from the group consisting of Lys, Arg and Orn; X₃, X₆, X₉ and Xj₂, each is independently selected from the group consisting of Ala, Val, He, Leu, Met, Asn, Gin, and Phe; X₄ and X₈, each independently selected from the group consisting of Ser, Thr, Ala, Met and Val; X_2; X₅, X₁₀, and X_11; each independently is any natural or synthetic amino acid. In another embodiment, the peptide of the invention comprises the peptide of formula I and additional amino acids appended to the carboxyl terminus, amino terminus, or both.

In another aspect, the present invention provides a synthetic or recombinant peptide consisting of 12-30 amino acids comprising general formula I, wherein X_\ and X₇, each is independently selected from the group consisting of Lys, Arg and Orn; X₃, selected from the group consisting of Val, He, Leu, and Met; X₆, and X₉ each independently selected from the group consisting of He, and Leu; X₁₂, selected from the group consisting of He, Leu, and Phe; X4 and X₈, each independently selected from Ser, Thr, and Ala; X_2i and X₅, each independently selected from Ala, and Gly; and X₁₀, and Xn_, each independently selected from the group consisting of: Ala, Thr, and Ser.

In yet another aspect, the present invention provides a synthetic or recombinant peptide consisting of 9-30 amino acids, comprising at least 9 contiguous amino acids derived from the sequence of general formula I, wherein Xi and X₇, each independently selected from the group consisting of Lys, Arg, and Orn; X₃, selected from the group consisting of Val, He, Leu, and Met; X₆, and X₉, each independently selected from the group consisting of lie, Asn, Gin, and Leu; X₁₂, selected from the group consisting of He, Leu, and Phe; X₄ and X₈, each independently selected from the group consisting of Ser, Thr, and Ala; X_2, and X₅, each independently selected from the group consisting of Ala, and Gly; and Xi₀, and X_11; each independently selected from the group consisting of Ala, Thr, Leu, and Ser.

In another aspect, there is provided a pharmaceutical composition containing an effective amount of the peptide, homologues or fragments as defined herein, and an excipient.

In additional aspect, the present invention provides a polynucleotide encoding an EPO derived peptide, homologues, and fragments thereof.

In another additional aspect, the present invention provides vector comprising the polynucleotide which encodes an EPO derived peptide, homologues, and fragments thereof. In another aspect, the invention provides a method for treating a subject afflicted with erythrocytosis, comprising administering to the subject an effective amount of the peptide, homologues or fragments thereof, thereby treating a subject afflicted with erythrocytosis.

According to another aspect, the present invention provides a method for altering binding of EPO to EPO-R in a cell, comprising the step of contacting the cell with the peptide, thereby altering binding of EPO to EPO-R.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 includes sequence alignments and predicted 3-D structure of mEPO-EPO-R complex. Sequence alignments of (A) hEPO-R and mEPO-R extracellular domains and (B) hEPO and mEPO. EPO helix predicted to exert the strongest interaction with EPO-R is designated in bold (residues 121-137 of mEPO) (C) Predicted 3-D structure of mouse EPO-EPO-R complex. 3-D structure prediction was utilized using the Discovery Studio program.

Figure 2 is an image demonstrating the non-symmetric interaction of EPO with EPO-R dimer. Arrows show amino acids on EPO that are within 5A from mEPO-R. The four helical structures on EPO are marked with the letters A to D.

Figure 3 is an image of predicted EPO-EPO-R interactions. Electrostatic surface potential prediction of EPO-R and its interaction with EPO was utilized using discovery studio software. The four helical structures on EPO are marked with letters A to D. Bold circle designates helix C which-a candidate for being EPO-R antagonist. Figure 4 is an image of predicted interactions of EPO antagonist

(EPOantO) with EPO-R. Predicted interaction of a 17 mer EPO derived peptide with EPO-R. Interaction is mediated through electrostatic surface potential of EPO-R with EPOantO.

Figure 5 includes bar graphs demonstrating EPOantO mediated inhibition of EPO binding to EPO-R. (A) 96-well plates were coated with soluble EPO-R (sol- EPO-R) over night. The wells were then washed and incubated with rHuEPO (5 U/ml) in the presence or absence of ΙΟμΜ EPOantO, or control peptide, for lh. (B) 96-well plates were coated with sol-EPO-R and then incubated with rHuEPO (50U/ml) and with increasing concentrations of EPOantO. EPO binding to sol-EPO-R was determined using HRP-conjugated anti-EPO antibodies. Absorbance, indicative of binding, was measured at 450 nm.

Figure 6 is a gel micrograph demonstrating that EPOantO inhibits EPO induced EPO-R activation in Ba/F3 cells. (A) A western blot image of lysates from Ba/F3 cells expressing EPO-R. Cells were pre-incubated with or without 10 μΜ of EPOantO or with control peptide followed by incubation with or without, rHuEPO (5 U/ml). Cell lysates were subjected to western blot analysis with antibodies against pEPO-R, pERKl/2 and actin that was used as control of loading. (B) A graph showing quantitative analysis of phosphorylation levels in cells stimulated for 5 min with EPO. Presented is an average of four independent experiments. Phosphorylation levels of cells treated with EPO alone were determined as 100%. Figure 7 includes an image and a bar graph demonstrating inhibition of

EPO-induced cell proliferation by EPOantO. EPO-R expressing Ba/F3 cells were incubated for three consecutive days with medium containing 0.01 U/ml EPO, and with EPOantO (10μΜ) or the control peptide. Proliferation was measured using MTT reagent. (A) Capture image of a representative proliferation assay of the third day of the assay. Cell proliferation is indicated by the intensity of the color; dark color indicates of higher number of live cells. (B) Bar graph showing fold increase average of 4 independent proliferation experiments, recorded on day 1, day 2 and day 3 of the experiment. A value of one was assigned to the proliferation of non-treated cells at day 1. Other values were normalized accordingly. Non treated cells, cells treated with EPOantO, and cells treated with control peptide are represented by white, black, and hatched columns, respectively.

Figure 8 includes FACS plots demonstrating that EPOantO inhibits EPO- induced cell survival. EPO-R expressing Ba F3 cells were cultured overnight with RPMI containing 10%o FCS and 0.1 U/ml of rHuEPO in the presence or absence of EPOantO. Following incubation cells were washed with PBS containing 2% FCS, and were then subjected to 0.1% Triton X-100, followed by staining with propidium-iodide (PI) as in order to assess their cell cycle profile. (A) Representative histogram of cells treated in the absence or presence of 10 μΜ EPOantO. Apoptotic cells appear in sub- Gl phase. (B) A graph summarizing quantitative analysis of the average of three independent experiments.

Figure 9 includes images and bar graphs demonstrating that EPOantO inhibits EPO induced cell migration. (A) MDA-MB-435 cells were grown to 100% confluence in a 6 well plate and a cross shaped scratch was created using a sterile tip. Cells were then washed and subjected to medium containing 0.1% serum, EPO (lOU/ml), and EPOantO (10μΜ). Cells incubated with medium containing 0.1% serum without EPO served as negative control, while cells subjected to medium containing 0.1% serum with EPO served as positive control. The left panel shows a representative image of the cross area was captured on day 0, 1 and 2. The right panel shows a bar graph summarizing the percent migration on day 1 compared to day 0. Percent migration was derived from the distance between the two sides of the scratch measured for each field and captured at 5 different points using Adobe illustrator. The percent of cell migration was calculated as 100% minus the distance between the two sides of the scratch on day 1 divided by the distance at the same location on day 0 for each treatment. (B) MDA-MB-435 cells were incubated with FCS free medium over night. The cells were then placed at the top of a transwell chamber and were subjected to medium containing 0.1% serum, EPO (l OU/ml), and EPOantO (10μΜ). Cells incubated with medium containing 0.1% serum without EPO served as negative control, while cells subjected to medium containing 0.1% serum with EPO served as positive control. Cells were allowed to migrate for 24h. Cells were imaged and counted. The left panel shows a representative experiment of migrating cells after 24h. Magnification X10, bar=50μm. The right panel, shows quantification analysis of the average of three independent experiments.

Figure 10 includes a table and bar graphs demonstrating the effect of EPOantO on blood count analysis. Six control mice and five peptide treated mice were injected for 10 days with either PBS or EPOantO (0.37mg/per day/per mouse), respectively. (A) Table summarizing an average of the body weight and spleen weight in grams. (B) Bar graph summarizing the blood hemoglobin levels. (C) Bar graph summarizing blood hematocrit levels. (D) Graph summarizing blood MCV levels. Figure 11 includes bar graphs and a table demonstrating that in vivo administration of EPOantO increases the mRNA of EPO-R in the bone marrow.

Total RNA was isolated from bone marrow and spleen cells using TRIzol reagent. Reverse transcription (RT) was performed using MMLV Reverse Transcriptase from 2 μg of total RNA. Real-time RT-PCR was performed using SYBR green with primers for EPO-R, to monitor double-stranded DNA (dsDNA) synthesis. The results were quantified using a comparative Ct method. TMB was used as control mRNA.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention is directed to peptides derived from highly conserved helical region of EPO. In another embodiment, the conserved helical region corresponds to residues 118 to 138 of mEPO, homologues, and fragments thereof capable of binding EPO receptor (EPO-R). In another embodiment, peptides of the invention bind EPO-R and interfere with EPO binding to EPO-R.

In some embodiments, the present invention provides compositions and methods utilizing the peptides described herein as inhibitors or modulators of EPO. In another embodiment, peptides of the invention are utilized as inhibitors or modulators of EPO activity in cancer cells such as but not limited to inhibiting or modulating undesirable cancer cell proliferation, migration, and/or survival. In another embodiment, the present invention provides compositions and methods for treating primary and secondary erythrocytosis.

It is to be emphasized that the peptides of the present invention are within or homologous to the conserved helical region of EPO, corresponding to residues 1 18 to 138 of mEPO. This region is highly conserved in human (homo sapiens) EPO (hEPO), mouse (Mus musculus) EPO (mEPO), cat (Felis catus) EPO (cEPO) and rat (Rattus norvegicus) EPO (rEPO), as can be seen hereinbelow, in the multiple sequence alignment of this EPO region:

hEPO VDKAVSGLRSLTTLLRA (SEQ ID NO: 16)

cEPO VDKAVSSLRSLTSLLRA (SEQ ID NO: 27)

rEPO and mEPO IDKAISGLRSLTSLLRV (SEQ ID NO: 25). In some embodiments, the synthetic or recombinant peptide consist 9-30 amino acids comprising at least 9 contiguous amino acids derived from the sequence of general Formula I: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12, wherein X₁ and X₇, each independently selected from the group consisting of Lys, Arg, and Orn; X₃, selected from the group consisting of Val, He, Leu, and Met; X₆, and X₉, each independently selected from the group consisting of He, Asn, Gin, and Leu; X_i2, selected from the group consisting of He, Leu, and Phe; X4 and X₈, each independently selected from the group consisting of Ser, Thr, and Ala; X₂_ and X₅, each independently selected from the group consisting of Ala, and Gly; and X₁₀, and Xn_, each independently selected from the group consisting of Ala, Thr, Leu, and Ser.

In other embodiments, the peptide comprises 12 amino acid residues according to general Formula I. In other embodiments, the peptide consists_of 12 contiguous amino acids according to general Formula I.

In other embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4; KAVSGLRSL (SEQ ID NO: 1); KAISGLRSL (SEQ ID NO: 2); SGLRSLTSL (SEQ ID NO: 3); SGLRSLTTL (SEQ ID NO: 4).

In other embodiments, the peptide comprises amino acid sequence according to general formula I, wherein

is Lys; X₂, selected from the group consisting of Ala, and Gly; X₃, selected from the group consisting of Val, He, Leu, and Met; X4 and X₈, is Ser; X₅, selected from the group consisting of Ala, and Gly; X₇ is Arg; X₉ is Leu; X10, and Xn_, each independently selected from the group consisting of Ala, Thr, and Ser; and X₆ and X₁₂, each independently selected from the group consisting of Leu, Met, Val, and lie.

In additional embodiments, the peptide comprises or consists an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-9; KAVSGLRSLTTL (SEQ ID NO: 5); KAISGLRSLTTL (SEQ ID NO: 6); KAVSGLRSLTSL (SEQ ID NO: 7); KAISGLRSLTSL (SEQ ID NO: 8); KAITANRTQTLF (SEQ ID NO: 9).

In additional embodiments, the peptide comprises or consists an amino acid sequence according to formula I, selected from the group comprising of SEQ ID NOs: 10-26; VDKAISGLRSLTSLLRV (SEQ ID NO: 10); IDKA VS GLRS LTS LLR V (SEQ ID NO: 11); IDKAISGLRSLTTLLRV (SEQ ID NO: 12); IDKAISGLRSLTSLLRA (SEQ ID NO: 13); VDKAVSGLRSLTSLLRV (SEQ ID NO: 14); VDKAVSGLRSLTTLLRV (SEQ ID NO: 15); VDKAVSGLRSLTTLLRA (SEQ ID NO: 16); VDKAIS GLRSLTSLLRA (SEQ ID NO: 17); IDKAVSGLRSLTTLLRA (SEQ ID NO: 18); IDKAISGLRSLTTLLRA (SEQ ID NO: 19); VDKAISGLRSLTTLLRA (SEQ ID NO: 20); IDKAVSGLRSLTTLLRV (SEQ ID NO: 21); IDKAVSGLRSLTSLLRA (SEQ ID NO: 22); VDKAISGLRSLTTLLRV (SEQ ID NO: 23); VDKAVSGLRSLTSLLRA (SEQ ID NO: 24); IDKAISGLRSLTSLLRV (SEQ ID NO: 25); AAKAITANRTQTLFQRA (SEQ ID NO: 26).

In other embodiments, the peptide comprises or consists of 12-17 amino acids residues according to formula I.

In other preferred embodiments, the peptide comprises or consists amino acid sequence according to formula I, selected fromjhe group comprising of SEQ ID NOs: 1-27.

Without being bound by any theory or mechanism of action the peptide antagonist may bind to the extracellular domain of EPO-R, thus inhibiting the binding of EPO to its receptor. According to preferred embodiments, the peptide inhibits the binding of human EPO to its receptor.

According to another aspect, the present invention provides a method for altering binding of EPO to EPO-R in a cell, comprising the step of contacting the cell with a peptide of the invention, thereby altering, modulating or inhibiting the binding of EPO to EPO-R. According to a specific embodiment, altering refers to inhibiting. According to another embodiment, altering refers to inhibiting activation of the EPO receptor by endogenous or exogenous EPO. According to yet another specific embodiment, altering refers to inhibiting EPO-induced EPO-R phosphorylation, cell proliferation, migration and survival.

According to some embodiments of the invention, the peptide is capable of inhibiting the phosphorylation of EPO-R induced or mediated by EPO. According to some other embodiments, the peptide inhibits EPO-mediated cell proliferation migration and survival. According to some embodiments, cell proliferation, migration and survival refers to a eukaryotic cell proliferation, migration and survival. According to some other embodiments, cell proliferation migration and increase of survival refers to cancer cell proliferation.

In another embodiment, the cell is a human cell. In another embodiment, the cell is a diseased cell. In another embodiment, the cell is characterized by aberrant proliferation. In another embodiment, the cell is derived from a mammal. In another embodiment, the cell is a rodent cell.

The peptide of the present invention may inhibit according to some embodiments, undesirable cell proliferation, migration and increase of survival. According to some embodiments, such undesirable cell proliferation migration and increase of survival may be the result of cells exposure to EPO. As used herein, the terms "undesirable proliferation" also refers to "proliferative disorder". In another embodiment, a "proliferative disorder" is any pathological or non-pathological physiological condition characterized by aberrant proliferation of at least one cell, including conditions characterized by unwanted cell proliferation, cell survival, or cell migration. In another embodiment, the peptide of the invention is utilized as therapy for conditions characterized by deficient or aberrant apoptosis, deficient or aberrant migration, as well as conditions characterized by aberrant or unwanted cell survival. In another embodiment, the peptide of the invention is utilized as therapy for erythrocyte sis.

According to some embodiments, erythrocytosis is selected from the group consisting of primary erythrocytosis and secondary erythrocytosis. Each and every possibility represents a different embodiment of the invention.

Primary erythrocytosis also referred to as primary polycythemia, or polycythemia vera is a myeloproliferative disorder in which the red blood cell (RBC) count increases without being stimulated by erythropoietin (EPO). Secondary erythrocytosis also referred to as secondary polycythemia is characterized by the increase in RBC counts due to an increase in EPO. Potential causes of this include: low blood oxygen, caused by heart disease or high altitude; continual exposure to carbon monoxide (heavy smoking of cigars or cigarettes); congenital (hereditary) disorders producing abnormal hemoglobin or an overproduction of EPO; and diseases such as kidney disease. In another embodiment, the peptide of the invention is used for preventing the diseased disclosed herein and target populations such as populations having or afflicted by low blood oxygen, heart disease or high altitude, continual exposure to carbon monoxide (heavy smokers), congenital (hereditary) disorders producing abnormal hemoglobin or overproduction/overexpression of EPO, and diseases such as kidney disease.

According to yet another aspect, the invention provides a method for inhibiting undesirable proliferation of cancer or malignant cells mediated by EPO, comprising administering to a subject in need thereof an effective amount of an EPO derived peptide comprising or consisting sequence set forth in SEQ ID NOs: 1-27 or a homologue or fragment thereof as defined herein. The inhibition of undesirable proliferation, migration and survival using the peptides of the invention will enable safe administration of EPO to cancer patients who require EPO for the treatment of anemia.

As used herein, the term "safe administration" or "safe treatment" refers to a treatment which yields a desired therapeutic response without undue adverse side effects commensurate with a reasonable benefit/risk ratio when used in the manner according to the embodiments of this invention.

According to one embodiment, EPO is administered concomitantly with at least one EPO derived peptide antagonist or a homolog or fragment thereof. According to another embodiment, EPO is administered before the administration of the EPO derived peptide _> antagonist; alternatively, EPO is administered after the administration of the EPO derived peptide antagonist.

According to another embodiment, the present invention further provides an EPO derived peptide targeted therapy to a subject in need thereof, comprising the step of administering a compound is comprising a targeting moiety and the peptide of the invention, wherein the targeting moiety is specific for cancer or malignant cells. In another embodiment, the peptide comprises or consists a sequence set forth in SEQ ID NOs: 1-27 or a homolog or fragment thereof as defined herein.

Erythropoietin derived peptide as used herein is a peptide derived from the highly conserved helical region of EPO that corresponds to residues 121-137 of mEPO, that is capable of binding EPO receptor (EPO-R). In some embodiments, an EPO derived peptide comprises at least 3 consecutive amino acids from residues 118 peptide comprises at least 4 consecutive amino acids from residues 118 to 138 of mEPO or a homologue thereof. In another embodiment, an EPO derived peptide comprises at least 7 consecutive amino acids from residues 118 to 138 of mEPO or a homologue thereof. In another embodiment, an EPO derived peptide comprises at least 9 consecutive amino acids from residues 118 to 138 of mEPO or a homologue thereof. In another embodiment, an EPO derived peptide comprises at least 12 consecutive amino acids from residues 118 to 138 of mEPO or a homologue thereof. In some embodiments, an EPO derived peptide comprises 5-21 consecutive amino acids from residues 1 18 to 138 of mEPO. In some embodiments, an EPO derived peptide comprises residues 1 18 to 138 of mEPO. In some embodiments, an EPO derived peptide consists at least 5 consecutive amino acids from residues 1 18 to 138 of mEPO. In some embodiments, an EPO derived peptide consists 21 consecutive amino acids from residues 118 to 138 of mEPO. In another embodiment, an EPO derived peptide comprises or consists of 9 to 30 amino acids.

In additional embodiments, an EPO derived peptide is selected from the group comprising SEQ ID NOs: 1-27 or an analog or a fragment thereof. In other additional embodiments, the peptide comprises or consists of 12 to 17 consecutive amino acids from residues 118 to 138 of mEPO or an analog or a fragment thereof. In another embodiment, analog is a mimetic. In another embodiment, a peptide of the invention comprises at least 7 amino acids. In another embodiment, a peptide of the invention comprises at least 9 amino acids. In another embodiment, a peptide of the invention comprises at least 12 amino acids. Each possibility represents a separate embodiment of the invention.

As used herein, a peptide comprises a synthetic peptide or a recombinant peptide which is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the peptide in nature. Typically, a preparation of a peptide contains the peptide in a highly purified form, i.e., at least about 70% pure, at least about 80% pure, at least about 90% pure, greater than 90% pure, or greater than 95% pure.

The term "peptide" as used herein is meant to encompass natural, non-natural and/or chemically modified amino acid residues, each residue being characterized by having an amino and a carboxy terminus, connected one to the other by peptide or non-peptide bonds. The amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art. The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.

The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three- letter codes according to IUPAC conventions. When there is no indication, either the L or D isomers may be used.

The present invention encompasses peptide derivatives and analogs having amino acid substitutions, and/or extensions. The term "analog" as used herein refers to peptides according to embodiments of the invention comprising altered sequences by amino acid substitutions or chemical modifications. The amino acid substitutions may be of conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of an all L-amino acid or diastereomeric peptides of the invention with amino acids of similar charge, size, and/or hydrophobicity characteristics. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are known as conservative substitutions. Non-conserved substitutions consist of replacing one or more amino acids of an all L-amino acid or a diastereomeric peptides with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, substitution of a glutamic acid (E) to valine (V). The amino acid substitutions may also include non-natural amino acids. In another embodiment, peptide homologues may be synthesized, said homologs being essentially based on the disclosed peptides as regards their amino acid sequence but having one or more amino acid residues deleted, substituted or added. When amino acid residues are substituted, such conservative replacements which are envisaged are those which do not significantly alter the structure or biological activity of the peptides. For example basic amino acids will be replaced with other basic amino acids, acidic ones with acidic ones and neutral ones with neutral ones. In addition to homologues comprising conservative substitutions as detailed above, peptides homologues comprising non-conservative amino acid substitutions are further envisaged, as long as said homologues essentially retain the biological activities of the peptides, as detailed herein. In another embodiment, the term "homologue" is directed to a peptides having at least 70%, preferably at least 80%, most preferably at least 90% sequence similarity to the amino acid sequence of the peptides in question, as defined, e.g., by the BLOSUM-80, 62 or 45 amino acid substitution matrices. It is well appreciated by the skilled artisan that the degree of homology may be calculated taking into account the overall length of the sequences being compared.

The following provides conservative substitutions for amino acids: 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 another embodiment, a homologue is composed of at least one substituted amino acid according to the present invention.

Amino acid extensions may consist of a single amino acid residue or stretches of residues. The extensions may be made at the carboxy or amino terminal end of peptides according to embodiments of the invention. Such extensions will generally range from 3 to 20 amino acids in length. Preferably, the peptide comprises not more than 30 amino acid residues in total. One or more such extensions may be introduced into a peptide so long as such extensions result in an active peptide as described herein.

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

A "chemical derivative" as used herein refers to peptide containing one or more chemical moieties not normally a part of the peptide molecule such as esters and amides of free carboxy groups, acyl and alkyl derivatives of free amino groups, phospho esters and ethers of free hydroxy groups. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Preferred chemical derivatives include peptides that have been phosphorylated, C-termini amidated or N-termini acetylated.

"Functional derivatives" of the peptide of the invention as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it and do not adversely affect the antigenic properties thereof. These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the peptide substantially retains the desired functional property. In some embodiments, the peptide of present invention is biochemically synthesized such as by using standard solid phase techniques. In some embodiments, these biochemical methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. In some embodiments, these methods are used when the peptides are relatively short (about 5- 15kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

In some embodiments, solid phase peptide synthesis procedures are well known to one skilled in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic peptides are purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing by methods known to one skilled in the art.

In some embodiments, recombinant protein techniques are used to generate the peptide of the present invention. In some embodiments, recombinant protein techniques are used for generation of relatively long polypeptides (e.g., longer than 18-25 .amino acid). In some embodiments, recombinant protein techniques are used for the generation of large amounts of the peptides of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307- 311, Coruzzi et al. (1984) EMBO J. 3: 1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

In another embodiment, peptide of the present invention is synthesized using a polynucleotide encoding a peptide of the present invention. In some embodiments, the polynucleotide encoding peptides of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the peptides of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue specific expression of the peptides of the present invention. In some embodiments, the cis- regulatory sequence is suitable for directing inducible expression of the peptides of the present invention.

In some embodiment, tissue-specific promoters suitable for use with the present invention include sequences which are functional in specific cell population, example include, but are not limited to promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1 :268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al, (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas- specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland- specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Inducible promoters suitable for use with the present invention include for example the tetracycline-inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase "a polynucleotide" refers to a single or double stranded nucleic acid sequence which be isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

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

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

In one embodiment, the polynucleotides of the present invention further comprise a signal sequence encoding a signal peptide for the secretion of the peptides of the present invention. In some embodiment, the signal sequence is N-terminal to the CTP sequence that is in turn N-terminal to the peptide sequence of interest; e.g. the sequence is (a) signal sequence- (b) CTP- (c) sequence-of-interest- (d) optionally 1 or more additional CTP sequences.

In one embodiment, following expression and secretion, the signal peptides are cleaved from the precursor proteins resulting in the mature proteins.

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

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

In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the peptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the peptides coding sequence; yeast transformed with recombinant yeast expression vectors containing the peptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the peptide coding sequence.

In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems such as CHO cells) to express the peptides of the present invention. In one embodiment, the expression vector used to express polynucleotides of the present invention in mammalian cells is pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene.

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

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

In one embodiment, the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric peptides.

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

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

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

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

In some embodiments, introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

In one embodiment, it will be appreciated that the peptides of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in-vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy). .

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

It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the peptides), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed peptide.

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

In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant peptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art. In some embodiments, depending on the vector and host system used for production, resultant peptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coll, or retained on the outer surface of a cell or viral membrane.

In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is effected.

In one embodiment, the phrase "recovering the recombinant peptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.

In one embodiment, peptides of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the peptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the peptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the peptide and the cleavable moiety and the peptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et ah, Immunol. Lett. 19:65-70 (1988); and Gardella et ah, J. Biol. Chem. 265:15854-15859 (1990)].

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

According to some embodiments, the peptide of the present invention may be either linear or cyclic. Each possibility represents a separate embodiment of the invention. The present invention encompasses cyclic peptides with improved specificity or improved metabolic stability, as well as all cyclic peptides whose activity was not impaired upon cyclization.

As used herein, the term "cyclic" or "cyclized" denote a peptide or polypeptide whose amino and carboxy termini are themselves linked together with a covalent bond, including but not limited to a peptide bond, forming a continuous (e.g. circular) chain. In general, such compounds form covalently closed circles, and thus are not "loop structures", such as may be formed by formation of a disulfide bond between cysteines in a polypeptide having more than 3 or 4 residues. The term "cyclic peptide" as used herein refers to the peptide, wherein the peptide is composed of naturally-occurring amino acids that are covalently linked to one another by peptide bonds, where a peptide bond is -(C=0)-(N-H)-. In other embodiments, the invention relates to cyclized peptides, which encompass peptides rendered cyclic by a lactam bridge. Lactams can be of several types, such as "head-to-tail" (carboxy terminus to amino terminus), "head-to-side chain" and "side chain-to-head" (carboxy or amino terminus respectively to a side chain amino or carboxyl group) and "side chain-to-side chain" (amino group of one side chain and carboxyl group of another side chain). In certain other embodiments, the cyclized peptides encompass peptides in which the two terminal amino acids are bonded together by a synthetic non-peptide bond such as a thioether, phosphodiester, disiloxane, azo or urethane bond. Every possibility represents a separate embodiment of the invention.

The peptide of the invention can be targeted to specific locations by using targeting moieties. For example, the peptide of the invention may be conjugated to an antibody or fragment thereof which is immunoreactive with a tumor marker as is generally understood in the preparation of immunotoxins in general, and thus may directly deliver the peptide of the present invention to cancer cells. The targeting moiety can also be a ligand. The targeting ligand can be suitable for a receptor which is present on the tumor. Any targeting ligand which specifically reacts with a marker for the intended target tissue can be used. Methods for coupling the invention peptide to the targeting moiety are well known in the art. According to some embodiments of the present invention, the targeting moiety may be coupled to the N-terminal, to the C-terminal, or to any other free functional group along the peptides chain, for example, to the ε-amino group of lysine. The terms "coupling" and "conjugation" are used herein refer to the chemical reaction, which results in covalent attachment of a targeting moiety to peptides to yield a conjugate. According to some embodiments, coupling of a targeting moiety to peptides is performed similarly to the coupling of an amino acid to peptides during peptides synthesis. Alternatively, the coupling of a targeting moiety to peptides may be performed by any coupling method known in the art. Alternatively, the inhibitory peptides conjugated to the targeting moiety may be produced as a fusion protein using recombinant biotechnology methods as are well known in the art.

The present invention also contemplates several drug delivery systems that includes, but not limited to a variety of different media, medical devices, microparticles, liposomes, polymers, nanospheres, nanocapsules and nanoparticles. Note that any medium or carrier may be combined with another medium or carrier; for example, in one embodiment a polymer microparticle carrier attached to the peptides of the present invention may be combined with a gel medium. Carriers or mediums contemplated by this invention includes, but not limited to gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2- hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin-resorcin- aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof. A medical device contemplated by this invention comprise, but not limited to, a reservoir comprising the peptides of the invention, a catheter, a sprayer, or a tube In a preferred embodiment the peptides would be administered via an osmotic pump, ensuring continuous release of the peptides.

"A peptide conjugate" according to the present invention, denotes a molecule comprising the peptide of the invention to which another moiety, either peptidic or non peptidic, is bound, directly or via a spacer.

The term "spacer" denotes a chemical moiety whose purpose is to link, covalently, a cell-permeability moiety and a peptide or peptidomimetic. The spacer may be used to allow distance between the permeability-enhancing moiety and the peptide, or it is a chemical bond of any type. Linker denotes a direct chemical bond or a spacer. Pharmaceutical compositions

The present invention provides pharmaceutical compositions comprising the EPO-R antagonist peptide of the invention and a pharmaceutically acceptable carrier. Hereinafter, the phrases "physiologically acceptable", "physiologically suitable" and "pharmaceutically acceptable", which may be used interchangeably, when used to describe carriers, excipients or diluents, refer to such materials that do not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The pharmaceutical compositions useful in the practice of the present invention comprise a peptide according to some embodiments of the invention optionally formulated into the pharmaceutical composition as a pharmaceutically acceptable salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide), which are formed with inorganic acids, such as for example, hydrochloric or phosphoric acid, or with organic acids such as acetic, oxalic, tartaric, and the like. Suitable bases capable of forming salts with the peptide of the present invention include, but are not limited to, inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like).

A pharmaceutical composition useful in the practice of the present invention typically contains a peptide according to some embodiments of the invention formulated into the pharmaceutical composition as a pharmaceutically acceptable salt form. Pharmaceutically acceptable salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.

Pharmaceutically acceptable salts may be prepared from pharmaceutically acceptable non-toxic bases including inorganic or organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylarninoethanol, ethanolamine, ethylenediamine, N- ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

Techniques for formulation and administration of drugs may be found in the latest edition of "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, which is herein fully incorporated by reference (Remington: The Science and Practice of Pharmacy, Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, Pa., 20th ed, 2000).

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

The pharmaceutical compositions of the invention are suitable for administration systemically or in a local manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. In particular embodiments, the peptide is administered by injection, e.g. subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally or by intralesional administration directly to a tumor for example. Each possibility represents a different embodiment of the present invention.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

A therapeutically effective amount of a peptide according to some embodiments of the invention is an amount that when administered to a patient is capable of exerting an inhibitory activity of EPO-induced EPO-R phosphorylation, cell proliferation, migration, and survival. According to some embodiments, a pharmaceutical composition of the present invention is useful for the treatment of erythrocytosis in a patient. According to some embodiments, erythrocytosis is a primary erythrocytosis. According to some embodiments, erythrocytosis is a secondary erythrocytosis. According to such embodiments, a therapeutically effective amount is an amount that when administered to a patient is sufficient to mediate, preferably to reduce red blood cell mass.

The pharmaceutical compositions of the present invention comprise at least one peptide according to the embodiments of the present invention, and methods of the present invention involve the administration of at least one peptide according to embodiments of the present invention.

It is to be further understood that the peptide according to embodiments of the present invention may be therapeutically used in combination with additional therapies.

As used herein, "treating" a disease or condition (or treating a subject with a disease, e.g. erythrocytosis) refers to taking steps to obtain beneficial or desired results, including but not limited to, alleviation or amelioration of one or more symptoms of the disease, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival and other beneficial results known in the art.

As used herein, the terms "inhibiting" or "reducing" refer to either statistically significant inhibition or reduction, or to inhibition or reduction to a significant extent as determined by a skilled artisan, e.g. the treating physician. It should be understood, that inhibition or reduction does not necessarily indicate a total elimination of the measured function or biological activity. A reduction in activity may be for example about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

MATERIALS AND METHODS

Peptides

All peptides were synthesized by GL Biochem and were at least 90% pure. The peptides were N-terminus acetylated in and C-terminus amidated.

Antibodies

Rabbit antibody directed against a glutathione S -transferase fusion protein containing the extracellular domain of the murine EPO-R (Cohen, J. et al. (1997) Biochem J 327 ( Pt 2):391-7; Ravid, O. et al. (2007) Proc Natl Acad Sci U S A 104: 14360-5) was used to bind solEPO-R. Anti EPO antibody was purchased from R&D systems (Minneapolis, MN) and anti-phospho-EPO-R (pY479) antibody was purchased from Santa Cruz (Santa Cruz, CA). The antibody against phosphorylated ERKl/2 was purchased from Cell Signaling Technology (Danvers, MA) and anti- actin antibody from Chemicon (Billerica, MA).

Generating the 3-D structure of mouse EPO-EPO-R complex

The alignment between the mouse and human sequences of EPO and EPO-R were generated using Blast software. The excision number used for and hEPO was P07321 and P01588, respectively. The excision number used for mEPO-R and hEPO- R was PI 4753 and PI 9235 respectively. The crystal structure of the human EPO- EPO-R complex that was used to generate the mouse model is at a resolution of 1.9 A

[Protein Data Bank (PDB) ID code 1EER] (Syed, R. S. et al, (1998) Nature 395:511- 6). All the modeling was performed using MODELER default parameters as integrated in Discovery studio (Accelrys).

Generation of soluble EPO-R

293 cells were transiently transfected with mouse solEPO-R cDNA using the calcium chloride method (Jordan, M. et al. (1996) Nucleic Acids Res 24:596-601). After 24 hours, the cells were washed and fresh serum-free DMEM medium was incubated for 5h at 37°C, 5% C0₂. The medium containing secreted solEPO-R was then collected and spun at 1500 RPM for 5 min. The supernatant was concentrated using a lOkDa membrane Centriocon filter unit (Millipore), and was used as the source for solEPO-R.

EPO binding Assay

96-well microtiter plates were coated with an antibody against the N terminus of EPO-R in phosphate buffered saline (PBS). Excess antibody was removed and wells were quenched with PBS- containing 1% bovine serum albumin (BSA).

Media containing solEPO-R were added to the wells and incubated overnight at 4 C . The wells were then washed 3x with Tris-buffered saline containing 0.05% tween-20 (TBS-T) and incubated at room temperature for 1 h with rHuEPO (5U/ml) in the absence or presence of 10μΜ EPOantO or control peptide. Following 3x wash with TBS-T the wells were probed with an antibody against EPO for 1 h and secondary antibody conjugated to HRP for an additional hour. Tetramethylbenzemidine (Sigma) was added and binding was followed by spectrophotometery (O.D: 450nm).

EPO induced EPO-R activation

Ba/F3 cells expressing EPO-R (2xl0⁶ cells for each time point) were starved for 1 h at 37 C by incubating the cells in RPMI medium without supplements. Subsequently, the cells were pre-incubated with or without 10μΜ EPOantO or control peptide followed by 5 minutes of incubation with rHuEPO (5 U/ml). Cells were then lysed at 4^°C in 200μ1 of lysis buffer (50 mM Tris pH 7.4, 1% Triton X-100, 5 mM iodoacetic acid, 5 mM EDTA, 150 mM NaCl) containing phosphatase (2mM ZnCl₂ , 2mM Vanadate, 50mM NaF, 20mM Na P₂0₇) and protease inhibitors (Complete Protease Inhibitors, Roche Diagnostics). Cell lysates were then spun at 14,000 rpm for 10 min and the supernatants were collected, separated on SDS-PAGE gel and subjected to Western blot analysis.

Proliferation assay

To determine the proliferation rate, BaF/3 cells (3x10 per well) were cultured in 96-well plates for 1, 2 and 3 days in the presence of rHuEPO 0.01 U/ml with or without 10μΜ EPOantO or control peptide. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) colorimetric assay. Absorbance at 570 nm was measured after 4 h incubation. Wound healing (scratch) assay

MDA-MB-435 cells were grown to 100% confluence in a 6 well plate and a cross shaped scratch was created using a sterile tip. The cells were then washed and transferred to medium containing 0.1% serum with or without EPO in the presence or absence of EPOantO. The cross area was captured on day 0 and on two consecutive days. Percent migration was derived from the distance between the two sides of the scratch measured for each field and captured at 5 different points using Adobe illustrator. The percent of cell migration was calculated as 100% minus the distance between the two sides of the scratch on day 1 divided by the distance at the same location on day 0 for each treatment.

Migration assay

MDA-MB-435 cells were incubated with FCS free medium over night. Cell migration assay was performed using modified Boyden chambers Transwells^®. Cells were placed at the top of a transwell chamber in the presence or absence of EPO (lOU/ml) and EPOantO (10μΜ) and allowed to migrate to the underside of the chamber for 24h. Cells were then fixed and stained. The stained migrated cells were captured and imaged. Migration was normalized to percent migration, with 100% representing migration without EPO.

Cell survival analysis.

Ba F3 cells stably expressing EPO-R were cultured overnight in RPMI containing 10% FCS and 0.1 U/ml of rHuEPO in the presence or absence of EPOantO. The cells were then put on ice, washed three times in cold PBS containing 2% FCS, subjected to 0.1% Triton X-100 and stained with propidium-iodide (PI) in order to assess their cell cycle profile. EPOantO effect on blood count

Six control mice and five peptide treated mice were injected for 10 days with either PBS or EPOantO (0.37mg/per day/per mouse) respectively. At termination body and spleen were weighed. Blood hemoglobin MCV and hematocrit levels were analyzed. EPO-R mRNA levels increase in EPOantO treated mice Total RNA was isolated from bone marrow and spleen cells using TRIzol reagent. Reverse transcription (RT) was performed using MMLV Reverse Transcriptase from 2 μg of total RNA. Real-time RT PCR was performed using SYBR green with primers for EPO-R, to monitor double- stranded DNA (dsDNA) synthesis. The results were quantified using a comparative Ct method. TMB was used as control mRNA.

EXAMPLE 1: Identification of potential EPO-R antagonists

In order to identify EPO-derived peptides that would competitively bind EPO- R, the crystal structure of EPO bound to the extracellular domain of human EPO-R (hEPO-R) was used (Livnah O. et al., 1996, Science 273:464-7; Syed R. S. et al., 1998, Nature 395:511-6). This structure was shown to have very few interaction points between EPO and EPO-R (Barbone F. P. et al., 1997, J Biol. Chem. 272:4985- 92; Elliott S. et al., 1997, Blood 89:493-502). The first predicted 3-D structure was that of murine EPO (mEPO) bound to mEPO-R. For that, the inventors have generated a sequence homology alignment of the mouse and human proteins using BLAST software (Fig. 1A and B). The homology between the human and mouse extracellular domain of EPO-R and the human and mouse EPO molecules is high (80% identity, 86% similarity for the mouse and human EPO-Rs, and 75% identity, 84%o similarity for the respective EPOs). The comparison refers to the entire EPO molecule (residues 27 to 192 of mEPO and residues 28 to 193 of hEPO). The aligned sequences and the 3-D structure of hEPO bound to the hEPO-R dimer, were introduced into the Discovery Studio software (Accelrys), and using default parameters, the 3-D model of the mEPO-mEPO-R complex was generated (Fig. 1C). As expected from the high sequence homology, the predicted structure of the mouse EPO-EPO-R complex was very similar to that of human complex.

Utilizing this model it was possible to identify potential regions in EPO that bind to EPO-R. Using the Discovery Studio software, all the residues in EPO that are located within a 5A distance from the EPO-R were identified (Fig. 2). As can be seen, EPO binds to the EPO-R dimer in a non-symmetrical manner. On the left side of the depicted structure, the area binding to the EPO-R has a random coil structure (Fig 2). The flexibility of this binding region prevents this area from being a good candidate for the design of an antagonist.

Without wishing to be bound by theory or mechanism of action, a peptide derived from a helical structure (unlike a peptide which is derived from a random coil) may be able to maintain or mimic its native structure within EPO upon binding and thus bind to the same location on EPO-R as the helical structure it was derived from. At the right side of the depicted dimer (Fig. 3), the inventors detected three helices on EPO that were predicted to interact with EPO-R. In order to identify the region based on which the most suitable EPO derived peptide can be selected, a surface map of the EPO-R was generated using the Discovery Studio software (Fig. 3). This surface map shows the electrostatic potential of the EPO-R surface in a 5 A distance from EPO. While most of the EPO-R area that is in contact with EPO is hydrophobic, the right side presents a pattern of negative and positive charges on the EPO-R surface. These residues have high potential for interaction with a peptide by creating electrostatic interactions. Careful analysis of the 3 helices region on EPO resulted with the prediction that the dashed circled helix, corresponding to residues 1 18 to 138 of mEPO would exert the strongest interaction with the EPO-R and would thus be the most promising candidate for designing an antagonist (Fig. 4).

A stretch of 17 amino acids (residues 121-137 of mEPO; IDKAISGLRSLTSLLRV (SEQ ID NO: 25) was selected as an exemplary inhibitory peptide for inhibiting EPO-EPO-R binding (Fig. 4A). This peptide was termed EPOantO (EPO antagonist Original). It is important to note that the human sequence, namely VDKAVSGLRSLTTLLRA (SEQ ID NO: 16) is similar but not identical to the mouse sequence (see Fig 1). Since these two sequences are about 80% identical, it is recognized that peptides that are homologous but not identical to EPOantO would have similar antagonistic activities. Furthermore, the inventors have recognized that amino acids in the a helical peptide that face the EPO-R (underlined: KAIS GLRSLTSL) are the most important for the antagonistic activity. Notably, these six amino acids are conserved in human (homo sapiens) EPO (hEPO), mouse (Mus musculus) EPO (mEPO), cat (Felis catus) EPO (cEPO) and rat (Rattus norvegicus) EPO (rEPO), as can be seen herein below, in the multiple sequence alignment of this EPO region:

hEPO VDKAVSGLRSLTTLLRA (SEQ ID NO: 16) cEPO VDKAVSSLRSLTSLLRA (SEQ ID NO: 27)

rEPO and mEPO IDKAISGLRSLTSLLRV (SEQ ID NO: 25).

According to some embodiments, the present invention is directed to peptides consisting of 9-30 amino acids, comprising at least 9 contiguous amino acids derived from the a helical peptide that face the EPO-R (KAISGLRSLTSL). According to further embodiments, 9 amino acids peptides derived from that region, having the amino acid sequence set forth in SEQ ID NOs: 1-4 are also expected to bind EPO-R and compete with EPO for binding to EPO-R. Additionally, 12 amino acids long peptide sequences having the amino acid sequence set forth in SEQ ID NOs: 5-9, are also included. Other examples include sequences that consists 17 amino acids, having the amino acid sequence set forth in SEQ ID NOs: 10-26.

EXAMPLE 2: EPOantO inhibits EPO binding to EPO-R

In order to evaluate the binding capacity of EPOantO to EPO-R, binding experiments of EPO to EPO-R in the presence or absence of the peptide were performed. Microtiter 96 well plates were coated with solEPO-R, which is the extracellular domain of mEPO-R, and incubated for 1 h with 5 U/ml rHuEPO in the presence or absence of 1 ΟμΜ EPOantO. Alternatively, solEPO-R was incubated with EPO in the presence or absence of a 17 mer control peptide (TQAQRJFATANALRKTA). The sequence of the control peptide was derived from the mouse EPO onto which several amino acid substitutions were introduced. As can be seen in Fig. 5, EPOantO, but not the control peptide, significantly inhibited EPO binding to solEPO-R, suggesting that the peptide is an antagonist for EPO-R.

EXAMPLE 3: EPOantO inhibits EPO-induced EPO-R activation

The ability of EPOantO to inhibit EPO-induced activation was further tested.

Ba/F3 cells expressing mEPO-R were incubated with serum and EPO depleted medium for 1 h. EPOantO was then introduced into the medium for 10 min. The cells were then incubated for additional 5 min with rHuEPO (5U/ml). EPO-induced EPO-R activation was measured using a selective antibody directed against phosphorylated EPO-R. Treatment with EPOantO reduced the activation levels of EPO-R in response to EPO stimulation compared with non-treated or control peptide treated cells (Fig. 6). In addition, the levels of phosphorylated ERK were also reduced. These results indicate that EPOantO is capable of inhibiting EPO-induced EPO-R activation and the activation of downstream effectors. As expected, treatment with EPOantO alone (without EPO) had no effect on EPO-R activity, ensuring that EPOantO has no agonistic effect on EPO-R (data not shown).

The effect of EPOantO on EPO-induced activation in the human cell line UT- 7 cells was further tested. UT-7 human cell line was established from the bone marrow of a patient with megakaryoblastic leukemia. The UT-7 human cells endogenously express EPO-R and may thus serve as a useful model for testing the effect of the antagonist on human EPO-R. Similarly to the results obtained from Ba/F3 cells, EPOantO inhibited the phosphorylation level of EPO-R in UT-7 cells (data not shown). EPOantO is derived from the sequence of mEPO and acts as an antagonist of HuEPO-R as well.

EXAMPLE 4: EPOantO inhibits EPO induced cell proliferation, survival, and migration.

Activation of EPO-R in response to EPO leads to cell proliferation (Richmond, T. D. et al., 2005, Trends Cell Biol 15: 146-55). To test whether activation of EPO-R by EPOantO induce cell proliferation, cell proliferation assay using MTT reagent was performed. EPO-R expressing BaF/3 cells were incubated with EPO in the presence or absence of 10μΜ EPOantO. Cells viability was measured for three consecutive days. As can be seen in Fig. 8, EPOantO inhibited EPO-induced cell proliferation. This effect was significant as of day 2 and was even more pronounced on day 3 (Fig. 8).

EPO also plays a key role in cell survival (Hedley BD et al., 2011, Clin Cancer

Res; 17(19); 1-12). To test whether inhibition of EPO-R by EPOantO would have a negative effect on cell survival a cell cycle analysis using FACS was performed. Cell cycle stage analysis in Ba/F3 cells that were cultured in a low concentration of EPO (0.1 U/ml), in the presence or absence of EPOantO were determined. Data depicted in Fig. 9 demonstrate that treatment of the cells with EPOantO significantly increased the percent of cells in sub-Gl : from 27.6±6% in untreated cells to 38±3% in cells treated with EPOantO (Fig. 9). Expression of EPO-R on cancer cells raises the concern that activation of EPO-R would lead to their undesirable proliferation (Fandrey, J. et al. 2009, Oncologist; 14:34-42) .This undesirable phenomenon could be overcome by using EPO-R blocking agents that would be targeted to the malignant cells, thereby enabling a safe administration of EPO for the treatment of cancer associated anemia. The breast cancer cell line MDA-MB-435 that was previously shown to express EPO- R (Shi Z et al. 2010, Mol Cancer Res; 8: 615-626) was utilized as to test the effect of EPOantO on cell migration. Cells migration was evaluated using both scratch and migration chamber assays. As can be seen in Fig. 10, treatment with EPO alone induced cell migration. EPO induced cell migration was inhibited in cells treated with EPO in combination with EPOantO, suggesting that said antagonist inhibits the undesirable EPO induced cell migration.

EXAMPLE 5: EPOantO inhibits MCV levels in mice BALB/C mice were injected intra-peritonealy (i.p) with either 0.375 mg/mouse of EPOantO or control (PBS) every day, for 10 days. At termination, mice were euthanized and blood samples were subjected for blood count analysis (Fig 10). In addition, body and spleen were weighed, showing no difference between the control and EPOantO treated mice. There was no significant difference in hematocrit (proportion of blood volume that is occupied by erythrocytes) and hemoglobin levels between the control and EPOantO treated mice. However, there was a significant decrease in the mean corpuscular volume (MCV) levels. Low values of MCV may indicate of anemia, suggesting that treatment with EPOantO induced anemia (Fig 10).

EPOantO increases EPO-R mRNA levels in the bone marrow and spleen of mice.

EPO starvation was shown to increase the mRNA levels of EPO-R (Nalbant D et al. J Pharmacol Exp Ther, 2010; 333:528-32). Thus, the hypothesis was that inhibition of EPO with EPOantO would lead to similar results. The spleen and bone marrow of mice treated with diluent (PBS) or EPOantO were collected and mRNA was isolated. Using real time PCR the mRNA levels of EPO-R of control and EPOantO treated mice were evaluated. EPO-R mRNA levels increased in both bone marrow and spleen although the latter was not statistically significant (Fig. 11). These results indicate that although minimal, EPOantO had an effect on blood counts, and on modulating the EPO-R mRNA expression.

The in vitro and in vivo results presented above indicate that EPOantO serves as an EPO-R antagonist capable of inhibiting the binding of EPO to EPO-R. Consequently, EPOantO inhibited EPO mediated EPO-R phosphorylation, as well as EPO-induced cell proliferation, migration, and survival. Furthermore, treatment with EPOanto increased mRNA expression of EPO-R in the spleen and bone marrow of mice, indicating of EPO-starvation. Finally, as demonstrated from MCV count analysis, by inhibiting binding of endogenous EPO to EPO-R, EPOantO induced at least to some extent anemia in mice.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A peptide comprising at least 9 contiguous amino acids derived from the sequence of general Formula I:

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12

wherein X and X₇, each independently selected from the group consisting of Lys,

Arg, and Orn;

X₃, selected from the group consisting of Val, He, Leu, and Met;

X₆, and X9, each independently selected from the group consisting of He, Asn, Gin, and Leu;

X4 and X₈, each independently selected from the group consisting of Ser, Thr, and

Ala;

X_2, and X₅, each independently selected from the group consisting of Ala, and Gly; and

Xio, and Xn_, each independently selected from the group consisting of Ala, Thr, Leu, and Ser, and,

X₁₂, selected from the group consisting of He, Leu, and Phe;

wherein said peptide is 9 to 30 amino acids long.

2. The peptide of claim 1, comprising 12 amino acid residues according to general Formula I.

3. The peptide of claim 1, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.

4. The peptide of claim 1, wherein is Lys;

X₂, selected from the group consisting of Ala, and Gly;

X₃, selected from the group consisting of Val, lie, Leu, and Met;

4 and X₈, is Ser;

X₅, selected from the group consisting of Ala, and Gly;

X₇ is Arg;

X₉ is Leu; Xio, and Xn_, each independently selected from the group consisting of Ala, Thr, and Ser; and,

X₆ and X₁₂, each independently selected from the group consisting of Leu, Met, Val, and He.

5. The peptide of claim 1 , wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-9.

6. The peptide of claim 1, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-26.

7. The peptide of claim 1, consisting 12 to 17 amino acids.

8. A peptide according to any one of claims 1-7 consisting a sequence selected from the group consisting of SEQ ID NOs: 1-26.

9. The peptide of claim 1 , wherein said peptide is cyclized.

10. A pharmaceutical composition comprising the peptide of claim 1, and an excipient.

11. The pharmaceutical composition of claim 10, wherein said peptide comprises 12 amino acid residues according to general Formula I.

12. The pharmaceutical composition of claim 10, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26.

13. The pharmaceutical composition of claim 10, wherein said peptide comprises amino acid sequence according to general Formula I, wherein,

X] is Lys;

X₂, selected from the group consisting of Ala, and Gly;

X₃, selected from the group consisting of Val, He, Leu, and Met;

X and X₈, is Ser;

X₅, selected from the group consisting of Ala, and Gly;

X₇ is Arg;

X₉ is Leu; Xio, and X_11; each independently selected from the group consisting of Ala, Thr, and Ser; and

X₆ and X₁₂, each independently selected from the group consisting of Leu, Met, Val, and lie.

14. The pharmaceutical composition of claim 10, wherein said peptide consists 12 to 17 amino acids.

15. The pharmaceutical composition of claim 10, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26.

16. A polynucleotide encoding the peptide of claim 1.

17. A vector comprising the polynucleotide of claim 16.

18. A method for treating a subject afflicted with erythrocytosis, comprising administering to said subject an effective amount of the peptide of claim 1, thereby treating a subject afflicted with erythrocytosis.

19. The method of claim 18, wherein said peptide comprises 12 amino acid residues according to general Formula I.

20. The method of claim 18, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26.

21. The method of claim 18, wherein said peptide comprises amino acid sequence according to general Formula I wherein,

X₁ is Lys;

X₂, selected from the group consisting of Ala, and Gly;

X₃, selected from the group consisting of Val, He, Leu, and Met;

and X₈, is Ser;

X₅, selected from the group consisting of Ala, and Gly;

X₇ is Arg;

X₉ is Leu;

X]0, and Xn_, each independently selected from the group consisting of Ala, Thr, and Ser; and X₆ and X₁₂, each independently selected from the group consisting of Leu, Met, Val, and He.

22. The method of claim 18, wherein said peptide consists 12 to 17 amino acids long.

23. A method for altering binding of EPO to EPO-receptor (EPO-R) in a cell, comprising the step of contacting said cell with the peptide of claim 1, thereby altering binding of EPO to EPO-receptor.

24. The method of claim 23, wherein said altering is inhibiting.

25. The method of claim 23, wherein said altering binding of EPO to EPO-R in a cell results in inhibiting EPO-R phosphorylation, inhibiting EPO induced cell proliferation, inhibiting EPO induced cell migration, inhibiting EPO induced cell survival, or any combination thereof.

26. The method of claim 23, wherein said peptide comprises 12 amino acid residues according to general Formula I.

27. The method of claim 23, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-26.

28. The method of claim 23, wherein said peptide comprises amino acid sequence according to general Formula I wherein,

X] is Lys;

X₂, selected from the group consisting of Ala, and Gly;

X₃, selected from the group consisting of Val, He, Leu, and Met;

X and X₈, is Ser;

X₅, selected from the group consisting of Ala, and Gly;

X₇ is Arg;

X₉ is Leu;

X10, and X_11; each independently selected from the group consisting of Ala, Thr, and Ser; and

X₆ and X₁₂, each independently selected from the group consisting of Leu, Met, Val, and He.

29. The method of claim 23, wherein said peptide consists 12 to 17 amino acids.