WO2011012306A2 - Use of emps for antagonising epo-stimulatory effects on epo-responsive tumors while maintaining erythropoiesis - Google Patents

Abstract

The present invention pertains inter alia to the use of a compound comprising an EPO mimetic peptide or a functional variant thereof for prophylactic or therapeutic treatment of a patient afflicted with or at risk of being afflicted with a disease wherein EPO adversely affects the mortality and/or disease progression.

Description

"Use of EMPs for antagonising EPO-stimulatory effects on EPO-responsive tumors while maintaining erythropoiesis"

Erythropoiesis stimulating agents (ESAs) are successfully used in clinical indications such as renal insufficiency and tumor-associated or chemotherapy-associated anemias to restore erythrocyte (hematocrit) and hemoglobin levels, and oxygen transport. Erythropoiesis is naturally regulated by the cytokine erythropoietin (EPO) which is synthesized by the kidney.

ESAs used in medicine are usually based on natural EPO but, due to the expression systems used, or due to modifications introduced intentionally, have modified properties

(reviewed in MacDougall et al., 2006; Elliot et al., 2008; Jelkmann 2008). New generation modified ESAs such as Darbepoietin alpha (Aranesp) and CERA were developed primarily because of their extended biological half-lives.

EPO, apart from inducing erythropoiesis in hematopoietic progenitor cells, has been described to have various effects on cell types and in vivo models. Thus, EPO was described to have pleiotropic activities outside of the hematopoietic lineages. Erythropoietin e.g. was described

- to act on neuronal cells;

- to stimulate tumor cell proliferation in vitro;

- to act anti-apoptotic in many systems and e.g. to inhibit cisplatin toxicity;

- to stimulate angiogenesis and to be potentially beneficial in wound healing;

- to be relevant or even a cofactor of tumor development in rodent models in vivo.

The effects listed above are exemplary and certainly not complete in this aspect. However, little was known about possible interferences of EPO pleiotropy with clinical situations. Cancer progression is influenced by multiple factors including the induction of tumor angiogenesis. EPO is the hematopoietic cytokine that regulates the formation of red blood cells by binding to the erythropoietin receptor (EPOR), a member of the cytokine receptor family that is expressed not only in erythroid cells, but also in many non-hematopoietic cell types including vascular endothelial cells and cancer cells. Recent clinical trials reported that recombinant EPO therapy in some cancer patients may negatively impact recurrence-free survival have raised concerns regarding potential adverse direct effects of erythropoietin in tumors, such as the stimulation of the proliferation of cancer cells and/or tumor angiogenesis E.g. for a long time EPO was explicitly not approved for the treatment of anemia in myeloic disorders like MDS and myeloic leukemias, as it was known from the very beginning that EPO can cause undesired tumor stimulation (stimulation of malignant blasts in the bone marrow). Myeloic diseases are explicitly excluded from ESA approvals. Recently, Erythropoietin and Aranesp have demonstrated adverse effects in a series of clinical trials on cancer patients, causing shortened survival and/or increased tumor growth in the groups receiving ESA compared to placebo groups (see e.g. Henke et al., 2003; Leyland-Jones and BEST Investigators and Study Group, 2003; Danish Head-and Neck Cancer Group, 2007; Wright et al., 2007). The FDA has issued warnings with regard to the use of ESAs in certain tumor types, in cancer patients not under chemo- or radiotherapy, and against the use of high doses ESAs intended to increase Hb values above 12 g/l (see e.g. Jenkins et al., 2007). ESAs also can pose a risk in the treatment of patients with myelodysplastic syndrome (MDS). MDS is a hematological disorder associated with poor hematopoiesis in various lineages, which frequently develops into acute myeloid leukaemia (AML). Treatment of anemic MDS patients with ESA, even in high dosages, often fails to restore erythropoiesis (see e.g. Moyo et al., 2008). At the same time treatment of MDS patients with EPO has been shown to be able to stimulate disease progression towards AML (Bunworasate et al., 2001).

It is the object of the present invention to provide improved prophylactic and therapeutic concepts.

It is a further object of the present invention to stimulate erythropoiesis in a patient while reducing EPO associated effects on diseases wherein EPO adversely affects the mortality and/or disease progression. SUMMARY OF THE INVENTION

The present invention is based on the finding that erythropoietin mimetic peptides (EMP) show a differential stimulating effect on normal erythropoietic progenitor cells on one hand and on malignant cells on the other hand. Cells of the first type, which, among other characteristics appear to express high levels of EPO receptor, are efficiently stimulated to differentiate and proliferate by EMPs. That EPO and EMP both may achieve the same maximum effect level was inter alia demonstrated in a model cell line that can be used to measure the effects of ESAs such as EPO and EMPs. The cell line UT7/EPO which shows high expression levels of EPO receptor is, just like erythropoietic progenitor cells, stimulated as efficiently by EMP as by EPO. Both EPO and EMP induce cellular signalling and cell proliferation with similar maximum effects in the cell line UT7/EPO. Thus, EMPs can - as EPO - efficiently stimulate their target cells and induce erythropoiesis.

However, surprisingly, it was now found that EMPs differ from EPO in their stimulatory effects on other, non-target cells. It was found by the inventors that EMPs do not efficiently induce cellular signalling and survival of malignant cell types which are, however, stimulated efficiently by EPO. In the performed assays malignant cell types were used for testing that do express EPO receptors but always at relatively low levels compared to those on erythroid progenitor cells or UT7/EPO cells. The maximum effect achievable with an EMP on those cell lines is much lower than the maximum effect achievable with EPO or other strong ESAs such as Aransep. Apparently, the EPO sensing system on these cells is responding weaker or not at all to EMPs. EMPs even show EPO-inhibitory effects on such cells and were found to reduce the stimulatory effects of EPO on the tested EPO responsive malignant cells to the maximal effect possible when using an EMP - even in the presence of EPO. In genetically engineered cell lines expressing differing levels of EPO receptors, the maximum response to EMP in comparison to the maximum response to EPO, correlated with the relative surface expression level of the EPO receptor. An equal responsiveness to EMP and to EPO was found only in cells expressing the highest EPO receptor levels. Cells that express relatively low amounts of the functional EPO receptor responded less strongly to EMPs than to EPO. The inventors thus showed that the expression level of the EPO receptor is one important determinant for the question of whether a cell type responds strongly to EMP or not. Thus, cancer cells derived from non-erythroid cell types, as well as endothelial cells as part of the vascular system feeding cancer cells in situ, which express in general very low, to almost undetectable levels of EPO receptor (Sinclair et al., 2010) respond less strongly to EMPs compared to EPO. The low relative EPO receptor surface levels found on the cell types tested in the examples which also responded only weakly to EMP in comparison to EPO, demonstrate that these cell types are good examples of most cancer cell types which express low relative EPO surface levels and accordingly, which are stimulated less efficiently by EMPs compared with EPO.

Therefore, the inventors found that while EMPs are adequate agonists of EPO in normal hematopoietic tissues thereby stimulating, respectively maintaining erythropoiesis, EMPs show EPO antagonising effects in extrahematopoietic tissues, especially in myeloid and non- myeloid malignancies or pre-malignancies. The stimulation, by EPO or by EMP, of the target erythroid progenitor cells is the desired effect of e.g. anaemia treatment, leading to increased red cell mass, haemoglobin, and oxygen transport capacity. However, in particular when treating tumor-related anaemias the co-stimulation of malignant or pre-malignant cells by EPO is of course an unwanted side- effect and a considerable risk of a respective EPO treatment. The differential stimulation pattern now found for EMPs is of important clinical value and opens up new and valuable prophylactic and therapeutic treatment possibilities. E.g. treatment of tumor-related anaemias with EMPs instead of EPO allows to avoid the drawbacks of regular EPO treatments, which are particularly the increased risks of mortality and/or tumor promotion, respectively tumor progression. Furthermore, treatment with EMPs also provides curative applications because it was shown by the inventors that EMPs can antagonize the undesired stimulatory effects of EPO in particular on EPO-responsive malignancies and/or pre-malignancies while maintaining functional erythropoiesis. Partial or complete removal of the EPO-based stimulatory potential with the concept of EMP-controlled erythropoiesis in these clinical situations is changing the focus of therapy of malignancy or pre-malignancy associated anemia from a purely supportive therapy concept to a therapeutic strategy with an inherent curative component by antagonising the harmful stimulatory effects of EPO (which is usually always present due to the endogenous EPO production) on said EPO-responsive malignancies and/or pre-malignancies.

Furthermore, many patients afflicted with a malignancy or pre-malignancy show elevated endogenous EPO levels and thus EPO levels that are above the level of a healthy person. Elevated endogenous EPO levels may also be a side effect of chemotherapy or other cancer related treatment methods. E.g. induction of EPO-expression is a normal reaction of the body as soon as the oxygen levels are falling, e.g. due to an anemic condition which is often observed as a side-effect of cancer therapy. Bleeding and direct myelosuppresion can occur in conjunction with a malignancy or treatment of a malignancy and both can cause mild to moderate anemia with endogenous EPO induction as a feedback reaction. It is also under discussion that tumors themselves lead to a rise in the endogenous EPO levels. This is especially true for those malignant diseases, which originate from the bone marrow, and thus particularly preleukemic and leukemic conditions (e.g. Myelodysplastic Syndrome (MDS), and leukemias as well as other premalignant and malignant diseases). In these cases, the bone marrow undergoes dysplastic modifications and is increasingly unable to produce enough erythrocytes and other blood components. The resulting anemia leads to increased EPO production, and e.g. in MDS patients extraordinarily high peak concentrations can be reached. Increased EPO concentrations set the patient at a higher risk of co-stimulation of its tumor and e.g. developing an acute myeloid leukemia (AML). This contributes to the bad prognosis and adds a building block to the pathogenetic chain, which is associated with severe anemia e.g. in MDS. Severity of anemia is one predictor of the outcome for an MDS patient. Severe anemia implies a bad prognosis. Physicians frequently treat the anemia with

EPO - though this is an off-label use - thereby ameliorating the feedback reaction of the body which is already overproducing EPO. Elevated EPO levels in a patient may thus also be the result of an EPO therapy. These elevated EPO levels in the patient also pose an increased risk of mortality and/or tumor promotion due to the respective stimulatory effects of EPO (see above and below). Also in such a clinical setting, the treatment with EPO mimetic peptides provides several advantages which will also be outlined briefly below.

EMPs can inhibit binding of EPO to the EPO receptor. The obtained data also demonstrates that EMPs do not stimulate, or stimulate at least to a considerably lesser extent than EPO, EPO-responsive malignant cells. The same applies to EPO-responsive pre-malignant cells. The potential of EMPs to inhibit binding of EPO to its receptor and the differential stimulation pattern of EMPs result in a reduced or even eliminated stimulatory/growth promoting effect of high EPO levels on EPO responsive malignancies and pre-malignancies while maintaining functional erythropoiesis. Therefore, the administration of EMPs has important therapeutic benefits for treating patients with elevated EPO levels or at risk of developing elevated EPO levels. Also prophylactic benefits are worth mentioning because the treatment with EMPs is also beneficial in the preparation of a patient for chemo- or radiotherapy, as such therapies pose a risk of developing elevated endogenous EPO levels and often causes a physician to administer EPO for anemia correction.

Furthermore, it is also known that malignancies - especially solid tumors like e.g. head and neck cancer, breast carcinoma, sarcoma and the like - can develop so-called autocrine loops. It is known that EPO-dependent autocrine loops can cause independent autonomous tumor growth. Autocrine loop implies that a tumor cell or tumor subclone is producing both, EPO receptor and EPO in small amounts. EPO is then locally acting back on the tumor cell itself. Such tumor clones are often independent from normal physiological EPO production. Autocrine loops in tumors will profit from high systemic EPO concentrations, but will be inhibited by the administration of EMPs, since EMPs will occupy the tumor cells' EPO receptors (preventing binding of the tumor produced EPO) while only weakly activating or not activating the tumor cells. Furthermore, the administration of the EPO mimetic peptides lead to a rise in the haemoglobin level, as EPO mimetic peptides do elicit the EPO functions at their normal targets, the erythropoietic progenitor cells and accordingly, do stimulate erythropoiesis. Indirectly, also the endogenous EPO levels can be reduced and/or the effects of EPO can be antagonised and accordingly, also the stimulatory effects of the endogenous EPO levels on the malignancies and pre-malignancies, in particular EPO responsive malignancies and pre- malignancies can be reduced. Thus, while bone marrow is responsive to an EMP, reduction of anemia will reduce endogenous EPO production and accordingly, decrease endogenous EPO levels. According to a first aspect of the present invention, a compound comprising an EPO mimetic peptide or a functional variant thereof is provided for prophylactic or therapeutic treatment of a patient afflicted with or at risk of being afflicted with a disease wherein erythropoietin (EPO) adversely affects the mortality or disease progression. Thus, a compound comprising an EPO mimetic peptide or a functional variant thereof is used for prophylactic or therapeutic treatment of a patient afflicted with or at risk of being afflicted with a disease wherein EPO adversely affects the mortality or disease progression. Also provided is the use of a compound comprising an EPO mimetic peptide or a functional variant thereof for the preparation of a medicament, said medicament being prepared for prophylactic or therapeutic treatment of a patient afflicted with or at risk of being afflicted with a disease wherein EPO adversely affects the mortality or disease progression.

According to a second aspect of the present invention a compound comprising an EPO mimetic peptide or a functional variant thereof is provided for therapeutic or prophylactic reduction of EPO levels in a patient while maintaining functional erythropoiesis. Thus, a compound comprising an EPO mimetic peptide or a functional variant thereof is used for therapeutic or prophylactic reduction of EPO levels in a patient while maintaining functional erythropoiesis. Also provided is the use of a compound comprising an EPO mimetic peptide or a functional variant thereof for the preparation of a medicament, said medicament being prepared for therapeutic or prophylactic reduction of EPO levels in a patient while maintaining functional erythropoiesis.

According to a third aspect of the present invention, a compound comprising an EPO mimetic peptide or a functional variant thereof is provided for prophylactic or therapeutic treatment for inhibiting and/or reducing the stimulatory effects of EPO on diseases wherein EPO adversely affects the mortality or disease progression. Thus, a compound comprising an EPO mimetic peptide or a functional variant thereof is used for prophylactic or therapeutic treatment for inhibiting and/or reducing the stimulatory effects of EPO on diseases wherein EPO adversely affects the mortality or disease progression. Also provided is the use of a compound comprising an EPO mimetic peptide or a functional variant thereof for the preparation of a medicament, said medicament being prepared for prophylactic or therapeutic treatment for inhibiting and/or reducing the stimulatory effects of EPO on diseases wherein EPO adversely affects the mortality or disease progression.

According to a fourth aspect of the present invention, a compound comprising an EPO mimetic peptide or a functional variant thereof is provided for therapeutic or prophylactic treatment for decelerating or stopping EPO responsive tumor cell growth and progression, for reducing the risk of developing EPO responsive tumors. Thus, a compound comprising an EPO mimetic peptide or a functional variant thereof is used for therapeutic or prophylactic treatment for decelerating or stopping EPO responsive tumor cell growth and progression and/or for reducing the risk of developing EPO responsive tumors. Also provided is the use of a compound comprising an EPO mimetic peptide or a functional variant thereof for the preparation of a medicament, said medicament being prepared for therapeutic or prophylactic treatment for decelerating or stopping EPO responsive tumor cell growth and progression and/or for reducing the risk of developing EPO responsive tumors.

According to a fifth aspect of the present invention, a pharmaceutical composition is provided comprising a compound for the pharmaceutical uses as defined in the first to the fourth aspects of the present invention.

Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

DEFINITIONS

As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

The term "erythropoietin mimetic peptide" (EMP, also referred to as EPO mimetic peptide) in particular refers to a compound which shares no sequence homology with erythropoietin but is capable of binding and activating the erythropoietin receptor thereby stimulating, respectively maintaining erythropoiesis. Preferably, the EMP is a peptide which is used in dimeric form and binds two subunits of the EPO-receptor at two identical binding sites of the receptor, thereby forming a symmetrical EPO-receptor dimer conformation connected by the dimeric EMP compound. As outlined in the further description, one characteristic of EMPs is the ability to stimulate the growth of the UT7/EPO cell line, and erythroid differentiation and proliferation of hematopoietic progenitor cells. Preferably, similar maximum effects can be achieved as with endogenous EPO. On the other hand, EMPs cause a lower maximum effect than EPO in stimulating survival of at least one cell line selected from the group consisting of UT7, F36-P, and TF- 1. Preferably, said maximum effect achieved with an EMP according to the present invention is less than 50%, less than 40%, less than 30%, less than 20%, less than 10% of the maximum effect achieved with recombinant erythropoietin, preferably erythropoietin alpha (Erypo) in stimulating survival of at least one cell line selected from the group consisting of UT7, F36-P, and TF- 1. A first EMP, EMP-1 , was introduced in 1996 (Wrighton et al., 1996). Optimization of the EMP, including dimerization and conjugation to polyethylene glycol (PEG), has led to the development of Hematide, which has passed clinical phase Il (Stead et al., 2006; MacDougall 2006). Furthermore, a dimeric recombinant protein containing the EMP-1 sequence and a human IgG Fc tail has also been tested in phase I studies under the name CNTO 528 (Bouman-Thio et al., 2008). Other EPO mimetic peptides are e.g. described in WO 96/40749; WO 96/40772; WO 98/25965; WO 01/38342; WO 04/101611 ; WO 04/101606; WO 06/050959; WO 06/136450; WO 07/101698; WO 04/002424; and are also described herein. EPO mimetic peptides as described in the state of the art can be regarded as monomeric binding domains recognizing the binding site of the erythropoietin receptor. However, as was pointed out by Wrighton et al. (Wrighton 1997), two of these binding domains are generally needed in order to homodimerize the EPO receptor and to induce signal transduction. Thus, a combination of two of these EPO mimetic peptides and hence the EPO receptor binding domains in one single dimeric molecule enhanced activity considerably. The potency of monomeric EPO mimetic peptides can be improved up to 1000-fold by dimerisation. Even some inactive monomeric peptides can be converted into agonists by dimerization. Thus, preferably, an EMP dimer is used according to the teachings of the present invention.

Several techniques are known to dimerize EMP monomers. Monomers can be dimerized e.g. by covalent attachment to a linker. A linker is a joining molecule creating a covalent bond between the polypeptide units of the present invention. The polypeptide units can be combined via a linker in such a way, that the binding to the EPO receptor is improved (Johnson et al. 1997; Wrighton et al. 1997). Alternatively, it is known to achieve dimerization by forming a diketopiperazine structure. Another alternative way to obtain peptide dinners known from prior art is to use bifunctional activated dicarboxylic acid derivatives as reactive precursors of the later linker moieties, which react with N-terminal amino groups, thereby forming the final dimeric peptide (Johnson et al, 1997). Monomers can also be dimerized by covalent attachment to a linker. Preferably the linker comprises NH-R-NH wherein R is a lower alkylene substituted with a functional group such as carboxyl group or amino group that enables binding to another molecule moiety. The linker might contain a lysine residue or lysine amide. Also PEG may be used as linker. The linker can be a molecule containing two carboxylic acids and optionally substituted at one or more atoms with a functional group such as an amine capable of being bound to one or more PEG molecules. A detailed description of possible steps for oligomerization and dimerization of peptides with a linking moiety is also given in WO 2004/101606. In a preferred embodiment, two EMP monomers are dimerised by way of a peptide linker, preferably a glycine or alanine linker as is also described e.g. in WO 07/101698. Alternative dimerisation strategies for EPO mimetic peptides are also appreciated.

The compound according to the present invention comprising an EMP or a functional variant thereof also includes embodiments wherein the EMP either in monomeric or dimeric form is coupled to another macromolecule in order to e.g. improve the pharmacokinetic properties of the resulting larger entity. Coupling can be accomplished by recombinant technology and e.g. by fusion of the peptide sequence to a suitable carrier e.g. an Fc fragment-like protein or a randomized sequence. With the recombinant approaches, the EMP is first encoded as part of a nucleic acid and then expressed as full size protein including the EMP moiety. Alternatively, chemical conjugation of a macromolecule to an EMP is known to the skilled artisan, e.g. coupling to polyethylene glycol or to polymeric sugars like dextrans, hydroxyalkylated starches, polyvinylalkohol and the like (see the references above, in particular WO 07/101698 and below). In an extension of the abovementioned concepts other covalent modifications or formulation additives e.g. PEG-liposomes, nanoparticular modification or formulation and the like with similar effects and intentions than those mentioned above are compounds according to the present invention.

The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of and "consist of.

The term "EPO responsive malignancy/malignancies", "EPO responsive pre-malignancy or pre-malignancies" or "EPO responsive tumor(s)" and corresponding terms as used herein, in particular refer to a malignancy or pre-malignancy such as a tumor that adversely reacts to EPO and wherein EPO particularly results in an increase of patient mortality and/or progression of the malignancy or pre-malignancy, respectively the disease. EPO responsive reactions include but are not limited to enhancement of cellular proliferation, inhibition of apoptosis, phosphorylation of cellular signaling proteins, resistance to cytotoxins, increase of angiogenesis, or decrease of life expectancy. According to one embodiment, at least a portion of the EPO-responsive malignant or pre-malignant cells, respectively the tumor cells express the EPO receptor. However, also malignancies, pre-malignancies and tumors are encompassed by and thus included in the term "EPO responsive", wherein EPO has an indirect stimulatory effect. Thus, EPO may have an indirect stimulatory effect on malignant, pre-malignant or tumor cells even if said cells itself do not express the EPO receptor or in cases wherein only a portion of the cells express the EPO receptor. EPO can elicit its stimulatory effects e.g via non-tumor cells that are associated with the malignant or pre- malignant cells such as e.g. endothelial cells or other cells that are associated with the malignant or pre-malignant cells and which express the EPO receptor. By stimulating said associated cells, EPO can also stimulate the progression or development of the malignant or pre-malignant cells, respectively the tumor cells, even if said cells themselves do not express the EPO receptor. The molecular mechanism, how EPO stimulates the malignancies or pre- malignancies, respectively the tumor cells is not relevant. Only the fact is decisive that EPO is known to be an important factor in the stimulation of tumor growth and progression. EPO is known as an angiogenic factor that e.g. regulates the induction of tumor cell-induced neovascularization and tumor growth during the initial stages of tumorigenesis. Thus, the terms "EPO responsive malignancy", "EPO responsive pre-malignancy" and/or "EPO responsive tumor" in particular include but are not limited to such malignancies, pre- malignancies, including tumors wherein EPO is substantially involved in the growth, viability, angiogenesis and progression. The term "EPO responsive malignancy/malignancies", "EPO responsive pre-malignancy", "EPO responsive disease" and similar terms used herein thus particularly refers to malignancy or pre-malignancy classes and or diseases in which the experience of physicians treating such patients, the outcomes of clinical trials testing large populations of patients, regulatory authorities judging applications for drug approvals, or the scientific community in general, consider treatment with EPO to be potentially harmful to the patient's life expectancy and/or the progression of the malignant disease and/or the development of a pre-malignant disease into a malignant disease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to novel uses of EMP in therapy including prophylactic treatments, in particular therapy of malignancies or pre-malignancies that are stimulated by EPO.

At a first glance EPO mimetics appear to have an identical way of action as EPO or other conventional ESAs, in that they bind the EPO receptor and thereby cause phosphorylation of signalling proteins such as JAK2, STAT5 and others (Fan et al., 2006; Bugelski et al., 2008). Surprisingly, it was now found that EMPs display much lower maximum activities than EPO in a series of in vitro assays utilizing a panel of leukemia-derived cell lines. A lower maximum activity in a functional or biochemical assay induced by EMP in comparison to EPO is defined here as lower responsiveness to EMP compared to EPO. Excess EMP concentrations even inhibited the stimulatory effects of EPO on these cell lines. Using a Schild-Analysis, it was clearly shown that inhibition was competitive at the same receptor, i.e. the EPO receptor. A common finding was that the cell lines in which the EMPs caused weaker effects than EPO expressed lower surface levels of the EPO receptor, but higher levels of CD131 , the common beta chain of the receptors for IL-3 and GM-CSF, than a cell line in which EMP constructs displayed full activity. In vitro assays using genetically engineered cells have, however, shown no direct relation between the expression of CD131 along with the EPO receptor, and the responsiveness to EMP compared to EPO. However, a strong correlation was found in murine BA/F3 cells stably transfected with EPO receptor expressing constructs, between expression levels, preferably cell surface expression levels, of the EPO receptor, and responsiveness to EMP compared to EPO.

Without wishing to be bound by theory, it is believed that signalling by the EPO receptor bound to EMP in principle is less efficient than that of the EPO receptor bound to EPO. In some cell types, including normal erythropoietic progenitor cells, this suboptimal signalling by EMP obviously is compensated for. The data acquired through UT7/EPO cells and EPO receptor transfected BA/F3 cells strongly suggest that compensation is associated with expression of high relative levels of surface EPO receptor and/or JAK2 protein in comparison to the levels of downstream signalling proteins. Another surprising association was with expression of CD131 , which in some unexpected way correlated with the submaximal signals caused by EMP in three leukaemia cell lines. Independent from mechanistic considerations, the surprising differences in the efficiencies of cellular signalling between EPO and EMPs demonstrated here have important implications for the use of EMPs in the clinic.

According to a first aspect of the present invention, a compound comprising an EPO mimetic peptide or a functional variant thereof is provided for prophylactic or therapeutic treatment of a patient afflicted with or at risk of being afflicted with a disease wherein EPO adversely affects the mortality of the patient and/or disease progression.

According to one embodiment, the patient is afflicted with or is at risk of being afflicted with a malignancy or pre-malignancy wherein EPO adversely affects the mortality and/or malignancy progression. Particularly, a patient can be treated that is afflicted with or is at risk of being afflicted with an EPO responsive malignancy or pre-malignancy. A risk of being afflicted with an EPO responsive malignancy e.g. describes a scenario wherein EPO might promote the development from a pre-malignant state of the disease to a malignant state of the disease. According to one embodiment, the patient is afflicted with or is at risk of being afflicted with a malignancy or pre-malignancy wherein at least a portion of the malignant or pre-malignant cells express the EPO receptor. However, also malignancies or pre- malignancies wherein EPO has an indirect stimulatory effect are encompassed and thus included in the term "EPO responsive". Thus, EPO may have an indirect stimulatory effect on malignant or pre-malignant cells even if said cells itself do not express the EPO receptor or in cases wherein only a portion of the cells express the EPO receptor. As discussed above, EPO is known as an angiogenic factor that e.g. regulates the induction of tumor cell-induced neovascularization and tumor growth during the initial stages of tumorigenesis. Therefore, the administration of the EMPs according to the present invention is useful for treating and/or preventing the progression or development of angiogenesis-dependent tumors and can be used e.g. to inhibit the EPO-stimulated induction or promotion of tumor angiogenesis and progression.

As is outlined above, due to the differential stimulating effects of EMPs and EPO it is possible to avoid the unwanted effects of EPO, in particular on malignancies such as MDS, leukemias or solid tumors. The administration of EMPs does not only avoid the side effects of an EPO therapy but may also inhibit the unwanted effects of EPO at the endocrine, paracrine and autocrine level and thus also the effects of endogenous or exogenous EPO on diseases, wherein EPO adversely affects the mortality or disease progression. Thereby, EPO mimetic peptides can elicit a curative effect on the progression of diseases, wherein EPO adversely affects the mortality of the patient and/or disease progression and in particular on the progression of EPO responsive malignancies. Furthermore, due to their erythropoietic characteristics, EPO mimetic peptides are also beneficial in treating anemia in the patient; anemia often occurs as a side effect of the actual tumor therapy. As is outlined above, the administration of the EMPs also reduces endogenous EPO levels indirectly and via the normal physiological feedback regulations. Thus, with the present invention also a compound is provided comprising an EPO mimetic peptide or a functional variant thereof for therapeutic or prophylactic reduction of EPO levels in a patient, preferably while maintaining functional erythropoiesis. This therapeutic use, namely the reduction of EPO levels in a patient has the therapeutic benefits that were already outlined above. Furthermore, a compound is provided comprising an EPO mimetic peptide or a functional variant thereof for therapeutic or prophylactic inhibition of an increase of EPO levels in a patient while maintaining functional erythropoiesis. This therapeutic use, namely the prevention of a rise of the endogenous EPO levels in a patient has the therapeutic benefits that were already outlined above. According to one embodiment, the patient shows elevated EPO levels, preferably being≥ two times above the standard deviation of the normal value in a healthy reference population. Any commercially available assay for validated determination of endogenous EPO-levels is suitable for this purpose. In particular, a compound is provided with the present invention comprising an EPO mimetic peptide or a functional variant thereof for prophylactic or therapeutic treatment for inhibiting and/or reducing the stimulatory effects of EPO on diseases wherein EPO adversely affects the mortality and/or disease progression. According to one embodiment the disease is characterised by a malignancy wherein EPO adversely affects the mortality of the patient and/or progression of the malignancy, in particular an EPO responsive malignancy. Said malignancy may also be characterised by the expression of the EPO receptor. By administering an EMP, administration of EPO for curative purposes is no longer necessary, endogenous EPO levels can be decreased and/or the stimulatory effects of EPO can be inhibited. In particular, the malignancy promoting effects of remaining endogenous EPO on the EPO responsive malignancies and/or pre-malignancies can be inhibited while maintaining functional erythropoiesis.

According to a preferred aspect, a compound comprising an EPO mimetic peptide or a functional variant thereof is provided for therapeutic or prophylactic treatment for decelerating or stopping EPO responsive tumor cell growth and progression and/or for reducing or preventing the risk of developing EPO responsive tumors, to minimize spread and growth, e.g. metastatic spread in hypoxic conditions, to support (chemo- or radiotherapeutic) treatment-based regression of an EPO responsive tumor and/or to inhibit EPO responsive tumor growth. According to one embodiment, the compound according to the present invention as described above for the different aspects of the invention is for the prophylactic or therapeutic treatment of a disease that is characterised by a malignancy or pre-malignancy having one or more of the following characteristics: a. it is a malignancy or pre-malignancy wherein EPO adversely affects the mortality or malignancy progression;

b. it is an EPO responsive malignancy or pre-malignancy; and/or

c. it is a malignancy or pre-malignancy which expresses the EPO receptor.

Details with respect to the advantages and effects associated with a respective EMP treatment were discussed above. It is referred to the above disclosure which also applies here. According to one embodiment, the compound according to the present invention as described above for the different aspects of the invention is for the prophylactic or therapeutic treatment of a patient having one or more of the following characteristics: a. he is afflicted with or is at risk of being afflicted with a malignancy or pre- malignancy, wherein EPO adversely affects the mortality or malignancy progression;

b. he is afflicted with or is at risk of being afflicted with an EPO responsive malignancy or pre-malignancy;

c. he is afflicted with or is at risk of being afflicted with a malignancy or pre- malignancy which expresses the EPO receptor; and/or

d. he is anemic.

Details with respect to the advantages and effects associated with a respective EMP treatment were discussed above. It is referred to the above disclosure which also applies here.

According to one embodiment, the compounds according to the present invention are used for suppressing tumor angiogenesis and/or tumor progression. According to one embodiment, the compounds according to the present invention are used for stimulating erythropoiesis and for the prophylactic or therapeutic treatment of anemia.

According to one embodiment, the compounds according to the present invention are used for inhibiting or reducing the stimulatory effects of EPO, preferably for preventing or treating diseases wherein EPO adversely affects the mortality and/or disease progression. According to one embodiment, the compounds according to the present invention are used for suppressing tumor angiogenesis and/or tumor progression of EPO responsive tumors by inhibiting or reducing the stimulatory effects of EPO on tumor angiogenesis and/or tumor progression while stimulating erythropoiesis.

According to one embodiment, the compounds according to the present invention are used for stimulating erythropoiesis and for inhibiting or reducing the stimulatory effects of EPO on EPO-responsive malignancies that have or are at risk of developing EPO-dependent autocrine loops. Details with respect to the development of autocrine loops and the advantages of EMP treatment are discussed above. It is referred to the above disclosure.

According to one embodiment, the compounds according to the present invention are used for the prophylactic and/or therapeutic treatment in combination with chemotherapeutic agents and/or radiation therapy. Preferably, the compound is administered before the chemotherapy or the radiation therapy begins. The treatment with the compound according to the present invention is preferably continued during the chemotherapy and/or the radiation therapy. The dosage of the compound according to the present invention can be increased when used in combination with the chemotherapeutic agent and/or the radiation therapy. Whether the patient has a malignancy or pre-malignancy as defined herein and in particular an EPO responsive malignancy or pre-malignancy does not need to be determined on the level of the individual patient. Conversely, it is sufficient when it is known from clinical studies or for classes of malignancies, in particular tumors that there is a risk that EPO adversely affects the patient's mortality and/or the progression of the malignancy in order to use an EMP for therapy as taught herein. In general the persons skilled in the art know from e.g. the reports in clinical literature as outlined e.g. by the FDA, which malignancies, respectively tumors are considered as being EPO responsive. This list can potentially grow over time as knowledge on EPO-dependent growth accumulates. Besides this type of deduction from the knowledge of the clinical community, biomarkers like EPO level and EPO-receptor expression level can be used to validate an indication for treatment in an individual patient. E.g. appropriate assays can be used to determine, whether the patient is afflicted with an EPO responsive tumor and accordingly, in particular whether elevated EPO levels are present and/or might pose a risk for the mortality and/or disease progression. In a direct form, endogenous EPO levels can be determined with routine methods known to the person skilled in the art, e.g. by a commercial ELISA or RIA. Thus, according to one aspect it is either alternatively or additionally determined whether the patient shows elevated EPO levels. Whether there is transcription from the EPO receptor gene in excised tumor cells or tumor cells isolated from circulating blood can be detected e.g. by PCR methods or methods of protein detection according to the state of the art. Furthermore, if desired, the effects of EMP, in comparison to those of EPO, to induce the effects described above on a certain malignancy or pre-malignancy, may be predicted based on experiments performed on isolated cells, cell lines or biopsies from this particular malignancy or pre-malignancy. Effects, in particular the maximum levels of effect induced by EPO compared to the EMP may be compared directly in functional assays as described above, but may also base on measurements of the expression level of the EPO receptor in these cells, cell lines or biopsies, determined by methods such as immunohistochemistry, flow cytometry, Western or Northern blotting or RT-PCR. In particular suitable for the treatment with the EMPs according to the present inventions are cells wherein when tested in an assay that measures and compares functional expression levels of the EPO receptor with other cellular factors including the common beta chain of the IL-3 and GM-CSF receptors (CD131) and/or one or more cytosolic proteins required for cellular signalling (for instance Jak2, STAT5, ERK) in biopsies, sections, lysates, cells or cell lines derived from malignancies or -premalignacies, the level of EPO receptor is limiting in comparison with the other cellular factor(s) involved in transducing the signal(s) induced by binding of the compound to the EPO receptor.

According to one embodiment, the patient is afflicted with a malignancy or pre-malignancy that is selected from the group consisting of myeloid malignancies and pre-malignancies, in particular leukemic malignancies, MDS, AML, non-myeloid malignancies and pre- malignancies, lymphoid malignancies and pre-malignancies, breast cancer, in particular metastatic breast cancer, mammary cancer, cervical cancer, ovarian cancer, head and neck cancer, epithelial tumors, in particular gastro-intestinal cancer, gastric cancer, prostate cancer, lung cancer, in particular non-small cell lung cancer and malignant tumors of mesenchymal origin, in particular sarcomas, glioblastoma, melanoma, esophagal ADC, choriocarcinoma, colon cancer, pancreatic cancer, uterine ADC, prostate cancer, leukemia and hepatoma. According to one embodiment, the malignancy or pre-malignancy is not MDS. According to one embodiment, the compound is used according to the present invention for the treatment of anemia in myeloic leukemia and/or for preventing the development or stimulation of EPO responsive malignancies and/or pre-malignancies. In particular, the compound is used according to the present invention for the prophylactic or therapeutic treatment of the anemic condition of a patient afflicted with MDS and for preventing the development of MDS into acute myeloid leukemia (AML).

According to one embodiment, an EPO mimetic peptide is used which comprises the following consensus sequence: X6X7X8X9X10X11X12X13X14X15 X16X17X18X19 wherein each amino acid is selected from natural or unnatural amino acids and

X₆ is an amino acid with a sidechain functionality capable of forming a covalent bond;

X₇ Js R₁ H, L₁ W, Y or S;

X₈ is M, F₁ 1₁ homoserinemethylether or norisoleucine;

X₉ is G or a conservative exchange of G;

X₁₀ is proline or a non conservative exchange of proline;

Xii is selected from any amino acid;

X₁₂ is an uncharged polar amino acid or A;

X₁₃ Js W, 1-nal, 2-nal, A or F;

X₁₄ Js D, E₁ I, L or V;

X₁₅ is an amino acid with a sidechain functionality capable of forming a covalent bond;

X₁₆ is independently selected from any amino acid; preferably G, K, L, Q, R, S,

Har or T and more preferred a positively charged amino acid;

X₁₇ is independently selected from any amino acid; preferably A, G, P, R₁ K, Y,

Har and more preferred a positively charged amino acid;

X₁₈ is independently selected from any amino acid; preferably L or Q;

X₁₉ is independently selected from any amino acid, preferably a charged amino acid, preferably a positively or negatively charged amino acid, or a functional variant of an EPO mimetic peptide defined by the above consensus sequence, that stimulates erythropoiesis and at least partially antagonises the stimulatory effects of EPO on EPO-responsive malignancies and/or pre-malignancies.

Peptides having a consensus sequence as defined above are known in the prior art as EPO (erythropoietin) - mimetic peptides (see for example WO 2007/101698). The numbering (X6 to X19) is chosen to simplify the comparison with the EPO mimetic peptides known in the prior art, which uses a corresponding numbering for discussing and discussing EPO mimetic peptides (see e.g. Johnson et al 1998, page 3699, right column, penultimate paragraph and page 3703, table 1). However, this numbering does not exclude that e.g. more or less amino acids are present and the chosen numbering does not indicate the overall length of the peptide but merely defines the consensus.

According to one embodiment, the amino acids in position X₆ and X₁₅ are chosen such that they are capable of forming an intramolecular bridge within the peptide by forming a covalent bond between their side chains. Preferably, said bridge is either a disulfide or a diselenide bridge. According to one embodiment, the amino acid in X₆ and or X₁₅ are selected from the group comprising cysteine, cysteine derivatives such as homocysteine and selenocysteine, thiolysine, K or E.

Preferably, X₁₃ is naphthylalanine.

Preferably, the EMP depicts a charged amino acid in position Xi₀, X₁7 and/or X_ig. According to one embodiment, the charged amino acid in position X₁₀, X₁7 and/or X_ig is either positively or negatively charged and is selected from the group consisting of natural amino acids, non- natural amino acids and derivatised amino acids.

According to one embodiment, the EMP depicts a positively charged amino acid in position X₁₀ or Xi₇. Preferably, said positively charged amino acid is selected from the group consisting of

natural positively charged amino acids, e.g. lysine, arginine, histidine or ornithine; - non-natural positively charged amino acids, which depict in position X₁₀ and/or X₁₇ preferably an elongated side chain such as in e.g. homoarginine;

originally negatively charged amino acids which are, however, derivatized with suitable chemical groups in order to provide them with a positively charged group. According to one embodiment, the EMP comprises a negatively charged amino acid in position X₁₉. Preferably, said negatively charged amino acid is selected from the group consisting of

natural negatively charged amino acids, especially D or E;

non-natural negatively charged amino acids, which preferably depict an elongated side chain such as Aad, 2-aminoheptanediacid, Asu;

- originally positively charged amino acids which are, however, derivatized with suitable chemical groups in order to provide them with a negatively charged group. As described above, the EMP may also be a functional variant of an EMP defined by the above consensus sequence, which exhibits a corresponding activity profile as a respective EMP. A functional variant in particular refers to an EMP as defined above which may comprise one or more amino acid mutation(s), like a substitution, deletion and/or addition of one or more amino acids or chemical modifications but which still exhibits the effects defined above, namely the stimulation of erythropoiesis and an at least partial antagonistic effect with respect to the stimulatory effects of EPO on EPO-responsive malignancies and/or pre- malignancies. Also encompassed by the term a compound comprising an EPO mimetic peptide or a functional variant thereof are fusion proteins, respectively polypeptides comprising an EMP. Preferably, the EMP is present in a dimeric form, i.e. comprising two EMP monomers. Preferably, each monomer of the EMP dimer comprises an EMP consensus sequence as defined above. The length of the EMP monomer is preferably between ten to forty or fifty or sixty amino acids. In preferred embodiments, the EMP consensus depicts a length of at least 10, 15, 18, 20 or 25 amino acids. Of course the consensus can be embedded respectively be comprised by longer sequences. A longer length can also be created by dimerising two monomeric EMPs of the above consensus. According to one embodiment, the compound comprises i) at least two dimeric EMP units and ii) at least one polymeric carrier unit; wherein said dimeric EMP units are bound to said polymeric carrier unit. The dimeric EPO mimetic peptide units used in this embodiment can be either homo- or heterogenic, meaning that either identical or differing EMP dimers are used for creating the compound. The same applies to the EMP monomers which can also be homo- or heterogenic. The monomeric EMPs are preferably cyclic. A cyclic molecule can be for example created by the formation of intramolecular cysteine bridges (see also WO 2007/101698).

The polymeric carrier unit comprises at least one natural or synthetic branched, linear or dendritic polymer. The polymeric carrier unit is preferably soluble in water and body fluids and is preferably a pharmaceutically acceptable polymer. Water soluble polymer moieties include, but are not limited to, e.g. polyalkylene glycol and derivatives thereof, including PEG, PEG homopolymers, mPEG, polypropyleneglycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end e.g. with an acylgroup; polyglycerines or polysialic acid; cellulose and cellulose derivatives, including methylcellulose and carboxymethylcellulose; starches (e.g. hydroxyalkyl starch (HAS), especially hydroxyethyl starch (HES) and dextrines, and derivatives thereof; dextran and dextran derivatives, including dextransulfat, crosslinked dextrin, and carboxymethyl dextrin; chitosan (a linear polysaccharide) heparin and fragments of heparin; polyvinyl alcohol and polyvinyl ethyl ethers; polyvinylpyrrollidon; alpha.beta- poly[(2-hydroxyethyl)-DL-aspartamide; and polyoxyethylated polyols. One example of a carrier unit is a homobifunctional polymer, of for example polyethylene glycol (bis-maleimide, bis-carboxy, bis-amino etc.). The polymeric carrier unit which is coupled to at least two dimeric EPO mimetic peptides preferably comprising monomeric consensus sequences as described herein can have a wide range of molecular weight due to the different nature of the different polymers that are suitable in conjunction with the present invention. There are thus no size restrictions. However, it is preferred that the molecular weight is at least 3 kD, preferably at least 1OkD and approximately around 20 to 500 kD and more preferably around 30 to 150 or around 60 or 80 kD. The size of the carrier unit depends on the chosen polymer and can thus vary. For example, especially when starches such as hydroxyethylstarch are used, the molecular weight might be considerably higher. The average molecular weight might then be arranged around 100 to 4,000 kD or even be higher. However, it is preferred that the molecular weight of the HES molecule lies around 50 to 500 kD, or 100 to 40OkD and preferably around 20OkD. The size of the carrier unit is preferably chosen such that each peptide unit is optimally arranged for binding their respective receptor molecules.

Details of this embodiment wherein at least two dimeric EMPs units are covalently or non- covalently bound to a polymeric carrier unit and the associated advantages are described in WO 2006/136450, herein incorporated by reference. According to one embodiment, at least 4 dimeric EMP units, preferably 2 to 20, more preferred 2 to 10 and most preferred 4 to 10 dimeric EMP units are coupled to the polymeric carrier unit.

Preferably, after the EMP dimers are created by combining the monomeric binding units to EMP dimers (either head to head, head to tail, or tail to tail) the polymeric carrier unit is connected to EMP dimers. The polymeric carrier unit is connected/coupled to the EMP dimers via a covalent or a non-covalent (e.g. a coordinative) bond. However the use of a covalent bond is preferred. The attachment can occur e.g. via a reactive amino acid of the peptide units e.g. lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine or the N-terminal amino group and the C-terminal carboxylic acid. In case the peptide does not carry a respective amino acid, such an amino acid can be introduced into the amino acid sequence. The coupling should be chosen such that the binding to the target is not or at least as little as possible hindered. Depending on the conformation of the peptide unit, the reactive amino acid is either at the beginning, the end or within the peptide sequence.

In case the polymeric carrier unit does not possess an appropriate coupling group, several coupling substances/linkers can be used in order to appropriately modify the polymer in order that it can react with at least one reactive group on the EMP dimer. Details are also described in WO 2007/101698.

According to a preferred embodiment, the EPO mimetic peptide shows the following activity profile: a) when tested in an assay that measures the potency of an agent to stimulate the proliferation or survival of the cell line UT7/EPO or to stimulate the development of erythroid colonies from human hematopoietic progenitor cells in semisolid medium, the maximum effect achieved with the EMP should be at least half as high, preferably as high as that achieved by recombinant erythropoietin, preferably epoetin alfa (Tradename: Erypo) and b) when tested in an assay that measures the survival of cell line UT7 or of cell line F36-P, the maximum effect achieved with the EMP should be less than half as high, preferably less than 40%, less than 30%, less than 20% or less than 10% as that achieved by recombinant erythropoietin, preferably epoetin alfa (Tradename: Erypo), preferably absent.

Furthermore, when tested in an assay that measures the potency of an agent to stimulate proliferation, survival, resistance to cytostatics, invasion, metastasis, phosphorylation of signalling proteins, or other effects, on cells or cell lines, stromal or parenchymal, isolated from malignancies or pre-malignancies, the maximum effect achieved with the EMP in its API format is preferably less than 50%, preferably less than 40%, less than 30% or less than

20%, less than 10% of the maximum effect achieved by recombinant erythropoietin, preferably epoetin alfa (Tradename: Erypo).

According to one embodiment of the therapeutic uses described above, the patient has received or will be receiving chemotherapy and/or radiotherapy. Also provided is a pharmaceutical composition comprising a compound as defined herein for the pharmaceutical uses as defined herein. Also covered is the use of a compound comprising an EPO mimetic peptide as defined herein in therapy as defined herein.

EXAMPLES

The examples were performed as follows: I. Materials and Methods

1. Sources of erythropoietin

Erythropoietin alpha from Ortho Biotech/Janssen-Cilag (Neuss, Germany, brand name: Erypo, stock solutions 16.8 μg/ml (2000 IE/ml) or 33.6 μg/ml (4000 IE/ml)) was used as a source of EPO in all experiments, except in some cases in which also Aranesp (Amgen, Munich, Germany, stock solution of 500 μg/ml) was used.

2. Peptide synthesis and conjugation to hydroxyethyl starch

The peptide sequence of AGEM400 is as follows (in which NaI stands for 1-Naphtylalanine; Ac: N-terminal acetylation; Am: C-terminal amidation):

AGEM400: Ac-GGTYSCHFGKLT-NaI-VCKKQRG-GGTYSCHFGKLT-NaI-VCKKQRG-

C(tBu)-Am

Further EMPs tested:

AGEM115: A less positively charged dimeric EMP variant, alternative to AGEM400. AGEM099:

Ac -GGLYACHMGPIT-Nal -VCQPLRG-GGLYACHMGPIT-Nal -VCQPLRG- C ( tBu) -Am

I I I I AGEM134: (analogous to a dimer of the published EMP-1 sequence (Wrighton et al., 1996))

Ac -GGTYSCHFGPLTWVCKPQGG-GGTYSCHFGPLTWVCKPQGG- C ( tBu) -Am

I I I I Internal disulfide bridges were closed between Cys(6)-Cys(15) and between Cys(26)- Cys(35).

Synthesis of peptides

AGEM400 was synthesized at a scale of 0.25mmol by microwave assisted solid phase peptide synthesis in an automated Liberty (CEM) unit. The growing peptide chain was assembled on PAL ChemMatrix resin from Matrix Innovation. Deprotection was achieved by adding 10 ml Piperidine (25% in DMF) and irridation with 65 W for 3 min. Coupling of the next amino acid was achieved using a five fold excess of reagents (amino acid, Pybop, DIEA) in 10 ml DMF and irridation with 40 W for 5 min. All couplings were done by a double coupling procedure including capping with Z-2-CI-OSu (40 equivalents,) and irridation with 40 W for 2 min. Acetylation of the peptide was achieved by introducing Ac-GIy-OH as last building block. After washing with DCM the peptide was cleaved off by adding 40 ml cleavage cocktail (94% TFA₁ 1.0% TIS, 2.5% H₂O, 2.5% DODT) and incubation at room temperature for 3 h. The peptide was then precipitated in cold ether, redisssolved in acetonitrile/water (2/1) and directly purified by LCMS using a Nebula (Gilson) purification system.

The first disulfide bond in AGEM400, was created as described in WO 2007/076993 using coffeine. Using coffeine has advantages compared to standard oxidation methods (oxygen, iodine) as no workup to remove the reagent prior purification is needed and as it leads to higher yields. In a typical experiment 10-20 mg of the peptide were dissolved in 10ml caffeine (room atmosphere). After 18-24 h at room temperature this solution was directly purified by LCMS using a Nebula (Gilson) purification system. To create the second disulfide bond in AGEM400, the monocyclic peptide (20mg) was dissolved in 40 ml 80% acetic acid. After addition of 0.512 ml 0.1 M HCI and 3.424 ml 50 mM iodine solution in acetic acid the solution was stirred for 2.5 h. Excess of iodine was removed by adding ascorbic acid until the solution was colourless. The solution was then diluted with water to a total volume of 400 ml and added onto a C18-SPE-Column, the column was washed with 200 ml water and the peptide was eluted with 50ml acetonitrile/water (95/5). The crude peptide was directly purified by LCMS using a Nebula (Gilson) purification system. Owing to the fact that the 41mer was purified 3 times by HPLC a high purity of the final product could be achieved (above 90%).

The other peptides were synthesised analogously.

Removal of the remaining Cys(tBu)-protection before conjugation to HES

The purified bicyclic AGEM400 peptide was transferred into a teflon vessel and anisole was added. The vessel was cooled down with dry ice/aceton and HF was condensed into the vessel (anisol:HF=1 :10). The deprotection was run at O⁰C for 75 min. HF was removed via recondensation followed by a nitrogen gas stream. The oily residue was precipitated with

TBME and removed by centrifugation. The precipitant was then washed 4 times with TBME, taken up with 0.1%TFA in water and lyophilized over night. The crude peptide was purified by LCMS using a Nebula (Gilson) purification system.

Conjugation to HES

A hydroxyethyl starch HES450/0.7 is fractionated in order to obtain a size fraction with an Mw=130±20kDa. A number of 30±3μmol/g active groups were introduced in a three step modification process. For the conjugation of the deprotected AGEM400, 80,1 mg (M=4657,5g/mol, 1.2eq, peptide/active thiol content=70%) of the peptide were dissolved in 10ml 5OmM phosphate buffer pH=6.5. 300mg of Supravalent-Maleimide-HES130/0.7 were dissolved in 20ml 5OmM phosphate buffer pH 6.5, added to the peptide solution and vigorously stirred for 2h at 37⁰C. The peptide excess was removed by size exclusion chromatography on a Superdex 200 column (250x50mm, 440ml bed volume) using a 5OmM phosphate buffer pH 6.5 with 50OmM NaCI as mobile phase. The macromolecular fractions were collected and desalted via a Sephadex G15 column (500x50mm, 880ml bed volume) with desalted water as mobile phase. The collected and combined product fraction was lyophilized and 265mg of a white solid product, AGEM400(HES), were obtained.

AGEM 400 was chosen as an example/model for an EPO mimetic peptide. The results are also applicable to EPO mimetic peptides in general as was found by further experiments with other EPO mimetic peptides.

3. Cell lines

Cell lines TF-1 (Kitamura et al., 1989), UT7 (Komatsu et al., 1991), and F36-P (Chiba et al., 1991) were all obtained from DSMZ. Cell line UT7/EPO, which is a subline of UT7 strictly dependent on EPO for its proliferation (Komatsu et al., 1993), was a kind gift of Prof. W. Jelkmann (University of Lϋbeck, Germany). TF-1 was grown in RPMI medium with Glutamax (Gibco, Invitrogen, Karlsruhe, Germany) supplemented with antibiotics (penicillin/streptomycin and amphotericin B, Invitrogen); 20 per cent fetal calf serum (FCS, Sigma Aldrich, Taufkirchen, Germany; and human IL-3 (Peprotech, Hamburg, Germany). UT7 was grown in alphaMEM + 20% FCS + 2.5 ng/ml hGM-CSF (R&D Systems, Wiesbaden, Germany). F36-P was grown in RPMI + 20% FCS + 2.5 ng/ml hGM-CSF. UT7/EPO cells were cultured in DMEM with Glutamax (Invitrogen) supplemented with antibiotics, 10 per cent FCS, and 3.4 ng/ml EPO. Cells were passaged every two to three days, always keeping cell densities between 200 000 and 1 500 000 cells/ml. Cell lines BA/F3, HeLa, M07e, and SupT1 were all obtained from DSMZ, and cultured according to the provider's instructions. M07e was grown in RPMI + 20% FCS + 10 ng/mL hlL-3, BA/F3 in RPMI + 10% FCS + 1 ng/ml mlL-3.

Before performing experiments with cytokine-dependent cell lines, they were starved for 72h (M07e), 48h (TF-1 , UT7/EPO), 2Oh (UT7), 2-4h (BA/F3 and transfectants) in culture medium without cytokine (and in case of TF-1 with a reduced FCS content: 5% instead of 20%). F36- P cells were starved in medium without GM-CSF for 48h before starting phosphorylation experiments and before determining receptor expression levels (e.g. by flow cytometry), but starved for 24h before starting MTS assays. SupT1 and HeLa cells were cultured in media without exogenously added cytokines. 4. Cell survival assays (MTS assays)

The dependence of cell lines on EPO or EMPs for survival was tested using the CellTiter 96 AQ non-radioactive cell proliferation assay (Promega). Starved cells were plated in multiwell plates in assay medium (the cell lines' starvation medium - the cell lines' culture medium without cytokine, and in case of TF-1 , with only 5% FCS) with differing concentrations of EPO or EMP. UT7/EPO were plated at 10,000 cells per well, TF-1 and F36-P at 15,000 cells per well, UT7 and BA/F3 and transfectants at 20,000 cells/well (100 μl/well). Every substance concentration was tested in triplicate. Assays were incubated for 72h (or 24h where indicated) before addition of 20 μl of MTS reagent per well, and cultured until brown formazan color developed (usually two to four hours). Cells were lysed with SDS (e.g. by the addition of 25μl of 10% SDS per well) and absorbance was measured at 492 nm.

5. Clonogenic assays on progenitor cells in methylcellulose

Development of erythroid colonies from bone marrow cells was studied in methylcellulose media (from StemCell Technologies, Grenoble, France) containing a cocktail of human cytokines (Methocult H4535: with hSCF, hGM-CSF, hlL-3, hlL-6, and hG-CSF). EPO or peptide dilutions were prepared in 20 x stocks in IMDM + 2 per cent FCS + antibiotics, and added as 150 μl volumes to 2.7 ml Methocult aliquots. Cryopreserved CD34-enriched human bone marrow cells were obtained from StemCell Technologies. After thawing and washing, cells were also added to aliquots of Methocult in 150 μl volumes of IMDM (Invitrogen) with 2

% FCS, yielding 3 ml volumes of each substance dilution. Human CD34-positive bone marrow cells were plated at 1000 cells per well, two 1.1 ml portions of each 3 ml substance dilution were plated. After 12 days the numbers of CFU-E colonies and BFU-E colonies were counted microscopically.

6. Liquid culture of progenitor cells

CD34-enriched human bone marrow cells were thawed, washed, and seeded at 10⁵ cells/mL in IMDM + 30% FCS; 1% BSA; 0.1 mM β-mercapto-ethanol; antibiotics; 50 ng/mL hSCF (Cellsystems, St. Katharinen, Germany); 20 ng/mL hGM-CSF (R&D Systems); 20 ng/mL hlL- 3 (Peprotech, Hamburg, Germany); 20 ng/mL hlL-6 (Peprotech); 20 ng/mL hG-CSF (Cellsystems) and either 10 ng/mL EPO, 200 ng peptide/mL AGEM400(HES), or no ESA. Fresh medium was added on day 3. Densities of viable cells were determined on days 3, 4, 5 and 6. Partly differentiated and expanded progenitor cells were submitted to flow cytometry to determine surface EPOR and CD131 as described below. Part of the cells cultured for six days in medium with EPO were transferred to the same medium as above but without any cytokines and incubated for another 24h. These starved cells were incubated for 30 minutes at 37⁰C with 10 ng/mL EPO, 200 ng peptide/mL AGEM400(HES), or no stimulus. Cells were subsequently lysed, electrophoresed and immunoblotted as described below. 7. Expression constructs

Plasmids containing the complete open reading frames (ORFs) of EPOR and common beta chain (CD131) were obtained from Invitrogen (Ultimate™ ORF clones; IOH29824 and IOH62492, respectively). The ORFs were amplified by PCR and ligated into mammalian expression vector pSI (Promega) yielding clones pSI-EPOR and pSI-CD131.

8. Transfectants of HeLa and BA/F3 cells

HeLa cells were transfected using the calcium precipitation method. Two days after transfection, HeLa cells were stimulated with EPO or EMP, lysed, and lysates were immunoblotted as described below. In parallel, transfected cells were trypsinized and stained for detection of EPOR and CD131 by flow cytometry, as described below. BA/F3 cells were electroporated with plasmid pSI-EPOR or pSI. After 48h of culture in culture medium containing mlL-3, it was replaced by medium containing 0.5 nM EPO. In flasks containing pSI-EPOR transfected cells, cell growth was noted after 6 days of EPO selection. These cells were cloned in multiwell plates by limiting dilution in EPO-containing medium, and 48 clones were amplified until analysis of EPOR levels by Western blotting (see below) was possible. Eight clones with varying EPOR levels were amplified further and these clones were used for performing additional experiments. 9. lmmunoblotting to determine receptor levels and phosphorylation of signalling proteins

Cells (starved if dependent on cytokine) were pelleted and resuspended in medium without cytokine or with given amounts of EPO or peptide, and incubated at 37°C for a given period of time. All subsequent steps were performed on ice. Cells were then pelleted, washed with PBS, and lysed for 30 min. on ice in 100 μl per 1 million cells lysis buffer: 15OmM NaCI, 5OmM Tris/HCI pH8, 1% Triton-X-100, protease inhibitor cocktail (Complete mini, Roche, Mannheim, Germany), and 1 mM orthovanadate. Insoluble material was pelleted, and supematants were supplemented with reducing Laemmli buffer (containing DTT). Samples were boiled for 5 minutes before application to SDS-PAGE gels and electrophoresis. Proteins were electroblotted to PVDF membranes (Invitrogen). Blots were washed, blocked, and incubated with antibodies in Tris-buffered saline with 0.05 per cent Tween 20 (TBST). After blocking with 3 per cent skim milk powder (blocking buffer), primary and peroxidase- conjugated secondary antibodies were also diluted in blocking buffer. Detection was performed using the ECL detection kit (GE Healthcare, Freiburg, Germany) and exposure of X-ray films (ECL hyperfilm, GE Healthcare). Antibodies used were: monoclonal mouse anti- P-STAT5 (Cell Signalling Technology, Beverly, USA; 9356) diluted 1 :10 000; monoclonal mouse anti-P-Erk (Cell Signalling 9106) diluted 1 :10 000; polyclonal rabbit anti-STAT5 (Cell Signalling 9310) diluted 1:5000; polyclonal rabbit anti-Erk (Cell Signalling 9102) diluted 1:25 000; rabbit-anti-P-Jak2 (Cell signalling 3771) diluted 1 :1000; rabbit -anti-mouse-HRP (Dako, Hamburg, Germany, P0260) diluted 1 :30 000; donkey-anti-rabbit-HRP ECL (GE Healthcare, NA934V) diluted 1 :10 000. Staining for EPOR was performed using a goat-anti-EPOR extracellular domain antibody conjugated to biotin (R&D Systems, BAF307, diluted 1:250). For staining CD131 , a goat-anti-common beta chain-biotin antibody was used (R&D Systems, BAF906, diluted 1 :250). Biotin was made visible by incubating with streptavidin- HRP (Dako, Hamburg, Germany, diluted 1 :5000) and chemo-luminescent detection. In indicated cases, protein content of cellular lysates was tested using the BCA protein assay kit (Pierce, Perbio, Bonn, Germany) before loading on gels.

10. Determination of surface receptors by flow cytometry

Starved cell lines, or cultured hematopoietic progenitor cells were washed once in PBS, and then stained for 30 minutes at in 20 μl volumes of PBS containing 5% FCS and 5% human serum with FITC-labelled anti-EPO-R antibody (diluted 1 :3, R&D Systems) and PE-labelled anti-CD131 antibody (diluted 1 :6, eBioscience, NatuTec, Frankfurt a. M., Germany), or with control mlgG1-FITC (diluted 1 :20) and mlgG2a-PE (diluted 1 :40, both from Sigma Aldrich). After staining, 0.5 ml PBS was added, cells were pelleted and resuspended in PBS with 10 μg/ml propidium idodie (Pl) for exclusion of dead cells. Analysis was performed by flow cytometry (FACScalibur, Beckton Dickinson, Heidelberg, Germany). Cellular debris was gated out in the FSC/SSC plot. The relative surface expression levels of the receptors on cell lines were determined by plotting the fluorescence histograms after staining for EPO-R or for CD131 in overlays with the histograms after control staining. Expression of EPO-R or CD131 on progeny of CD34-positive bone marrow cells in liquid culture was determined in dot-plots by setting a quadrant around control-stained cells, which was copied to the dot-plots of receptor-stained cells. Cells found beyond the borders of the quadrant were considered receptor-positive. II. Figures

Fig. 1 a): Comparing the efficacies of EPO, Aranesp, and EPO mimetics in stimulating growth/survival of human UT7/EPO cells in an MTS assay (top panel, shown are means and SDs of triplicate measurements); and in clonogenic assays to measure development of red colonies from human CD34-enriched bone marrow cells in methylcellulose medium under the influence of erythropoieis stimulating agents after 12 days (bottom panel; CFU-E, dotted lines; and BFU-E, continuous lines). ■: EPO; D: Aranesp; A: AGEM400 (peptide); •: AGEM400(HES). Typical examples are shown of experiments performed at least three times. Concentrations of agents are shown as ng/ml protein or peptide, considering the peptide content of AGEM400(HES) to be 10% w/w.

Fig.1 b): Comparing the efficacies of EPO, and alternative EMPs in stimulating growth/survival of human UT7/EPO cells in an MTS assay. Top:■: EPO; A: AGEM115;•: AGEM400; ▼: AGEM099. All peptides were predissolved at 10 mg/ml in DMSO before diluting them into assay medium. Bottom:■: EPO;•: AGEM400; T: AGEM134. All peptides were predissolved at 10 mg/ml in DMSO before diluting them into assay medium. Fig. 2: Assays comparing the efficacies of EPO, Aranesp, and EPO mimetics in stimulating growth/survival of human UT7 cells (top panels); TF-1 cells (left hand bottom panel); or F36- P cells (right hand bottom panel) in MTS assays. UT7 assays were evaluated either after 72h (left hand top panel) or 24h (right hand top panel). ■: EPO; D: Aranesp; A: AGEM400 (peptide); •: AGEM400(HES). Shown are means and standard deviations of triplicate measurements. Typical examples are shown of experiments performed at least three times. Concentrations of agents are shown as ng/ml protein or peptide, considering the peptide content of AGEM400(HES) to be 10% w/w. Fig. 3: Assays comparing the efficacies of EPO and alternative EMPs in stimulating growth/survival of human UT7 cells (left hand panels); or F36-P cells (right hand panels) in MTS assays. All assays were evaluated after 72h. Top: ■: EPO; A: AGEM115; •: AGEM400; T : AGEM099. All peptides were predissolved at 10 mg/ml in DMSO before diluting them into assay medium. Bottom:■: EPO;•: AGEM400; T : AGEM134. All peptides were predissolved at 10 mg/ml in DMSO before diluting them into assay medium.

Fig. 4: Western blot analysis studying STAT5 phosphorylation and ERK phosphorylation (in Fig. 4 continued: also Jak2 phosphorylation in UT7/EPO) in four starved cell lines in response to incubations for 30 minutes at 37⁰C with increasing concentrations of EPO or AGEM400(HES). Concentrations are in ng protein or peptide/ml.

Fig. 5: Antagonism of AGEM400(HES) against the activity of EPO in the UT7 MTS assay. Top: Right-shifting dose-response curves of EPO in the presence of increasing concentrations of AGEM400(HES). AGEM400(HES) concentrations are depicted as molarities of AGEM400 peptide moieties, considering the peptide content of AGEM400(HES) to be 10 per cent, and the molecular mass of one peptide 4.6 kDa. Middle: Schild representation of the EC50 shifts of EPO as a function of the concentrations of AGEM400 peptide (A) or AGEM400 peptide as part of AGEM400(HES) (•). Slopes of the linear regressions were: 1.006 and 0.956; while pA₂ values were -9.17 and -9.63, for AGEM400 and AGEM400(HES), respectively. This is a typical example of an experiment performed three times. Bottom: Influence of excess (100 ng peptide/ml) AGEM400(HES) on the survival-promoting effect of moderate concentrations of EPO, IL-3, or GM-CSF on UT7 cells. Cytokine concentrations are depicted in ng/ml. Fig. 6: Schild representations of the EC50 shifts of EPO as a function of the concentrations of alternative EMPs. Top: AGEM115(HES) (T) in comparison to AGEM400(HES) (•); Middle and bottom: AGEM099 peptide (A), AGEM134 peptide (♦) in comparison to AGEM400 peptide (■); peptides were predissolved at 10 mg/ml in DMSO before diluting them into assay medium. Slopes of the linear regressions and pA₂ values (indicating the affinities of the peptides for the EPO receptor) are depicted in the figures. Fig. 7: Influence of EPO (E), AGEM400(HES) (H), or both agents added together, after a 30 minute incubation period, on the phosphorylation levels of STAT5 and - where applicable- ERK and Jak2 (Fig. 7 continued), in UT7/EPO cells; UT7 cells; TF-1 cells; and F36-P cells. EPO concentrations were chosen as low as still eliciting a maximum or near-maximum effect (see Fig. 4): 3.4 ng/ml in all cell lines. AGEM400(HES) was used in excess in order to see potential antagonistic effects: 200 ng peptide/ml for UT7/EPO, UT7 and TF-1, and 250 ng peptide/ml for F36-P. Note that in all lines but UT7/EPO addition of AGEM400(HES) along with EPO caused weaker phosphorylation signals than EPO alone. Fig. 8: Analysis of the expression levels of EPO receptor and CD131 (common beta chain) on six hematopoietic cell lines and transfected HeLa cells. A. Flow cytometry. Starved myeloid/erythroleukemic cell lines UT7/EPO, UT7, TF-1 , F36-P, and M07e, as well as lymphoid cell line SupT1 were stained as live cells with fluorescent antibodies against the two receptor chains and tested by flow cytometry. Histograms showing fluorescence caused by staining with EPOR antibody (left, filled) or CD131 antibody (right, filled) were overlayed with histograms after control staining (gray, open). B. Flow cytometry on transfected HeLa cells. HeIa cells were transiently transfected with expression constructs containing EPOR (top) or CD131 (bottom) inserts and analysed by flow cytometry for expression of the receptor chains (left), with control staining shown on the right. The data was plotted as dot- plots with the fluorescence caused by anti-EPOR in FL-1 and the fluorescence caused by anti-CD131 in FL-2. C. Western analysis. Lysates of the same cells shown in A and B (symbols: UE: UT7/EPO; U: UT7; T: TF-1 ; F: F36-P; M: M07e; S: SupT1 ; H: naive HeLa cells; HE: HeLa cells transfected with EPOR construct; HC: HeLa cells transfected with CD131 construct) were electrophoresed, blotted, and incubated with antibodies against EPOR (top) or CD131 (center). After the latter staining a non-specific band of 98 kDa is present in lysates of most cells, while the 95 kDa band representing CD131 protein is indicated by an arrowhead. Coomassie staining of the blots (bottom) indicates a higher loading density of the lane with lysate of naive HeLa cells. All experiments in this Figure were repeated twice with similar results.

Fig. 9: Comparison of the levels of signalling proteins in four cell lines. Lysates of starved cells were assayed for protein content, after which equal protein amounts were loaded on SDS PAGE, electrophoresed and blotted. After immunostaining with antibodies against total Jak-2, total STAT5, and total ERK, blots were stained with Coomassie to check for equal loading density once again.

Fig. 10: Analysis of bone marrow progenitor cells cultured for six days in liquid medium containing a cocktail of cytokines. A: Growth curves of CD34-positive cells cultured for 6 days in the presence of 10 ng/mL EPO (■, black line); 200 ng peptide/mL AGEM400(HES) (•, gray line), or no ESA (α, discontinuous line). B: Flow cytometry of CD34-positive cells after six days of culture in cytokine cocktail including EPO. Top: dot plot after staining with control antibodies; bottom: after staining with FITC-labeled anti-EPO receptor antibody (FL1 channel) and PE-labeled anti-CD131 antibody (FL2 channel). C: Western blot of lysates from CD34-positive cells after six days of culture in cytokine cocktail including EPO, one day without cytokines, and subsequently incubated for 30 min. with 10 ng/mL EPO (E); 200 ng peptide/mL AGEM400(HES) (H), or no stimulus (-). Blots were incubated with antibodies against phospho-STAT5, STAT5, and EPO receptor. The data shown in this Figure are typical examples of experiments performed three times with cells from different donors each time. Continuation of Fig. 10: Flow cytometric analysis of surface expression of EPO receptor and CD131 , and Western analysis of STAT5 phosphorylation, in bone marrow progenitor cells cultured for six days in liquid medium containing a cocktail of cytokines including EPO. Left panel: dot plot after staining with control antibodies; middle panel: dot plot after staining with FITC-labeled anti-EPO receptor antibody (FL1 channel) and PE- labeled anti-CD131 antibody (FL2 channel); right panel: Western blot demonstrating STAT5 phosphorylation after incubation of cells cultured for six days with cytokines including EPO, and one day without cytokines, with 10 ng/ml EPO or 100 ng peptide/ml AGEM400(HES) for 30 minutes at 37⁰C. Total STAT5 protein was difficult to detect on these blots, but Coomassie staining of the blot indicates equal loading densities in all lanes. Note: High level EPOR expression on cultured CD34-positive cells as seen in this flow cytometry experiment was observed only in one occasion.

Figure 11. Levels of STAT5 phosphorylation induced by EPO or AGEM400(HES) as a function of EPO receptor and CD131 expression. HeLa cells were transfected with expression constructs containing EPOR insert, CD131 insert, mixtures of two receptor expression constructs, or expression vector pSI only. A: Two days after transfection cells were analysed for surface EPOR (FITC-labeled antibody, FL-1 channel) and CD131 (PE- labeled antibody, FL-2 channel) expression by flow cytometry. B: Two days after transfection cells were incubated for 30 minutes with 10 ng/mL EPO (E); 100 ng peptide/mL AGEM400(HES) (H); or no stimulus (0). Lysates were blotted and incubated with antibodies against EPO receptor, CD131 (top panels) and phospho-STAT5 and STAT5 (bottom panels). Note that the levels of STAT5 phosphorylation remained equally strong after stimulation with

EPO or AGEM400(HES) irrespective of the amount of CD131 plasmid admixed with EPO receptor plasmid. This experiment was performed three times with similar results.

Figure 12. Levels of STAT5 phosphorylation and cell survival induced by EPO or AGEM400(HES) as a function of EPO receptor expression. BA/F3 cells were stably transfected with an EPO receptor expression construct, after which eight clones were isolated and analysed. A: Flow cytometric determination of surface EPO receptor expression on maternal BA/F3 cell line (B); and BA/F3-EPOR clones 2.2 and 3.3. Histograms showing fluorescence caused by staining with EPOR antibody (filled) were overlayed with histograms after control staining (open). B: Western blot analysis of EPO receptor levels in the lysates of the above cell lines. Loading density of the lanes was checked by Coomassie staining of the blot. C: The above cell lines, starved for 4h, were incubated for 30 min. with 10 ng/mL EPO (E); 100 ng peptide/mL AGEM400(HES) (H); or no stimulus (0). Lysates were blotted and incubated with antibodies against phospho-STAT5 and STAT5. D: MTS assays using the above cell lines, testing the effects on survival of increasing concentrations of murine IL-3 (T , gray interrupted lines); EPO (■, black lines); and AGEM400(HES) (•, gray lines). All data in A - D were reproduced twice with similar outcomes. E: Correlation between the relative surface expression level of EPO receptor as measured by flow cytometry ((mean fluorescence with anti-EPOR antibody minus mean fluorescence with control antibody) divided by (mean fluorescence with control antibody)) and the maximum effect ratio AGEM400(HES)/EPO (maximum effect induced by AGEM400(HES) (plateau absorbance minus baseline absorbance) divided by the maximum effect induced by EPO in the MTS assay), of seven different BA/F3-EPOR clones (clone 3.3, with the maximum effect ratio AGEM400(HES)/EPO but with an EPO receptor surface expression level that was way out of range with the other clones, was omitted in this graph). The linear regression is shown as the straight line. R² of the linear regression was 0.620 in this experiment, and 0.674 in a different experiment.

III. Results AGEM400, as well as AGEM400(HES) (which means AGEM400 conjugated to HES) has growth stimulating effects on EPO-dependent cell line UT7/EPO. The potency of AGEM400(HES) to stimulate erythropoiesis in semisolid media from bone marrow derived progenitor cells are shown in Fig. 1. The efficacy of AGEM400(HES) in comparison to those of EPO (or Aranesp) was different in different assay systems. The potency (measured by the EC50 expressed as ng/ml peptide or protein) of AGEM400(HES) in the UT7/EPO MTS assay was similar to that of EPO, while in the clonogenic assay the EC50 of AGEM400(HES) was about 10 fold higher than that of EPO. The plateau heights achieved by AGEM400(HES) were always equally high as those achieved by EPO or Aranesp in both assay systems. Also alternative dimeric EMPs, including some with prolines in positions 10 and 17, induced equally high plateaus as EPO in the UT7/EPO MTS assay (Fig. 1. b)).

However, in other EPO responsive cell models, all derived from leukaemia, AGEM400(HES) shows only some EPO-like activity, in varying efficacies as compared with EPO, but always with a lower maximum effect than that elicited by EPO. E.g. UT7, the cell line UT7/EPO was derived from, demonstrated improved survival in three-day MTS assays in the presence of EPO or Aranesp. In contrast, AGEM400 or AGEM400(HES) did not show any effect on the survival of UT7 cells in three-day assays. UT7 assays in which MTS was added already after 24h of incubation with the substances did show an effect of AGEM400(HES) which was, however, very weak in comparison with the effect elicited by EPO (Fig. 2). In cell line F36-P, AGEM400(HES) showed very modest effects in MTS assays in comparison with EPO or

Aranesp. In cell line TF- 1 , which is a standard cell line for testing activities of erythropoietins (Hammerling et al., 1996), AGEM400 and AGEM400(HES) reproducibly showed survival- promoting activity, in efficacies comparable to those of EPO or Aranesp, but always leading to plateaus that were somewhat lower as those achieved with EPO or Aranesp (Fig. 2). So, depending on the cell type used, AGEM400(HES) displays full EPO-like activity (UT7/EPO, clonogenic assay on progenitor cells), somewhat lower maximum activity than EPO (TF-1), or very weak to absent EPO-like activity (UT7, F36-P). Thus, the EPO mimetic peptide showed differential maximum activities on the different cell lines. Also alternative dimeric EMPs, including some with prolines in positions 10 and 17, showed low maximum activity, or absent activity, in the UT7 and F36-P MTS assays (Fig. 3).

Next, the potential of AGEM400(HES) in comparison with EPO to cause phosphorylation of signalling molecules in the different cell models was studied. Phosphorylation of STAT5 and ERK in UT7/EP0 cells caused by 30 minute incubations with either EPO or AGEM400(HES), was equally strong provided the concentrations of the stimuli were high enough. However, in UT7, TF-1 and F36-P cells, the maximum STAT5 phosphorylation level caused by AGEM400(HES) was reproducibly lower than that caused by EPO (Fig. 4). An increase in phosphorylated ERK was seen in F36-P cells only with EPO but not with AGEM400(HES) (Fig. 4). Phosphorylation of ERK was, however, inconsistent between experiments in cell lines UT7, TF-1 and F36-P, and therefore in the following this data is only shown in cases where significant increases in ERK phosphorylation were seen.

In search of an explanation for the observed differences in the maximum effects of AGEM400(HES) versus EPO in various cell models, we first tested whether EPO and AGEM400(HES) compete for the same cellular receptor in UT7 cells. In three-day UT7 MTS assay, EPO shows a survival-stimulating activity, while AGEM400(HES) and other EMPs have no activity at all (see Figs. 2 and 3). As is shown in Fig. 5 increasing concentrations of AGEM400 or AGEM400(HES) reduced the efficacy of EPO in the UT7 MTS assay dose- dependently, demonstrating some kind of receptor antagonism of the EPO mimetic peptide AGEM400(HES). When the increasing EC50 of EPO were plotted as the log(r-1) (in which r = EC50 with antagonist/EC50 without antagonist) against the log concentration of peptide antagonist (Schild plots), straight lines appeared with a slope of 1.0, indeed indicating receptor antagonism (Fig. 5, central panel). The intercepts of the Schild plots with the X-axis (where log(r-1) = O, thus where r=2), the so-called pA₂, represent the affinities of the peptide antagonist for the receptor. The pA₂ of AGEM400 was -9.2 (630 pM), while that of AGEM400 peptides conjugated to HES AGEM400(HES) was -9.6 (250 pM). The specificity of the inhibition of EPO by AGEM400(HES) in the UT7 MTS assay is demonstrated in Fig. 5, bottom panel. Excess AGEM400(HES) (100 ng/ml peptide) was added to moderate concentrations of EPO, IL-3 or GM-CSF. The survival-promoting activity of EPO was completely blocked by AGEM400(HES), while AGEM400(HES) did not inhibit the activities of IL-3 and GM-CSF at all. AGEM400(HES) even had a very mild stimulating effect on UT7 cells incubated with IL-3 or GM-CSF. Fig. 6 shows the Schild plots generated with alternative EMPs, including some with prolines in positions 10 and 17. Fig. 6 shows that also other EMPs display receptor antagonism with EPO, albeit sometimes with lower affinities for the EPO receptor than that displayed by AGEM400. The inhibitory effect of AGEM400(HES) on the activity of EPO was also demonstrated on the level of STAT5 phosphorylation (and where applicable ERK phosphorylation). In UT7 cells, TF-1 cells, and F36-P cells, AGEM400(HES) added in high concentrations not only caused lower levels of phosphorylation than EPO, AGEM400(HES) also caused low phosphorylation levels when added in excess along with EPO (Fig. 7). In contrast, UT7/EPO cells incubated with EPO, AGEM400(HES), or both, showed similar phosphorylation levels of STAT5 and ERK.

The four cell lines were tested for expression levels of EPOR and CD131 , the common beta chain of the IL-3 and GM-CSF receptors, which has been suggested to be involved in EPO signalling or to occur in complex with the EPOR. Live starved cells were stained with fluorescent antibodies and analysed by flow cytometry. The relative surface expression levels of EPOR were highest in UT7/EP0 cells, and were lower but still detectable in cell lines UT7, TF-1 and F36-P. Expression of surface CD131 was demonstrated on UT7, TF-1 , F36-P, and M07e cells (human acute megakaryoblastic leukemia cells dependent on IL-3, but not responsive to EPO), but was not detectable on UT7/EP0 cells (Figure 8A). Specificity of the antibodies in this application is demonstrated: 1) by the absence of reactions with SupT1 (human T-cell lymphoma), HeLa, and with anti-EPOR also M07e cells; 2) staining of EPOR on all cells responsive to EPO, and on EPOR-transfected HeLa cells (Figure 8B); and 3) staining of CD131 on all cells dependent on IL-3 or GM-CSF and on CD131-transfected HeLa cells. The cell lines on which AGEM400(HES) had a weaker effect than EPO thus demonstrated lower surface levels of EPOR but higher levels of CD131 than cell line UT7/EP0 in which AGEM400(HES) displayed maximum effects similar to EPO. Lysates of the same cells were also analysed by Western blotting using different antibodies than in flow cytometric analysis (Figure 8C), but leading to a similar picture for CD131 expression: a 95 kDa band was visible (arrowhead) only in UT7, TF-1 , F36-P and M07e lysates, as well as in CD131-transfected HeLa cells. The EPOR level in UT7 lysates was a little higher than expected based on the flow cytometric data. A major 55 kDa band representing the EPOR protein was visible in UT7/EPO and UT7 lysates and in EPOR-transfected HeLa cells, while a much weaker 55 kDa band was seen in TF-1 and F36-P lysates. TF-1 , however presented with a major 45 kDa EPOR band, which is caused by a genomic mutation (Winkelmann et al., 1995). Other bands may represent degradation products of EPOR or unspecific bands.

The relative levels of intracellular signalling proteins were also compared between cell lines. Normalized amounts of cellular lysates of starved UT7/EP0, UT7, TF-1 , and F36-P cells were blotted and immunostained with antibodies against total Jak2, total STAT5, and total

ERK protein. While the levels of ERK protein were very comparable between cell lines, there were differences in the levels of Jak2 and STAT5 proteins. TF-1 cells had a clearly higher level of STAT5 protein than the other three lines, while it had a lower level of Jak2 protein than the other lines. UT7/EPO had a somewhat higher level of Jak2 protein than the other three cell lines (Fig. 9).

We tried to find out whether the same relationship between responsiveness to AGEM400(HES) and expression levels of EPOR and CD131 can be observed in human erythroid progenitor cells. Such cells, whether from bone marrow or other sources, are difficult to obtain in amounts high enough to perform Western analysis. Liquid culture of CD34-enriched bone marrow cells for 6 days in media containing a cocktail of various cytokines, supplemented with EPO, AGEM400(HES) or no ESA, yielded expanded numbers of partially differentiated progeny cells sufficient for functional studies. Numbers of live cells in cultures with EPO or AGEM400(HES) increased faster than in cultures without ESA, with no large differences between EPO and AGEM400(HES) noted after three independent experiments (Figure 1OA). Flow cytometry of progenitor cells expanded for six days with EPO (Figure 10B) usually did not show a clearly EPOR-positive population, although in one out of four experiments a population of cells clearly EPOR-positive was observed (continuation of Fig. 10). A population of CD131 -positive cells was always visible, however. The same results were obtained after culture with AGEM400(HES) or without ESA, and also after five days of culture with EPO, and after culture with EPO for six days followed by 24 h of cytokine starvation after (not shown). Cells cultured for six days with EPO and starved for 24h, did have functional EPOR, because STAT5 was efficiently phosphorylated after incubation with EPO or AGEM400(HES), with phospho-STAT5 levels induced by AGEM400(HES) being similar or only slightly lower than those induced by EPO (Figure 1OC, and continuation of Fig. 10). Staining of cell lysates for EPOR yielded several bands of 50-55 kDa, 45 kDa and 40 kDa, but due to absence of controls, it cannot be proven that at least the larger bands actually represent EPOR (Figure 1OC).

To study whether coexpression of CD131 along with EPOR can suppress the response of cells to AGEM400(HES) in comparison to EPO₁ we used a transient transfection system that allows for co-expression of two transgenes. HeLa cells were transfected with expression constructs encoding EPOR, CD131 , mixtures of the EPOR and CD131 constructs, or empty vector. Flow cytometry showed that after mixing the EPOR and the CD131 construct in ratios 75/25 or 50/50, almost all cells that presented as EPOR-positive were also CD131 -positive (Figure 11A). Stimulation of the transfected cells with 10 ng/mL EPO or 100 ng peptide/mL AGEM400(HES) yielded similar phospho-STAT5 levels, independent of whether EPOR was expressed on its own or co-expressed with CD131 (Figure 11 B). Using ratios of EPOR/CD131 of 25/75 or lower caused EPOR, and also phospho-STAT5 after stimulation, to become undetectable (not shown). Also expression of CD131 on its own, or transfection with empty expression plasmid, did not lead to phosphorylation after EPO or AGEM400(HES) stimulation (Figure 11 B). To study whether the relative response of cells to AGEM400(HES), in comparison to the response to EPO, depends on the expression level of EPOR₁ we generated stable EPOR- transfected BA/F3 clones. EPOR-transfected cells were selected by their potential to grow in medium lacking mlL-3 but containing human EPO, and clones were isolated by limiting dilution. Eight clones expressing differing levels of EPOR were tested for their responses to EPO and AGEM400(HES). BA/F3-EPOR clones 2.2 and 3.3, and maternal cell line BA/F3, are compared in Figure 12A-D. Whereas BA/F3 cells showed no (human) EPOR, clone 2.2 expressed a small amount, while clone 3.3 expressed a large amount of EPOR, as determined by flow cytometry and Western blotting (Figure 12A-B). In BA/F3, no STAT5 phosphorylation could be demonstrated upon stimulation with EPO or AGEM400(HES). In clones 2.2 and 3.3 phospho-STAT5 was clearly demonstrable after stimulation with 10 ng/mL EPO, but stimulation with 100 ng peptide/mL AGEM400(HES) only induced detectable phospho-STAT5 in clone 3.3, not in clone 2.2 (Figure 12C). In MTS assays (Fig. 12D), BA/F3 cells responded to mlL-3. Survival-promoting effects on BA/F3 cells of EPO or AGEM400(HES) could not be demonstrated. BA/F3-EPOR clones 2.2 and 3.3 still responded to mlL-3, while expression of human recombinant EPOR made them strongly responsive to EPO as well. Clone 3.3 also responded well to AGEM400(HES), while the response of clone 2.2 to AGEM400(HES) in the MTS assay was very weak. A plot of the relative surface expression levels of EPOR (measured by flow cytometry) and the ratio of AGEM400(HES)- versus EPO-induced plateau heights in the MTS assays of all 8 clones, clearly yielded a correlation between EPOR surface level and relative response to the EMP (Figure 12E).

The results thus show that there are different maximum levels of activity of AGEM400(HES) as an example of an EPO mimetic peptide in comparison to EPO in various cellular models. AGEM400(HES) displayed full activity on the cells it is intended to work on: human hematopoietic progenitor cells developing into erythrocytes. Full activity was also observed in stimulating growth of the UT7/EP0 cell line. This cell line is a subline of UT7 which has become completely dependent on EPO (Komatsu et al., 1991 ; Komatsu et al., 1993). Unlike UT7 cells, UT7/EPO do not grow in IL-3 of GM-CSF anymore, and have a relatively high level of surface EPO-R expression with no or very little expression of CD131. The maximum level of STAT5-phosphorylation caused by AGEM400(HES) was also just as strong as that caused by EPO in cultured progenitor cells and in UT7/EPO cells, while at least in UT7/EP0 cells this also was true for the maximum level of ERK phosphorylation. In contrast, AGEM400(HES) was only partially agonistic in a different set of leukaemia cell lines. In TF-1 cells, F36-P cells, and UT7 cells the maximum effect of AGEM400(HES) in survival assays was always lower than that of EPO, while maximum levels of STAT5 phosphorylation achieved by AGEM400(HES) were also lower than by EPO in these cell lines. The partiality of the effect of AGEM400(HES) in these assays was also evident with the AGEM400 peptide on its own or with alternative EMPs (see Figs. 2 and 3), so this was neither caused by the conjugation of the peptide to HES, nor a specific property of the AGEM400 sequence.

Surprisingly, in cell lines in which AGEM400(HES) caused weaker effects than EPO, incubation with EPO along with excess AGEM400(HES), AGEM400, or different EMPs led to a decrease of the effects of EPO. AGEM400(HES) partially inhibited phosphorylation of

STAT5 by EPO in UT7, TF-1 and F36-P cells, and completely inhibited the effects of EPO on the survival of UT7 cells. This latter inhibition was shown to be the result of competitive receptor binding of EPO and AGEM400(HES). In UT7 cells the replacement of EPO by EMP from the EPO receptor obviously led to a complete abolishment of the survival-promoting effects of EPO.

This work shows that EPO and AGEM400(HES) do not have identical activities in all cell types, which means that AGEM400(HES) will not show the same severity of side-effects associated with EPO. Based on the generated in vitro data, AGEM400(HES) appears to have full activity on the intended target cells, but weak to almost absent activity on a series of cell lines which are all of leukemic origin. TF-1 is an acute erythroleukemia cell line (Kitamura et al., 1989), UT7 and F36-P are both from acute myeloid leukemia (AML; Komatsu et al., 1991 ; Chiba et al., 1991). F36-P actually was derived from a patient who developed AML secondary to myelodysplastic syndrome (MDS), subtype refractory anaemia with excess blast cells (RAEB).

One of the problems associated with treating anaemic patients with MDS is the relatively poor response rate of erythropoiesis to EPO, necessitating the use of high levels of EPO in MDS patients. Also, endogenous EPO levels are often quite high in MDS patients (Wallvik et al., 2002; Moyo et al., 2008). Treatment of MDS with EPO is considered being risky, since EPO can induce AML in MDS patients (Bunworasate et al., 2001). Moreover, there are general concerns in other myeloid diseases on the disease-promoting effects of EPO-based anemia treatment. The generated in vitro data indicate that treatment of MDS, and potentially other myeloid or non-myeloid malignancies with AGEM400(HES) or EMPs in general will have a similar efficacy as EPO in stimulating erythropoiesis, but that EMPs such as AGEM400(HES) have a much milder stimulating effect, or even no effect, on the MDS blast cells that tend to progress towards leukaemia. In this case, AGEM400(HES), as an example of EMPs, is a safer choice for treating anaemia in MDS patients than EPO and additionally supports treatment of the underlying disease by inhibiting the EPO-stimulatory effects. The antagonism of AGEM400(HES) towards EPO, as shown in UT7 MTS assays and in phosphorylation experiments on TF-1 , F36-P, and UT7 cells, shows that treatment with AGEM400(HES) as an example of EMP, counteracts potential adverse effect of endogenous high-level EPO on disease progression in MDS patients. Furthermore, if anaemia in MDS patients is effectively cured by EMPs such as AGEM400(HES), it is also expected to indirectly reduce endogenous EPO levels. A similar response to AGEM400(HES) as in UT7 cells was found in BA/F3-EPOR clone 2.2 with a low level of EPOR expression: a weak response to AGEM400(HES) in comparison to EPO, and inhibition by AGEM400(HES) of the actions of EPO. In contrast, BA/F3-EPOR clone 3.3, with a high level of EPOR expression behaved much like cell line UT7/EP0. When comparing eight different BA/F3-EPOR clones, a direct correlation was found between EPOR expression levels and the relative response to the EMP. This finding is an important clue in helping to explain the differences in activity spectrum between EPO and EMP.

The absolute number of EPORs per cell is probably not the only parameter that determines the responsiveness to AGEM400(HES) per se. Erythroid progenitor cells and their progeny were fully responsive to AGEM400(HES), yet in most cases it was impossible to demonstrate a population of clearly EPOR-positive progenitor cells by flow cytometry. Previous studies have shown that progenitor cells in various stages of erythroid development express several hundreds EPORs per cell, while cell lines UT7 and TF- 1 express several thousands of EPORs per cell (Winkelmann et al., 1995; Broudy et al., 1991 ; Shinjo et al., 1997). However, the latter cell lines were found here to respond weakly to AGEM400(HES). It is also worth noting that only EPOR present on the cell membrane is functional, and high levels of EPOR found in cellular lysates do not predict high surface expression of EPOR because maturation and trafficking steps can inhibit surface expression. This might also explain the apparent difference between the relative EPOR level in UT7 cells detected by Western and by flow cytometry (Figure 8).

We hypothesize that the response of an EPO-responsive cell to AGEM400(HES) may at least partially depend on the ratio of functional surface EPOR to some other cellular factor. Some authors have speculated on the role of CD131 as a cofactor in signalling effects of EPO, and that CD131 occurs in complex with EPOR, both in hematopoietic and in neuronal cell types (Brines et al., 2004; Hanazono et al., 1995; Jubinski et al 1997; Blake et al., 2002). CD131 may, however, not be involved in signalling effects of EMPs. Thus, the ratio of surface EPOR to surface CD131 may determine the responsiveness of a cell to AGEM400(HES). Note that the BA/F3-EPOR clones were still responsive to mlL-3 so they did express functional murine homolog of common beta chain/CD131 along with recombinant EPOR. Therefore this, theoretically, can play a role in these models. Transfection experiments with HeLa cells expressing EPOR with or without CD131 , however, have not shown such a relation.

An alternative explanation is that the ratio of surface EPOR to downstream signalling protein(s) may determine the responsiveness of a cell to AGEM400(HES). In general, an EPOR complexed with a symmetrical dimeric EMP may have a signalling efficiency much lower than an EPOR asymmetrically complexed with EPO (Syed et al., 1998). In cases where the EPOR surface level is very high compared to the levels of (one or more) signalling proteins, the signalling cascade may still be saturated by many EPOR/EMP complexes all signalling with low efficiency, thus leading to compensation of the presumed weaker signalling efficiency of the symmetrical complex. Determining whether either surface EPOR, or one or more signalling proteins (JAK2, STAT5, ERK, etc.) is limiting in a certain cell type is a challenging task. However, measuring and comparing expression levels of (cell surface bound) EPO receptor and one or more signalling proteins, might develop into a method for predicting the responsiveness of a cell type or a malignancy to EMP in comparison to EPO.

Except from the use of EMPs in leukemic and preleukemic diseases such as MDS₁ as discussed above, the data presented here open other intriguing clinical possibilities. One of the side effects of EPO most frequently discussed is the increased mortality rate observed in cancer patients treated with EPO variants compared with control groups (Henke et al., 2003; Leyland-Jones and BEST Investigators and Study Group, 2003; Danish Head-and Neck Cancer Group, 2007; Wright et al., 2007). The etiology of these unwanted effects is still under debate, but it is assumed that they result from direct effects of EPO on tumor cells, or from indirect effects, e.g through angiogenesis. It is an interesting issue to consider, in light of our findings, that EPOR levels, if detected at all on non-hematopoietic cells (tumor or endothelial), appear to be very low (Sinclair et al., 2010). Thus, the responsiveness of such cancer cells and endothelial cells to EMPs is much weaker than to EPO, and accordingly, EMPs have much less severe effects on the tumor stimulation than EPO. In addition, the observed antagonism of AGEM400(HES) towards EPO shows that EMPs such as AGEM400(HES) may counteract deleterious effects of ectopic EPO production (e.g. autocrine EPO loops in tumor tissue) or high endogenous EPO levels in anemic cancer patients having undergone radiation or chemotherapy.

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Claims

1. A compound comprising an EPO mimetic peptide or a functional variant thereof for stimulating erythropoiesis and for prophylactic or therapeutic treatment of a patient afflicted

5 with or at risk of being afflicted with a disease wherein EPO adversely affects the mortality and/or disease progression.

2. A compound comprising an EPO mimetic peptide or a functional variant thereof for therapeutic or prophylactic reduction of EPO levels in a patient while maintaining functional0 erythropoiesis.

3. A compound comprising an EPO mimetic peptide or a functional variant thereof for stimulating erythropoiesis and for prophylactic or therapeutic treatment of a patient for inhibiting and/or reducing the stimulatory effects of EPO on diseases wherein EPO adversely5 affects the mortality and/or disease progression.

4. A compound comprising an EPO mimetic peptide or a functional variant thereof for stimulating erythropoiesis and for therapeutic or prophylactic treatment for decelerating or stopping EPO responsive tumor cell growth and/or tumor progression and/or for reducing the O risk of developing EPO responsive tumors.

5. The compound according to any one of claims 1 to 4, wherein the disease is characterised by a malignancy or pre-malignancy having one or more of the following characteristics:

5

a. it is a malignancy or pre-malignancy wherein EPO adversely affects the mortality or malignancy progression;

b. it is an EPO responsive malignancy or pre-malignancy; and/or

c. it is a malignancy or pre-malignancy which expresses the EPO receptor.

0

6. The compound according to any one of claims 1 to 5, wherein the patient has one or more of the following characteristics: a. he is afflicted with or is at risk of being afflicted with a malignancy or pre-5 malignancy, wherein EPO adversely affects the mortality or malignancy progression;

b. he is afflicted with or is at risk of being afflicted with an EPO responsive malignancy or pre-malignancy;

c. he is afflicted with or is at risk of being afflicted with a malignancy or pre- o malignancy which expresses the EPO receptor; and/or

d. he is anemic.

7. The compound according to one or more of the claims 1 to 6, for suppressing tumor angiogenesis and/or tumor progression.

8. The compound according to one or more of the claims 1 to 7, for stimulating erythropoiesis and for the prophylactic or therapeutic treatment of anemia.

9. The compound according to one or more of the claims 1 to 8 for inhibiting or reducing the stimulatory effects of EPO, preferably for preventing or treating diseases wherein EPO adversely affects the mortality and/or disease progression.

10. The compound according to one or more of the claims 1 to 9, for suppressing tumor angiogenesis and/or tumor progression of EPO responsive tumors by inhibiting or reducing the stimulatory effects of EPO on tumor angiogenesis and/or tumor progression while stimulating erythropoiesis.

11. The compound according to one or more of claims 1 to 10, wherein the patient shows elevated EPO levels, preferably being≥ two times above the standard deviation of the normal value with the respective assay in a healthy reference population.

12. The compound according to one or more of claims 1 to 11 , wherein it is determined prior to the treatment with the compound that a. the patient is afflicted with and/or is at risk of being afflicted with an EPO responsive malignancy or pre-malignancy; and/or

b. the patient has elevated EPO levels.

13. The compound according to one or more of the claims 1 to 12, wherein the disease is characterised by a malignancy or pre-malignancy that is selected from the group consisting of myeloid malignancies and pre-malignancies, in particular leukemic malignancies, MDS and AML, non-myeloid malignancies and pre-malignancies, lymphoid malignancies and pre-malignancies, breast cancer, in particular metastatic breast cancer, mammary cancer, cervical cancer, ovarian head and neck cancer, epithelial tumors, in particular gastro-intestinal cancer, gastric cancer, prostate cancer, lung cancer, in particular non-small cell lung cancer and malignant tumors of mesenchymal origin, in particular sarcomas, glioblastoma, melanoma, esophagal ADC, choriocarcinoma, colon cancer, pancreatic cancer, uterine ADC, prostate cancer, leukemia and hepatoma.

14. The compound according to one or more of the claims 1 to 13, for the prophylactic or therapeutic treatment of the anemic condition of a patient afflicted with MDS and for preventing the development of MDS into acute myeloid leukemia (AML).

15. The compound according to one or more of the claims 1 to 14, for the prophylactic or therapeutic treatment in combination with chemotherapeutic agents and/or radiation therapy.

16. The compound according to one or more of the claims 1 to 15, wherein the EPO mimetic peptide comprises the following consensus sequence:

X6X7X8X9X10X11X12X13X14X15 X16X17X18X19 wherein each amino acid is selected from natural or unnatural amino acids and

X₆ is an amino acid with a sidechain functionality capable of forming a covalent bond;

X₇ is R, H, L, W, Y or S;

X₈ is M, F, I₁ homoserinemethylether or norisoleucine;

X₉ is G or a conservative exchange of G;

X₁₀ is proline or a non conservative exchange of proline;

X₁₁ is selected from any amino acid;

X₁₂ is an uncharged polar amino acid or A;

X₁₃ Js W₁ 1-nal, 2-nal, A or F;

X₁₄ Is D, E, I, L or V;

X₁₅ is an amino acid with a sidechain functionality capable of forming a covalent bond;

X₁₆ is independently selected from any amino acid; preferably G₁ K, L₁ Q, R, S, Har or

T and more preferred a positively charged amino acid;

X₁₇ is independently selected from any amino acid; preferably A₁ G₁ P, R, K, Y, Har and more preferred a positively charged amino acid;

X₁₈ is independently selected from any amino acid; preferably L or Q;

X₁₉ is independently selected from any amino acid, preferably a positively charged amino acid.

17. The compound according to one or more of the claims 1 to 16, wherein the EPO mimetic peptide shows the following activity profile: a) when tested in an assay that measures the potency of an agent to stimulate the proliferation or survival of the cell line UT7/EPO, the maximum effect achieved with the EMP in its API format should be at least half as high as that achieved by recombinant erythropoietin, preferably epoetin alfa (Tradename:

Erypo); and b) when tested in an assay that measures the survival of cell line UT7 or of cell line F36-P, the maximum effect achieved with the EMP should be at least less than half as high as that achieved by recombinant erythropoietin, preferably epoetin alfa (Tradename: Erypo).

18. The compound according to one or more of claims 1 to 17, wherein the patient has received or will be receiving chemotherapy and/or radiotherapy.

19. The compound according to one or more of claims 1 to 18, wherein said compound is administered for reducing and/or inhibiting EPO-induced stimulatory effects on EPO responsive tumor cells.

20. The compound according to one or more of claims 1 to 19, for antagonizing EPO for reducing or inhibiting EPO-induced stimulatory effects on EPO responsive tumor cells.

21. A pharmaceutical composition comprising a compound as defined in one or more of the claims 1 to 20 for the pharmaceutical uses as defined in one or more of the claims 1 to 20.

22. The use of a compound comprising an EPO mimetic peptide as defined in one or more of the claims 1 to 20 in therapy as defined in one or more of the claims 1 to 20.