WO2008113597A1

WO2008113597A1 - Peptides for treating multiple myeloma

Info

Publication number: WO2008113597A1
Application number: PCT/EP2008/002271
Authority: WO
Inventors: Amparo Hausherr-Bohn; Guenther Krause; Michael Hallek
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt Gmbh; Universitaet Koeln
Priority date: 2007-03-22
Filing date: 2008-03-20
Publication date: 2008-09-25

Abstract

The present invention is directed to peptides for treating multiple myeloma. The invention furthermore is directed to a nucleic acid sequence encoding same and a pharmaceutical composition comprising said peptides and/or said nucleic acid sequence. Moreover, the invention concerns the use of those peptides in the treatment of multiple myeloma.

Description

Peptides for treating multiple myeloma

Background of the Invention

Multiple Myeloma

Multiple Myeloma (MM) is a clonal B -cell neoplasm. The disease is characterized by the accumulation of terminally differentiated, antibody producing plasma cells in the bone marrow. MM is associated with bone lesions, renal failure and immunodeficiencies (Yasui et al., 2006).

Despite conventional and high dose chemotherapies as well as novel biological therapeutics the disease remains incurable. The median survival time is 4-5 years. MM is responsible for 20% of all deaths caused by hematopoetic malignancies and 2% of all cancer deaths. The annual incidence of MM is approximately 3.8 per 100 000 population (-15 000 new cases of MM/year in the USA) and approximately 1.5 per 100 000 in Europe. The disease peaks are between the age of 65 to 70. MM is 1.5 times more common in male versus female. MM is almost twice as common in the black versus Caucasian population. One reason for the increased incidence of MM in African-Americans could, at least in part, be related to increased serum cholesterol levels since cholesterol is an essential component of MM cell membranes, it has been implicated in disease pathogenesis, and is associated with higher mortality in black versus Caucasian men. Independent of the genetic background of the population, a prospectively studied cohort of more than 900,000 US adults showed that increased body weight is associated with increased death rates from cancers, including MM (Greenstein et al., 2003; Kuehl & Bergsagel, 2002; Podar & Anderson, 2006).

Novel therapeutics include Thalidomide and its immunomodulatory derivatives (IMiD) like lenalidomide/Relimid™, as well as the proteasome inhibitor Bortezomib/Velcade™ show promising results in the clinic even in relapsed refractory MM (Hayashi et al., 2003; Hideshima & Anderson, 2002; Hideshima et al., 2004; Roccaro et al., 2006)

The role of the cytokine IL-6 in Multiple Myelom

IL-6 is one of the major growth factors for IL-6 in vivo and in vitro. IL-6 is produced by the MM cells themselves, autocrine loop, and the Bone marrow stromal cells (BMSCs), paracrine loop. The secretion of TNF-α by the MM cells induces the production and secretion of IL-6 by the BMSC. Besides IL-6, IGF-I and IL-10 stimulate the proliferation of MM cells (Drexler & Matsuo, 2000). Other factors like VEGF, SDF- lα, and TNF-α influence the survival of MM cells via different pathways (Anderson, 2001; Burger et al., 2001; Mitsiades et al., 2002; Yasui et al., 2006).

In the clinic, serum EL-6 and IL-6 receptor (IL-6R) levels are the most important prognostic factors to determine the proliferative fraction of the MM cells and the tumor mass within the patient (Bataille et al., 1989).

On the membrane of a target cell, IL-6 first interacts with a specific membrane bound IL-6- receptor (IL-6R, IL-6Roc, gp80 or CD 126). The so formed hexameric IL-6-IL-6R complex associates with signal-transducing transmembrane receptor, gpl30 (IL-6Rβ or CD130). Binding of the IL-6-IL-6R complex promotes dimerization of gpl30 and subsequent initiation of intracellular signaling (Boulanger et al., 2003; Schroers et al., 2005; Skiniotis et al., 2005; Ward et al., 1994; Ward et al., 1996).

IL-6 based treatment strategies include monoclonal antibodies against IL-6 or IL-6R and the superantagonist Sant7. All three strategies are designed to antagonize the downstream signaling from the IL-6 receptor complex. In clinical trail using the IL-6R antibody a transient response to the treatment is shown (Yasui et al., 2006). Disease stages and IL-6 dependence

In the progress of the disease MM cells can acquire secondary mutation (c-myc, pl6, ras, p53 and others) which makes them independent of growth factors like IL-6. The isolation and cultivation of cell lines is difficult and generally more successful from late disease stages (Kuehl & Bergsagel, 2002). Currently, out of 112 described MM cell lines only 27 (-25%), are IL-6-dependent (Drexler & Matsuo, 2000). On IL-6-independent cell lines like MMl. S cells, IL-6 has a positive effect on proliferation. The amount of cells which are in the S-Phase of the cell cycle is increased by IL-6 stimulation. For further information it is also referred to Fig.8 showing the stages of Multiple Myeloma from Kuehl & Bergsagel, 2002.

IL-6 influences Proliferation and Survival via different pathways:

1) The activation of the JAK, STAT pathway leads to the up-regulation of anti-apoptotic proteins like Bcl-xL and McI-I, resulting in survival and resistance to apoptotic stimuli induced by chemotherapeutic drugs. 2) The activation of the MAPK pathway via SHP-2, SHC, Grb2, SOS, RAS, RAF, MEK or via GAB and Src Family Kinases. 3) The PI3K/Akt pathway leads to the phosphorylation and inactivation of various anti-apoptotic proteins like BAD, Caspase-9, GSK3β and FKHR. Furthermore Akt phosphorylation leads to the activation of NF- KB. NF- KB induces the transcription of pro-survival/anti-apoptotic mediators like IL-6 itself, cell adhesion molecules, Bcl-xL, IAPs and Cyclin Dl. A cross talk between the PD K/ Akt pathway and the MAPK pathway is known. 4) The activation of SFKs by CD45 leads to the activation of PLCγ2, Ca²⁺ release form the ER and the activation of PKC-β, which was shown to be independent of JAK/STAT and SHP-2/Erkl/2 signaling (Anderson, 2003; Greenstein et al., 2003; Hallek, 1995; Hallek et al., 1998; Hallek et al., 1997; Hideshima et al., 2004; Ishikawa, 2006; Podar et al., 2004). To sum up: IL-6 pushes the equilibrium between survival/proliferation and apoptosis/cell death towards survival and proliferation.

Most of the above described signaling pathways can be analyzed in cell culture experiments. MMl. S is a well characterized model cell line for the analysis of IL-6 signaling pathways and the effects of novel potential therapeutic molecules on IL-6 induced signaling pathways. Besides IL-6, IGF-I is known to move the equilibrium towards survival and proliferation by signaling via the PI3K/Akt and MAPK pathways. IL-6 and IGF-I both protect MMl. S cells against glucocorticoid induced Apoptosis. Glucocorticoids especially dexamethasone (Dex) are used in standard chemotherapeutic regimes in patients. However, the mechanism how these cytokines confer to drug resistance is not fully understood. Besides various cytokines, cell-cell contacts and cell adhesion molecules lead to the activation of pathway which results in survival and proliferation. However, a model integrating the different stimuli and signaling pathways with respect to spatial and temporal information is not available.

Recently the inventors were able to show that a membrane permeable peptide, named peptide 18AD, which is derived from the Src Family Kinase (SFK) interacting domain of the signal transducing subunit of the IL-6 receptor complex, is able to inhibit IL-6-induced proliferation and to induce apoptosis in Myeloma cells. Peptide 18AD inhibits the association of the SFK Hck with gpl30 and reduces the IL-6-induced activities of the SFKs Hck, Lyn and Fyn. (Hausherr et al., 2007; Schaeffer et al., 2001).

However, there is still a need existing to provide new targeted therapies of the MM. In particular, there is a need to develop a therapeutic agent which can - independently of growth factor IL-6 - successfully defeat myeloma cells.

Summary of the invention

Therefore, it is an object of the invention to provide a new peptide, which effectively can inhibit growth and proliferation and/or induce apoptosis of myeloma cells. It is in particular an object of the invention to provide a protein or therapeutic agent, which can be used for targeted treatment of Multiple Myeloma also independently of IL-6. Or, in other words, to provide a means which may also be used for defeating myeloma cells which are independent from IL-6. It is a further object of the invention to provide a pharmaceutical composition containing said protein. A still further object of the invention can be seen in effectively treating a Multiple Myeloma in a mammal, preferably a human patient.

These objects are achieved by the subject-matter of the independent claims. Preferred embodiments are set forth in the dependent claims. The inventors derived a novel peptide from peptide 18AD (mentioned above), namely peptidelA (comprising SEQ ID NO: 1 = ASAARPAA as a core sequence). Two major changes were introduced in the new peptide with respect to peptide 18AD.

Although peptidelA was originally developed in a screen to identify amino acids which were responsible for the effects seen by peptide 18AD treatment (Hausherr, 2005), the underlying mechanisms by which peptide 18AD and peptidelA induce cell death, are entirely different.

For comparison, an additional peptide termed peptide OB has been derived from peptide 18AD, in respect of which in peptidelA all acidic residues in the core sequence are changed to alanine. See, in this respect, Figure 1 for further information.

1) Peptide 18AD affects only IL-6-dependent MM cells or cells which transduce a pro- proliferative signal via the gpl30-Hck axis by inhibiting the interaction between SFKs and gpl30, while peptidelA targets IL-6-dependent and IL-6-independent myeloma cells. 2) By peptide 18AD, SFK activities downstream of gpl30 are inhibited, while STAT3 and Akt pathway are unaffected. PeptidelA treatment impairs STAT and Akt pathways. 3) The kinetic of growth inhibition and apoptosis induction is in the range of days by peptide 18AD treatment while it is in the range of one to two hours when using peptidelA. 4) In a mutant receptor the eight amino acids of the core sequence were deleted. This mutant receptor was unable to interact with the SFK Hck. It was shown that the core sequence is necessary for the interaction between SFK and receptor. PeptidelA does not carry the sequence of these eight amino acids and it is unlikely that it acts as a specific inhibitor for the interaction between Hck and gpl30.5) Peptide 18AD treatment does not show the same changes in morphology, which are detected after peptidelA treatment.

Based on these findings, peptidelA may interfere with the assembly of signaling complexes at the plasma membrane. It could be possible that the peptide changes the membrane structure in a way that the association of functional receptor complexes is impaired. Looking at the peptidelA treated cells which were stimulated with IL-6, IL-6 induced Akt and STAT3 phopsphorylation and activation is impaired, figure 5. Akt associates after stimulation with its Pleckstrin-Homology (PH)-domain via phosphoinositides with the plasma membrane. STAT3 associates with the membrane via gpl30, which is localized in the plasma membrane. The effects of further downstream kinases like Erkl/2 are less pronounced. In the experiments, the IL-6 pathway is used as one example, other pathways emerging from different stimuli on the plasma membrane should also be impaired.

Podar K. et al 2003, 2006 analyzed the effects of cholesterol inhibitors on MM cells. In contrast to other cells of the hematopoetic system MM cells express Caveolin-1 (Cav-1). Cav- 1 is responsible for the assembly of so called caveole. Caveoles are a sort of lipid rafts, a membrane structure which is characterized by its high content of cholesterol. Active signaling complexes like the IL-6 receptor complex are present in these caveoles. In MM cells which were pretreated with cholesterol inhibitors like β-cyclodexin, the phosphorylation and activation of Akt and STAT3 after IL-6 stimulation is inhibited. Treatment with β-cyclodexin in concentration of 4mM induces cell death even in the presence of IL-6 or IGF-I. In the same line of evidence it was shown that a phospholipids analogue, Perifosine has cytotoxic effects on myeloma cells in vitro and in vivo. In myeloma cells the activation of Akt is inhibited by Perifosine treatment (Hideshima et al., 2006).

As mentioned above as one of the objects of the present invention, with respect to MM therapy, the treatment should target MM cells specifically and it should kill IL-6-dependent and -independent MM cells and the treatment should be resistant to the above described pro- survival factors. So far in the cell culture system, peptidelA shows all of these characteristics.

Therefore, the invention has been completed by the inventors.

Detailed description of the invention

In a first aspect, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, wherein said variant comprises one or more insertions, substitutions and/or deletions as compared to the sequence of SEQ ID NO: 1, and wherein the biological activity of said variant is substantially equal to the activity of the peptide comprising the unmodified amino acid sequence of SEQ ID NO: 1. In particular variants of the peptide, for example deletions, insertions and/or substitutions in the sequence, which cause for so-called "silent" changes, are considered to be part of the invention.

Preferably are such amino acid substitutions the result of substitutions which substitute one amino acid with a similar amino acid with similar structural and/or chemical properties, i.e. conservative amino acid substitutions.

Amino acid substitutions can be performed on the basis of similarity in polarity, charges, solubility, hydrophobic, hydrophilic, and/or amphipathic (amphiphil) nature of the involved residues. Examples for hydrophobic amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar, neutral amino acids include glycine, serine, threonine, cysteine, thyrosine, asparagine and glutamine. Positively (basic) charged amino acids include arginine, lysine and histidine. And negatively charged amino acids include aspartic acid and glutamic acid.

"Insertions" or "deletions" usually are in the range of one amino acid. This in order to avoid a too high unpredictability as regards the resulting biological function of the peptide. The allowed modifications of the original sequence can be experimentally determined via methodically applied insertions, deletions or substitutions of amino acids in a polypeptide molecule using recombinant DNA methods. The resulting variants can be tested for their biological activity.

As mentioned above, the biological activity in a broader sense may be defined as the effect of the peptide to induce cell death in myeloma cells. In a narrower sense, it means the effect of inducing cell death in IL-6 dependent as well as independent myeloma cells.

In an embodiment, the peptide of the invention additionally comprises further sequences which provide a means for coupling to other molecules, which allow for a reduced degradation of the peptide in vivo or which allow for cell membrane permeation.

As a preferred example for sequences, which allow for a reduced degradation of the peptide in vivo, one can name D amino acid comprising sequences. Preferably, the three amino acid residues next to the N- and the C-terminus can take the form of D-isomeres. The D-isomeres do not occur in vivo, therefore, peptides build of D-isomeres are more stable inside the cells because they are not recognized and degraded by intracellular proteases and peptidases.

To avoid structural changes in the core sequence (SEQ ID NO: 1) of the peptide, these amino acid residues preferably are not changed.

Said further sequences preferably are sequences flanking the amino acid sequence of SEQ ED NO: 1, i.e. which are present at the N- and C-terminus of the core sequence of SEQ ED NO: 1.

In a further preferred embodiment, a lysine residue is provided at the N-terminus of the peptide in order to allow coupling to other molecules. The primary amine of the ε-amino- group of the lysine residue can serve as an acceptor for coupling fluorescence dyes or other markers to the peptides. A fluorescent dye generally may be defined as a dye that consists of molecules that selectively absorb light in the visible range or spectrum. The dye is fluorescent because upon absorbing light, it instantly emits light at a longer wavelength than the light absorbed. Examples of fluorescent dyes include fluorescein, tetramethylrhodamine and carboxy-x-rhodamine. However, also other known dyes may be used without any limitation. Further possibilities include coupling to succinimides or biotin etc.

Diagnostically relevant modifications of the peptide include a radioactive labelling of the peptide or a labelling with scintigraphic markers in order to allow an in vivo detection by means of imaging methods.

As further molecules, which are coupled to the peptide of the present invention (wherein coupling is not restricted to the above mentioned lysine residue), drugs might be named, which can be used for the targeted therapy of myelomas. As an example, the following can be used: thalidomide, Relimid^® (lenalidomide), Velcade^® (bortezomib), pamidronate, and Zometa® (zoledronic acid), or cytostatics as vincristine, doxorubicine or cyclophosphamide, among others. As a summary, those molecules may serve as an additional tool for use in therapy or in diagnosis/research in order to elucidate the molecular mechanism of action.

In a further embodiment, a myristoyl residue is added to the N terminus of the peptide in order to allow for membrane permeation of the peptide.

In a preferred embodiment, the peptide of the invention comprises the amino acid sequence of SEQ ID NO: 2 (TQPLLASAARP AALQLVD). This sequence contains the core sequence of SEQ ID NO: 1 and, additionally, two short sequences which are flanking this core sequence at the N- and C-terminus, respectively. These flanking sequences are derived from peptide 18AD, mentioned above, having the sequence of TQPLLDSEERPEDLQLVD.

In a more preferred embodiment, the peptide of the present invention comprises the amino acid sequence of SEQ ID NO: 3 (KTQPLLAS AARP AALQL VD). This sequence corresponds to SEQ ID NO: 2 and additionally contains the above mentioned lysine residue at its N- terminus.

In a most preferred embodiment, the peptide of the invention takes the form of: myr- ktqPLLASAARPAALQlvd, wherein capital letters denote L amino acids and small letter denote D amino acids. It is noted that this embodiment comprises all peptides, which comprise that sequence, but also a peptide, which consists of this sequence. In this embodiment, all above mentioned advantages are combined, i.e. the characteristics of treating MM, the possibility for coupling further molecules via the lysine residue, decreased degradation by proteases and peptidases and better membrane permeation.

In a second aspect, the invention provides a nucleic acid sequence coding for the peptide as described above.

The invention comprises also such variants which hybridize to the nucleic acids according to the invention at stringent or moderately stringent conditions.

Stringent hybridization and wash conditions are in general the reaction conditions for the formation of duplexes between oligonucleotides and the desired target molecules (perfect hybrids) or that only the desired target can be detected. Stringent washing conditions mean 0.2 x SSC (0.03 M NaCl, 0.003 M sodium citrate, pH 7)/0.1% SDS at 65⁰C. For shorter fragments, e.g. oligonucleotides up to 30 nucleotides, the hybridization temperature is below 65°C, for example at 50⁰C, preferably above 55°C, but below 65°C. Stringent hybridization temperatures are dependent on the size or length, respectively of the nucleic acid and their nucleic acid composition and will be experimentally determined by the skilled artisan. Moderate stringent hybridization temperatures are for example 42°C und washing conditions with 0.2 x SSC/0.1% SDS at 42°C.

The respective temperature conditions can vary dependent on the chosen experimental conditions and to be tested nucleic acid probe, and have to be adapted appropriately. The detection of the hybridization product can be done for example using X-Ray in the case of radioactive labeled probes or by fluorimetry in the case of fluorescent labeled probes.

In a third aspect, the invention comprises a pharmaceutical composition, comprising the peptide or nucleic acid sequence as defined above in combination with a pharmaceutically acceptable carrier and/or diluent. The pharmaceutical composition preferably takes the form of a parenteral composition.

The peptides of the present invention are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease (MM) can effectively be treated. Such a composition can (in addition to the peptide) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.

The term "pharmaceutically acceptable" carrier is defined as non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application.

Techniques for the formulation or preparation and application/medication of the peptides of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. An appropriate application can include parenteral application, including intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, or intraperitoneal injections. The intravenous injection is the preferred treatment of a patient.

A typical composition for an intravenous infusion can be produced such that it contains 250 ml sterile Ringer solution and a sufficient amount of the peptide of the invention. See also Remington's Pharmaceutical Science (15. edition, Mack Publishing Company, Easton, Ps., 1980).

In a further aspect, the peptide of the invention is used for the treatment of multiple myeloma in a mammal. Preferably, the mammal is a human patient.

The present invention will be further described with reference to the following figures and examples; however, it is to be understood that the present invention is not limited to such figures and examples.

In the figures, the following is shown:

Figure 1: Sequence of peptid OB and peptid IA. CAPITAL LETTERS, L-isomers, small caps D-isomers.

Figure 2: Growth curves. A.) Proliferation of INA-6 cells in the presence of 0 - lOOμM peptidelA or peptide OB after 36h. B.) Growth curves of the experiment shown in A., p<0,001.

Figure 3: Concentration dependent effects of peptidelA in different cell lines. A.) 0,5xl0⁵ INA-6, 7TDland MMlS cells were treated with 0 - 80μM peptidelA. Peptide induced cell death was monitored by FSC/SSC-Analysis 48h after peptide addition. Cells treated with DMSO were chosen as a reference value. The medium of INA-6 and 7TDl cells was supplemented with lng/ml and 125pg/ml IL-6, respectively. Data from one representative experiment are shown. B.) Proliferation of INA-6, MMlS and 7TDl cells was monitored for 48h in the presence of 0 - 80μM peptidelA. Growth rates were calculated. As in A., DMSO treated cells were use as the reference value. The mean value of three independent experiments is shown. (A. and B. each experiment was performed in triplicates). Figure 4: Growth curves at high cellular densities A.) INA-6 cells, B.) MMlS in the presence of 50μM PeptidelA .

Figure 5: Peptide iΛ-induced cell death. A.) Annexin-V-staining of INA-6 cells which were either grown in the presence or abscence of IL-6 for 48h; or treated for 48h with 50μM of peptides. B.)The percentage of Annexin-V-positive cells was monitored in INA-6 cells which were treated for 2h, 4h and 8h with 0,8%o DMSO or with 40μM or 80μM peptide. C.) Form the cells analyzed in B. an additional sample was treated for 16h with the indicated concentrations of the peptide. Samples from all time points were lysed, equal amounts of protein were loaded on an SDS-PAGE and analyzed for PARP cleavage and tubulin expression by western blot. The ratio of cleaved/ full-length PARP was calculated from densitometry data generated from the western blot shown above. INA-6 cells were treated for 3h with D.) 0,8%o DMSO and E.) 80μM peptide and were analyzed by light microscopy.

Figure 6: Signaling. A.) Global tyrosine-phosphorylation-profile and STAT3-phosphorylation in INA-6 cell-lysates. B.) STAT3, Akt and Erkl/2 phosphorylation in MMlS cell-lysates. For A. and B.: Prior to lysis cells were serum deprived for 16h, treated for 2h with 0,6%o DMSO (as control), 30 or 60μM of peptide and stimulated for the indicated timepoints with lng/ml IL-6. The expression of β-tubulin was analyzed as a loading control. C.) INA-6 cells were left untreated, 0 or treated for 2h either with 0,8%o DMSO or with 20, 40,60 or 80μM peptide and lysed. The phosphorylation of STAT3, Erkl/2 and p38 was analyzed.

Figure 7: Different cell lines. Cell lines derived from different types of human or murine Myeloma, Lymphoma or Leukemia were treated with 50μM of peptides or DMSO for 48h, respectively. A.) the percentage of living, Annexin V negative cells is shown, calculated as the percentage of the DMSO treated cells. Red: 0-9%, orange: 10-49%, light green: 50-89% and green: 90-100% living cells. Annexin-V-Assays as well as FSC/S SC- Assays served as basis for the data shown in A. B.) A representative Annexin- V-Assay.

Figure 8: Stages of multiple myeloma (from Kuehl & Bergsagel, 2002). Figure 9: Left side: Sequences of peptide IA and peptide sc-01. CAPITAL LETTERS, L- isomeres, small caps D-isomeres. Right side: Predicted 3D-structures of peptide IA and peptide sc-01 are based on secondary structure predictions done with the open source program Predict Protein. Visualization and calculation of the electrostatic potentials are analyzed using the program SwissPDBViewer. The myristoyl modification is not included. For the calculation L-isomers of the amino acids are taken into account.

Figure 10: INA-6 are treated for 48 h with 10 μM to 120 μM peptides. The part of living cells as percentage of control (DMSO) treated cell is shown. The experiments are done in triplicates, N=2-l l.

Figure 11: A.) INA-6 and MMl. S cells are treated with peptide IA or Trail alone or in combination with Z-VAD or IL-6. The activation of caspase-3 is analyzed by western blot using an antibody which recognizes the active, cleaved form of caspase-3. Cleaved PARP is detected with a specific antibody against the cleaved form of the enzyme. Beta-tubulin is used as the loading control. B.) Aliquots from cells of the above described experiment are taken after 3 h peptide IA or Trail treatment and the cellular viability is analyzed by FSC/SSC analysis.

Figure 12: Cells are incubated with a.) DMSO, b.) 80 μM peptide lAbio or c.) peptide sc- Olbio. Peptides are visualized with Avidin-Alexa-488 (green), nuclei with DAPI (blue) and lipid rafts with Cy3 (red). The lower part shows interference contrast pictures of the respective cells.

Figure 13: Cells are incubated with 80 μ M peptide lAbio, the peptide is visualized with Avidin-Alexa-488 (green), the nuclei are stained with DAPI (blue) and lipid rafts with Cy3 (red). The right side shows interference contrast pictures of the respective cells. A cut through the cell along the white line is show.

Figure 14: Cell lines derived from solid tumor and non-tumor tissue are treated with 60 μM peptide IA or 0.06% DMSO for 48 h. The uptake of PI at 4⁰C in the cells is measured by FACS. The percentage of Pi-positive cells is shown. N=3. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples:

Results

Cellular effects: Concentration dependent effects of peptidelA on cellular viability and proliferation of myeloma cells

The effects of peptide treatment were analyzed on the human, IL-6-dependent cell line INA-6 (Burger et al., 2001). In INA-6 cells treated with peptidelA a dose dependent decrease in proliferation can be observed. The control peptide, peptide OB did not show a dose dependent inhibitory effect on proliferation, figure 2A. While growth curves of INA-6 cells treated with 0-100μM peptide OB showed growth kinetics comparable to those of cells treated with DMSO as a control, the curves of INA-6 cells treated with concentrations of peptidelA above 40μM diverged from the curves of peptide OB or DMSO treated cells during the first 20 hours of the treatment, figure 2B.

Concentration dependent effects of peptidelA on cellular viability were assayed in human IL- 6-dependent INA-6 cells, IL-6-independent MMlS cells and murine IL-6-dependent 7TDl. Cells were treated for 48h with 0-80μM peptide or DMSO. Peptide effects were evaluated by FSC/SSC-analysis, figure 3A. In both human Myeloma cell lines, INA-6 and MMlS, the amount of living cells decreased with increasing concentration of peptide added. From this observation it can be concluded, that the effect of peptidelA on Myeloma cells is dose- dependent. The IC50 is 31 ±3 μM for INA-6 cells and 48±7μM for MMlS cells. The IC50s were calculated from three different experiments. The dose-dependent effect of the peptide on the viability of 7TDl cells can be detected with higher concentrations of peptide, only. Concentration dependent effects on cellular proliferation were analyzed. INA-6, MMlS and 7TDl cells were treated with 0-80μM of peptide IA the proliferation was monitored for 48h by counting the total number of cells. Growth rates for the different peptide concentrations were calculated from the obtained growth curves. To compare the effects of the different peptide concentrations on the different cell lines the growth rates of DMSO treated cells were set to 100%, respectively, figure 3B. INA-6, MMlS and 7TDl cells proliferate less in the presence of increasing concentrations of peptide IA. The IC50s are 40±12μM, for INA cells, 36±7μM for MMlS cells and 57±4μM for 7TDl cells. While the effects on cellular viability of 7TDl cells could only be detected at higher peptide concentrations, figure 3B shows that peptide IA inhibited proliferation of 7TDl cells in a concentration dependent way.

Influence of cellular density on the effects of peptidelA

Five to Six times higher seeding concentrations did not protect the cells against peptidelA induced cell death. INA-6 or MMlS cells were seeded with a density of ~2xlO⁵ cells/ml and proliferations was monitored in the presence of 50μM peptidelA, peptide OB or DMSO. The antiproliferative effect of peptidelA became visible in the growth curves within the first 24h of treatment, figure 4B.

Mechanism of peptidelA induced cell death

Annexin-V-binding to phosphatidyl serine shows the disruption of the plasma membrane asymmetry which is an early marker for Apoptosis. IL-6 depletion in INA-6 cells leads to the induction of Apoptosis. INA-6 cells were either supplemented with IL-6 alone or were supplemented with IL-6 and treated with 50μM peptidelA or peptide OB. As a control the cells were deprived from IL-6. Cells were stained with Annexin-V-PE for Phosphatidylserin in the outer leaflet of the membrane after 48h incubation time. 26,4% of INA-6 cells incubated in the presence of IL-6 alone or 30,1% of the cells kept with IL-6 and 50μM peptide OB were Annexin-V-positive. 85,5% of the IL-6 deprived INA-6 cells and 83,4% of the cells incubated with IL-6 and peptidelA were Annexin-V-positive. PeptidelA but not peptide OB induces exposure of phosphatidyl serin in the outer leaflet of the plasma membrane, figure 5A. Already 2h after peptide addition, the amount of total Annexin-V-postive cells increased tremendously from 26% to 68%. Two effects could be observed during that time: an increase of Annexin-V-single positive cells as well as an increase of Annexin-V- and 7AAD-double- positive cell. During the following hours the Annexin-V-single-positive population became double positive. With 40μM peptidelA only a transient increase of Annexin-V-positive cells was found, figure 4B and data not shown.

Besides the disruption of plasma membrane asymmetry, the activity of caspases is a marker for Apoptosis. PARP is one of caspases' -3 substrates which is cleaved when cells undergo Apoptosis. Figure 4C shows that the ratio of cleaved- to full-length-PARP increased more than 20-fold during 8h of treatment with 80μM of peptidelA. However, this effect is caused by an increase of cleaved-PARP mainly during the first 4h of the treatment, followed by a decrease in full-length protein after incubation time. Although equal amounts of protein were loaded, the amount of β-tubulin in the lysates decreased during the time of peptide treatment. The effect of peptidelA on the cells could also be visualized by light microscopy. INA-6 cells were treated for 3h with DMSO or 60μM peptidelA., figure 5D and 5E. Loss of membrane integrity, cellular shrinkage and clump formation was seen in the microscope.

INA-6 and MMl. S cells are treated with Trail (Tumor necrosis factor related apoptosis inducing ligand) or with peptide IA to induce cell death. The effects of peptide treatment on caspase-3 activation is analyzed. In addition it is assessed if the caspase inhibitor Z-VAD (Z- VAD-fmk) or the growth factor IL-6 can modulate peptide IA induced caspase activation. As a positive control, cells are treated with Trail. Trail induced caspase activation can be inhibited by pre-incubation of the cells with Z-VAD. The presence of an excess of IL-6 can not protect the cells against Trail-induced apoptosis. Trail-treated cells can be used as positive controls, Z-VAD and Trail treated cells can be used as negative controls to investigate the effects of peptide IA on caspase activation. In the experiment the activation of caspase-3 and the cleavage of PARP are analyzed.

To find out if caspase-3 is one of the mediators which participates in the mechanism of peptide lA-induced cell death, cells are treated for 1 h with 50 μM Z-VAD or DMSO as a control before they are incubated with 60 μ M peptide IA. In addition an excess of IL-6 (2 ng/ml) is added to the cells to find out whether IL-6 can inhibit peptide- induced caspase activation. Figure HA shows induction of caspase-3 activation in INA-6 and MMl. S. The active (cleaved) form of caspase-3 is characterized by a double band at 17kDA. In cells which are pre-treated with Z-VAD, the cleaved form of caspase-3 is hardly detectable. In INA-6 cells some active caspase-3 can be seen in control cells. An antibody directed against the cleaved form of PARP shows that PARP-cleavage can be induced by Trail or peptide IA treatment and that the caspase inhibitor Z-VAD can inhibit the cleavage induced by Trail or peptide IA in both cell lines tested.

Incubation of INA-6 cells with peptide IA leads to activation of caspase-3 and digestion of PARP by caspase-3. The peptide- induced activation of caspase-3 can be blocked by pre- treatment of the cells with Z-VAD. MMl. S cells show similar results. DMSO treated MMl. S cells show less basal caspase-3 activation and less PARP cleavage. The addition of IL-6 can neither prevent Trail- nor peptide lA-induced activation of caspase-3 and subsequent PARP cleavage. While the amount of cleaved PARP is comparable in Trail and peptide treated cells, the amount of active caspase-3 is higher in Trail treated cells. This could indicate that different proteases are activated by peptide IA. While INA-6 cells are already lysed after 3 h incubation with peptide IA or Trail, MMl. S. are lysed after 16 h. The control staining with an anti-beta-tubulin-antibody reveals that the treatment with Trail leads to the proteolytic digestion of beta-tubulin.

In parallel to the experiment shown in figure HB, the viability of peptide IA and Trail treated cells in the presence or absence of the caspase inhibitor Z-VAD is assessed. In Trail treated MMl. S cells 35% more dead cells can be found than in Z-VAD + Trail treated cells. In peptide IA treated cells 26% more dead cells are detected, compared to peptide IA + Z- VAD treated cells, figure HB.

Sequence specificity of peptide IA and hypothetical structure

Sequence specificity is confirmed and unspecific effects of the peptide IA are excluded by the use of a control peptide which has the same amino acid composition as peptide IA but an arbitrarily scrambled primary sequence. The physicochemical properties of both peptides are comparable. The two peptide sequences differ mainly in the distribution of charges within the peptides. This is visualized by possible hypothetical structures with the corresponding electrostatic potentials, figure 9.

Concentration dependent effects - peptide IA vs. control peptide sc-01

Concentration dependent effects of peptide IA compared to peptides sc-01 are assessed. Peptide concentrations between 10 μM to 120 μM are used. The cells are treated for 48 h with the peptides. The percentage of living cells is determined.

While peptide IA shows the expected concentration dependent effects on cellular viability of INA-6 cells, no such effect is visible with peptide sc-01. The IC50 of peptide IA in INA-6 cells is -31 μM which is comparable with the IC50 from previous experiments, figure 10.

Effects of peptidelA on IL-6 induced signaling

To exemplify the effects of peptide treatment on pro-survival and pro-proliferative stimuli, Myeloma cells were stimulated with the cytokine BL-6. INA-6 and MMlS cells were stimulated with IL-6 in the presence of different concentrations of peptide! A. The effects on downstream signaling molecules which are known to be essential for the anti-apoptotic and pro-proliferative effects of IL-6 were analyzed. Compared to INA-6 cells which were treated with DMSO, cells treated with 60μM peptidelA led to a reduced global tyrosin- phosphorylation profile, figure 6A.

hi the presence of 60μM the stimulation with IL-6 did not show the typical increase in phosphorylation of several IL-6 induced proteins. One key-player in the signal transduction from the stimulated IL-6 receptor to the nucleus is STAT3, phosphorylated at Tyrosine 705. IL-6 induced STAT3 activation was already reduced by a treatment with 30μM peptidelA, with 60μM of peptidelA STAT3 tyrosine-phosphorylation was nearly blocked completely in INA-6 cells, figure 6A. Similar effects of peptidelA on MMlS cells which were stimulated with IL-6 for 1 - 15min were found. In MMlS cells IL-6-induced Akt phosphorylation was impaired by the treatment with 60μM of peptidelA, while the effects on Erkl/2 were less pronounced, figure 6B. Basal phosphorylation of the MAPK-family members Erkl/2 and p38 was reduced in INA-6 cells which were kept in the presence of peptide IA concentrations higher than 40μM. Minor effects could be seen on STAT3 phosphorylation, figure 6C.

In conclusion: IL-6 induced phosphorylation of downstream signaling molecules as well as basal phosphorylation of signaling molecules is impaired in the two human multiple myeloma cell lines INA-6 and MMlS by peptide IA concentrations higher than 30^0μM.

Localization of peptide IA

To analyze the localization of peptide IA inside the cell, a biotin moiety is covalently linked to the epsilon-amino-group of the N-terminal lysine residue. A linker of six C-atoms separates the biotin from the peptide. Peptide IA and peptide sc-01 are biotinylated in that way. The biotinylated peptides are termed peptide lAbio and peptide sc-Olbio. Using fluorescence- labeled Avidin or Strepavidin the peptide can be visualized.

INA-6 cells are incubated for 1 h with peptide lAbio and peptide sc-Olbio. The localization of the peptides is visualized by confocal microscopy. Peptides are stained with Avidin- Alexa- 488 (green), nuclei are stained with DAPI (blue) and the plasma membrane is visualized by lipid raft staining (red) using Choleratoxin-B (CTX-B).

DMSO treated cells show a clear lipid raft staining. As expected the cells are only very faintly green, this background can be attributed to cellular biotin. In peptide lAbio treated cells co- localization between peptide and lipid rafts is only rarely detected. The peptide seems to be inside the cell. The cut through the cell shown in figure 13 underlines this finding. Peptide sc- Olbio seems to be predominantly localized at the plasma membrane, the lipid raft staining is hardly detectable in these cells. After 1 h of peptide IA treatment the peptide is not localized inside the nucleus.

PeptidelA induced cell death is Myeloma specific

The ability of peptide IA to kill neoplastic cells was analyzed in 18 different cell lines. The cell lines were derived from human mantle cell lymphoma (MCL), other lymphomas, acute myeloid leukemia (AML), T-cell acute lyphocytic leukemia (T-ALL) and Multiple Myeloma. In addition, the IL-3-dependent pro-B-cells BaF3, the EPO-dependent BaF-EH and the IL-6- depentent hybridoma cells 7TDl from murine origin were analyzed for their sensitiveness to the peptide.

Cells were treated for 48h with 50μM peptidelA, peptide OB or DMSO, respectively. The part of living cells was determined by FSC/SSC-analysis and Annexin-V-staining. As shown in figure 6 the human Myeloma cell lines INA-6 and MMlS were most sensitive to peptidelA treatment. Only 6,6% of INA-6 an 23,3% of MMlS cells were not killed by the peptide. Less pronounced effects were seen in, MM-6 and NCEB. It should be mentioned that medium requirements of the assayed cell lines were different.

hi addition the effects of peptide IA on cellular viability of cells, which are derived from solid tumors and on cells which are not derived from tumor tissue are analyzed.

The mammary carcinoma cell line, MCF-7, the glioblastoma/asterocytoma cell line, U373, the renal cell carcinoma cell line, A34, and the neuroblastoma cell line, Kelly are used as tumor derived adherent growing cell lines. Human embryonic kidney cells, HEK 293, and mouse embryonic fibroblasts, MEF, are used additionally. These latter two cell lines are used with respect to potential side effects and general toxicity of the peptide. The cells are stained with PI and analyzed by FACS. PI can only enter cells with a destroyed plasma membrane. The peptide has no effects on the viability of the cell lines tested.

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Claims

Peptides for treating multiple myeloma

1. A peptide comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, wherein said variant comprises one or more insertions, substitutions and/or deletions as compared to the sequence of SEQ ID NO: 1, and wherein the biological activity of said variant is substantially equal to the activity of the peptide comprising the unmodified amino acid sequence of SEQ ED NO: 1.

2. The peptide of claim 1, which comprises further sequences which provide a means for coupling to other molecules, which allow for a reduced degradation of the peptide in vivo or which allow for cell membrane permeation.

3. The peptide of claim 1 or claim 2, wherein the further sequences comprise D amino acids.

4. The peptide of one or more of the preceding claims, wherein the further sequences are sequences flanking the amino acid sequence of SEQ ID NO: 1.

5. The peptide of one or more of the preceding claims, wherein a lysine residue is provided at the N terminus of the peptide in order to allow coupling to other molecules.

6. The peptide of claim 5, wherein the other molecules are selected from marker molecules, preferably fluorescence dyes.

7. The peptide of one or more of the preceding claims, wherein a myristoyl residue is added to the N terminus of the peptide in order to allow for membrane permeation.

8. The peptide of one or more of the preceding claims, which comprises the amino acid sequence of SEQ ID NO: 2.

9. The peptide of one or more of the preceding claims, which comprises the amino acid sequence of SEQ ID NO: 3.

10. The peptide of one or more of the preceding claims, which comprises or consists of myr-ktqPLLASAARPAALQlvd, wherein capital letters denote L amino acids and small letter denote D amino acids.

11. A nucleic acid sequence coding for the peptide of one or more of claims 1-10.

12. A pharmaceutical composition, comprising the peptide of one or more of claims 1-10, in combination with a pharmaceutically acceptable carrier and/or diluent and preferably takes the form of a vaccine.

13. Use of the peptide of one or more of claims 1-10 for the manufacture of a medicament for the treatment of multiple myeloma in a mammal.

14. The use of claim 13, wherein the mammal is a human patient.