WO2008059066A1

WO2008059066A1 - New chemokine derivatives of ccl14, synthesis and use as drugs for the treatment of allergic diseases

Info

Publication number: WO2008059066A1
Application number: PCT/EP2007/062507
Authority: WO
Inventors: Sylvia Escher; Jörn ELSNER; Wolf-Georg Forssmann; Shipra Gupta
Original assignee: Haemopep Pharma Gmbh
Priority date: 2006-11-17
Filing date: 2007-11-19
Publication date: 2008-05-22

Abstract

The present invention relates to a polypeptide capable of binding to chemokine receptors and has the sequence R1-N1N2XB-R2, wherein N1 and N2 represent equal or different neutral amino acids selected from the group consisting of A, N, C, Q, G, I, L, M, F, P, S, T, W, Y or V, X represents any amino acid and B represents a basic amino acid, selected from the group consisting of R, H or K; wherein R1 and R2 comprise at least 15 amino acids.

Description

New chemokine derivatives of CCL14, synthesis and use as drugs for the treatment of allergic diseases

The present invention relates to polypeptides capable of binding to chemokine receptors. Further, a method of inhibiting the extravasation of cells and the use of the polypeptides is disclosed.

Chemokines are a family of small cytokines, or proteins secreted by cells. Proteins are classified as chemokines according to shared structural characteristics such as small size (they are all approximately 8-10 kDa in size), and the presence of four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. The name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines. However, these proteins have historically been known under several other names including the SIS family of cytokines, SIG family of cytokines, SCY family of cytokines, Platelet factor-4 superfamily or intercrines. Some chemokines are considered pro-inflammatory and can be induced during an immune response to promote cells of the immune system to a site of inflammation, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development. Chemokines are found in all vertebrates, some viruses and some bacteria, but none have been described for other invertebrates. These proteins exert their biological effects by interacting with G-protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells.

The major role of chemokines is to guide the migration of cells. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine. Some chemokines control cells of the immune system during processes of immune surveillance, such as directing lymphocyte to the lymph node so they can screen for invasion of pathogens by interacting with antigen-presenting cell residing in these tissues. These are known as homeostatic chemokines and are produced and secreted without any need to stimulate their source cell(s). Some chemokines have roles in development; they promote angiogenesis or guide cells to tissues that provide specific signals critical for cellular maturation. Other chemokines are inflammatory and are released from a wide variety of cells in response to bacteria infection, virus and agents that cause physical damage such as silica or the uric acid that occur in gout. Their release is often stimulated by pro- inflammatory cytokines such as interleukin 1. Inflammatory chemokines function mainly as chemoattractants for leukocyte, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate an immune response or promote wound healing. They are released by many different cell types and serve to guide cells of both innate immune system and adaptive immune system (extravasation).

The extravasation of leukocytes into the inflamed tissue is one of the most important functions of the natural immunity. The extravasation can be basically divided into four steps (cf. Janeway et al., Die angeborene Immunitat, 2002, 37- 96) :

In the first step, the leukocytes depart from the central bloodstream by vasodilatation which is induced by several inflammation mediators. Thus, the leukocytes obtain a short contact with endothelium. P- and E-selectins are expressed on the surface of endothelial cells depending on the degree of activation by various cytokines and other inflammation mediators (Leukotriene B4, histamine, LPS etc.). These bind reversibly to adequate carbohydrate ligands of the leukocytes. Since these bindings cannot resist the shear forces of the bloodstream, the cells move slowly along the wall of the vessels, that is why this phenomenon is called "rolling".

In the second step, the leukocytes bind with their specific leukocyte integrins, LFA-I and Mac-1, to adhesion molecules of the endothelial cells, ICAMs (intracellular adhesion molecules). The additional binding to chemokines like CXCL8 causes a change in the conformity of the leukocyte integrins, wherein the adhesion is increased and the leukocyte binds to an endothelial cell.

In the third step, diapedese, the inflamed cells are migrating through the endothelium by means of proteolytic enzymes after binding to PECAMs (platelet endothelium adhesion molecules) and destruction of the basement membrane. In the fourth step, the leukocytes are migrating along a concentration gradient through tissue to the inflammation site.

Members of the chemokine family are categorized into four groups depending on the spacing of their first two cysteine residues.

The CC chemokines (or β-chemokines) have two adjacent cysteines near their amino terminus. There have been at least 27 distinct members of this subgroup reported for mammals, called CC chemokine ligands (CCL)-I to -28; CCLlO is the same as CCL9. Chemokines of this subfamily usually contain four cysteines (C4- CC chemokines), but a small number of CC chemokines possess six cysteines (C6-CC chemokines). C6-CC chemokines include CCLl, CCL15, CCL21, CCL23 and CCL28. CC chemokines induce the migration of monocytes and other cell types such as natural killer cells and dendritic cells. An example of a CC chemokine is CCL2 (MCP-I or CCL2) which induces monocytes to leave the bloodstream and enter the surrounding tissue to become tissue macrophages. CC chemokines induce cellular migration by binding to and activating CC chemokine receptors, ten of which have been discovered to date and called CCRl-10. These receptors are expressed on the surface of different cell types allowing their specific attraction by the chemokines. The N-terminal domain (amino acids 1-7) and N-loop (amino acids 10-17) is responsible for receptor activation and binding respectively. Mutations of the N-terminus causes still highly specific binding to chemokine receptors, but fails to activate them, whereby the receptors are being blocked (Hartley et al., Proc. Natl. Acad. Sci. USA, 2004, 101, 16460-16465).

Chemokine receptors associate with G-proteins to transmit cell signals following ligand binding. Activation of G proteins, by chemokine receptors, causes the subsequent activation of Phospholipase C (PLC). PLC cleaves Phosphatidylinositol (4,5)-bisphosphate (PIP2) into inositol triphosphate and diacylglycerol (DAG) that trigger intracellular signaling events; DAG activates Proteinkinase C (PKC), and IP3 triggers the release of calcium from intracellular stores. These events promote many signaling cascades such as the MAP kinase pathway that generate responses like chemotaxis, degranulation, release of superoxide anions and changes in the avidity of cell adhesion molecules called integrins within the cell harbouring the chemokine receptor.

CCL14 is a chemokine consisting of 74 amino acids and is constitutively expressed in various tissues. It binds and activates the chemokine receptor CCRl with a decreased affinity than CCL3.

When examining various N-terminal truncated CCL14 derivatives, it was found that NONANOYL-CCL14 is the most effective derivative. NONANOYL-CCL14 induces chemotaxis of monocytes, eosinophilic granulocytes and T-cells. Based on these studies, NONANOYL-CCL14 has been synthesized. The exchange of the N-terminal glycine by nonanoylic acid causes an agonistic activity at human CCR3.

It is significantly more effective than the natural occurring CCL14(9-74) and comparable to the agonist of CCR3, CCLIl, with regard to CCR3-internalization, the release of intracellular calcium and radical oxygen-species as well as chemotaxis. Moreover, it has been shown in in vivo experiments in mice that a decreased accumulation of eosinophilic granulocytes in lung tissue is caused after an i. v. application (Forssmann et al., J. Immunol., 2004, 173, 3456-3466).

In further studies it has been shown that NONANOYL-CCL14 internalizes and desensitizes human as well as murine chemokine receptors CCRl and CCR5. Murine CCR3 cannot be internalized by incubation with NONANOYL-CCL14 in contrast to human CCR3 (Gupta et al., J. Allergy Clin., Immunol., 2006, 117(2) : 250(A)).

Unfortunately, all known chemokines and their derivatives cause local inflammatory reactions and can only be administered via the i. v. route.

Surprisingly, the polypeptide according to claim 1 is able to modulate leukocyte functions by binding to chemokine receptors with significantly reduced induction of an inflammatory reaction at the injection site.

This polypeptide is capable of binding to chemokine receptors and has the sequence R¹N¹N²XBR², wherein N¹ and N² represent equal or different neutral amino acids selected from the group consisting of A, N, C, Q, G, I, L, M, F, P, S, T, W, Y or V, X represents any amino acid and B represents a basic amino acid, selected from the group consisting of R, H or K; wherein R¹ comprises at least 10 or at least 15 amino acids and R² comprises at least 10 or at least 15 amino acids.

In yet another embodiment R¹ comprises 40 and R² comprises 21 amino acids.

In one embodiment of the present invention, the polypeptide is capable of binding to the chemokine receptors CCRl, CCR2, CCR3 and CCR5.

In a further embodiment the polypeptide internalizes the chemokine receptors CCRl, CCR2, CCR3 and CCR5.

In yet another embodiment, R¹ comprises the sequence PYH PSECCFTYTTYKI PRQRI M DYYETNSQCSKPGIVFIT and/or R² comprises the sequence SVCTNPSDKWVQDYIKDMKEN.

In an additional embodiment, the polypeptide according to the invention has the sequence (SEQ-ID No. 1) NONANOYL-

PYHPSECCFTYTTYKIPRQRIMDYYETNSQCSKPGIVFITGAGHSVCTNPSDKWVQDYIKD

MKEN or is a derivative thereof.

Herein it is also called NONANOYL-CCL14(G50, A51).

The blocking of chemokine receptors by NONANOYL-CCL14(G50, A51) results in a significantly reduced extravasation of leukocytes from the blood vessel into the inflamed tissue in vivo. NONANOYL-CCL14(G50, A51) may therefore play an important role in the treatment of allergic diseases like asthma.

The term "derivative" as used herein refers to a chemically modified polypeptide derived from the polypeptides according to claim 1. For instance, a "derivative" may be generated by processes such as altered phosphorylation, or glycosylation, or acetylation, or lipidation, or esterification, or amidation, or halogenation, or hydrohalogenation. The polypeptide according to the invention was synthesized. The synthesis can be carried out using Fmoc-chemistry (9-Fluorenmethyloxycarbonyl) by stepwise solid phase chemistry (solid phase peptide synthesis). The two disulfide-bridges can be introduced using orthogonal side chain-protecting groups.

Other synthesized derivatives are the following :

SEQ-ID No: 2 PASVPTASCFTYTTYKI

SEQ-ID No: 3

FTYTTYKIPRQRIMDYYETNS

SEQ-ID No: 4

NONANOYL-

PASVPTTCCFTATTYKIPRQRIMDYYETNSQCSKPGIVFITKRGHSVCTNPSDKWVQDYIK DMKEN

SEQ-ID No: 5

NONANOYL-

PASVPTTCCFTYTTAKIPRQRIMDYYETNSQCSKPGIVFITKRGHSVCTNPSDKWVQDYIK DMKEN

SEQ-ID No: 6

NONANOYL- PASVPTTCCFNLANRKIPRQRIMDYYETNSQCSKPGIVFITKRGHSVCTNPSDKWVQDYIK DMKEN

SEQ-ID No: 7 NONANOYL-

PASVPTTCCFTYTTYKIPRQRIMDYYETNSQCSKPGIVFITKRGHSVCTNPSDKWVQDYIKD

MKEN

SEQ-ID No: 8

KIPRQRIMDYYETNSQCSKPGIVFITGAGHSVCTNPSDKWVQDYIKDMKEN

In one specific embodiment of the invention, the basic amino acids Lysine 50 and Arginine 51 of N0NAN0YL-CCL14 were substituted by the aliphatic amino acids Glycine and Alanine respectively. The resulting chemokine was termed NONANOYL-CCL14(G50, A51), which showed no induction of an inflammatory reaction at injection sites. Moreover, after injection, it was available in the circulation.

One embodiment of the peptide according to the present invention is a method of inhibiting the extravasation of cells from intravascular compartment into tissue (or through any membrane limiting any body compartment from another), characterized in that chemokine receptors of these cells are blocked by an agonist, which binds to said chemokine receptors and is therefore not able to build up an haptotactic concentration gradient on said membrane.

In particular, the cells of this method are blood-circulating cells and the intravascular compartment is the bloodstream.

Further, said cells can be leukocytes.

A method is one further embodiment of the invention, wherein an agonist used to inhibit the cell migration is a chemoattractant binding to a corresponding receptor.

Moreover, said agonist can be the polypeptide according to claim 1.

It is one further embodiment of the present invention to manufacture a medicament comprising the polypeptide according to the invention. In one embodiment, said polypeptide is used for the manufacturing of a medicament for the treatment of allergic airway inflammation and/or of the Acquired Immune Deficiency Syndrome (AIDS) and/or of diseases associated with migration of blood cells from blood into tissue.

In a further embodiment, the medicament according to the invention comprises the polypeptide in concentration of 0.01 ng polypeptide per kg bodyweight to 100 mg polypeptide per kg bodyweight. The medicament can be delivered as ampule, capsule, elixir, emulsion, fluid, grain, drop, injection, solution, powder, suspension, syrup or tablet.

Based on the mechanisms of action, NONANOYL-CCL14(G50, A51) can be given via different systemic routes (i. v., s. c, intrapulm. etc) without causing a local inflammatory reaction unlike compound NONANOYL-CCL14 which can be given only via the i. v. route.

Description of the figures:

Fig. la-d show the activation of and the mobilization of intracellular calcium in

B300.19- CCRl ⁺ , -CCR3+ and CCR5+ and PBMCs (Peripheral Blood Mononuclear Cells). In comparison to NONANOYL-CCL14, the potency of NONANOYL- CCL14(G50,A51) is 2-fold lower on CCRl and 4-fold lower on CCR3, but does not change on CCR5. Moreover, potency of NONANOYL-CCL14(G50, A51) is in the nanomolar range on PBMCs.

Fig. 2a-c disclose the internalization of human CCRl, CCR3 and CCR5 of 300.19- CCRl⁺ , -CCR3+ and -CCR5+ cell lines, monocytes and eosinophils (37 ⁰C, 30min). NONANOYL-CCL14(G50, A51) efficiently internalizes both CCRl and CCR5 but loses the ability to downmodulate CCR3 as effectively as NONANOYL- CCL14. Fig. 3a-b show the chemotaxis of monocytes and eosinophils in a neuropore chamber (37 ⁰C, 60 min). The chemotaxis of monocytes and eosinophils is reduced by NONANOYL-CCL14(G50, A51).

Fig. 4 discloses the internalization of NONANOYL-CCL14(G50, A51) on murine peripheral blood leukocytes. It internalizes surface CCR5 on different lymphocyte subsets in mouse blood, but it does not internalize mCCR3 on blood eosinophils alike NONANOYL-CCL14.

Fig. 5 shows that systemic treatment with NONANOYL-CCL14 and NONANOYL-

CCL14(G50,A51) down-modulates CCR5 from the surface of lymphocytes (DX5+) in vivo in BALB/c mouse. The rapid down - modulation is followed by a gradual in vivo CCR5 re-expression over 3 hours.

Example 1 :

Chemokines

The chemokine derivatives CCL14(9-74), NONANOYL-CCL14 and NONANOYL- CCL14(G50, A51) were synthesized according to the method by Escher et al. (Escher et al., J. Pept. Res., 2004, 63, 36-47). CCL14(9-74) and NONANOYL- CCL14 were employed in the experiments as references.

Example 2:

Cell isolation and cell cultures

The cell isolation and cultivation as well as the preparation of monocytes and granulocytes was conducted according to the method disclosed in Rieder (Rieder S., dissertation, Tierarztliche Hochschule Hannover, 2006). Stably transfected murine pre-B 300.19 cells were obtained from Bernhard Moser (Theodor Kocher Institute, University of Berne, Switzerland). Example 3:

Chemotaxis

The chemotaxis assays were conducted in 48-well Boyden micro-chemotaxis- chambers. Peptide dilutions of 0.1 to 1000 nM were employed. The cells were diluted in RPMI 1640 medium and 0.5% BSA to yield a concentration of 10⁶ cells per ml. The adherence of this concentration is of great importance for the alignment of the cells as a monolayer.

Per well 30 μl dissolved and diluted chemokines were pipetted in the bottom of the chamber. Upon the filled bottom a Polyvinyl-Pyrrolidon-free filter (25 x 80 mm with 5 μm pores (Nucleophore, Neurobe)) was positioned with the splendent side faced upwards. A rubber mat was sited on that, the upper part of the chamber was put on carefully, the bolts were tightened and the upper wells were filled with 50 μl cell suspension each (equivalent to 5 x 10⁴ cells). The chamber was incubated for one hour at 37°C and 5% CO₂. Subsequently, the upper side of the filter was washed and wiped off with PBS for removal of the not migrated cells. The filter was air-dried and after that dyed with DIFFQuick dye (Dade Diagnostika) according to the manufacturer's manual (fixation : 30 sec. in methanol, dye: with eosin and haematoxyline, 2 min each). The dried filters were fixed to microscope slides with entellan (toluene and alkyl alcylate). Subsequently, five areas were counted under the microscope with a 1000 x magnification to determine the number of the migrating cells. The migration indices were determined by taking into account the negative control.

Example 4:

Receptor internalization

The internalization was determined according to prior studies (Eisner et al., J. Biol. Chem., 275, 7787-7794). A FACScan was used for the determination of the fluorescence intensity. The receptor internalization was conducted with CCR3⁺- and CCR5⁺-transfected 300.19 cell lines as well as natural cell lines (eosinophiles and monocytes). In case of the cell lines, the suspension obtained from culture bottles was diluted in PBS + 0.5% BSA and once in this and twice in buffered RPMI 1640 medium washed. The natural cells were isolated as described above.

After that, the cells were counted and adjusted to a concentration of 5 x 10⁵ cells/150 μl RPMI and HEPES. 150 μl of the cell suspension were each conveyed to 15 ml tubes and 1.5 μl stimulus was added instantaneously. To enable receptor internalization, an incubation for 30 min followed at 37 ⁰C and 5% CO₂. After 30 min, the reaction was immediately interrupted on ice and the tubes were filled with cold PBS and 0.5% BSA to 5 ml. The suspension was centrifuged at 300 x g and 4°C for 5 min. All following steps were performed on ice. For determination of the indirect fluorescence intensity, the cell suspension was transferred to cooled FACS tubes. Subsequently, the isotype controls and the primary antibodies anti-CCRl, -CCR3 or -CCR5 mAk respectively were added and the suspension was incubated for 30 min on ice. Then, 1-2 ml PBS with 0,5% BSA was added again, the cells were isolated by centrifugation (200 x g, 5 min, 4° C), decanted, resuspended in 0.2 ml BSA and put on ice until measurement. The fluorescence intensity was determined by FACScan and the relative fluorescence intensity in % was calculated by the following equation : (median channel fluorescence (stimulus) - median channel fluorescence (isotype control) * 100/ (median channel fluorescence (buffer) - median fluorescence (isotype control).

Example 5:

Intracellular calcium mobilization

The increase of the intracellular calcium concentration was measured in a FLIPR (Fluorometric Imaging Plate Reader)-system with a laser intensity of 0.4 W. This measurement system consists of four components: personal computer, argon laser, 96 well pipetting system and a cooled CCD-camera. Fluo-4-AM was used as calcium indicator. This substance is able to permeate the cellular membrane in this form during the incubation time of 30 min at 37 ⁰C. Fluo-4-AM is cleaved intracellular^ by cytoplasmatic esterases, whereby a free acid is produced. This acid is able to bind to calcium ions, but it is not capable of crossing cellular membranes. This newly formed free acid is activated by the argon laser. Thus, the increase of intracellular calcium can be detected reliably. The argon laser emits radiation on the side of the plated cells to minimize the scattered light.

Corresponding to receptor internalization, CCR3+- and CCR5+-transfectants were employed. These were washed twice with buffer, then diluted in 1 ml buffer and counted. Depending on the number of cells, 2-3 μl Fluo-4 (acetoxymethyl) were added and this cell suspension was incubated for 30 min at 37°C. In advance, an anti foaming agent, Pluronic F127, was added to the dye Fluo-4 in a ratio of 1 : 1. The cells were washed again twice after the incubation and were resuspended in a sufficient amount of buffer to arrive at a final concentration of 4 x 10⁵ cells in 150 μl. After that, the cell suspension was plated on a black 96 well plate (Costar, 4 x 10⁵ cells/well) and for 1 min at 200 x g centrifuged. Each 60 μl of the chemokines were presented onto an application plate in four concentrations. The plate was then placed in a FLIPR-system, the measurement was started and the changes of the fluorescence was measured after automatic chemokine application. 50 μl of the presented 60 μl were pipetted to the cell suspension during the measurement.

The measurement was conducted in three consequential steps. The first ten pictures were taken every six seconds, the next ten in 10 seconds and finally, twenty were taken every six seconds (the stimuli were added after the 12^th picture).

Example 6:

Biological activity

The biological activity of NONANOYL-CCL14(G50, A51) was investigated using either CCRl, CCR3 and hCCR5-stably transfected cell lines or in freshly isolated eosinophils and monocytes. Receptor internalization was measured by flow cytometry, mobilization of intracellular calcium was determined using the FLIPR method and in vitro chemotaxis was carried out by microchemotaxis assays. The GAG-deficient mutant of CCL14, NONANOYL-CCL14(G50, A51), showed only a moderate reduction of its activity on CCRl which was more evident in the measurement of intracellular calcium fluxes than in receptor internalization. While the activity of NONANOYL-CCL14(G50, A51) on CCR3 was lost completely, all investigated biological functions mediated via CCR5 were unaltered.

Claims

1. A polypeptide capable of binding to chemokine receptors that has the sequence R¹N¹N²XBR², wherein N¹ and N² represent equal or different neutral amino acids selected from the group consisting of A, N, C, Q, G, I, L, M, F, P, S, T, W, Y or V, X represents any amino acid and B represents a basic amino acid, selected from the group consisting of R, H or K; wherein R¹ and R² comprise at least 15 amino acids.

2. The polypeptide according to claim 1, wherein R¹ comprises 40 amino acids and R² comprises 21 amino acids.

3. The polypeptide according to claim 1 and/or 2, characterized in that it is capable of binding to the chemokine receptors CCRl, CCR2, CCR3 and CCR5

4. The polypeptide according to at least one of claims 1-3, characterized in that it internalizes the chemokine receptors CCRl, CCR2, CCR3 and CCR5.

5. The polypeptide according to at least one of claims 1-4, characterized in that R¹ comprises the sequence

PYH PSECCFTYTTYKI PRQRI M DYYETNSQCSKPGIVFIT and/or R² comprises the sequence SVCTNPSDKWVQDYIKDMKEN.

6. The polypeptide according to at least one of claims 1-5, characterized in that it has the sequence (SEQ-ID No. 1) Nonanoyl- PYHPSECCFTYTTYKIPRQRIMDYYETNSQCSKPGIVFITGAGHSVCTNPSDKWVQ DYIKDMKEN or is a derivative thereof.

7. A method of inhibiting the extravasation of cells from intravascular compartment into tissue (or through any membrane limiting any body compartment from another), characterized in that chemokine receptors of these cells are blocked by an agonist, which binds to said chemokine receptors and is therefore not able to build up an haptotactic concentration gradient on said membrane.

8. The method according to claim 7, characterized in that the cells are blood circulating cells and the intravascular compartment is the blood stream.

9. The method according to claim 7, characterized in that said cells are leukocytes.

10. The method according to at least one of claims 7-9, characterized in that said agonist is a polypeptide according to claim 1.

11. A medicament comprising the polypeptide according to at least one of claims 1-6.

12. A medicament according to claim 11, which can be administered via different systemic routes, in particular intravenously, subcutaneously or intrapulmonary, without causing a local inflammatory reaction.

13. Use of a polypeptide according to at least one of claims 1-6 for the manufacturing of a medicament for the treatment of allergic airway inflammation.

14. Use of a polypeptide according to at least one of claims 1-6 for the manufacturing of a medicament for the treatment of the Acquired Immune

Deficiency Syndrome (AIDS).

15. Use of a polypeptide according to at least one of claims 1-6 for the manufacturing of a medicament for the treatment of diseases associated with migration of blood cells from blood into tissue.

16. The medicament according to claim 11 and/or 12, characterized in that it comprises the polypeptide according to at least one of claims 1-6 in concentration of 0.01 ng polypeptide per kg bodyweight to 100 mg polypeptide per kg bodyweight.