EP2729487A2 - Novel sdf-1-based glycosaminoglycan antagonist and methods of using same - Google Patents

Abstract

The present invention relates to novel SDF-1 mutant polypeptides with increased GAG binding affinity and reduced GPCR activity compared to wild type SDF-1 protein wherein said SDF-1 polypeptide is modified in a structure-conserving way by replacing glutamine at position 60 by a basic or electron donating amino acid and by replacing at least one additional amino acid at position 29, 39, 49, 67 and/or 68 according to the numbering of SEQ ID NO 1 by at least one basic and/or electron donating amino acid and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 polypeptide is modified by addition, deletion and/or replacement of at least one amino acid and methods for producing these mutants and to their use for preparing medicaments for the treatment of cancer.

Description

Novel SDF-1 -based glycosaminoglycan antagonist and methods of using same

The present invention relates to novel mutants of human stromal cell-derived factor-1 i.e. SDF-Ι or SDF-Ιβ or SDF-lgamma or any variants thereof comprising an amino acid replacement at position 59, exhibiting (i) increased glycosaminoglycan (GAG) binding affinity and (ii) inhibited or down-regulated GPCR activity compared to wild type SDF-1, and to their use for therapeutic treatment of cancer.

Chemokines are small (8-11 kD) soluble chemoattractant cytokines. They consist of four families, depending on the relative position of their cysteine residues. The a chemokines have one non-conservative amino acid, separating the first two cysteine residues (CXC-motif) , whereas the β-chemokines have their first two cysteines adjacent to each other (CC-motif) . Fractalkine is the only representative of a chemokine family with three non-conservative amino acids, separating the first two cysteine residues (CX3C-motif) and lymphotactin the only member of a family with a total of only 2 cysteines (C-motif) , compared to 4 cysteines in the other three families (Baggiolini et al . , Adv. Immunol, . 55, 97 179 (1994)). Stromal cell-derived factor-la (SDF-1, CXCL12) belongs to the a-chemokines although it is no typical

representative of this class. The sequence identity to other human CXC- and CC-chemokines is only 27% and 21%, respectively. Despite this equally distinct relationship to both large

chemokine families, mature SDF-1 shows very high similarities between different species. With approx. 99% sequence identity between human and murine and 100% amino acid sequence identity between human and feline SDF-1, it is the most conserved cytokine known so far (Shirozu et al . , Genomics, 28, 495-500 (1995)).

Orthologues have actually been found in agnatha, the most primitive vertebrate branch. Another characteristic of SDF-1 is its gene SCYB12, which is located on chromosome 10, whereas all other CXC chemokines and most CC chemokines are clustered on chromosome 4 and chromosome 17.

SCYB12 contains four exons . SDF-Ι , the most common splice isoform, is derived from exon 1-3, while SDF-Ιβ also contains additional sequence from exon 4 resulting in four additional amino acids at the C-terminus. The three dimensional structure of SDF-1 shows a core of three anti -parallel β- strands and a C- terminal -helix (Crump et al . , EMBO J., 16, 6996-7007 (1997)).

In contrast to the tertiary properties, the quaternary structure has always been controversial. A conflict of statements was evident between a monomeric form in NMR-Studies, a dimeric form in crystallographic measurements or a supposed monomer-dimer equilibrium according to sedimentation velocity ultracentri- fugation and dynamic light scattering. It was found out later, that SDF-Ι in fact exists in a monomer-dimer equilibrium and that dimer association is strongly dependent on both the presence of stabilizing counterions and the solution pH . It was further found that glycosaminoglycans (GAGs) promote SDF-Ι dimer

formation (Veldkamp et al . , Protein Sci . , 14, 1071-1081 (2005)).

GAGs are linear polysaccharides comprising repeating disaccharide units that vary in linkage, composition, as well as N- and 0- sulfation patterns (Capila & Linhardt, Angew. Chem. Int. Ed.

Engl., 41, 391-412 (2002)). Most of them are covalently attached to a protein core thereby forming the so called proteoglycans. Examples of GAGs are heparan sulfate (HS) , heparin, keratan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronic acid. Because of the presence of sulfate and carboxylate groups GAGs are highly negatively charged. This drives the binding of the positively charged chemokines which bind soluble glycosamino- glycans as well as GAGs attached on cell surfaces or extracellular matrix. Heparan sulfate, similar to heparin but less continuously charged, is considered fundamental to the biological activity of chemokines. It is produced by most cell types and several core proteins carry HS-chains, or instance, the syndecans and the glypicans at the cell surface and agrin and perlecan in the extracellular matrix. It is worth noting that SDF-Ι binds to syndecan-4, but not to syndecan-1, syndecan-2, CD44 or beta- glycan. It was shown that Lys24, Lys27 and to a lesser degree Arg41 and Lys43 are essential for the binding of SDF-1 to HS (Sadir et al . J. Biol. Chem. , 276, 8288-8296 (2001)). These basic amino acids are located in a small opening between the β- strands of the SDF-Ι dimer (Lortat-Jacob et al . , Proc . Natl. Acad. Sci . , 99, 1229-1234 (2002) ) .

GAGs lead to the retention of chemokines on cell surfaces and therefore increase the local concentrations of chemokines, in spite of shear forces caused by blood flow. If not for this interaction chemokines would be washed away and could not

establish a chemotactic gradient for leukocytes or stem cells to follow. GAG binding is therefore a pre-requisite for in vivo activity. Chemokines with mutations in GAG binding sites are not able to recruit cells in vivo. Structural changes of chemokines leading for example to oligomerization are driven by binding to GAGs. Binding to HS or heparin can protect SDF-1 also from proteolytic cleavage by dipeptidyl peptidase IV (DPP IV) /CD26 in vitro .

The traditionally well known receptor for SDF-1 is CXC chemokine receptor 4 (CXCR4) (Y.R. Zou, A.H. Kottmann, M. Kuroda, I.

Taniuchi, D.R. Littman, Nature, 393, 595-599 (1998)) for which, reciprocally, SDF-1 is the only known endogenous ligand (M.K. Schwarz, T.N. Wells, Curr . Opin. Chem. Biol., 3, 407-417 (1999)). CXCR4 belongs to the pertussis toxin sensitive G-protein coupled receptors (GPCR) , characterized by seven transmembrane domains. A two-step binding model for SDF-1 and CXCR4 was proposed (M.K. Schwarz, T.N. Wells, Curr. Opin. Chem. Biol., 3, 407-417 (1999)). Only recently, a second receptor for SDF-1 has been identified, namely CXCR7 (Burns JM et al . , J Exp Med. 2006 ; 203 (9) : 2201-13. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.

CXCR7 is typically membrane-associated and is expressed by many tumour cell lines as well as by activated endothelial cells and by fetal liver cells. Contrary to most other chemokine receptors, ligand activation of CXCR7 does not cause Ca2+ mobilization or cell migration. It does provide CXCR7 -positive cells with a growth and survival advantage and increased adhesion properties.

Molecular dynamics simulation confirmed the two-step binding mechanism of SDF-1 to CXCR4 which is not characterised in this detail for CXCR7. SDF-1 is a highly potent chemokine and attracts lymphocytes and monocytes, as well as hematopoietic stem and progenitor cells (K. Hattori et al . , Blood, 97, 3354-3360

(2001) ) . WO2005/054285 describes the modification of GAG binding proteins to increase the GAG binding affinity.

WO2008/074047 describes chemokine chaperone fusion proteins comprising at least one chemokine and a peptide derived from a chaperone .

In view of the role of SDF-1 in chemokine attraction and lymphocyte and monocyte attraction and the constant need for new drugs for cancer treatment it is an object of the invention to provide novel proteins for modifying chemokine receptor interaction.

The invention is based on engineering a higher GAG binding affinity into human SDF-1, which can be SDF-1 alpha or SDF-Ιβ or SDF-1 gamma or any variants thereof, and simultaneously knocking out or down regulating the GPCR activity specifically the CXCR4 and CXCR7 activity of the chemokine . This is accomplished by selectively introducing basic amino acids into the GAG binding site of SDF-1 in order to increase GAG binding affinity, and by modifying the N-terminus of the chemokine either by truncation of 8 amino acids or by replacing/deleting the first two amino acids, or by fusion to other proteins in order to inhibit CXCR4 and CXCR7 binding. Said SDF-1 mutants exhibit a minimum of five- fold, preferably a minimum of 7.5-fold, preferably a minimum of tenfold improved Kd for standard GAGs (heparin or heparan sulfate) and they show a reduced chemotactic activity in a standard Boyden chamber using CXCR4- or CXCR7 -positive cells. Biophysical and cell biological proof for these characteristics is provided.

Subject matter of the present invention is to inhibit CXCR4- positive cell migration and CXCR7 activation, more specifically stem cells and metastatic cells, by antagonising the GAG interaction with an SDF-1 -based mutant in the context of tumour growth, spreading and neovascularisation processes. Anti- tumorigenic characteristics are shown in a murine xenotransplant model using human breast cancer cells.

The invention therefore provides new SDF-1 mutant polypeptides with increased GAG binding affinity and reduced GPCR activity compared to wild type SDF-1 protein wherein an SDF-1 protein is modified in a structure-conserving way by replacing glutamine at position 60 by a basic or electron donating amino acid and optionally by replacing at least one additional amino acid at position 29, 39, 49, 67 and/or 68 according to the numbering of SEQ ID NO 1 by at least one basic acid and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 protein is modified by addition, deletion and/or replacement of at least one amino acid. The present invention also provides isolated polynucleotides encoding the polypeptides and expression vectors containing the isolated polynucleotides. Accordingly, the invention provides methods and compositions for treatment and prevention of diseases or conditions associated with the polypeptides of the invention, as well as the polynucleotides encoding the polypeptides.

According to the invention the modification in a structure conserving way results in a maximum deviation of the modified structure from wild type SDF-1 structure of less than 30%,

preferably less than 20% as measured by far-UV-CD spectroscopy.

In a preferred embodiment of the invention, the basic amino acids are selected from the group consisting of Arg, Lys, His.

Specifically, the SDF-1 mutant polypeptide has eight amino acids of the N-terminal region of said SDF-1 polypeptide deleted. More specifically, the first two amino acids of the N-terminal region of said SDF-1 polypeptide are replaced and/or deleted, preferably the amino acids are replaced by amino acids selected from the group consisting of Lysine, Arginine, Proline or Glycine.

Even more specifically, the inventive SDF-1 mutant polypeptide comprises an N-terminal Met.

In a specific embodiment, the amino acids at positions 29 and 39 of the inventive polypeptide are replaced by basic amino acids.

In an alternative embodiment, an SDF-1 mutant polypeptide is provided, wherein the amino acid sequence is described by the general formula:

(M) _n (XI ) _m (X2 ) _pVSLSYRCPCRFFESHVARANVKHLKI (X3 ) NTPNCALQI (X4 ) ARLKNNNRQ (X5) CIDPKLKWIQ (X6) YLEKAL (X7) (X8) (GRREEKVGKKEKIGKKKRQKK RKAAQKRKN) ₀ according to the numbering of SEQ ID NO 1

wherein XI is a K or R residue,

wherein X2 is a P or G residue,

wherein X3 is selected from L, R, K or H residues, preferably it is K,

wherein X4 is selected from V, R, K, H residues, preferably it is K,

wherein X5 is selected from V or A preferably it is A,

wherein X6 is selected from R, K or H, preferably it is K

wherein X7 is selected from N, R, K or H, preferably it is K wherein X8 is selected from R, K or H, preferably it is R

and wherein n and/or m and/or p and/or o can be either 0 or 1 and wherein position X6 is modified and optionally at least one of positions XI, X2 , X3 , X4 , X7 or X8 are modified (SEQ ID No. 6) and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 protein is modified by addition, deletion and/or replacement of at least one amino acid.

In a specific embodiment, the SDF-1 mutant polypeptide is of the structure Met-SDF-1 Δ8 E60K, Met-SDF-1 Δ8 E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K, Met-SDF-1 Δ8 L29K V39K E60K N67K, Met-SDF-1 Δ8 L29K V39K V49A E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K N67K K68R, Met-SDF-1 Δ8 L29K V39K E60K K68R.

Additionally, the invention also provides an isolated polynucleic acid molecule, coding for the inventive protein.

Also a vector is provided, comprising the isolated polynucleic acid molecule and a recombinant non-human cell, transfected with the vector.

The present invention also provides a pharmaceutical composition, comprising the inventive mutant SDF-1 polypeptide or a polynucleotide coding for said polypeptide, or a vector comprising the isolated polynucleic acid molecule and a pharmaceutically acceptable carrier.

Said SDF-1 mutant polypeptide or the polynucleotide or the vector can be used for preparing a medicament, specifically for use in the treatment of cancer.

Figures :

Figure 1: Sequence of SDF-1 mutants, mutations with respect to the wild type chemokine are underlined

Figure 2 : Isothermal titration calorimetry data- SDF1 mutants titrated with Enoxaparin, a) PA1009, b) PA1010, c) PA1011, d) PA1016

Figure 3: Chemotactic activity of wtSDF-1 and mutant proteins as observed in the modified Boyden Chamber.

Figure 4: Nucleotide sequences of expression plasmid pJExpress411 having the coding sequences underlined of mutants

a) PA1009, b) PA1010, c) PA1011, d) PA1016

It has been shown that increased GAG binding affinity can be introduced by increasing the relative amount of basic amino acids in the GAG binding region (WO 05/054285, incorporated in total herein by reference) leading to a modified polypeptide that acts as strong competitor with natural GAG binding polypeptides. This was particularly shown for interleukin- 8.

The location of GAG binding regions and their modification by introducing basic amino acids was disclosed in WO2009/053064. The present mutant SDF-1 polypeptide comprising the specific

modification of amino acid position 60 shows a highly increased GAG binding affinity compared to the SDF-1 mutants according to WO2009/053064 and a comparable chemotactic activity. Thus, surprisingly, the modification of the specific amino acid at position 60 leads to a four-fold increased glycosaminoglycan binding affinity, specifically towards enoxaparin. A modification of amino acid position 67 alone, i.e. without modifying amino acid 60, did not result in such increase of GAG binding affinity compared to the mutants of WO2009/053064.

Although WOOl/85196 discloses CXCR4 antagonists and their

possible use for treatment of hematopoietic cells, there is no indication to improve GAG binding affinity of an SDF-1 molecule. WOOl/85196 discloses SDF-1 derived peptides wherein part thereof can be covalently joined by a linker sequence to a C-terminal fragment of SDF-1 including those domains of SDF-1 binding to GAGs which thereby should increase the antagonistic effect of the peptides. In this publication nothing is said about increasing the GAG binding affinity of SDF-1 by any means. Like with the other examples of GAG binding proteins stated in this

publication, reference is made by this means to the general principle of protein-GAG binding and not to the specific

technology of introducing higher GAG-binding affinity into SDF-1.

The present invention now provides a SDF-1 mutant polypeptide with increased GAG binding affinity and reduced GPCR activity compared to wild type SDF-1 polypeptide wherein a GAG binding site or the vicinity of said GAG binding site SDF-1 polypeptide is modified in a structure-conserving way by replacing glutamine at position 60 by a basic amino acid and by replacing at least one additional amino acid at position 29, 39, 49, 67 and/or 68 according to the numbering of SEQ ID NO 1 by at least one basic amino acid and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 polypeptide is modified by addition, deletion and/or replacement of at least one amino acid. Said SDF-mutant polypeptides may be derived from SDF-la or SDF-Ιβ or SDF-lgamma or any variants thereof.

Specifically, the SDF-1 mutant polypeptide further comprises an amino acid replacement at position 67, preferably said amino acid is replaced by Lysine.

According to an alternative embodiment, the SDF-1 mutant polypeptide further comprises an amino acid replacement at position 68, preferably said amino acid is replaced by Arginine .

The term "variant of SDF-1" means any fragment or derivative or variant of an SDF-1 polypeptide comprising the amino acid

modifications according to the invention and still having the chemokine's remaining properties.

The terms "polypeptide", "peptide", and "protein", can be used interchangeably herein and may refer to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics , and polypeptides having modified, cyclic, or bicyclic peptide backbones. The term also includes single chain proteins as well as multimers. The term also includes conjugated proteins, fusion proteins, including, but not limited to, glutathione S-transferase (GST) fusion proteins with heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, fusion proteins with or without N-terminal methionine residues, pegylated proteins, and immunologically tagged or his-tagged proteins. The term also includes peptide aptamers . The term "vicinity" as defined according to the invention

comprises amino acid residues which are located within the conformational neighbourhood of the GAG binding site but not positioned directly in the GAG binding sites. Conformational neighbourhood can be defined as either amino acid residues which are located adjacent to GAG binding amino acid residues with respect to the amino acid sequence of a polypeptide, or amino acids which are located adjacent to GAG binding amino acid residues as a consequence of the three dimensional structure (or fold) of the protein.

To be able to exert their biological function, proteins fold into one, or more, specific spatial conformations, called domains, which is driven by a number of non-covalent interactions such as hydrogen bonding, ionic interactions, Van der Waals' forces and hydrophobic packing. Three dimensional structures can be determined by known methods like X-ray crystallography or NMR

spectroscopy .

Identification of native GAG binding sites can be accomplished, for example, by mutagenesis experiments. GAG binding sites of proteins are characterized by basic residues located mainly at the surface of the proteins. To test whether these regions define a GAG binding site, these basic amino acid residues can be mutagenized, for example by replacement by Alanine residue (s), and decrease of heparin binding affinity can be measured. This can be performed by any affinity measurement techniques as known in the art .

Rationally designed mutagenesis by insertion or substitution of basic or electron-donating amino acids can be performed to introduce foreign amino acids in the vicinity of the native GAG binding sites which can result in an increased size of the GAG binding site and in an increase of GAG binding affinity.

The GAG binding site or the vicinity of said site can also be determined by using a method as described in WO2010/122176 , comprising :

(a) providing a complex comprising the polypeptide and the GAG ligand molecule, for example heparan sulfate (HS) , heparin, keratin sulfate, chondroitin sulfate, dermatan sulfate and hyaluronic acid etc. bound to said polypeptide;

(b) contacting said complex with a cleavage reagent like a protease, e.g. trypsin, capable of cleaving the polypeptide, wherein said GAG ligand molecule blocks polypeptide cleavage in region of the polypeptide where the GAG ligand molecule is bound and whereby said polypeptide is cleaved in regions that are not blocked by said bound GAG ligand molecule; and

(c) separating and detecting the cleaved peptides, wherein the absence of cleavage events in a region of the polypeptide indicates that said GAG ligand molecule is bound to that region. Detection can be for example by LC-MS, nanoHPLC-MS/MS or Mass Spectrometric Methods .

A protocol for introducing or improving a GAG binding site is fo example partially described in WO05/054285 and can be as follows

- Identify a region of the protein which is involved in GAG binding

- Design a new GAG binding site by introducing (replacement or insertion) basic or electron donating amino acids, preferably Arg, Lys, His, residues at any position or by deleting amino acids which interfere with GAG binding

- Check the conformational stability of the resulting mutant protein in silico

- Clone the wild-type polypeptide cDNA (alternatively: purchase the cDNA)

- Use this as template for PCR-assisted mutagenesis to introduce the above mentioned changes into the amino acid sequence

- Subclone the mutant gene into a suitable expression system (prokaryotic or eukaryotic dependent upon biologically required post-translational modifications)

- Expression, purification and characterization of the mutant polypeptide in vitro

Criterion for an increased GAG binding affinity: K_d ^GAG (mutant) < lOuM.

- Check for structural conservation by far-UV CD spectroscopy or 1-D NMR spectroscopy.

A deviation of the modified structure as measured by far-UV CD spectroscopy from wild type structure of less than 30%,

preferably less than 20% is defined as structure conserving modification according to the invention.

Basic amino acids can be selected from the group consisting of Arg, Lys or His.

If the native amino acids replaced by said basic amino acids are basic amino acids, the substituting amino acids preferably are more basic amino acids or introduce more or less structural flexibility into the native protein structure.

Structural flexibility according to the invention is defined by the degree of accommodating to an induced fit as a consequence of GAG ligand binding.

According to a specific embodiment of the invention the native amino acids replaced by basic and/or electron donating amino acids are non-basic or less basic amino acids.

According to the present invention the SDF-1 mutant polypeptide can comprise a modification in a structure conserving way wherein the deviation of the modified structure as measured by far-UV CD spectroscopy from wild type SDF-1 structure is less than 30%, preferably less than 20%, preferably less than 10%.

According to an alternative embodiment, the structure conserving modification is not located within the N-terminus of the SDF-1 polypeptide .

The invention covers a SDF-1 mutant polypeptide with increased GAG binding affinity and reduced GPCR activity wherein the a GAG binding region of the SDF-1 polypeptide modified in a structure- conserving way by replacing glutamine at position 60 by a basic amino acid and by replacing at least one additional amino acid at position 29, 39, 49, 67 and/or 68 according to the numbering of SEQ ID NO 1 by at least one basic amino acid and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 polypeptide is modified by

addition, deletion and/or replacement of at least one amino acid.

According to the embodiment of the invention a SDF-1 mutant polypeptide with increased GAG binding affinity and reduced GPCR activity compared to wild type SDF-1 protein is provided wherein said SDF-1 polypeptide is modified in a structure-conserving way by replacing glutamine at positions 29, 39 and 60 by a basic amino acid and by optionally replacing at least one further amino acid at position 49, 67 and/or 68 according to the numbering of SEQ ID NO 1 by at least one basic amino acid and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 protein is modified by addition, deletion and/or replacement of at least one amino acid.

Specifically, the invention covers a SDF-1 mutant polypeptide wherein the glutamine at position 60 is substituted by a basic amino acid and amino acids at position 29 and/or 39 according to the numbering of SEQ ID NO 1 are substituted by at least one basic amino acid and/or wherein at least eight amino acids are deleted at the N-terminus and/or wherein a Methionine is

introduced at the N-terminus of said polypeptide.

As an alternative, the invention covers a SDF-1 mutant polypeptide wherein glutamine at position 60 is substituted by a basic amino acid and amino acids at position 29 and/or 39 and/or 67 according to the numbering of SEQ ID NO 1 are substituted by at least one basic amino acid and/or wherein at least eight amino acids are deleted at the N-terminus and/or wherein a Methionine is introduced at the N-terminus of said polypeptide.

As a further alternative, the invention covers a SDF-1 mutant polypeptide wherein glutamine at position 60 is substituted by a basic amino acid and wherein amino acids at position 29 and/or 39 and/or 67 and/or 68 according to the numbering of SEQ ID NO 1 are replaced by at least one basic amino acid and/or wherein eight amino acids are deleted at the N-terminus and/or wherein a

Methionine is introduced at the N-terminus of said polypeptide.

As a further alternative, the invention covers a SDF-1 mutant polypeptide wherein the glutamine at position 60 is substituted by a basic amino acid and amino acid at position 29 and/or 39 and/or 68 according to the numbering of SEQ ID NO 1 are substituted by at least one basic amino acid and/or wherein at least eight amino acids are deleted at the N-terminus and/or wherein a Methionine is introduced at the N-terminus of said polypeptide.

According to a specific embodiment, the N-terminal amino acids can be selected from the group consisting of Lysine, Arginine, Proline or Glycine.

According to an alternative embodiment of the invention the SDF-1 mutant polypeptide can contain an N-terminal Met.

Alternatively, the amino acid sequence of the inventive SDF-1 mutant polypeptide can be described by the general formula:

(M) _n (XI ) _m (X2 ) _pVSLSYRCPCRFFESHVARANVKHLKI (X3 ) TPNCALQI (X4 ) ARLKNNNRQ (X5) CIDPKLKWIQ (X6) YLEKAL (X7) (X8) (GRREEKVGKKEKIGKKKRQKK

RKAAQKRKN) ₀

wherein XI is a K or R residue,

wherein X2 is a P or G residue,

wherein X3 is selected from L, R, K or H residues, preferably it is K,

wherein X4 is selected from V, R, K, H residues, preferably it is K,

wherein X5 is selected from V or A preferably it is A,

wherein X6 is selected from R, K or H, preferably it is K

wherein X7 is selected from N, R, K or H, preferably it is K wherein X8 is selected from R, K or H, preferably it is R

and wherein n and/or m and/or p and/or o can be either 0 or 1 and and optionally at least one of positions XI, X2 , X3 , X4 , X5, X7 or X8 are modified (SEQ ID No. 6) and optionally wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 protein is modified by addition, deletion and/or replacement of at least one amino acid.

As a further alternative, the amino acid sequence of the

inventive SDF-1 mutant polypeptide can be described by the general formula:

(M) _n (XI ) _m (X2 ) pVSLSYRCPCRFFESHVARANVKHLKI (X3 ) NTPNCALQI (X4 ) ARLKNNNRQ VCIDPKLKWIQ (X5 ) YLEKALNK (GRREEKVGKKEKIGKKKRQKK RKAAQKRKN) ₀

wherein XI is a K or R residue,

wherein X2 is a P or G residue,

wherein X3 is selected from R, K or H residues, preferably it is R or K,

wherein X4 is selected from R, K, H residues, preferably it is R or K,

wherein X5 is selected from R, K or H, preferably it is K

and wherein n and/or m and/or p and/or o can be either 0 or 1 and optionally at least one of positions XI, X2 , X3 , X4 are modified (SEQ ID No. 15) and optionally wherein eight amino acids of the N-terminal region of the wild type SDF-1 protein are deleted.

As a further alternative, the amino acid sequence of the

inventive SDF-1 mutant polypeptide can be described by the general formula:

(M) _nKPVSLSYRCPCRFFESHVARANVKHLKI (X3 ) NTPNCALQI (X4 ) ARLKNNNRQVCIDPKL KWIQ (X5) YLEKALNK (GRREEKVGKKEKIGKKKRQKK RKAAQKRKN) ₀

wherein X3 is selected from R, K or H residues, preferably it is R or K,

wherein X4 is selected from R, K, H residues, preferably it is R or K,

wherein X5 is selected from R, K or H, preferably it is K,

and wherein n and/or o can be either 0 or 1 and optionally at least one of positions X3 and X4 are modified (SEQ ID No. 17) and optionally wherein eight amino acids of the N-terminal region of the wild type SDF-1 protein are deleted.

As a further alternative, the amino acid sequence of the invent - tive SDF-1 mutant polypeptide can be described by the general formula :

(M) _n (XI ) m (X2 ) pVSLSYRCPCRFFESHVARANVKHLKI (X3 ) NTPNCALQI (X4 ) ARLKNNNRQ VCIDPKLKWIQ (X5) YLEKAL (X6) (X7) (GRREEKVGKKEKIGKKKRQKK RKAAQKRKN) ₀ wherein XI is a K or R residue,

wherein X2 is a P or G residue,

wherein X3 is selected from L, R, K or H residues, preferably it is R or K, wherein X4 is selected from V, R, K, H residues, preferably it is R or K,

wherein X5 is selected from R, K or H, preferably it is K, wherein X6 is selected from N, R, K or H, preferably it is K, wherein X7 is selected from R, K or H, preferably it is R, and wherein n and/or m and/or p and/or o can be either 0 or 1 and optionally at least one of positions XI, X2 , X3 , X4 , X6 or X7 are modified (SEQ ID No. 16) and optionally wherein eight amino acids of the N-terminal region of the wild type SDF-1 protein are deleted .

As a further alternative, the amino acid sequence of the invent- tive SDF-1 mutant polypeptide can be described by the general formula :

(M) _nKPVSLSYRCPCRFFESHVARANVKHLKI (X3 ) NTPNCALQI (X4 ) ARLKNNNRQVCIDPKL KWIQ (X5) YLEKAL (X6) (X7) (GRREEKVGKKEKIGKKKRQKK RKAAQKRKN) ₀

wherein X3 is selected from L, R, K or H residues, preferably it is R or K,

wherein X4 is selected from V, R, K, H residues, preferably it is R or K,

wherein X5 is selected from R, K or H, preferably it is K, wherein X6 is selected from N, R, K or H, preferably it is K, wherein X7 is selected from R, K or H, preferably it is R, and wherein n and/or o can be either 0 or 1 and optionally at least one of positions X3 , X4 , X6 or X7 are modified (SEQ ID No. 18) and optionally wherein eight amino acids of the N-terminal region of the wild type SDF-1 protein are deleted.

According to a specific embodiment, the present invention also provides a SDF-1 mutant polypeptide of the structure Met-SDF-1 Δ8 E60K, Met-SDF-1 Δ8 L29K V39K E60K, Met-SDF-1 Δ8 E60K N67K, Met- SDF-1 Δ8 L29K V39K E60K N67K, Met-SDF-1 Δ8 L29K V39K V49A E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K N67K K68R, Met-SDF-1 Δ8 L29K V39K E60K K68R.

As an alternative, without limiting the embodiment of the invention, the SDF-1 mutant polypeptides provide the structure of Met-SDF-1 Δ8 E60R, Met-SDF- 1 Δ8 E60H, Met-SDF-1 Δ8 L29R V39R E60K, Met-SDF-1 Δ8 L29K V39R E60K, Met-SDF-1 Δ8 L29R V39K E60K, Met-SDF-1 Δ8 L29R V39R E60H, Met-SDF-1 Δ8 L29K V39K E60H, Met- SDF-1 Δ8 E60R N67K, Met-SDF-1 Δ8 E60R N67R, Met-SDF-1 Δ8 E60K N67R, Met-SDF-1 Δ8 L29R V39R E60R N67R, Met-SDF-1 Δ8 L29R V39R V49A E60R N67R, Met-SDF-1 Δ8 L29R V39R E60R N67R K68K, Met-SDF-1 Δ8 L29K V39K E60K K68R, and any mutant SDF-1 polypeptides wherein the amino acids at said positions comprise a replacement of R, K or H.

The term "Δ" in combination with a number is meant to have the amino acids deleted.

A further aspect of the present invention is an isolated poly- nucleic acid molecule which codes for the inventive polypeptide as described above. The polynucleic acid may be DNA or RNA.

Thereby the modifications which lead to the inventive SDF-1 mutant polypeptide are carried out on DNA or RNA level. This inventive isolated polynucleic acid molecule is suitable for diagnostic methods as well as gene therapy and the production of inventive SDF-1 mutant polypeptide on a large scale.

Specifically said polynucleic acid molecules are DNA molecules which may comprise one of the following sequences:

ATGTGCCCATG CCGCTTCTTC GAATCCCACG TCGCTCGCGC TAACGTTAAG CATCTGAAGA TCAAAAACAC CCCGAATTGT GCGCTGCAGA TCAAAGCGCG TCTGAAGAAC AACAATCGTC AGGTGTGTAT TGACCCGAAG CTGAAATGGA TCCAAAAGTA TCTGGAGAAA GCATTGAACA AG (PA1009, SEQ ID No . 7) ATGTGCCCATG CCGCTTCTTC GAATCCCACG TCGCTCGCGC TAACGTTAAG CATCTGAAGA TCAAAAACAC CCCGAATTGT GCGCTGCAGA TCAAAGCGCG TCTGAAGAAC AACAATCGTC AGGTGTGTAT TGACCCGAAG CTGAAATGGA TCCAGAAGTA CCTGGAGAAA GCATTGAAAA AG (PA1010, SEQ ID No . 8)

ATGTGCCCATG CCGCTTCTTC GAATCCCACG TCGCTCGCGC TAACGTTAAG CATCTGAAGA TCAAAAACAC CCCGAATTGT GCGCTGCAGA TCAAAGCGCG TCTGAAGAAC AATAACCGTC AGGTGTGTAT TGACCCGAAG CTGAAATGGA TCCAAAAGTA TCTGGAGAAA GCATTGAAAC GC (PA1011, SEQ ID No . 9)

ATGTGCCCATG CCGCTTCTTC GAATCCCACG TCGCTCGCGC TAACGTTAAG CATCTGAAAA TCAAAAACAC CCCGAATTGT GCGCTGCAGA TCAAAGCACG TCTGAAAAAC AATAATCGTC AAGTGTGTAT TGACCCGAAG CTGAAGTGGA TTCAGAAGTA CTTGGAGAAA GCGCTGAACC GT (PA1016, SEQ ID No. 10)

According to alternative embodiments, sequences that are at least 95% identical, preferably at least 98%, more preferred at least 99%, more preferred at least 99.5% identical to said sequences are also covered by the invention.

Still preferred, the isolated polynucleic acid molecule

hybridises to the above defined inventive polynucleic acid molecule under stringent conditions. Depending on the hybridisation conditions complementary duplexes form between the two DNA or RNA molecules, either by perfectly matching or also comprising mismatched bases (see Sambrook et al . , Molecular Cloning: A

laboratory manual, 2^nd ed., Cold Spring Harbor, N.Y. 1989). Probes longer than about 50 nucleotides may accomplish up to 25 to 30% mismatched bases. Smaller probes will accomplish fewer mismatches . The tendency of a target and probe to form duplexes containing mismatched base pairs is controlled by the stringency of the hybridisation conditions which itself is a function of factors, such as the concentrations of salt or formamide in the hybridisation buffer, the temperature of the hybridisation and the post-hybridisation wash conditions. By applying well known principles that occur in the formation of hybrid duplexes conditions having the desired stringency can be achieved by one skilled in the art by selecting from among a variety of

hybridisation buffers, temperatures and wash conditions. Thus, conditions can be selected that permit the detection of either perfectly matching or partially matching hybrid duplexes. The melting temperature (Tm) of a duplex is useful for selecting appropriate hybridisation conditions. Stringent hybridisation conditions for polynucleotide molecules over 200 nucleotides in length typically involve hybridising at a temperature 15-25°C below the melting temperature of the expected duplex. For oligonucleotide probes over 30 nucleotides which form less stable duplexes than longer probes, stringent hybridisation usually is achieved by hybridising at 5 to 10 °C below the Tm. The Tm of a nucleic acid duplex can be calculated using a formula based on the percent G+C contained in the nucleic acids and that takes chain lengths into account, such as the formula

Tm = 81.5-16.6 (log [NA⁺] ) + 0.41 (% G+C) - (600/N), where N = chain length .

A further aspect relates to a vector comprising an isolated DNA molecule according to the present invention as defined above. The vector comprises all regulatory elements necessary for efficient transfection as well as efficient expression of polypeptides. Such vectors are well known in the art and any suitable vector can be selected for this purpose.

A further aspect of the present invention relates to a recombinant cell which is transfected with an inventive vector as described above. Transfection of cells and cultivation of recombinant cells can be performed as well known in the art. Such a recombinant cell as well as any therefrom descendant cell comprises the vector. Thereby a cell line is provided which expresses the SDF-Ι mutant polypeptide either continuously or upon activation depending on the vector.

It also provides a method of producing a polypeptide by providing the nucleic acid as described above and expressing the nucleic acid molecule in a cell free expression system to produce the polypeptide. The cell free expression system can be chosen from a wheat germ lysate expression system, a rabbit reticulocyte expression system, and an E. coli lysate expression system.

A further aspect of the invention relates to a pharmaceutical composition comprising a SDF-Ι mutant polypeptide, a polynucleic acid or a vector according to the present invention as defined above and a pharmaceutically acceptable carrier. Of course, the pharmaceutical composition may further comprise additional substances which are usually present in pharmaceutical compositions, such as salts, buffers, emulgators, colouring agents, etc.

Specifically the pharmaceutical composition comprises at least one of the mutant polypeptides of the structure Met-SDF-1 Δ8 E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K N67K, Met-SDF-1 Δ8 L29K V39K V49A E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K N67K K68R, Met-SDF-1 Δ8 L29K V39K E60K K68R.

The modified SDFl protein of the present invention may be used as a medicament .

A further aspect of the present invention relates to the use of the SDF-1 polypeptide, a polynucleic acid or a vector according to the present invention as defined above in a method for inhibiting or suppressing the biological activity of the respective wild-type polypeptide. As mentioned above, the SDF-1 mutant polypeptide of the invention will act as a specific antagonist whereby the side effects which occur with known recombinant polypeptides are inhibited with the inventive SDF-1 mutant polypeptide. By increasing the GAG binding affinity the modified SDF- 1 polypeptide will act as specific antagonist and will compete with the wild-type GAG binding polypeptide for GAG binding.

In this case this will be particularly the biological activity involved in cancer development .

Specifically, the modified SDF-1 polypeptide may be used in the treatment of B-cell deficiency, platelet deficiency, stimulating lymphocyte growth or proliferation, diabetes, promoting angio- genesis, modulating immune response, suppression of

inflammation/autoimmune diseases, treatment of pulmonary diseases such IPF (idiopathic pulmonary fibrosis) , treating rheumatoid arthritis, osteoarthritis or psoriasis.

Therefore, a further use of the SDF-1 polypeptide, a polynucleic acid or a vector according to the present invention as defined above is in a method for producing a medicament for the treatment of cancer. In particular, it will act as antagonist without side effects and will particularly be suitable for the treatment of cancer disease by at least partially inhibiting tumour growth and spreading processed. Therefore, a further aspect of the present invention is also a method for the treatment of cancer diseases, wherein the SDF-1 mutant polypeptide according to the invention, the isolated polynucleic acid molecule or vector according to the present invention or a pharmaceutical preparation according to the invention is administered to a patient.

"Cancer" is any abnormal cell or tissue growth, for example, a tumor, whether malignant, pre-malignant , or non-malignant. It is characterized by uncontrolled proliferation of cells that may or may not invade the surrounding tissue and, hence, may or may not metastasize to new body sites. Cancer encompasses carcinomas, which are cancers of epithelial cells; carcinomas include

squamous cell carcinomas, adenocarcinomas, melanomas, and

hepatomas. Cancer also encompasses sarcomas, which are tumors of mesenchymal origin; sarcomas include osteogenic sarcomas, leukemias, and lymphomas. Cancers may involve one or more

neoplastic cell type.

"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating

material, formulation auxiliary, or excipient of any conventional type. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

"Medicament" herein refers to a composition that usually contains an active ingredient, a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic or prophylactic purposes.

The present invention is further illustrated by the following examples, however, without being restricted thereto.

Exam les :

Example 1 : Increase in GAG binding affinity

The engineered increased GAG binding affinity of the SDF-la mutants with respect to wtSDF-la was determined by isothermal titration calorimetry (ITC, see Figure 2) . ITC measurements were conducted on a VP- ITC Microcalorimeter (MicroCal) . Both the sample (SDF-1 variants; 15 μΜ) and the titrant (GAGs; 72 μΜ to 360 μΜ) were transferred to test tubes (borosilicate glass, 73500650; Chase Scientific) and degassed for 2 x 5 minutes at 20°C in the Thermo Vac (MicroCal) . The cell was loaded with the sample and the auto-pipette was loaded with the titrant. The cell temperature was set to 25 °C, reference power to 11.8 uCal/sec, stirring speed to 270 rpm, the feedback mode was set to 'high' , equilibration set to 'Fast Equil', first injection volume was 2 μΐ , followed by 8 μΐ injections every 250 seconds with a total of up to 38 injections and a filter period of 2 seconds. Origin 8.0 (OriginLab) was used for data evaluation and Kd calculation. A clear improvement in heparin (Enoxaparin) affinity with respect to wtSDF-la was observed for most of the mutants.

Example 2 : Knock-down of GPCR activity

SDF-la wild type and mutant directed cell migration was investigated using a 48-well Boyden chamber system (Neuroprobe) equipped with 5μπι PVP-coated polycarbonate membranes. SDF-1 variants were diluted in HBSS +/+ buffer to 10/100/1000/10000 nM concentrations and 28.5 μΐ were pipetted into each bottom well (including HBSS +/+ for background and SDF-lwt for positive reference) . A polycarbonate membrane (25x80 mm, 5 μπι pore size, PFB5; Neuroprobe) was carefully put on top and after assembling of the chamber 50 μΐ containing 2 x 105 Jurkat cells in HBSS +/+ were pipetted into the upper wells. After incubation at 37 °C, 5% C0₂ for 2 hours the chamber was disassembled. Non-migrated cells from the upper side were washed off and migrated cells stuck to the lower side of the membrane were put after drying in 1)

Fixation solution (Hemacolor^® solution 1, 1.11955; Merck) 2) Haematoxylin solution according to Mayer (51275; Sigma-Aldrich) and 3) Eosin Y solution with phloxine (HT110316; Sigma-Aldrich) with wash steps after step 2) and step 3) . The subsequently dried membrane was fixed onto microscopy slides for evaluation. The cells in 5 different areas per well (middle, left from middle, right from middle, above middle and below middle) were counted. Each concentration of each sample is measured in quadruplicate. Migration towards SDF-lwt is highest at 100 nM, which was

therefore set to 100% migration for comparison of different SDF-1 variants in different assays. Substances tested for their influence on chemotaxis were: PA517 (DS1014), Heparin (LMW Heparin; Iduron) , HS (GAG-HS01; Iduron) and cellulose sulphate (177810500; Acros Organics) .The migrated cells of five 400 x magnifications per well were counted and the mean of three independently conducted experiments was background corrected and the chemotactic index (CI) was calculated (see Figure 3) . The Met-SDF-1 mutants Met-SDF-1 Δ8 L29R V39K E60K (PA1009, 1.2%), Met-SDF-1 Δ8 L29K V39K E60K N67K (PA1010, 2.3%), Met-SDF-1 Δ8 L29K V39K E60K N67K K68R (PA1011, 2.3%), Met-SDF-1 Δ8 L29K V39K E60K K68R (PA1016, 1.4%) exhibited a clearly reduced chemotaxis in vitro compared to the wild type protein.

Example 3 : Stability towards guanidine unfolding

SDF-1 mutants were mixed with guanidine chloride and unfolding of the proteins was determined. The SDF-1 mutants showed high stability towards guanidine treatment compared to wild type SDF-1 SDF-1 wt (3.56M Guanidine), PA1009 (1.04M), PA1010 (1.31M

Guanidine), PA1011 (0.99M Guanidine), PA1016 (1.05M Guanidine)

Example 4 : Inhibition of breast cancer metastasis

For the purpose of investigating the inhibitory activity of SDF- la mutants on cancer metastasis, a murine xenograft model is used. 10 week old female immunodeficient SCID mice are used for the in vivo experiments. They are kept in isolation care during the whole course of the experiments. The animals are left for a few days after delivery to settle down before the experiments started .

LMD-231 cells are used in this xenograft model. They are cultured from lung metastasis of mice inoculated with MDA-MB-231. These cells are known to express high levels of CXCR4 and are routinely used in xenograft experiments. These adherent cells are cultured in complete Minimum Essential Medium and complete RPMI mixed in a 50:50 ratio and cell flasks are split in a ratio of 1:3. The cells are cultured at 37°C, 5% C0₂ in a humidified atmosphere.

For in vivo experiment, 120,000 LMD-231 cells are injected into the tail vein of each mouse. Just before injection, the cells are either mixed with PBS for the control group or the mutant chemokine in the desired concentration. (The cells and chemokine are not formally incubated, and the time duration they are mixed together before injection is 5-10 minutes on average.) A total volume of 100 μΐ is injected into each mouse.

On day 28, the animals are killed humanely and organs harvested. Half the liver and a lung from each mouse are snap frozen for future work. The other half of the liver and one lung are fixed in formalin, and later embedded in paraffin wax. Sections are then cut from the liver (and lung) . 2 sections from each mouse are stained with a cocktail of cytokeratin markers which are specific for human epithelial cells (breast cancer) .

Assessment of liver metastases is accomplished by calculating the total areas of each section using an LCM laser capture microscope and integrated software called. The total numbers of metastases in each section are then counted and average metastases per square mm are calculated.

The Met-SDF-1 Δ8 mutants show a clear inhibitory effect on metastasis migration into the liver at 20ug per dose.

Claims

Claims

1. SDF-1 mutant polypeptide with increased GAG binding affinity and reduced GPCR activity compared to wild type SDF-1 protein wherein said SDF-1 polypeptide is modified in a structure- conserving way by replacing glutamine at position 60 by a basic amino acid and

by replacing at least one additional amino acid at position 29, 39, 49, 67 and/or 68 according to the numbering of SEQ ID NO 1 by at least one basic amino acid and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 protein is modified by addition, deletion and/or replacement of at least one amino acid.

2. SDF-1 mutant polypeptide according to claim 1, wherein amino acids at position 29 and 39 are replaced by basic amino acids.

3. SDF-1 mutant polypeptide according to claim 1 or 2, wherein the modification in a structure conserving way is a deviation of the modified structure from wild type SDF-1 structure of less than 30%, preferably less than 20% as measured by far-UV-CD spectroscopy .

4. SDF-1 mutant polypeptide according to any one of claims 1 to

3, wherein the basic amino acids are selected from the group consisting of Arg, Lys, His.

5. SDF-1 mutant polypeptide according to any one of claims 1 to

4, wherein eight amino acids of the N-terminal region of said SDF-1 polypeptide are deleted.

6. SDF-1 mutant polypeptide according to any one of claims 1 to

5, wherein the first two amino acids of the N-terminal region of said SDF-1 polypeptide are replaced and/or deleted, preferably the amino acids are replaced by amino acids selected from the group consisting of Lysine, Arginine, Proline or Glycine.

7. SDF-1 mutant polypeptide according to any one of claims 1 to

6, containing an N-terminal Met.

8. SDF-1 mutant polypeptide according to any one of claims 1 to

7, wherein amino acids at positions 29 and 39 are replaced by basic and/or electron amino acids.

9. SDF-1 mutant polypeptide, wherein the amino acid sequence of the modified SDF-1 molecule is described by the general formula: (M) _n (XI ) m (X2 ) pVSLSYRCPCRFFESHVARANVKHLKI (X3 ) TPNCALQI (X4 ) ARLKNNNRQ (X5) CIDPKLKWIQ (X6) YLEKAL (X7) (X8) (GRREEKVGKKEKIGKKKRQKK

RKAAQKRKN) _o according to the numbering of SEQ ID NO 1,

wherein XI is a K or R residue,

wherein X2 is a P or G residue,

wherein X3 is selected from L, R, K or H residues, preferably it is K,

wherein X4 is selected from V, R, K, H residues, preferably it is K,

wherein X5 is selected from V or A preferably it is A,

wherein X6 is selected from R, K or H, preferably it is K, wherein X7 is selected from N, R, K or H, preferably it is K, wherein X8 is selected from R, K or H, preferably it is R, and wherein n and/or m and/or p and/or o can be either 0 or 1 and optionally at least one of positions XI, X2 , X3 , X4 , X5, X7 or X8 are modified (SEQ ID No. 6) and wherein at least one amino acid of the first 1 to 10 amino acids of the N-terminal region of the wild type SDF-1 protein is modified by addition, deletion and/or replacement of at least one amino acid.

10. SDF-1 mutant polypeptide of the structure Met-SDF-1 Δ8 E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K, Met-SDF-1 Δ8 L29K V39K E60K N67K, Met-SDF-1 Δ8 L29K V39K V49A E60K N67K, Met-SDF-1 Δ8 L29K V39K E60K N67K K68R, Met-SDF-1 Δ8 L29K V39K E60K K68R.

11. Isolated polynucleic acid, coding for a polypeptide according to any one of claims 1 to 10.

12. Vector, comprising an isolated polynucleic acid molecule according to claim 11.

13. Recombinant non-human cell, transfected with a vector according to claim 12.

14. Pharmaceutical composition, comprising a polypeptide

according to any one of claims 1 to 10, or a polynucleic acid according to claim 11, or a vector according to claim 12 and a pharmaceutically acceptable carrier.

15. SDF-1 mutant polypeptide according to any one of claims 1 to 10 or a polynucleotide according to claim 11 or a vector

according to claim 12 for preparing a medicament.

16. SDF-1 mutant polypeptide according to claim 15 for use in the treatment of cancer.