WO2004048409A2

WO2004048409A2 - Mutants of the tumor necrosis factor alpha with deletion of n-terminal amino acids

Info

Publication number: WO2004048409A2
Application number: PCT/SI2003/000041
Authority: WO
Inventors: Viktor Menart; Vladka Gaberc-Porekar; Maja Kenig; Irena Fonda
Original assignee: Lek Pharmaceuticals D.D.
Priority date: 2002-11-22
Filing date: 2003-11-21
Publication date: 2004-06-10
Also published as: AU2003283947A8; AU2003283947A1; WO2004048409A3; SI21373A

Abstract

The present invention relates to new protein analogues, especially TNF-alpha analogues in which the biological activity is modulated.

Description

Title of the invention

Protein analogues with modulated biological activity

Field of the invention

The present invention relates to new protein analogues with modulated biological activity. The principle of the present invention can be applied to a wide range of heterologous proteins to modulate their biological activity although it will be mainly described by referring to new tumour necrosis factor-alpha (TNF-alpha) analogues.

Summary of the invention

It is an object of the invention to provide new protein analogues, e.g., TNF-alpha analogues, which have modulated biological activity. The biological activity of these proteins is modulated in a way that the interactions of the proteins with the extracellular matrix or the cell surface components are modulated. Modulation of the biological activity can be achieved by introducing changes into the protein structure, the changes relating to the region (Arg/Lys)XYZ(Arg/Lys). The sequence XYZ is defined below and comprises small, flexible, polar and charged amino acids, selected from the group comprising Ser, Gly, Ala, Thr, His, Pro, Lys, Arg, Gin and Asn. Either deletion of one or more regions (Arg/Lys)XYZ(Arg/Lys) from the protein structure and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys) into the protein structure can lead to modulated biological activity of the proteins due to modulated interaction of the proteins with the extracellular matrix or the cell surface components.

Background of the invention

Human TNF-alpha is a non-glycosylated protein, which belongs to the family of cytokines. Primarily it is produced by monocytes and macrophages as a response to the stimulation with endotoxin and other stimuli. Soluble TNF-alpha can be also produced by other cells. It is an essential element in the cascade of factors, which are involved in cell immune response and is involved in the pathogenesis of different acute infections and chronic immune or inflammatory diseases. In general, it has a pleiotropic activity in both, healthy and unhealthy organism.

TNF-alpha was discovered in 1975 in the serum of mice infected with mycobacterium Calmett-Guerin (BCG) and treated with endotoxin (Carswell EA et al Proc Natl Acad Sci U S A 1975 Sep;72(9):3666-70).

Due to extensive anti-tumour effect TNF-alpha is a potential anti-cancer agent. However, it is highly toxic and causes severe side effects by systemic use and is only used in a local therapy (Taguchi in Sohmura Biotherapy 1991 ;3(2): 177-86; Lejeune et al Curr Opin Immunol 1998 Oct;10(5):573-80; Eggermont et al., Ann Surg. 1996 Dec;224(6):756-64; discussion 764-5.; Eggermont et al., J Clin Oncol. 1996 Oct;14(10):2653-65).

In many prior art studies there were attempts to design TNF-alpha analogues with conserved or even increased anti-tumour activity with less side effects. The analogues with modified N-terminal amino acid sequence with higher in vitro cytotoxicity and higher binding activity to receptors of tumour cells are described in Nakamura S et a/ Agric. Biol. Chem. 55: 53-57, 1991 ; WO8602381 , 1986; Creasey A.A et al. Cancer Res. 47: 145-149, 1987; Carlino J.A et al. J. Biol.Chem 262:958- 961 , 1987; Shikama H. et al., J Interferon Cytokine Res. 1995 Aug;15(8):677-84.; Kuroda K. et al., Int J Cancer 1995 Sep 27;63(1 ): 152-7; Atarashi Y. Et al., Hepatology. 1998 Jul;28(1):57-67.; Nakamura S et al Int. J. Cancer 48,744-748, 1991. In general the studies were basically focused on in vitro cytotoxicity and in vivo anti-tumour activity of analogues comprising deletions of N-terminal amino acids or the substitutions of N-terminal amino acids with more basic or/and hydrophobic amino acids. There are less data on in vivo anti-tumour activity as well as on systemic toxicity.

The changes in in vitro cytotoxicity due to changes in C-terminal end of TNF-alpha molecule are described in Masegi et al., Jpn J Cancer Res. 1993 Apr;84(4):451-4. in Guo D. Et al., Biochem Biophys Res Commun. 1995 Feb 27;207(3):927-32. Introduction of mutations which cause changes in the TNF-alpha receptor binding specificity is described in Barbara JA et al, EMBO J. 1994 Feb 15;13(4):843-50, EP0486908, 1992; van Ostade X. et al Nature. 1993 Jan 21 ;361 (6409):266-9, Atarashi Y et al, Hepatology. 1998 Jul;28(1):57-67.

The influence of mutation in the tip region of a trimeric mouse TNF-alpha on the binding specificity to certain oligosaccharides is described in Lucas R. et al., Science. 1994 Feb 11 ;263(5148):814-7; US5891679, 1999. The triple alanine mutant in this region was declared to have decreased systemic toxicity in mouse (Lucas R. et al., Infect Immun. 1997 Jun;65(6):2006-10). The addition of amino acid sequence ArgGlyAsp in the N-terminal part of human TNF-alpha led to conserved anti-tumour activity but with less influence on the metastases formation (Miyata k. Et al., J Interferon Cytokine Res. 1995 Feb;15(2):161-9; US5519119, 1996). In Novakovic S. et al., Cytokine. 1997 Aug;9(8):597-604 the analogue of human TNF-alpha with increased cytotoxic activity in vitro and reduced systemic toxicity in mouse models with implanted tumour is described, indicating the improved therapeutic characteristics for cancer therapy.

Decreased systemic toxicity of TNF-analogues with added basic amino acids in the N-terminal part of the molecule is described in (Nakamoto T et al. Anticancer Res

2000 Nov-Dec;20(6A):4087-96; de Wilt JH et al. Anticancer Res 2000 Sep- Oct;20(5B):3491-6).

Interaction of TNF-alpha with fibronectin and laminin is described in Alon R, Cahalon L. Et al., J. Immunol. 152:1304-1313,1994; the interaction of TNF-alpha with heparin is described in Lantz M, et al J. Clin. Invest. 88:2026-2031 , 1991. It is known that many proteins interact with heparin and the prior art suggests that the basic amino acids are responsible for binding of these proteins on heparin molecule (Fromm JR et al Arch Biochem Biophys 1995 Nov 10;323(2):279-87), Cardin AD et al Arteriosclerosis 1989 Jan-Feb;9(1 ):21-32, Rastegar-Lari G et al. Biochemistry. 2002 May 28;41 (21 ):6668-78; Soncin F, et al. J Biol Chem. 1997 Apr 11 ;272(15):9818-24); Zhao M et al J Biol Chem 1998 Nov 20;273(47):31153-9; Chen VC et al ό Biol Chem.

2001 Jan 12;276(2):1276-84; Whinna HC et al J Biol Chem. 1991 May 5;266(13):8129-35). In general, the heparin binding sites in different proteins differ and may not be predicted.

Neither in patent nor in scientific literature the binding sites of TNF-alpha or TNF- alpha analogues for heparin, heparan sulphate, glycosaminoglycans or other cell surface components or extracellular matrix are described. Accordingly, no report on modulated biological activity of TNF-alpha or its analogues based on specific amino acid sequences responsible for binding to the above-mentioned components has been found.

Description of the preferred embodiments

The present invention will be described in the following in further detail with respect to both the basic concept and the preferred embodiments of the invention. The present invention is however not limited to the specifically described embodiments, and the skilled person will understand that variations and modifications exist within the scope of the annexed claims.

The present invention provides new protein analogues in accordance with claims 1 , 6, 8, and 10. Preferred embodiments are set forth in the sub-claims. The present invention also provides a process according to claim 11 , the method according to claim 12, a pharmaceutical composition according to claim 17 and uses according to claims from 18 to 24.

It has been surprisingly found that deletion of one or more regions (Arg/Lys)XYZ(Arg/Lys) from the protein structure and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys) into the protein structure led to modulated interaction of proteins e.g., TNF-alpha, TNF-alpha analogues and other proteins, with the extracellular matrix or cell surface components.

The term '(Arg/Lys)' as used herein, refers to the replacement of Lys with Arg or vice versa in all possible combinations, i.e. ArgXYZArg, ArgXYZLys, LysXYZLys and LysXYZArg. In the sequence 'XYZ' as used herein, X, Y and Z refer to small, flexible, polar and/or charged amino acids, which are selected from the group comprising Ser, Gly, Ala, Thr, Pro,His, Lys, Arg, Gin and Asn. Among the regions (Arg/Lys)XYZ(Arg/Lys) the preferred regions in the present invention are ArgSerSerSerArg and ArgGlnHisProLys. The region ArgSerSerSerArg is the most preferred.

The term "extracellular matrix" as used herein, refers to the group of compounds selected from the group comprising laminin and fibronectin. The term "cell surface components" as used herein, refers to the group of compounds comprising proteoglycans e.g., glycosaminoglycans e.g. heparin and heparan sulphate and also other glycosaminoglycans, which occur on the surface of many cells (e.g., endothelial, tumour, liver, etc).

The present invention relates to the protein analogues which differ from the starting protein in one or more regions (Arg/Lys)XYZ(Arg/Lys). The protein analogues were obtained from the starting protein when one or more regions (Arg/Lys)XYZ(Arg/Lys) (e.g., ArgSerSerSerArg) were deleted from the starting proteins structure and/or one or more regions (Arg/Lys)XYZ(Arg/Lys) were introduced into the starting protein structure. Therefore the protein analogues of the present invention relate to the protein analogues in which one or more regions (Arg/Lys)XYZ(Arg/Lys) were deleted and/or one or more regions (Arg/Lys)XYZ(Arg/Lys) were introduced. The region (Arg/Lys)XYZ(Arg/Lys) can be deleted from different sites in the starting protein structure or it can be introduced into different sites of the starting protein structure, the sites being selected from the group comprising flexible termini (N- and C-), outer surface of the protein structure, totally flexible amino acid chains and partially flexible amino acid chains.

The protein analogues of the present invention have modulated biological activity, the modulation being a result of different interaction of the obtained analogue with the extracellular matrix or the cell surface components in comparison to the starting protein. The strength of binding of the protein to the extracellular matrix or the cell surface components can be increased by increasing the number of the introduced regions (Arg/Lys)XYZ(Arg/Lys) into the protein structure and can be decreased by increasing the number of the deleted regions (Arg/Lys)XYZ(Arg/Lys).

The term 'protein analogue' as used herein, refers to a polypeptide with certain mutations, deletions, and insertions or in general with any changes in amino acid sequence of a native protein.

The term 'native protein' as used herein, refers to a protein with native amino acid sequence having full biological activity. The term 'starting protein' as used herein, refers to a protein from which the protein analogues are obtained. The starting proteins are selected from the group of native proteins and protein analogues which either already comprise one or more regions (Arg/Lys)XYZ(Arg/Lys) or which do not comprise this region. Examples of starting proteins encompassed within the definition herein include mammalian proteins, such as, e.g., growth hormones, growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins, alpha-1-antitrypsin; insulin A-chain; insulin B-chain, proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagons; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; plasminogen activators, such as urokinase or tissue-type plasminogen activator (t-PA, e.g., Activase, TNKase, Retevase); bombazine; thrombin; ligands of tumor necrosis factor family, e.g., TNF-alpha, TNFbeta, TRAIL, FasL, soluble receptors from TNF family receptors, enkephalinaze; RANTES; macrophage inflammatory protein (MIP-1 -alpha); relaxin A-chain; relaxin B-chain, prorelaxin; gonadotropin-associated peptide; Dnase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrins; rheumatoid factors; neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurothropin-3,-4,-5, or -6 (NT-3, NT-4, NT-5, NT-6), or a nerve growth factor such as NGF-beta; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor; transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-beta1 , TGF-beta2, TGF-beta3, TGF-beta4 or TGF-beta5; insulin-like growth factor-l and-ll (IGF1 and IGF2), des (1-3)-IGF-l (brain IGF-I); insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; a bone morphogenetic protein (BMP); an interferon such as INF — alpha, beta and gamma, colony stimulating factors (CSFs), e.g., M-CSF, GM- CSF and G-CSF; interleukins (Ms), e.g., IL1-to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor (DAF); vaccines, viral antigen such as, portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; therapeutic antibodies; and biologically active fragments or variants of any of the above-listed polypeptides. The present invention also relates to a process for preparation of the protein analogues of the present invention, the process comprising either deletion of one or more regions (Arg/Lys)XYZ(Arg/Lys) from the starting protein structure and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys) into the protein structure.

The present invention also relates to the method for modulation of biological activity, the method comprising a modulated interaction of the protein analogue of the present invention with the extracellular matrix or the cell surface components. The method for modulation of biological activity may represent a new way of modulating the targeting on certain cells, e.g. tumour cells, endothelial cells, liver cells, etc. From the prior art it is known that the major problems for systemic application of TNF- alpha its analogues and many other proteins are: high systemic toxicity, short plasma half life time, no obvious targeting of, e.g. tumours, organs, cell types, tissues. The method for modulation of the biological activity of the present invention enables modification of the biological activity of TNF-alpha, its analogues and other proteins in such a way that the above stated problems may be solved.

The term TNF-alpha' as used herein, refers to the polypeptide of TNF-alpha with native human amino acid sequence. The biologically active TNF-alpha has a structure of a compact trimer with three equivalent N-terminal amino acid regions

ArgSerSerSerArg.

The term TNF-alpha analogue' as used herein, refers to a polypeptide with certain mutations, deletions, insertions or in general with any changes in amino acid sequence of TNF-alpha.

Deletion of a region (Arg/Lys)XYZ(Arg/Lys) from TNF-alpha or TNF-alpha analogues and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys) into the protein structure leads to changes in the biological activity of the proteins and analogues. The obtained TNF-alpha analogues are the subject of the present invention. For example deletion of the sequence ValArgSerSerSerArg from TNF-alpha (resulting in the analogue dN6-TNF-alpha) can lead to reduced interaction of dN6- TNF-alpha with the components of extracellular matrix, which may also reduce local binding of leukocytes. This may mean that the analogues with deleted region ValArgSerSerSerArg could have reduced proinflammatory potential and could therefore be safer for therapy. However, other different or additional interpretations are also possible e.g. changed systemic toxicity of these analogues could be derived from the changed interaction with some crucial cells or organs, but the exact site is not known. Potentially, plasma half-life time could .also be improved due to reduced affinity to alpha2 macroglobulin, which is one of putative natural scavengers and removers of biologically active TNF-alpha from the blood circulation. The term 'dN6-TNF' as used herein, refers to TNF-alpha, in which the N-terminal region ValArgSerSerSerArg is deleted.

Therapeutic value of any other starting protein defined above could be modulated by deletion and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys).

Deletion of one or more regions (Arg/Lys)XYZ(Arg/Lys) from the protein structure may therefore have different physiological consequences i.e. TNF-alpha analogues with the deletion dN6 may have increased anti-tumour activity in comparison to the native TNF-alpha and may therefore be more suitable for the treatment of cancer.

The biological activity of the TNF-alpha analogues which already have introduced mutations for the improvement of the therapeutical value can be additionally modulated by deletion and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys), representing an essential element for the interaction with the extracellular matrix or the cell surface components. Such analogues have a potential applicability for development of new approaches for treatment of cancer or other diseases.

An example of such TNF-alpha analogue is LK-805 in which the therapeutic value has been improved by introducing the mutation Glu107Lys. The biological activity of this analogue was further modulated by the deletion of the region ValArgSerSerSerArg from the N-terminal part of LK-805. Thus the analogue obtained, dN6-LK-805 is a subject of the present invention. The term 'LK-805' as used herein refers to the TNF-alpha analogue, in which the mutation Glu107Lys is introduced.

The term 'dN6-LK-805' as used herein refers to LK-805, in which the N-terminal region ValArgSerSerSerArg is deleted.

Another example is TNF-alpha analogue LK-802 in which the introduced mutations enable spontaneous formation of disulphide bridges between the neighbouring subunits. The deletion of the sequence ValArgSerSerSerArg from the N-terminal part of this analogue leads to the TNF-alpha analogue dN6-LK-802, which is also subject of the present invention. In this way the non-dissociable TNF trimer is obtained, which also has the improved stability in vitro and is therefore suitable for the preparation of pharmaceutical compositions with improved stability. The term 'LK-802' as used herein refers to the TNF alpha analogue in which the mutations Ser95Cys and Gly148Cys are introduced. LK-802 was described in the prior art in EP846169.

The term 'dN6-LK-802' as used herein refers to the TNF-alpha analogue, in which the N-terminal peptide sequence ValArgSerSerSerArg is deleted and mutations Ser95Cys and Gly148Cys are introduced.

Another protein analogue of the present invention is TNF-alpha analogue dN6-LK- 805/LK-802, which contains firstly the deletion of the sequence ValArgSerSerSerArg, secondly the introduced mutation Glu107Lys what leads to the reduced systemic toxicity and thirdly the mutations Ser95Cys and Gly148Cys what leads to the increased stability in vitro.

The term 'dN6-LK-805/LK-802' refers to the TNF alpha analogue, in which the N- terminal peptide sequence ValArgSerSerSerArg is deleted and three mutations Ser95Cys, Gly148Cys and Glu107Lys are introduced.

The protein analogues of the present invention can be used in a process for the manufacture of a pharmaceutical composition comprising them as an active ingredient. The pharmaceutical composition comprises an amount of the protein analogue e.g. TNF-alpha analogue that is therapeutically effective to treat a desired disease in a patient. Suitable pharmaceutically auxiliary substances can be added and are selected from the group of suitable diluents, adjuvants and/or acceptable carriers useful in the therapy.

The protein analogues i.e. TNF-alpha analogues of the present invention can be used as medicaments and can be also used for preparation of medicaments, in which the analogues and the proteins would act as active pharmaceutical ingredients. The medicaments may be used for modulation of targeting on certain cells selected from the group comprising tumour cells, endothelial cells, liver cells, and other cells and for modulation of systemic toxicity, for treatment of cancer and other possible indications.

TNF-alpha analogues and the proteins of the present invention can be used for modulation of targeting on certain cells, e.g. tumour cells, endothelial cells, liver cells, etc., for modulation of systemic toxicity, for treatment of cancer and other possible indications.

Brief description of the drawings

Figure 1 : Chromatographic behavior of TNF-alpha and TNF-alpha analogues on the affinity Heparin-Sepharose column. Peak A refers to dN6-TNF-alpha, whereas peak B refers to TNF-alpha.

Figure 2: Isoelectric focusing of LK-805, dN6-LK-805 and their equimolar mixture in dependence of time.

Figure 3: Chromatographic separation of the equimolar mixture of LK-805 and dN6- LK-805 after 4 day storing at temperature +4 °C, on the affinity Heparin-Sepharose column. Peak A refers to the TNF-alpha analogue dN6-LK-805, whereas peak B refers to the mixed trimers and to LK-805.

Figure 4: Isoelectric focusing analysis of the fractions from the peak B of Figure 3. The following examples illustrate the present invention without, however, limiting the same thereto.

Examples

Example 1 : Preparation of TNF-alpha analogues

We prepared TNF-alpha analogues of the present invention from the commercially available synthetic gene coding for TNF-alpha containing the codons for high expression in the bacterium Escherichia coli (E. coli). The synthetic gene was excised from the carrier plasmid BBG4 (British Biotechnology) by using suitable enzymes. It was excised by using suitable restriction enzymes and subcloned in the selected expression plasmids. The mutations were introduced using the plasmid pCYTEXPI and site directed oligonucleotide mutagenesis (Kunkel et al. 1987, Methods in Enzymology, Recombinant DNA, Part, Eds. R Wu in L. Grossman; Academic Press Inc., 367-382). TNF-alpha and its analogues with conserved (complete) N-terminus were expressed in E. coli using plasmid pCYTEXPI (Medac), which was modified from the termoinducible to constitutive plasmid pCydcl by the deletion of a part of repressor gene cl857. This enabled essentially higher accumulation of TNF-alpha and all of the TNF-alpha analogues. The optimal accumulation of target protein (10 - 25 %) was obtained by cultivation at about 30°C in known E. coli strains e.g.: NM522, DHδalfa, etc. After cell disruption in the high pressure homogeniser EmulsiFlex C-5 (Avestin), the majority of nucleic acids was precipitated with the addition of polyethyleneimine (PEI) in the final concentration of 0.1 %. The proteins were salted out from the clear supernatant by the addition of solid ammonium sulphate (AS) to the final concentration of 65 % of the saturation. pH value was simultaneously adjusted to 6.5 to 7.5 using 12.5 % NH₄OH. In the AS precipitate the content of TNF-alpha and TNF-alpha analogues increased, in general representing 35% to 50 % of the total proteins. The purified proteins were obtained by dissolving AS precipitate in suitable buffers, followed by the chromatographic isolation. The pure proteins were dissolved in phosphate buffer in the concentration range from 30 mM to 50 mM with the pH value at about 7.3 containing 0.5 M NaCI and then stably stored at - 80 °C. dN6-TNF-alpha and TNF-alpha analogue containing the deletion dN6 at the N- terminus (dN6-LK-802, dN6-LK-805, dN6-LK-802/LK-805) were expressed as fusion proteins with oligohistidine tags at N-terminus by using the commercially available plasmid TAGZyme pQE-2 (Qiagen) in the strain E. coli BL21 (DE3). Pure proteins were isolated by using the immobilised metal-ion affinity chromatography (Ni-NTA) following the producers' (Qiagen) instructions. The oligohistidine tag was then cleaved-off by the enzyme DAPase, and the N-terminal amino acid sequence was confirmed by N-terminal sequence analysis.

Example 2: Chromatography on Heparin-Sepharose - comparision of TNF-alpha analogues dN6-LK-805 and LK-805

We tested the affinity of the proteins with the complete N-terminus and of the dN6- proteins towards the Heparin-Sepharose by using the column HR 5/5 (Amersham

Pharmacia Biotech, column volume: 0.8 mL), filled with chromatographic matrix

Heparin-Sepharose 6 Fast Flow (Amersham Pharmacia Biotech). Each protein solution was dialyzed against buffer P1 and loaded onto the column, which had previously been equilibrated with the same buffer. The loaded amount of each protein sample was about 100 μg. The column was washed with buffer P1 for 10 minutes. The bound proteins were then eluted using 20 minutes linear gradient from

0 to 500 mM NaCI. Between the consequtive loadings the column was washed for 5 minutes with buffer P2 and equilibrated again with buffer P1. The flow rate was 1 mL/min, the whole chromatographic procedure was performed at room temperature

(-25 °C).

Buffers:

P1 : 50 mM TRIS/HCI, pH 7.4

P2: 50 mM TRIS/HCI, pH 7.4, 1 M NaCI

Figure 1 shows the chromatographic behavior of different proteins TNF-alpha and

TNF-alpha analogues on the affinity matrix Heparin-Sepharose 6 Fast Flow

(Amersham Bioscience).

Solid line ( ) represents the chromatogram recorded at 280 nm. Dashed line ( ) shows the gradient of buffer P2.

Peak A: flow through fraction - the proteins which do not bind to the column (dN6- TNF-alfa)

Peak B: elution: TNF-alpha.

Peak A shows the behavior of TNF-alpha analogues with shortened N-terminus - the amino acid region ValArgSerSerSerArg is deleted (dN6-TNF-alpha). These TNF- alpha analogues do not bind to the affinity column and occur in the flow through fractions.

Peak B shows the chromatographic behaviour of proteins having N-termini conserved (TNF-alpha). The protein comprising the region ArgSerSerSerArg binds to the Heparin-Sepharose column.

Similar results were obtained by the chromatographic analysis of TNF-alpha analogues LK-805 and dN6-LK-805, where dN-LK-805 was found in the peak A and LK-805 was found in the peak B (results not shown).

The results show that the region ValArgSerSerSerArg represents the essential amino acid sequence for the binding of TNF-alpha and TNF-alpha analogues to the immobilised heparin (e.g., Heparin-Sepharose column).

Example 3: Preparation of hybrid (mixed) trimers and their separation on Heparin- Sepharose column

In this example we showed that already a single ArgSerSerSerArg sequence enables binding of the protein to the Heparin-Sepharose column. Native, biologically active TNF-alpha is a compact trimer having three equivalent N-terminal sequences ValArgSerSerSerArg.

The incubation of equimolar amounts of two pure proteins, the first one being LK-805 (containing three equivalent sequences ValArgSerSerSerArg, per trimeric molecule) and the second one being dN6-LK-805 (all three N-terminal sequences ValArgSerSerSerArg are deleted), were incubated following the method described in the prior art (Menart V et al. 1996, Eur. Cytokine Network, Vol 7, No 2, p. 172). The final protein mixture was composed of proteins LK-805 and dN6-LK-805 as well as newly formed hybrid (mixed) trimers. Calculation of the ratio of components: a - subunit (monomer) of TNF-alpha analogue dN6-LK-805 (having deleted N- terminal sequence ValArgSerSerSerArg) b - subunit (monomer) of TNF-alpha analogue LK-805 (conserved N-terminus) aaa - trimer of dN6-LK-805 (pi = 6,7 ) bbb - trimer of LK-805 (pi = 8,9 ) aab - hybrid (mixed) trimer (pi = 8,0) abb - hybrid (mixed) trimer (pi = 8,7)

According to probability theory the ratio of components in the mixture formed from equimolar amounts of starting trimers aaa and bbb is as follows: aaa : aab : abb : bbb = 1 : 2 :2 : 1.

Preparation of hybrid (mixed) trimers

300 μg LK-805 (600 μL, C = 0.5 mg/mL) and 300 μg dN6-LK-805 (600 μL, C = 0.5 mg/mL) were mixed. Both proteins were dissolved in the buffer PBS containing 0.5 M

NaCI.

PBS (Phosphate buffer solution): 2.7 mM KCI, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄

(Maniatis).

The mixture was incubated for 4 days at temperature 4 °C. The formation of hybrid

(mixed) trimers was proved by using isoelectric focusing. The gel was stained with

Coomassie R250 (Figure 2).

Figure 2 shows the isoelectric focusing analysis of the starting equimolar mixture of two TNF-alpha analogues LK-805 and dN6-LK-805 and final protein mixture after the incubation. Polyacrylamide gel with the immobilized pH range 3.5 to 9.0 was used.

Legend:

1. Standard for isoelectric focusing (Bio-Rad)

2. dN6-LK-805 (Load: 1 μg)

3. LK-805 (Load: 1 μg)

4. Equimolar mixture of dN6-LK-805 and LK-805 (Load: 2 μg) in time 0 (prepared just before the analysis).

5. Mixture of dN6LK-805 and LK-805 (Load: 2 μg) after 4 days at temperature +4 °C. The results show that the mixed trimers with intermediate pi values are formed from the equimolar mixture of both TNF-alpha analogues at temperature 4 °C.

These results therefore reveal that after certain time the mixture contains trimeric molecules of TNF-alpha, in which the subunits differ in the number of conserved N- termini .

Chromatography on Heparin-Sepharose column:

The sample (solution of the mixed trimers) was desalted by using PD-10 (Amersham

Pharmacia Biotech) before loading onto the column. 1.2 mL of the solution was diluted to 2.5 mL with buffer P3 (10 mM TRIS/HCI, pH 8.0), loaded onto the column

PD-10 and 3.5 mL of the desalted eluate was captured (protein concentration was

0.17 mg/mL).

The column HR 5/5 (Amersham Pharmacia Biotech, column volume: 0.8 mL)

(chromatographic matrix: Heparin-Sepharose 6 Fast Flow (Amersham Pharmacia

Biotech)) was equilibrated with buffer P3. About 100 μg of proteins were loaded onto the column (590 μL, C = 0.17 mg/mL). The bound proteins were eluted by using NaCI gradient in the concentration range from 0 to 0.5 M NaCI with the gradient length of

20 column volumes, with the flow of 0.5 mL/min and the 0.5 mL fractions were collected. The whole chromatographic procedure ran at room temperature (~ 25 °C).

The chromatographic analysis resulted in two peaks (Figure 3).

Figure 3 shows the chromatographic separation of protein mixture of LK-805 and dN6-LK-805 after 4 days of incubation at temperature +4 °C on the affinity column Heparin-Sepharose 6 Fast Flow (Amersham Bioscience).

Solid line ( ) represents the chromatogram of purification recorded at 280 nm.

Dashed line ( ) shows the gradient of buffer P4.

Peak A: elution of dN6-LK-805

Peak B: elution of mixed trimers and of LK-805

Peak A represents the unbound fraction and contains only dN6-LK-805 in which the amino acid sequence ValArgSerSerSerArg at all three N-termini is deleted

Peak B which was eluted in the concentration range from 100 to 150 mM NaCI, contains three forms of trimers which contain either one or two or three conserved N- termini. The individual consecutive fractions of the peak B were analysed by isoelectric focusing (Figure 4).

Figure 4 shows the analysis of fractions from peak B (see chromatogram in Figure 3) using isoelectric focusing. Polyacrylamide gel with the immobilized pH range 3.5 to

9.0 was used and stained with Coomassie R250 after the analysis.

Legend:

1 : Standard for isoelectric focusing (Bio-Rad).

2: Mixture of LK-805 and dN6-LK-805 (Load: 2 μg), after 4 days of incubation at temperature +4 °C (before the separation on Heparin-Sepharose column).

3 - 7: Consecutive fractions of the peak B from the chromatogram in Figure 3

(maximal loading: 20 μL).

The analysis of single fractions showed that on Heparin-Sepharose column the trimers with lower isoelectric points (pi) were eluted first (Figure 3, peak B, first fraction). In the following fractions the amount of trimers with higher pi values was increasing. The later fractions contained mainly trimers with conserved N-termini. The ratio of surfaces of peak A (pure trimer aaa) and peak B (mixture of trimers aab, abb, bbb) on the chromatogram in Figure 3 is approximately 1:5.1 which is very close to the theoretical ratio (1 :5.0). This means that the unbound fraction contained only the trimer aaa (dN6-LK-805). All other types of trimers (either with one or with two or with three conserved N-termini) were able to bind and are therefore found in the peak B.

To conclude, a single ArgSerSerSerArg enables the binding of TNF-alpha analogue to the Heparin-Sepharose column. By increasing the number of such sequences the affinity of the protein to the Heparin-Sepharose matrix is also increasing. These conclusions were experimentally confirmed by the analysis of the chromatographic fractions shown in Figure 4. Example 4: Measurement of specific cvtotoxycitv of TNF-alpha and its analogues in vitro

Method for the immobilized cells

Specific cytotoxic activity was measured using the L-929 cell line (mouse fibroblasts) according to a modified procedure of Flick and Gifford (Flick, D.A., Gifford, G.E. 1984 68: 167-175). 2x10⁴ cells in 100 μl culture medium were seeded in each well of a microtiter plate and incubated for 24 hours (37°C, 5 % CO₂). After incubation the serial dilutions of internal TNF-alpha standard (prepared using WHO TNF-alpha standard 87/650) and TNF-alpha analogues were added into the wells in the presence of 2 μg/ml actinomycin D. The plates were incubated again for 18 - 20 hours ( 37 °C, 5 % CO₂ ). The viable cells were then fixed with 2.5 % glutaraldehyde and stained with 0.5 % crystal violet in 20 % methanol. The plates were dried and 100 μl of 1 % SDS were added in each well. After 10 minutes of shaking at room temperature (~ 25 °C), the optical density was measured at 570 nm. From the measured optical density the natural logarithm of the cell concentration was obtained. The specific cytotoxic activity of TNF-alpha and its analogues was determined by comparing the dilution of the standard and the samples yielding 50 % of maximal cytotoxicity i.e. 50% cells survived regarding the number of cells in the control wells without the protein (negative control)

Method for the suspension cells

Specific cytotoxic activity was measured using the cell line KYM-1 D4 (cells isolated from human rhabdomyosarcoma) according to the procedure described in Meager, A. 1991. J Immunol Meth 144: 141-143. The serial dilutions of the TNF-alpha internal standard (prepared using WHO TNF-alpha standard 87/650) and TNF-alpha analogues were prepared in the microtiter plates. In each well 2x10⁴ cells in 50 μl culture medium were added. After 18 -19 hours of incubation (37 °C, 5 % CO₂)_, 20 μl of MTT stain (Thiazolyl blue) was loaded in each well of the plate and the plate was incubated again for 1 hour. Insoluble formazan crystals, which were formed in the metabolically active cells, were dissolved with 10 % SDS and 0.02 M HCl. The optical density was measured at 590 nm. The cytotoxic activity of the proteins was determined using the same principle as described for immobilized cells.

using mouse and human cell lines. (Estimation': due to low number of the parallel samples only estimation (and not the real value) could have been obtained.)

The results in Table 1 show that the deletion of the amino acid region ValArgSerSerSerArg from TNF-alpha or from TNF-alpha analogue leads to the increased in vitro cytotoxicity at least with one cell line. In accordance with this result, the corresponding effects and/or modulation of biological activity in vivo may be expected.

Claims

Patent claims

1. A protein analogue, wherein the protein analogue differ from the starting protein in one or more regions (Arg/Lys)XYZ(Arg/Lys).

2. The protein analogue according to claim 1 in which: one or more regions (Arg/Lys)XYZ(Arg/Lys) was/were deleted and/or one or more regions (Arg/Lys)XYZ(Arg/Lys) was/were introduced.

3. The protein analogue according to claim 2, wherein the starting protein is selected from the group comprising: growth hormones, growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins, alpha-1-antitrypsin; insulin A-chain; insulin B-chain, proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagons; clotting factors; anti-clotting factors; atrial natriuretic factor; lung surfactant; plasminogen activators; bombazine; thrombin; ligands of tumor necrosis factor family; RANTES; macrophage inflammatory protein; relaxin A-chain; relaxin B- chain, prorelaxin; gonadotropin-associated peptide; Dnase; inhibin; activin; vascular endothelial growth factor; receptors for hormones or growth factors; integrins; rheumatoid factors; neurotrophic factor; nerve growth factor; platelet- derived growth factor; fibroblast growth factor; epidermal growth factor; transforming growth factor; insulin-like growth factor-l and-ll; insulin-like growth factor binding proteins; CD proteins; erythropoietin; thrombopoietin; osteoinductive factors; a bone morphogenetic protein; an interferon; colony stimulating factors; interleukins; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; therapeutic antibodies; and biologically active fragments or variants thereof.

4. The protein analogue according to claim 3, wherein the starting protein is selected from the group comprising TNF-alpha and TNF-alpha analogues.

5. The protein analogue of claim 4, wherein the analogue is a TNF-alpha analogue.

6. TNF-alpha analogue in which the N-terminal region ValArgSerSerSerArg is deleted and the mutation Glu107Lys is introduced.

7. TNF-alpha analogue according to claim 6 in which mutations Ser95Cys in Gly148Cys are introduced

8. TNF-alpha analogue in which the N-terminal region ValArgSerSerSerArg is deleted and the mutations Ser95Cys in Gly148Cys are introduced.

9. TNF-alpha analogue according claim 8 in which mutation Glu107Lys is introduced.

10. TNF-alpha analogue in which the N-terminal region ValArgSerSerSerArg is deleted and the mutations Ser95Cys, Gly148Cys in Glu107Lys are introduced.

11. A process of preparation of the proteins analogues, wherein the process comprises deletion of one or more regions (Arg/Lys)XYZ(Arg/Lys) from the starting protein structure and/or introduction of one or more regions (Arg/Lys)XYZ(Arg/Lys) into the starting protein structure.

12. A method for modulation of biological activity, wherein the method comprises a modulated interaction of the protein analogue according to claims from 1 , 6, 8 and 10 with the extracellular matrix or the cell surface components.

13. The method according to claim 12, wherein the extracellular matrix refers to compounds selected from the group comprising laminin and fibronectin.

14. The method according to claim 13, wherein the cell surface components are selected from the group comprising proteoglycans.

15. The method according to claim 14, wherein the proteoglycans are selected from the group comprising glycosaminoglycans.

16. The method according to claim 15, wherein the glycosaminoglycans are selected from the group comprising heparin and heparan sulphate.

17. A pharmaceutical composition comprising a therapeutically effective protein analogue according to claims 1 , 6, 8, and 10 and pharmaceutical auxiliary substances selected from the group comprising diluents, adjuvants and acceptable carriers.

18. The protein analogue according to claim 1 for use as a medicament.

19. The protein analogue according to claim 6 for use as a medicament.

20. The protein analogue according to claim 8 for use as a medicament.

21. The protein analogue according to claim 10 for use as a medicament.

22. Use of protein analogue according to claims 1 , 6, 8, and 10 as an active pharmaceutical ingredient.

23. Use of the protein analogues of claims 1, 6, 8, and 10 for the preparation of medicaments for targeting on the cells selected from the group comprising tumour cells, liver cells and endothelial cells.

24. Use of the protein analogues of the claims 1 , 6, 8, and 10 for the preparation of medicaments for modulation of systemic toxicity and for treatment of cancer.