Interferon analogs

Abstract

The invention relates to the field of medicine. Among others, it relates to biologically active analogs of interferons (IFNs) which show less unwanted side-effects and to the therapeutic uses thereof. Provided is an IFN analog, wherein the moiety mediating binding to its natural receptor is at least functionally disrupted and wherein the analog comprises a signaling moiety capable of mediating intracellular IFN activity, said signaling moiety being provided at its N-terminus, optionally via a linker, with at least one targeting domain capable of binding to a cell surface receptor other than the IFN receptor.

Claims

1. A conjugate comprising a compound of interest conjugated to a targeting domain, said targeting domain comprising the amino acid sequence X.sub.1 SRNLIDX.sub.2 (SEQ ID NO: 21)-linker-X.sub.3SRNLIDX.sub.4(SEQ ID NO: 21), wherein each of X.sub.1, X.sub.2, X.sub.3 and X.sub.4 is a cysteine and wherein the pair of X.sub.1 and X.sub.2 and the pair of X.sub.3 and X.sub.4 can form a bond, such that a bicyclic structure is formed wherein the sequences SRNLID are each part of a ring, and wherein the linker is an amino acid sequence of 2 to 7 amino acid residues.

2. A conjugate comprising a compound of interest conjugated to a targeting domain, said targeting domain consisting of the amino acid sequence X.sub.1SRNLIDX.sub.2 (SEQ ID NO: 21)-linker-X.sub.3SRNLIDX.sub.4 (SEQ ID NO: 21), wherein each of X.sub.1, X.sub.2, X.sub.3 and X.sub.4 is a cysteine and wherein the pair of X1 and X2 and the pair of X3 and X4 can form a bond, such that a bicyclic structure is formed wherein the sequences SRNLID are each part of a ring, and wherein the linker is an amino acid sequence of 2 to 7 amino acid residues.

Description

LEGEND TO THE FIGURES

(1) FIG. 1: cloning of mimetic IFN from the mouse. Splenocytes were stimulated with PHA and RNA was isolated. RT and PCR using specific primers yielded a PCR product of the expected size. Expression of the construct in BL21 cells and Western blotting confirmed production of the mimetic.

(2) FIG. 2: Recombinant mimetic IFN shows anti-fibrotic effects in mouse 3T3 fibroblasts. Cells are stained for -Smooth muscle actin which is a fibrosis marker.

(3) FIG. 3: Construction of pET39b-BiPPB encoding a bicyclic PDGF-receptor targeting domain.

(4) FIG. 4: Construction of pET39b-BiPPB-IFNgamma encoding a PDGFR-targeted IFN conjugate.

(5) FIG. 5: Construction of pET39b-BiPPB-mimeticIFN.

(6) FIG. 6: Dot Blot analysis of IFN-BiPPB and Mimetic IFN-BiPPB showing the coupling of pPB-peptide to INF.

(7) FIG. 7: Binding study in human hepatic stellate cell line LX2 cells. Cells were stained for PDGFR-binding peptide. Staining indicates the binding of pPB-containing constructs to the target cells.

(8) FIG. 8: Blood count analysis to study side effects of the treatment in acute liver injury mouse model. WBC=white blood cell count. LYM=lymphocytes. PLT=platelet count. RBC=red blood cell count. For details see Example 4.

(9) FIG. 9: Real Time PCR analysis of the liver fibrosis markers MMP13 and TIMP1. For details see Example 4.

(10) FIG. 10: Effect of mimetic IFN-PEG-BiPPB in the acute CC14-induced liver-injury mouse model. Quantitation of a-SMA (A), collagen-I (B) and desmin (C)staining of liver sections obtained from olive oil-treated normal mice and CC14-treated mice receiving either IFN, mimetic IFN, mimetic IFN-PEG, mimetic IFN-PEG-BiPPB (5 g/mice) or PBS alone. Quantitation was done using computerized Cell-D imaging software. Data represent the meanSEM from 5 mice per group. #P<0.05 versus PBS-treated olive oil group; *P<0.05, **P<0.01 versus PBS treated-CC14 group. For details see Example 6.

(11) FIG. 11: Real Time PCR analysis of markers for liver fibrosis in mice, 3 days after CCl4 administration or olive oil. Mice were treated with different compounds as indicated and Collagen 1a1 (A), a-SMA (B), Desmin (C) and TIMP1 (D) mRNA levels were measured. For details see Example 6.

(12) FIG. 12: Blood count analysis in whole blood of mice, 3 days after CCl4 administration or olive oil. Mice were treated with different compounds as indicated and Platelet count (PLT: fig A), white blood cell count (WBC, fig B) and lymphocyte count (C) were measured using a coulter counter. For details see Example 6.

EXPERIMENTAL SECTION

(13) Liver Fibrosis is characterized by the excessive accumulation of the extracellular matrix components that leads to hepatic scars. To date, no successful therapy is available for the treatment of liver fibrosis. Hepatic stellate cells (HSCs) and fibroblasts are the key effector cells involved in the progression of the disease, which are activated by crucial growth factors like Platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF). Interferon gamma (IFN) has been shown to have various beneficial effects in-vitro and in-vivo during liver fibrosis. However, IFN displays a strict species specificity and has a short circulating half life which limits its potential clinical use. Moreover, INF has serious adverse effects on the immune system, on endothelial cells and on the Central Nervous System (e.g. causing depressions) that all lead to frequent withdrawal of patients from clinical trials and consequently to failure of these trails. To circumvent these drawbacks, the present inventors produced a stable peptide mimetic of IFN that lacks the extracellular receptor recognition site and contains a signalling moiety which interacts directly at the downstream IFN signalling cascade, thereby retaining the prospective functions of IFN.

(14) To increase the specificity of IFN and IFN mimetic peptide, IFN and mimetic peptide fused to BiPPB (Bicyclic peptide against the PDGF-beta-receptor) were been generated, since PDGF receptor expression is highly upregulated during liver injury particularly in HSCs.

Example 1: Cloning of Mimetic IFN

(15) Mouse splenocytes were isolated from fresh spleens and were cultured in the presence of PHA (Phytohemagglutinin) to stimulate cytokine production. After 24 hrs of stimulation, RNA was isolated and cDNA was synthesised using gene specific reverse primer followed by PCR amplification by phusion DNA polymerase using mimetic IFN specific forward and reverse primers. The obtained fragment was cloned in pET42a (prokaryotic expression vector) at PshA1/EcoRI site and the positive clones were checked by restriction digestion analysis. See FIG. 1.

(16) The 5->3 nucleotide sequence of the truncated mouse Interferon gamma (NCBI Reference sequence: NM_008337.2) (nt 457-nt 572) is as follows:

(17) TABLE-US-00006 (SEQIDNO:49) 457gccaagtttgaggtcaacaacccacaggtccagc gccaagcattcaatgagctcatccgagtggtccaccagct gttgccggaatccagcctcaggaagcggaaaaggagtcgc tg572a.

(18) The last nucleotide has been added in order to insert the stop codon before cysteine, which is the last amino acid in the sequence. Cysteine is removed from the sequence to provide appropriate folding of the peptide and also to avoid inappropriate folding due to disulfide bonds in the fusion protein (with BiPPB).

(19) The encoded amino acid sequence is

(20) TABLE-US-00007 (SEQIDNO:50) AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR* *Denotes Stop codon

(21) Cloning of IFN-BiPPB and mimetic IFN-BiPPB A nucleic acid sequence encoding the bicyclic PDGF targeting domain BiPPB was generated by amplification of two fragments using 4 primers (2 for each fragment) and then ligated using inbuilt Bam HI restriction site and was then cloned in pET39b vector at ScaI/NotI site. IFN and Mimetic IFN was PCR amplified using peptide fusion primer (forward) and IFN or mimetic IFN reverse primer. The amplified fragment was digested and ligated in pET39b-BiPPB vector at NotI/XhoI site and the positive clones were checked by restriction digestion analysis. See FIGS. 4 and 5. The sequences of all the constructs were further confirmed by automated DNA sequencing.

(22) BiPPB

(23) Nucleotide sequence for BiPPB:

(24) TABLE-US-00008 (SEQIDNO:51) tgttctagaaacctcatcgattgtaagggatccgga ggttgttcacgtaatctaatagattgttca

(25) Amino acid sequence for BiPPB: CSRNLIDCKGSGGCSRNLIDCS (SEQ ID NO: 52) (see also FIG. 3)

(26) Interferon Gamma (Full Length)

(27) Nucleotide sequence: mouse Interferon gamma (NCBI Reference sequence: NM_008337.3)

(28) TABLE-US-00009 (SEQIDNO:53) gcggccgca457gccaagtttgaggtcaacaaccca481 caggtccagcgccaagcattcaatgagctcatccgagtgg tccaccagctgttgccggaa541tccagcctcaggaagcggaa aaggagtcg569ataa

(29) Amino Acid Sequence for Mouse IFNgamma

(30) TABLE-US-00010 (SEQIDNO:54) MNATHCILALQLFLMAVSGCYCHGTVIESLESLNNYFNSSGIDVEEKSLF LDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLIT TFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLR KRKRSRC.

(31) Nucleotide sequence of the fused protein (BiPPB-IFN gamma) tgt tct aga aac ctc atc gat tgt aag gga tcc gga ggt tgt tca cgt aat cta ata gat tgt tca gcggccgca (SEQ ID NO: 55) Interferon gamma sequence (NM_008337.3).

(32) Amino Acid Sequence for BiPPB-IFNgamma

(33) TABLE-US-00011 (SEQIDNO:56) CSRNLIDCKGSGSGGCSRNLIDCSAAAMNATHCILALQLFLMAVSGCYC HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQII SFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEV NNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC

(34) Italics denotes BiPPB; normal text denotes IFNgamma mimetic; Bold denotes linker or spacer. See also FIG. 4.

(35) Mimetic Interferon Gamma Fused to BiPPB

(36) Nucleotide sequence of the fused protein (BiPPB-IFN gamma mimetic)

(37) TABLE-US-00012 (SEQIDNO:57) tgttctagaaacctcatcgattgtaagggatccgga ggttgttcacgtaatctaatagattgttcagcggccgca 457gccaagtttgaggtcaacaacccacaggtccagc gccaagcattcaatgagctcatccgagtggtccaccagct gttgccggaatccagcctcaggaagcggaaaaggagtcg 569ataa.

(38) Amino Acid Sequence for BiPPB-IFNgamma Mimetic

(39) TABLE-US-00013 (SEQIDNO:58) CSRNLIDCKGSGSGGCSRNLIDCSAAA AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR*.

(40) Italics denotes BiPPB; normal text denotes IFNgamma mimetic; Bold denotes: linker or spacer. See also FIG. 5.

(41) The nucleic acids were then transformed into BL21 cells (E. coli) for the expression using IPTG induction. The expressed protein was analysed by SDS-PAGE and Western blot analysis or Dot Blot analysis using anti-IFN antibody and/or anti-PPB antibody (see FIG. 6). The expressed protein (with His Tag) was then purified under native conditions through Ni-NTA column chromatography. The tags were proteolytically cleaved and further purified using sepharose column chromatography.

Example 2: Anti-Fibrotic Effects of Cleaved and Active Mimetic IFN

(42) We evaluated the anti-fibrotic effects of cleaved IFN mimetic peptide in mouse NIH3T3 fibroblasts as assessed by immuno-cytochemistry (-SMA staining). Briefly, 3T3 fibroblasts were seeded in 24 well plates at the density of 610.sup.4 cells/well, After 24 hrs, cells were starved in 0.5% FBS containing medium for overnight. Thereafter, cells were incubated in starvation medium with different compounds (PDGF 50 ng/ml, TGF 10 ng/ml, mimetic IFN 1 g/ml, 50 ng/ml PDGF+1 g/ml mimetic IFN and 10 ng/ml TGF+1 g/ml mimetic IFN). After 48 hrs of incubation, cells were washed, fixed with ethanol:acetone (1:1) and stained for -SMA (marker of activated fibroblasts).

Example 3: Binding Study of BiPPB and Mimetic IFN-BiPPB in Human LX2 Cells

(43) We determined the binding of BiPPB and mimetic-BiPPB in LX2 cells (human HSCs). Briefly, LX2 cells were plated in 48 well plates at the cell density of 310.sup.4 cells/well. After 24 hrs, cells were starved in medium (-FBS) for overnight. Then, cells were incubated with different compounds (BiPPB, PPB-HSA and BiPPB-Mimetic IFN) for 2 hrs at room temperature for binding. After binding, cells were extensively washed with PBS, fixed with ethanol:acetone (1:1) and stained for PPB. Results are presented in FIG. 7.

Example 4: In-Vivo Effect Study in Acute CCl4-Induced Liver Injury in Rake

(44) Recombinant IFN, mimetic IFN, recombinant fusion protein IFN-BiPPB and recombinant fusion protein mimetic IFN-BiPPB were tested for anti-fibrotic effects in acute CCL.sub.4-induced liver injury mouse model. At day 1, the animals were given a single intra-peritoneal dose (1 ml/kg) of carbon tetrachloride (CCl.sub.4) in olive oil or olive oil (controls n=6). After 24 hrs of CCl.sub.4 injection, at day 2 and 3, animals were treated either with PBS (n=6), 50,000 U/mice of IFN (n=6), 50,000 U/mice of mimetic IFN (n=5), 50,000 U/mice of IFN-BiPPB (n=6), 50,000 U/mice of Mimetic IFN-BiPPB (n=6). Thereafter, at day 4, animals were sacrificed and blood counts were performed and anti-fibrotic effects (See Hemmann S, Graf J, Roderfeld M, Roeb. J Hepatol. 2007 May; 46(5):955-75) were evaluated using quantitative PCR. Results are presented in FIGS. 8 and 9.

(45) The data presented in FIG. 8. demonstrate that mimetic-BiPPB is more effective than unmodified INF in attenuating the upregulation of white blood cell count (WBC) and lymphocyte count (lym) in whole blood associated with CCl.sub.4-induced liver fibrosis (p<0.01). In contrast, the reduction in platelet count (PLT) associated with INF treatment (which is a well known adverse effect of INF) is less severe when animals receive mimetic-INF-BiPPB instead of unmodified INF (p<0.0025).

(46) The data presented in FIG. 9 show that mimetic-INF-Bi-PPB is endowed with antifibrotic activity: it attenuates CCl.sub.4-induced upregulation of matrix metalloproteinases (MMP13). The ratio MMP-13 versus Tissue Inhibitor of Metalloproteinases (TIPM1) is similar to that obtained with native INF, but better than mimetic INF (p<0.03), indicating that coupling of Bi-PPB to mimetic INF is beneficial. Collectively the data in FIGS. 8 and 9 show that mimetic INF-BiPPB is more potent compared to native INF and has less side effects.

Example 5: Chemical Synthesis of Mimetic Interferon Gamma Conjugates

(47) Mimetic IFN-PEG-BiPPB Conjugate:

(48) 0.111 mol Bicyclic PDGFR recognizing peptide (BiPPB, 2223.2 Da, Genosphere Biotechnologies) was coupled with 0.337 mol maleimide-PEG-succinimidyl carboxy methyl ester (Mal-PEG-SCM, 2 KDa, Creative PEGworks) for 3 hrs. Thereafter, lysine (0.337 mol) was added to block free groups of Mal-PEG-SCM. After 1 hr of reaction, the synthesized product BiPPB-PEG-MAL (0.112 mol) was reacted with 0.56 mol of Mimetic IFN-ATA (4689 Da, Genosphere Biotechnologies) in the presence of deacetylating reagent for overnight at room temperature. Finally, the synthesized Mimetic IFN-PEG-BiPPB conjugate (8828.2 Da) was extensively dialyzed against PBS using 7 KDa slide-a-lyzer G2 dialysis cassettes (Thermo scientific).

(49) Mimetic IFN-PEG Conjugate.

(50) 0.107 mol Mimetic IFN-ATA (4689 Da) was reacted with 0.321 mol of Poly (ethylene glycol)-succinimidyl -methylbutanoate (mPEG-SMB, 2 KDa, Nektar therapeutics) for 2 hrs and subsequently the product was dialyzed extensively.

(51) a) Mimetic IFN-PEG-BiPPB

(52) ##STR00001##

(53) b) Mimetic IFN-PEG

(54) ##STR00002##

Example 6: Effect on Fibrotic Parameters after Intravenous Administration of mimeticIFN-PEG-BiPPB in Acute Liver Injury Mouse Model

(55) Analysis of Fibrotic Parameters at the Protein Level:

(56) Mice were intraperitoneally injected with CCl.sub.4 at day 1 to induce liver injury. At day 2 and 3, mice were treated with IFN (5 g/dose), mimetic IFN-PEG, mimetic IFN-PEG-BiPPB (5 g/dose) or PBS alone. At day 4, animals were sacrificed; livers and different organs were collected for further analysis. Liver-sections were fixed with acetone, dried and rehydrated with PBS. Then, the sections were incubated with primary antibody (collagen, SMA and Desmin) for 1 hr. Thereafter, the sections were blocked with 0.03% H.sub.2O.sub.2 for endogenous peroxidase activity for 30 min. Subsequently, sections were incubated with secondary antibody HRP conjugated rabbit anti-goat antibody (1:100, DAKO) followed by HRP conjugated goat anti-rabbit antibody (1:100, DAKO) for 30 min. The peroxidase activity was developed using AEC (Sigma) for 20 min and nuclei were counterstained with hematoxylin (Fluka). The sections were mounted with Kaiser's gelatin and visualized under the light microscope (Olympus). For quantitative analysis, 27 microscopic pictures were captured and positively-stained areas were quantified using computerized Olympus Cell D imaging software. Results are shown in FIG. 10

(57) Analysis of Fibrotic Parameters at the Gene Expression Level:

(58) Total RNA from liver tissues was isolated using RNeasy mini kit (Qiagen) according to the manufacturer's instructions. The RNA concentration was quantitated by a UV spectrophotometer (NanoDrop Technologies, Wilmington, Del.). Total RNA (1.6 g) were used for reverse transcription in total volume of 50 l with the cDNA synthesis kit (Promega). All primers were purchased from Sigma-Genosys (Haverhill, UK). 10 ng of cDNA was used for quantitative real time PCR analysis. The reactions were performed using SYBR green PCR mix (Applied Biosystems) according to manufacturer's instructions. The samples were analyzed by ABI 7900HT sequence detection system (Applied Biosystems). Finally, the threshold cycle numbers (Ct) were calculated for each gene and relative gene expression was calculated after normalizing for expression of the reference gene GAPDH. Results are shown in FIG. 11. Analysis of adverse effects is depicted in FIG. 12.

Example 7: Effect on Fibrotic Parameters after Intravenous Administration of mimeticIFN-PEG-BiPPB in Established Advanced Liver Fibrosis Mouse Model

(59) Analysis of Fibrotic Parameters at the Protein Level:

(60) Male balb/c mice (20-22 g) were treated with olive oil or increasing doses of CCl.sub.4 (week 1: 0.5 ml/kg; week 2: 0.8 ml/kg and week 3-8: 1 ml/kg prepared in olive oil) twice weekly by intra-peritoneal injections for 8 weeks. In week 7 and 8, mice were treated intravenously with PBS, mimeticIFN-PEG or MimeticIFN-PEG-BiPPB (5 g/mice, thrice per week). All mice were sacrificed at week 8; blood and liver samples were collected for subsequent measurements. The liver sections were stained for Collagen I and desmin, CD68, 33D1 and MHC class II. It was found that the targeted truncated form of IFN (mimIFN-biPPB) induced substantial reduction in the fibrotic parameters in this chronic liver fibrosis model in mice; both collagen I and desmin staining was profoundly reduced in mimIFN-biPPB-treated CCl4 mice compared to untreated CCl4-mice. In contrast, treatment with untargeted mimIFN, lacking the receptor binding site, induced no effect on these parameters while full length mouse IFN induced only a small reduction in collagen I and desmin staining. The native (mouse) IFN induced infiltration of inflammatory cells (CD68.sup.+ macrophages, neutrophils, 33D1.sup.+ dendritic cells) as well as increased MHCII expression. In contrast, MimIFN-BiPPB did not induce this increased inflammatory response in livers.

Example 8: Truncated IFN Analog Targeted to the PDGF Receptor is Active but Causes Less Side Effects

(61) A targeted conjugate of BiPPB chemically coupled mimetic-IFN was synthesized and characterized using Western blot analyses and for its anti-fibrotic effects in vitro in mouse 3T3 fibroblasts. In vivo, the targeted conjugate was examined in 4 days (acute) and 8 weeks (chronic) liver fibrosis models induced with CCl.sub.4 in mice. Several fibrotic parameters and infiltration of inflammatory cells were assessed in the livers using immunohistochemistry and gene expression analysis.

(62) Results:

(63) The successfully synthesized conjugate caused inhibition of collagen expression in TGFbeta-induced mouse fibroblasts. In vivo, the targeted peptidomimetic of IFN (mimIFN-biPPB) induced substantial reduction in the fibrotic parameters in both acute and chronic liver fibrosis models in mice. Treatment with untargeted mimIFN, which lacks a receptor binding domain, showed no effect and unmodified mouse full length IFN showed only a moderate reduction. This mouse full length IFN induced infiltration of inflammatory cells (CD68.sup.+ macrophages, neutrophils, 33D1.sup.+ dendritic cells) as well as increased MHCII expression. In contrast, MimIFN-BiPPB did not induce an inflammatory response (data not shown).

Example 9: Study with MimeticIFN-PEG-BiPPB in Subcutaneous Tumor Model in Mice

(64) Materials and Methods

(65) Normal male C57BL/6 and Balb/c mice weighing 20 to 25 g were obtained from Harlan (Zeist, the Netherlands). They were kept at a 12:12-hour light/dark cycle and received ad libitum normal diet. All experimental protocols for animal studies were approved by the Animal Ethics Committee of the University of Groningen. To induce subcutaneous tumors, C26 cells were cultured in 125-mm.sup.3 flasks a day before injection in animals to keep them in the growth phase. Cells were detached by trypsanization, and trypsin was removed by centrifugation. The cell pellet was resuspended in PBS. A total of 110.sup.6 cells (B16 and C26 cells) suspended in 100 l of PBS were injected subcutaneously in the flank of Balb/c mice. Tumor growth was followed by measuring tumor size using a digital Vernier caliper. Tumor volume was established using the formula: ab2/2, where a denotes tumor length and b denotes the tumor width. C26 tumors were induced in mice as described. The treatment was started on day 5 when the tumor volume was reached the range of 50 to 100 mm.sup.3 because this tumor size has been shown as an optimum tumor size for the start of the treatment. Animals (n=4 per group) were injected intravenously with six doses of either vehicle (PBS), mimeticIFN-PEG (5 g/dose), mimeticIFN-PEG-BiPPB (5 g/dose) on alternative days under anesthesia (02/isoflurane). Tumor size was measured under anesthesia. The animals with C26 tumors were killed on day 20 because no effect of the treatment was observed. Animals were killed under gas anesthesia (O.sub.2/isoflurane), and tumors were isolated and fixed in cold isopentane for cryosections.

(66) 4-m-thick cryostat sections were prepared from snap-frozen tissue and stained for CD31 according to standard immunoperoxidase methods. We analyzed CD31 staining (endothelial cell marker) for the determination of the blood vessel lumen area and blood vessel density in tumor sections of C26 tumors. Results showed significant angiogenesis in untreated tumors and in tumors of mice treated with mimetic IFN, while mice treated with mimetic IFN-BiPPB displayed a strong reduction in angiogenesis in their tumors (data not shown).

Example 10: Effect of PDGF-Receptor Targeted Truncated INF on Pulmonary Fibrosis

(67) Background and Rationale for INF-Based Therapies in IPF Patients:

(68) Idiopathic pulmonary fibrosis (IPF) is a progressive parenchymal lung disease with a median survival of only 3-5 years following diagnosis.sup.1, 2. Besides IPF also other types of lung fibrosis have a poor prognosis, in particular fibrosing non-specific interstitial pneumonia (NSIP) and end-stage fibrosis of for example several auto-immune diseases and extrinsic allergic alveolitis. There are currently no effective therapies for lung fibrosis due to poor understanding of the disease mechanisms.

(69) The (myo)fibroblast has a central role in all types of lung fibrosis.sup.3. Ongoing damage to the lungs leads to initiation of dysregulated repair mechanisms with recruitment of fibroblasts and their transformation to myofibroblasts. Myofibroblasts are the key effector cells in fibrosis producing excessive amounts of extracellular matrix components like collagens. Myofibroblasts are currently thought to be the most important therapeutic target for treatment of lung fibrosis.sup.3. Key growth factors in the proliferation and transformation of myofibroblasts are transforming growth factor beta (TGF), platelet-derived growth factor (PDGF), and endothelin-1 and many new therapies have focussed on inhibiting these factors or their receptors. However, most of the clinical trials based on modulating myofibroblast behaviour have shown disappointing results.sup.1.

(70) Interferon gamma (IFN) is probably the most-studied anti-fibrotic mediator in clinical trials of IPF. It mediates myofibroblast growth arrest and apoptosis through a STAT-1 (Signal Transducers and Activators of Transcription-1)-dependent pathway, which is important for resolution of fibrogenic responses.sup.4. Despite an initial promising start, the large INSPIRE trial concluded that IFN did not prolong survival5. IFN, however, was administered subcutaneously and since IFN receptors are found on almost all cells in the body, it has severe doselimiting side effects. These include influenza-like illness and fatique.sup.5. Inhaled administration can partly circumvent this problem and one trial is currently registered to study the effects of aerosolized IFN in IPF.

(71) Another way to increase the concentration of IFN within myofibroblasts, and thus the efficacy, is to use the concept of drug targeting: drugs are coupled to cell-selective drug carriers that are specifically taken up by target cells, the drugs are released within those cells thereby reducing systemic side effects while attaining high local concentrations in target cells. Myofibroblasts for instance specifically upregulate PDGF-receptors that can be used for drug targeting purposes.sup.6.

(72) Although CCl.sub.4 induces liver fibrosis, the lungs also have low expression of Cytochrome P450 activity and CCL.sub.4 (turned into toxic compounds by this enzyme) is therefore also slightly activated in lung tissue. These leads to activation of profibrotic cells which in turn express the PDGF receptor, which initiates profibrotic activity. This model has been used to as a model of idiopathic pulmonary fibrosis (IPF, see ref 8 and 9). While exploring the antifibrotic effects of INF in liver tissue, a significant change in lung tissue was noted by the examiners. The weight of lungs from CCl.sub.4-treated mice was significantly higher than lungs of normal mice (0.750.05% of body weight in normal versus 2.030.15% of b.w. in untreated CCl4-treated mice; P<0.01) yet this increase was significantly reduced by treatment with PPB-PEG-INF (1.530.11% of b.w.; P<0.05 vs untreated CCl4 mice). Microscopical examination revealed that the lungs of CCl.sub.4-treated mice were affected by diffuse alveolitis, a condition which can precede fibrosis, and lungs displayed an enhanced collagen staining. When these mice were treated with IFN coupled to PDGF-receptor recognizing peptides, we found a significantly reduced alveolitis in lungs and a reduced collagen deposition. Of note, these beneficiary effects of targeted IFN were attained after establishing the lung disease.

(73) We therefore conclude that PDGF-receptor-targeted INF is able to accumulate in any tissue with significant PDGF-receptor expression, and is able to exert an antifibrotic effect in other tissues as well as illustrated by its effect in lungs. The INF analogs of the invention may thus be used for the treatment of idiopathic fibrosis and other forms of fibrosis or sclerosis in other tissues characterized by enhanced expression of the PDGF-receptor.

Example 11: Targeting of Truncated IFNgamma to Various Receptors

(74) In this experiment it is shown that an analog of the invention comprising the signaling moiety of murine IFN (mimIFN) and a cell surface targeting domain can be efficiently targeted not only to the PDGF receptor but also to other cell surface receptors. Exemplary targeting domains tested include lactose (ligand for the Asialoglycoprotein (ASGP)), mannose (ligand for the mannose receptor (CD 206) and the tripeptide RGD (ligand for the receptor v3 integrin receptor). The sequence of mimIFN consisted of

(75) TABLE-US-00014 (SEQIDNO:36) FEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR.
Various cell types and different parameters for testing IFN activity used in the study. The control samples were exposed to intact murine INN. For details see the Table below.

(76) TABLE-US-00015 TABLE 1 Parameter measured Target (details IFN analog receptor Cells used see text) Results Lactose- Asialoglyco- Human ICAM 1 enhanced HSA-PEG- protein Hepatocytes expression expresion vs mimIFN (ASGP) (HepG2) control INF.sub.Y receptor Mannose- mannose Mouse RAW MHC II equal HSA-PEG- receptor macrophages expression expression vs mimIFN (CD 206) control INF.sub.Y RGD-PEG- v3 - Endothelial In vitro Enhanced mimIFN receptor cells angiogenesis effectivity vs assay control INF.sub.Y

(77) Experiments with Lactose-HSA-PEG-IFN:

(78) Synthesis

(79) Lactose-HSA (25 lactose molecules coupled to human serum albumin) was conjugated to bifunctional PEG molecule (2 KDa). Following dialysis, lactose-HSA-PEG was coupled to SATA-modified truncated IFN derived from mouse. The synthesized product (Lac-HSA-PEG-mimIFN) was extensively dialysed against PBS.

(80) Experiment in Human Hepatocytes.

(81) Human Hepatocytes (HepG2) were plated in 12 well plates (110.sup.5 cells/well). The cells were grown overnight in 5%/37 C./CO.sub.2 incubator. Subsequently, cells were incubated with medium, mouse IFN (1 ug/ml), Human IFN (1 ug/ml), mouse IFN derived Lac-HSA-PEG-IFN (1 ug/ml), Lac-HSA or Lac-HSA-PEG-IFN (1 ug/ml) after 2 hrs of blocking with Lac-HSA. After 24 hrs of incubation, cells were lysed, RNA was isolated and reverse transcribed. The cDNA was used for the analysis of Inter-Cellular Adhesion Molecule 1 (ICAM1) expression, which is known to be induced in response to IFN. 18srRNA was used a housekeeping control.

(82) Results:

(83) As expected, neither mouse truncated IFN nor Lac-HSA induced ICAM1 expression in human hepatocytes, while both human IFN and mouse derived Lac-HSA-PEG-IFN upregulated ICAM1 expression in human hepatocytes. Furthermore, Lac-HSA-PEG-IFN-induced ICAM1 expression was almost completely blocked by excess of Lac-HSA.

(84) Conclusions:

(85) The species-specificity of INF was confirmed by showing that mouse (truncated) INF was ineffective in human hepatocytes. However, this cytokine was turned into a bioactive compound in human cells after coupling to the target moiety lactose, which is known to enter hepatocytes via the Asialoglycoprotein (ASGP) receptor. Specificity for the ASGP-receptor was shown by blockade of this receptor by Lac-HSA. This demonstrates that the signaling part of INF (which is not species specific) can be delivered into the cytoplasm of other target cells than fibroblasts via another target receptor than the PDGF-receptor using a sugar moiety instead of a peptide. Hepatocyte targeting is particularly relevant for the treatment of Hepatitis B and C and ICAM-1 upregulation is a physiological response to enhance antiviral activity. This experiment supports the use of cell-specific-truncated INF according to the invention as an antiviral compound.

(86) Similarly, mannose was coupled to truncated mouse INF according to standard techniques (L. Beljaars et al J. of Hepatology 29: 579-588, 1998) and bioactivity of this mannosylated INF was demonstrated in mouse macrophages with MHC class II expression as read-out parameters (rtPCR methods).

(87) The endothelial binding peptide RGD- or its control peptide RAG, that does not bind to these cells, were also coupled to truncated mouse INF according to standard methods. The resulting analogs were evaluated for their biological activity in cultures of endothelial cells (H5V) by measuring tube formation which is a parameter reflecting angiogenesis. Tube formation by H5V cells was inhibited most potently by RGD-mimINF in this assay (data not shown).

(88) Conclusions:

(89) Modification of truncated mouse INF by conjugation to a targeting moiety (e.g. an oligosaccharide or peptide) is feasible without disturbing the bioactivity of the signaling part of this cytokine. Delivery of an INF analog which is defective in binding to its natural receptor but which instead can bind to a distinct cell surface receptor mediating cellular uptake of the analog allows for more effective therapeutic applications with less side-effects.

REFERENCES

(90) 1. du Bois R M. Strategies for treating idiopathic pulmonary fibrosis. Nat Rev Drug Discov; 9(2):129-40. 2. Wilson M S, Wynn T A. Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol 2009; 2(2):103-21. 3. Scotton C J, Chambers R C. Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest 2007; 132(4):1311-21. 4. Bonner J C. Mesenchymal cell survival in airway and interstitial pulmonary fibrosis. Fibrogenesis Tissue Repair; 3:15. 5. King T E, Jr., Albera C, Bradford W Z, et al. Effect of interferon gamma-1b on survival in patients with idiopathic pulmonary fibrosis (INSPIRE): a multicentre, randomised, placebo-controlled trial. Lancet 2009; 374(9685):222-8. 6. Beljaars L, Weert B, Geerts A, Meijer D K, Poelstra K. The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue. Biochemical pharmacology 2003; 66(7):1307-17. 7. Homma S, Nagaoka I, Abe H, et al. Localization of platelet-derived growth factor and insulin-like growth factor I in the fibrotic lung. Am J Respir Crit Care Med 1995; 152(6 Pt 1):2084-9. 8. Paakko P, Anttila S, Sormunen R, et al. Biochemical and morphological characterization of carbon tetrachloride-induced lung fibrosis in rats. Arch Toxicol 1996; 70(9)(540-52. 9. Mizuguchi S, Takemura S, Minamiyama Y, et al. S-allyl cysteine attenuated CCl4-induced oxidative stress and pulmonary fibrosis in rats. Biofactors 2006; 26(1):81-92.

Example 12: Binding Study of BiPPB to Cultured or Freshly Isolated Primary Cells

(91) This example shows that Bi-PPB either produced chemically or through recombinant techniques specifically binds to the PDGF-receptor. Receptor specificity is demonstrated by blocking the binding with specific antiPDGF--receptor antibodies. BiPPB is species non-specific as it binds to Myo-fibroblast-like cells of rat, mouse and human. Receptor interaction requires at least two cyclic peptides, as the monocyclic form does not bind to the target receptor.

(92) Sequence of BiPPB:

(93) TABLE-US-00016 C(1)SRNLIDC(1)GGGDGGC(2)SRNLID C(2)(SEQIDNO:59): Cys(1)-Cys(1)andCys(2)-Cys(2)disulfidebridge cyclisations

(94) Methods:

(95) The binding of BiPPB was performed in primary freshly isolated rat hepatic stellate cells. Cells were seeded in the 8-well glass plates (Lab-Tek, Nunc, Naperville, Ill.) at 30,000 cells/well in the culture medium. After overnight incubation at 37 C./5% CO2, cells were washed with PBS and subsequently incubated with FITC-labeled PPB (monocyclic) or BiPPB (bicyclic: 10 g/ml) at room temperature. To block the binding, anti-PDGF-R IgG was added to the cells 1 h before FITC coupled PPB or BiPPB. After 2 h, cells were washed 3 to 4 times with cold PBS and fixed with 4% paraformaldehyde. The nuclei were counterstained with DAPI and mounted in citifluor (anti-fade reagent) and visualized under fluorescent microscope.

(96) Similar binding experiments were performed in primary freshly isolated human myofibroblasts, mouse 3T3 fibroblasts and human hepatic stellate cells (LX2). For human Hepatic stellate cells, the sequence of BiPPB was as follows:

(97) TABLE-US-00017 C(1)SRNLIDC(1)KGSGSGGC(2)SRNLIDC(2) (SEQIDNO:60): Cys(1)-Cys(1)andCys(2)-Cys(2)disulfidebridge cyclisations

(98) Results:

(99) The FITC-coupled monocyclic PPB did not show any binding as the PDGF-receptor requires dimeric interaction. FITC-coupled BiPPB showed significant binding to the cell type tested, which was almost completely blocked by PDGF receptor antibody, showing the receptor specificity of the binding to these cells (data not shown).