Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

• the invention concerns a method for diagnosing a cancer type, whereby the expression of anti-apoptotic cytokines in the tumour cells is determined.
• the differential diagnosis of the present invention is used to classify tumour disorders and to recommend the required treatment and to monitor the progress and response to the treatment.
• the balance between cell survival and cell death is controlled by pro- apoptotic and anti-apoptotic factors, whose dysregulation contributes to the development of several pathological conditions, including cancer.
• High expression of anti-apoptotic factors is commonly found in human cancers and contributes to both neoplastic cell expansion and resistance to the therapeutic action of cytotoxic drugs. It has already been reported that autocrine production of anti-apoptotic cytokines by tumour cells strongly modulates the susceptibility to the receptor and chemotherapy-induced apoptosis.
• IL-4 and IL-10 act as autocrine growth factor in cancer cells inducing upregulation of anti- apoptotic proteins, which protect the tumour cells from the death induced by chemotherapeutic drugs (Stassi et al., Cancer Res. 63, 6784-90 (2003), Todaro et al., Cancer Res. 66, 1491-9 (2006)).
• Tumours are composed of a heterogeneous combination of cells, with different therapeutic characteristics and different proliferative potentials.
• cancer cells may give rise to phenotypically diverse progeny of cells, either endowed with a definite proliferative potential or having a limited or no proliferative potential.
• cancer stem cells CSC
• CSC cancer stem cells
• the International Application PCT/IT2005/000523 discloses a method for isolation and culturing of stem cells from solid tumours. This subpopulation of cancer cells can self-renew and give rise to a population of heterogeneous cells which exhibit diverse degrees of differentiation. Moreover, it has recently been found that these cancer stem cells are significantly resistant to drug-induced apoptosis, thus escaping anti-tumour therapies and this being probably the underlying reason for chemotherapy inefficiency.
• CSC predominantly produce IL-4 and IL- 10 and are responsible for the above mentioned alteration of sensibility to drug-induced cell death.
• solid tumours may be differentiated in respect of anti-apoptotic cytokine expression level and/or profile.
• the expression of anti-apoptotic cytokines differs between individual tumours of the same organ and even within cells or portions of a single tumour.
• an object of the present invention was to provide a method which allows the identification and diagnosis of cancer types and cancer cells which express anti-apoptotic cytokines.
• the present invention provides a method for diagnosing tumour types, especially solid tumour types, using the anti-apoptotic cytokines as a target.
• the invention refers to a method for diagnosing a cancer type comprising the steps of:
• tumour cells (a) providing a sample from a solid tumour comprising tumour cells, (b) determining the expression of at least one anti-apoptotic cytokine in said tumour cells, and
• the invention concerns the differential diagnosis of cancer types by means of the determination and/or quantification of the expression profile and/or level of anti-apoptotic cytokines in the tumour sample.
• anti- apoptotic cytokines IL-4 and/or IL-10, particularly IL-4, is preferred.
• the differential diagnosis according to the invention allows to classify tumour types and to identify those which show expression of anti-apoptotic cytokines and which are refractory to treatment with chemotherapeutic agents.
• the expression of anti-apoptotic cytokines is a significant marker for tumour classification which allows a selection of targeted therapeutic strategies.
• the method of the invention may be useful to predict whether a patient suffering from a certain cancer type would be resistant or susceptible to a certain therapy and to provide an optimised treatment strategy.
• cancer types can be classified as non-cytokine-expressing tumours or as cytokine-expressing tumours.
• the solid tumours When determining the expression of IL-4 and/or IL-10, more particularly, the expression of IL-4, the solid tumours may be classified with regard to their expression of either only IL-4 or only IL-10 or both IL-4 and IL-10. Therefore, the method according to the present invention allows the differentiation between solid tumour classified as IL-4-expressing tumours or IL-4 non- expressing tumours, solid tumour classified as IL-10-expressing tumours or IL-10 non-expressing tumours and solid tumour classified as IL-4 and IL-10- expressing tumours or non-IL-4 and non-IL10 expressing tumours.
• the method of the present invention is preferably performed on solid tumours and in particular on epithelial tumours.
• Said epithelial tumours may be chosen from the group consisting of thyroid, breast, prostate, bladder, colon, gastric, pancreas, kidney, liver and lung cancer. More preferably, the epithelial tumour is a colon, gastric, breast, lung, bladder or prostate cancer.
• the diagnostic method of the present invention may be performed on various cell samples from a solid tumour.
• the test sample is preferably a cell sample from primary tumour and/or from the tumour environment isolated from a subject, e.g. a human patient.
• tumour cell tissue obtained by biopsy, resection or other techniques can be tested.
• the tumour sample comprises tumour cells.
• the expression of anti-apoptotic cytokine in the tumour cells is preferably determined on primary tumour cells and/or cancer stem cells.
• the determination of the expression, in particular of the overexpression, of the anti-apoptotic cytokine in the tumour cell is conducted by the detection of said cytokine on the protein level and/or the nucleic acid level.
• cytokine proteins may be performed in the tumour cells or in the tumour microenvironment.
• Methods to determine the presence and amount of cytokine proteins in a given sample are well known to the person skilled in the art and may be immunochemical methods such as immunohistochemistry, Western blotting, immunoprecipitation and ELISA methods. Further methods based on massspectrometry, comprising MALDI- MS, can be used to determine presence and amount of cytokine proteins.
• Cytokine nucleic acids are detected and quantified herein by any of means well known to those skilled in the art. Hybridization techniques together with optional amplification methods are frequently used for detecting nucleic acids. Expression of cytokine mRNAs may for example be detected by Northern blot analysis or by reverse transcription and subsequent amplification by PCR. The method according to the invention may comprise the further step of
• the viability of the tumour cells exposed to said chemotherapeutic or pro-apoptotic agents in the absence and/or presence of cytokine neutralizing agents may be measured.
• Methods for determining the sensitivity of the tumour cells to a given agent are well known by those skilled in the art (e.g. as described in Examples).
• the method according to the present invention may further comprise the step of
• the invention is based on the observation that solid tumours may be differentiated by their expression or degree of expression of anti-apoptotic cytokines and in particular IL-4 and IL-10 cytokines. Since the expression of IL-4 and IL-10 anti-apoptotic cytokines in tumours or tumour cells is responsible for refractoriness to treatment, e.g. with chemotherapeutic and/or pro-apoptotic agents, the anti-apoptotic cytokines should be neutralized in order to increase the sensitivity of the tumour towards treatment. Thus, the invention may also encompass an examination of the sensitivity or resistance to chemotherapeutic and/or pro-apoptotic agents in combination with antagonists of a cytokine expressed by the tumour.
• the sensitivity assay performed in step (d) of the method leads to the determination of a chemotherapeutic or a pro-apoptotic agent against which the cell of the cytokine-expressing tumours are particularly sensitive. Consequently, according to step (e) of the present invention, a successful tumour type-specific treatment may be selected comprising the administration of a combination of a cytokine-neutralizing agent and a chemotherapeutic or pro-apoptotic agent.
• a cytokine-neutralizing agent may be any compound which reduces the amount and/or activity of a cytokine.
• the cytokine neutralizing agent may be an agent which inhibits a signal transduction pathway triggered by the cytokine autocrinely expressed by the tumour cells.
• any agent is contemplated that is capable of modulating the expression and/or function of a cytokine directly and/or indirectly, namely affecting the expression and/or function of the respective cytokine protein and/or cytokine receptor.
• the cytokine neutralizing agent is an IL-4 and/or IL-10 neutralizing agent, i.e. any agent which is able to inhibit the signal transduction pathway triggered by the autocrine expression of IL-4 and/or IL- 10.
• Cytokine neutralizing agents may be selected, among others, from agents that inhibit and/or reduce the expressed cytokine protein activity, agents which degrade the expressed cytokine protein and agents that inhibit the cytokine production.
• Agents that block the cytokine activity are, for example, antagonists which block the cytokine receptors, e.g. peptides, small molecules, muteine variants of the cytokines which show an antagonistic activity compared to the original signal of the cytokine. Examples for such muteins are in particular IL-4 muteins such as Aerolast ® from Aerovance and Pitrakinra ® and BAY-36-1677 from Bayer. Further antibodies against the cytokine receptor or antibodies against the cytokine protein may be used.
• the antibody is preferably an antibody against IL-4 and/or IL-10, e.g. antibodies from Amgen and Immunex or an antibody against the IL-4 receptor and/or the IL-10 receptor, e.g. the antibody Pascolizumab ® from Glaxo.
• the antibody may be a complete antibody, e.g. an IgG antibody, or an antigen-binding fragment thereof.
• the antibody is a monoclonal chimeric or humanized antibody which has human constant domains, e.g. human constant IgGI, lgG2, lgG3 or lgG4 domains. More preferably, the antibody is a humanized antibody which additionally comprises human framework regions. Also preferred are antibody fragments, e.g.
• the antibody may be a recombinant antibody, e.g. a single chain antibody or a fragment thereof, e.g. an scFv fragment.
• Soluble cytokine receptors can also be used as agents blocking the cytokine activity.
• These soluble receptors are, for example, from Regeneron, in particular IL-4R/IL-13R-Fc fusion proteins, and soluble receptors from Amgen and Immunex, in particular Nuvance ® and Altrakincept ® .
• Specific examples of soluble receptors comprise the extracellular domain (ECD) of a human IL-4 receptor, e.g. from a shortened ECD of human IL-4R alpha amino acid 24 to amino acid 224, 225, 226, 227, 228, 229 or 230 and optionally further domains, e.g. the extracellular domain of a human 11-13 receptor and/or a human Fc immunoglobulin domain.
• agents that degrade the expressed cytokine protein designer proteases can be mentioned in the context of the present invention.
• the production of the cytokine proteins can, on the other hand, be inhibited for example by agents acting on the nucleic levels such as antisense nucleic acids, siRNA molecules and/or ribozymes.
• cytokine antagonists are described in the international patent application WO 2004/069274.
• Antibodies directed against cytokines are preferably used as cytokine-neutralizing agents.
• Anti-IL-4 antibodies disclosed in European patent application EP-A-O 730 609 are especially suitable as cytokine-neutralizing agents of the method of the present invention.
• the antibody derived from the monoclonal antibody 6A1 produced by hybridoma cell line ACC93100620 or an antigen-binding fragment thereof is used as cytokine-neutralizing agent.
• the chemotherapeutic agent used in steps (d) and/or (e) is selected from antimetabolites, DNA-fragmenting agents, DNA-cross-linking agents, intercalating agents, protein synthesis inhibitors, topoisomerase I and Il inhibitors, micro-tubule-directed agents, kinase inhibitors, hormones and hormone antagonists.
• the chemotherapeutic agent is selected from cisplatin, carboplatin and oxaliplatin.
• pro-apoptotic agents TRAIL and CD95 ligand can be selected.
• a therapeutic strategy can be developed based on a specific combination of drugs which has proven to be effective.
• a further object of the present invention is therefore the use of a combination of a cytokine-neutralizing agent and a chemotherapeutic or pro-apoptotic agent and the manufacture of a medicament for the tumour treatment, such as a first line tumour treatment or as second or third line tumour treatment, e.g. for the treatment of refractory tumours, such as tumours which have become refractory against one or more anti-tumour agents.
• a further aspect of the present invention is the use of a combination of (i) at least one cytokine-neutralizing agent and
• tumour cells One of the main causes of drug resistance in tumour cells is based on the observation that a surviving small population of tumour cells, and in particular of tumour stem cells, after an apparently complete regression or surgical excision of the primary tumour could renew the tumour and contribute to the so called minimal residual disease (MRD).
• MRD minimal residual disease
• a further aspect of the present invention is the use of a combination of (iii) at least one cytokine-neutralizing agent and (iv) at least a chemotherapeutic or pro-apoptotic agent for the manufacture of a medicament for the treatment of minimal residue disease.
• the use of a combined therapy of the above agents (i) and (ii) can further be in combination with surgery and/or irradiation therapy.
• the medicament combination is for simultaneous, separate or sequential combination therapy with surgery and/or irradiation therapy.
• the administration of agent (i) and agent (ii) is started simultaneously.
• the combination therapy can be started stepwise.
• the start of the administration of the cytokine-neutralizing agent (i) is ⁇ 1 week before the administration of the chemotherapeutic or pro-apoptotic agent (ii).
• the administration of the chemotherapeutic or pro-apoptotic agent (ii) may in turn start ⁇ 1 week before the administration of the cytokine-neutralizing agent (i).
• Still a further embodiment of the invention is a soluble IL-4 receptor polypeptide or fusion polypeptide comprising a C-terminally shortened extracellular domain, e.g. a domain shortened by 1 , 2, 3, 4, 5, 6, 7, 8 or more amino acids or a nucleic acid molecule encoding such a polypeptide.
• the shortened extracellular domain may be derived e.g. from human IL-4 receptor alpha (NCBI accession NP_000409) which C-terminally ends at amino acid 230, 229, 228, 227, 226, 225 or 224.
• the C-terminal end is amino acid 224.
• the polypeptide may comprise at least one further domain, e.g.
• an N-terminal signal peptide e.g. an IL-13 receptor extracellular domain, an Fc immunoglobulin domain, and/or a purification domain.
• a further effector domain e.g. an IL-13 receptor extracellular domain, an Fc immunoglobulin domain, and/or a purification domain.
• An example of a shortened IL-4R polypeptide is described in Example 4.
• the shortened IL-4R polypeptide is suitable for pharmaceutical applications, e.g. for the treatment of tumours, particularly for the treatment of IL-4-associated tumours as described above.
• anti-IL-4 and anti-IL-10 cancer cells were treated with oxaliplatin (100 ⁇ M) or doxorubicin (5 ⁇ M) or cisplatin (300 ng/ml), or taxol (5 ⁇ M) (Sigma) or etoposide (1 ⁇ M; Biomol, Plymouth Meeting, PA).
• human purified cancer cells were plated in 96- well plates in triplicate at 15,000 cells/well and cultured. The number of viable cells was detected by CellTiter Aqueous Assay Kit (Promega Corporation, Wl, USA) following the instructions of manufacturer. HuT78 cells plated at 2 x 150/ml and treated with CD95-activating antibody CH11
• lmmunohistochemical analysis lmmunohistochemistry was performed on 5 ⁇ m thick paraffin-embedded colon, gastric, prostate, breast, lung, liver, pancreas, kidney and bladder normal and tumour sample sections. Dewaxed sections were treated for 10 min in microwave oven in 0.1 M citrate buffer. Then, sections were incubated for 10 min with Tris Buffer Saline (TBS) containing 10% AB human serum to block the unspecific staining.
• TBS Tris Buffer Saline
• IL-4 B-S4 mouse IgGI , Caltag Laboratories, Burlingame, CA
• IL-10 B-N10 mouse IgG 23 , Caltag
• IL-4R ⁇ C-20 rabbit IgG Santa Cruz Biotechnology Inc, Santa Cruz, CA
• IL-10R C-20 rabbit IgG Santa Cruz Biotechnology
• TRAIL-R1 HS101 mouse IgGI , Alexis Biochemicals, Lausen, CH
• TRAIL-R2 HS201 mouse IgGI , Alexis
• isotype-matched controls at appropriate dilutions.
• GAPD gene was amplified from the same RNA preparations as housekeeping control (coding sequence 5'-TGA CAT CAA GAA GGT GGT GA-3 1 nucleotides 843-863 and 5'-TCC ACC ACC CTG TTG CTG TA-3 1 complementary to nucleotides 1033-1053; NM-002046 accession number). Thirty-five cycles were performed, each consisting of the following conditions: 94°C, 30 sec; 58°C, 30 sec; 72°C, 30 sec.
• Abs specific for actin Abs specific for actin
• Ab-1 mouse IgM, Calbiochem, Darmstadt, Germany
• CD95L G247-4, mouse IgGI , PharMingen, San Diego
• CD95 C-20, Santa Cruz Biotechnology
• cFLIP NF6 mouse IgGI , Alexis Biochemicals, Switzerland
• PED/PEA-15 rabbit IgG kindly provided by G.
• BcI- 2 124, mouse IgGI , Upstate Biotechnology Inc.
• BcI-Xi H-5, mouse IgGI , Santa Cruz Biotechnology
• Epithelial cancer cells express high levels of anti-apoptotic proteins. Colon, breast, gastric and lung cancer cells are resistant to death ligand- and to chemotherapy-induced cell death. To determine the mechanism responsible for this refractoriness, it was investigated whether aberrant expression of anti-apoptotic factors could be implicated in the impaired "extrinsic” and "intrinsic” apoptotic signal pathway generated by death ligands or chemotherapy. It was found by immunohistochemistry and Western blot analyses that epithelial carcinoma cells express CD95, TRAIL- R1 and TRAIL-R2 (Fig. 2a and b).
• the inventors of the present invention evaluated the presence and measured the expression levels of cFLIP, PED/PEA-15, Bcl-xL and Bcl-2 in colon, breast, gastric and lung normal and cancer cells. While cFLIP and PED/PEA-15 levels were approximately three fold higher in freshly purified cancer cells, as compared with normal colon, breast and lung cells (Fig. 2a), Bcl-xL levels were four fold higher. Bcl-2 expression levels were only two fold higher in all the cancer cells analyzed, as compared with normal cells. Thus, anti-apoptotic genes upregulation in colon, breast, gastric and lung cancer cells may confer resistance to CD95- TRAIL- and chemotherapy-induced apoptosis.
• IL-4 increases survival, growth of epithelial neoplastic cells.
• IL-4 receptor expression in cancer cells was evaluated. Immunohistochemistry on paraffin embedded sections showed that IL-4 receptor was expressed in all the cancer tissues analysed. The results are shown in the following Table 2 and in Fig. 3a. Table 2. IL-4R expression in cancer cells
• IL-4 significantly increased the growth rate of colon, breast and lung normal cells (Fig. 3b). Furthermore, to determine the involvement of IL-4 in the refractoriness of cancer cells to CD95, TRAIL and chemotherapeutic agents, normal colon, breast, gastric and lung cells were pre-incubated with IL-4 and then analyzed for expression of those anti-apoptotic proteins implicated in the death ligands and chemotherapy cell death resistance. IL-4 increased the protein levels of cFLIP, PED/PEA-15, Bcl-xL and Bcl-2 in normal colon, breast (Fig.
• IL-4 autocrine IL-4 production might protect cancer cells from chemotherapy and death receptor stimulation, up regulating anti-apoptotic factors.
• IL-4 neutralization promotes growth arrest and cell death induced by CD95, TRAIL and chemotherapy in cancer cells
• IL-4 neutralization blocked colon, breast, gastric and lung tumour cell growth up to 15 days (Fig. 5) and down-modulated the protein expression levels of cFLIP, PED/PEA-15, Bcl-xL and Bcl-2. These data indicate that autocrine production of IL-4 might play an important role in growth control and is specifically required for survival of cancer cells.
• Tissue specimens from freshly operated tumour patients were screened for IL-4 and IL-10 expression by a variety of standard methods such as RT-
• colon CSC apoptosis-inducing death ligand TRAIL (200 ng/ml).
• Primary (adherent) cells from human colon cancer specimens showed some sensitivity in vitro to all three drugs tested, whereas colon CSC were significantly resistant, confirming that CSC are relatively inert to drug-induced apoptosis (Fig. 6a). This suggests that CSC might escape anti-tumour therapies and could be the underlying reason for chemotherapy inefficiency.
• IL-4 production in colon CSC is responsible for up- regulation of anti-apoptotic proteins and therefore therapy refractoriness
• CSC were pre-treated for two days with IL-4-neutralising antibodies and then measured cell death and anti-apoptotic expression.
• Proteins levels of c- FLIP, Bcl-xL and PED, anti-apoptotic proteins previously shown to be regulated by IL-4 in cancer decreased by -two-fold in CSC exposed to anti- IL-4 (Fig. 6b-c). More important, following IL-4 blockade CSC cell death was significantly increased by the treatment with chemotherapeutic drugs or TRAIL (Fig. 6d-e).
• IL-4 protects colon cancer generated by CSC from chemotherapeutic drugs
• Tumours were allowed to grow for 10 days (size -0.2 cm 3 ) and then treated intra-tumourally with neutralising antibodies against IL-4 or control IgG twice a week for 3 weeks.
• intraperitoneal (i.p.) treatment with oxaliplatin once a week for 4 weeks, combined with control IgG reduced tumour size in mice, the efficacy of chemotherapy treatment was significantly enhanced by IL-4 neutralizing antibody (Fig. 7a and 7b).
• the signal-peptide and the extracellular domain of IL4-Receptor-alpha (aa1- aa231 of NCBI accession NP_000409) was fused N-terminally to the IL13- receptor alpha extracellular domain (aa27-aa343 of NCBI accession NP_001551)
• Two point mutations were introduced into the IL4R-alpha1- sequence (Gly2->Val2 and Cys207->Ser207) and a single point mutation was introduced into the IL13R-alpha1 -sequence (Cys46->Ala46).
• the enumeration of the point mutations also refers to NCBI-database entries NP_000409 for IL4R-alpha1 and NP_001551 for IL13R-alpha1.
• This IL4RIL13R protein-sequence was fused to the Fc-part of human IGHG1 (aa254-aa479 of NCBI accession AAH69020). Additionally, a flexible linker element and a Flexstreptag-ll motif (SSSSSSAWSHPQFEK) was added C- terminally.
• the amino acid sequence of the resulting IL4RIL13R-Fc- construct as shown below was backtranslated into a synthetic DNA- sequence and its codon usage optimised for mammalian cell-based expression. Gene synthesis was done by ENTELECHON GmbH (Regensburg, Germany). The final expression cassette was subcloned into pCDNA4-HisMax-backbone, using the unique Hind-Ill- and Not-l-sites of the plasmid.
• IL4R-IL13R-Fc fusion polypeptide Modifications of the IL4R-IL13R-Fc fusion polypeptide may be as follows:
• shortened IL-4R ECD e.g. without or with different mutations, particularly point mutations
• the signal-peptide and a shortened extracellular domain of IL4-Receptor- alpha (aa1-aa224 of NCBI accession NP_000409) was fused N-terminally to the Fc-part of human IGHG1 (aa250-aa479 of NCBI accession AAH69020).
• Two point mutations were introduced into the IL4R-alpha1 -sequence (Gly2- >Val2 and Cys207->Ser207).
• a single glycine was inserted inbetween the two domains and Lys251 of human IGHG1 in the hinge region was mutated to arginine.
• the enumeration of the described mutations also refer to NCBI- database entries NP_000409 for IL4R-alpha1 and NCBI accession AAH69020 for lGHG1 ).
• a flexible linker element and a Flexstreptag-ll motif was added C-terminally.
• the amino acid sequence of the resulting IL4R-Fc-construct as shown below was backtranslated into a synthetic DNA-sequence and its codon usage optimised for mammalian cell- based expression.
• Gene synthesis was done by ENTELECHON GmbH (Regensburg, Germany).
• the final expression cassette was subcloned into pCDNA4-HisMax-backbone, using the unique Hind-Ill- and Not-l-sites of the plasmid.
• VKPRAPGNLT VHTNVSDTLL LTWSNPYPPD NYLYNHLTYA VNIWSENDPA DFRIYNVTYL
• Aal-aa23 IL4R-alphal signal peptide
• Aa24-aa224 IL4R-alphal ECD
• Aa225-aa455 Fc part of IGHGl Aa456-aa470: Flexstreptag-II
• Modifications of the shortened IL-4R fusion polypeptide may be as follows: - absence of a signal peptide or presence of a heterologous signal peptide; presence of the different, e.g. shortened IL-4R ECD, e.g. without or with different mutations, particularly point mutations, presence of a different Fc domain, - a different fusion region between the IL-4R ECD and the Fc domain, e.g. deletion of one or more amino acids of the sequence RSC (positions
• Hek 293T cells grown in DMEM + GlutaMAX (GibCo) supplemented with 10 % FBS, 100 units/ml Penicillin and 100 ⁇ g/ml Streptomycin were transiently transfected with plasmids encoding IL4R-Fc and IL4R-IL13R-Fc, respectively.
• Cell culture supernatants containing recombinant proteins were harvested three days post transfection and clarified by centrifugation at 300 g followed by filtration through a 0.22 ⁇ m sterile filter.
• Streptactin Sepharose was packed to a column (gel bed 1 ml), equilibrated with 15 ml buffer W (100 mM Tris-HCI, 150 mM NaCI pH 8.0) and the respective cell culture supernatant was applied to the column with a flow rate of 4 ml/min. Subsequently, the column was washed with buffer W and bound IL4R-Fc or IL4R-IL13R-Fc was eluted stepwise by addition of 6 x 1 ml buffer E (10O mM Tris HCI, 15O mM NaCI, 2.5 mM Desthiobiotin pH 8.0).
• IL4R-Fc For determination of the apparent molecular weight under native conditions a Superdex 200 column was loaded with standard proteins of known molecular weight. Based on the elution volume of the standard proteins a calibration curve was calculated and the apparent molecular weight of purified IL4R-Fc was determined to be 137 KDa which fits well to the molecular weight observed by SDS-PAGE. The theoretical molecular weight based on the amino acid sequence of IL4R-Fc is 52.8 Kda for the monomelic protein. Based on the biochemical analysis IL4R-Fc very likely is expressed as a protein dimer.
• IL4R-IL13R-Fc the apparent molecular weight based on SEC was calculated to be about 600 KDa. Based on SDS-Page analysis the protein runs as a single band with about 250Kda. The theoretical molecular weight based on the amino acid sequence of IL4R-IL13R-Fc is 87.7 KDa. In principle the construction of the molecule should result in a stable dimeric protein with a theoretical molecular weight of about 180Kda. The high apparent molecular weight seen by SEC therefore either indicates an unusual behavior in SEC or further oligomerisation of the protein.
• IL4-pull down assay To test for specific IL4 binding of IL4R-Fc and IL4R-IL13R-Fc, 4 ⁇ g of both proteins, respectively, were immobilized to Streptactin Sepharose via their Strep-Tag. The immobilized proteins were subsequently incubated for 60 min with 400 ng of recombinantly expressed human Interleukin4 (IL4) in a total volume of 400 ⁇ l phosphate buffered saline. Subsequently the beads were washed and bound proteins were specifically eluted with desthiobiotin in a total volume of 40 ⁇ l elution buffer. Eluted proteins were finally analysed via SDS-PAGE and Silver staining. As shown in Figure 10 both IL4R-Fc and IL4R-IL13R-Fc show specific binding of human IL4 indicated by the presence of IL4 protein (12Kda) that could not be seen in control experiments.
• FIG. 11A shows the immunofluorescence analysis of breast cancer spheres pre-treated with PBS (w/o) or 10 ⁇ g of IL4R-Fc, IL4R-IL13R-Fc or anti IL4-antibody for 24 hrs and successively exposed for another 24 hrs to 5 ⁇ M doxorubicin.
• the cells were stained with orange acridine/ethidium bromide (red: dead cells; green: viable cells).
• both IL4R-Fc and IL4R-IL13R-Fc are able to sensitise breast cancer stem cells for doxorubicin induced apoptosis in the same range as shown for an IL4 specific antibody, that was used as a positive control in this experiment.

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  • 2007-06-21 RU RU2009101783/15A patent/RU2009101783A/ru not_active Application Discontinuation
  • 2007-06-21 DK DK07764767.5T patent/DK2041576T3/da active
  • 2007-06-21 ES ES07764767T patent/ES2371287T3/es active Active

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