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WO2004111085A1 - Facteur angiogenique et son utilisation medicale - Google Patents

Facteur angiogenique et son utilisation medicale Download PDF

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Publication number
WO2004111085A1
WO2004111085A1 PCT/EP2004/006270 EP2004006270W WO2004111085A1 WO 2004111085 A1 WO2004111085 A1 WO 2004111085A1 EP 2004006270 W EP2004006270 W EP 2004006270W WO 2004111085 A1 WO2004111085 A1 WO 2004111085A1
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Prior art keywords
sep
derivative
ssep
cells
functional active
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PCT/EP2004/006270
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English (en)
Inventor
Hendrik Gille
Beate Gawin
Rolf Schäfer
Stephan Hess
Christian Korherr
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Xantos Biomedicine Ag
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Publication of WO2004111085A1 publication Critical patent/WO2004111085A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a new angiogenic factor and its use in pharmaceutical and diagnostic compositions.
  • Angiogenesis the growth of new capillaries from pre-existing ones, is critical for normal physiological functions in adults [Carmeliet, P., Mechanisms of angio- genesis and arteriogenesis. Nat Med, 2000 6 (4) 389-95]. Abnormal angiogenesis can lead to impaired wound healing, poor tissue regeneration in ischemic conditions, cyclical growth of the female reproductive system, and tumor development [Carmeliet, P. and R. K. Jain, Angiogenesis in cancer and other diseases. Nature, 2000 407: 249-257].
  • angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis.
  • the angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. Because of the crucial role of angiogenesis in so many physiological processes, there is a need to identify and characterize factors which will promote angiogenesis.
  • VEGF-A and FGF -2 have been considered as a possible approach for the therapeutic treatment of ischemic disorders.
  • VEGF is an endothelial cell-specific mitogen and an angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ.
  • angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ.
  • both animal studies and early clinical trials with VEGF angiogenesis have encountered severe problems [Carmeliet, Nat Med, 2000 6 1102- 3;Yancopoulos et al., Nature, 2000 407 242-8; Veikkola et al, Semin Cancer Biol 1999 9 211-20; Dvorak et al., Semin Perinatol 2000 24 75-8; Lee et al., Circulation, 2000 102 898-901].
  • VEGF-A stimulated microvessels are disorganized, sinusoidal and dilated, much like those found in tumors [Lee et al., Circulation 2000 102 898-901; and Springer et al., MoI. Cell 1998 2 549-559]. Moreover, these vessels are usually leaky, poorly perfused, torturous and likely to rupture and re- gress. Thus, these vessels have limited ability to improve the ischemic conditions. In addition, the leakage of blood vessels induced by VEGF-A (also known as Vascular Permeability Factor) could cause cardiac edema that leads to heart failure.
  • VEGF-A also known as Vascular Permeability Factor
  • VEGF not only stimulates vascular endothelial cell proliferation, but also induces vascular permeability and angiogenesis.
  • Angiogenesis which involves the formation of new blood vessels from preexisting endothelium, is an important component of a variety of diseases and disorders including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, retinopathy, hemangiomas, im- mune rejection of transplanted tissues, and chronic inflammation.
  • angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment to the growing solid tumor. [Folkman, et al., Nature 339:58 (1989)]. Angiogenesis also allows tumors to be in contact with the vascular bed of the host, which may provide a route for metastasis of the tumor cells. Evidence for the role of angiogenesis in tumor metastasis is provided, for example, by studies showing a correlation between the number and density of microvessels in histologic sections of invasive human breast carcinoma and actual presence of distant metastases. [Weidner, et al., New Engl. J. Med. 324:1 (1991)].
  • the problem underlying the present invention therefore lies in providing an angiogenic agent which does not exhibit the deficiencies of VEGF as depicted above.
  • the human protein disclosed in the NCBI database entries BAA86585, AAH44952 (see SEQ ID NO: 4) and XP_045472 (see SEQ ID NO: 6) exhibits an important role in angiogenesis both in its membrane bound form (BAA86585, AAH44952 and XP_045472) as well as in a soluble form.
  • This protein was named SEP, and its soluble, not membrane bound form was named sSEP.
  • the corresponding cDNA sequences of the membrane bound form are given in the NCBI database entries BC044952 and XM_045472 (SEQ ID NO: 3 and 5).
  • SEP and sSEP are a novel angiogenic factors of a to-date unknown novel family.
  • the corre- sponding mouse sequences of SEP (mSEP) are given in SEQ ID NO: 1 (DNA) and 2 (protein).
  • soluble SEP soluble SEP
  • functional active soluble derivative thereof a soluble SEP or a functional active soluble derivative thereof.
  • Example 8 it is demonstrated that transfection of cells with DNA encoding SEP leads to the production of VEGF, and Example 17 shows that other angiogenic factors like IL-8 and RANTES are induced by SEP.
  • sSEP relates to any soluble SEP, wherein the amino acid sequence of SEP as demonstrated in the database has been manipulated with the consequence that the manipulated protein is soluble.
  • sSEP relates both to artificial as well as to naturally occurring proteins.
  • the sSEP of the invention does not comprise a transmembrane domain.
  • the transmembrane domain of SEP extends at least from amino acid 514 to amino acid 535 of the human SEP as disclosed in the data base entries AAH44952 (see SEQ ID NO: 4) and XP_045472 (see SEQ ID NO: 6).
  • An sSEP can therefore be produced by changing the amino acid sequence in this putative transmembrane region, e.g. by exchanging hydrophobic amino acids with hydrophilic amino acids.
  • Example 9 clearly demonstrates that sSEP has angiogenic properties.
  • Methods for the production of proteins starting from a cDNA include e.g. the expression of the protein in appropriate cells or the production by subsequent addition of amino acids to a starting amino acid (Current Protocols, John Wiley & Sons, Inc., New York (2003)).
  • the term "functional active derivative" of a polypeptide within the meaning of the present invention refers to polypeptides which have a sequence homology, in particular a sequence identity, of about at least 25 %, preferably about 40 %, in par- ticular about 60 %, especially about 70 %, even more preferred about 80 %, in particular about 90 % and most preferred of 98 % with the polypeptide, which has essentially the biological function(s) as the corresponding protein. In the case of SEP or sSEP, this may be an angiogenic activity as demonstrated in Examples 2 and 3. A test for the determination of the angiogenic activity of a putative sSEP derivative is demonstrated in Example 2.
  • Such derivatives are e.g. the polypeptide homologous to sSEP or SEP, which originate from organisms other than human.
  • Other examples of derivatives are polypeptides which are encoded by different alleles of the gene, of different indi- viduals, in different organs of an organism or in different developmental phases.
  • Functional active derivatives preferably also include naturally occurring muta- tions, particularly mutations that quantitatively alter the activity of the peptides encoded by these sequences. Further, such variants may preferably arise from differential splicing of the encoding genes.
  • functional active soluble derivative refers to a soluble, i.e. not membrane-bound, "functional active soluble derivative” as defined above.
  • the term "functional active derivative” or “functional active soluble derivative” include derivatives with single nucleotide polymorphism (SNP) at at least one of the positions 383 (G to C), 699 (A to C), 1332 (T to C), 1778 (C to T), 2260 (C to A) and/or 2896/7 (TT to GA) of the nucleotide sequence given in SEQ ID NO: 3 (BC044952).
  • SNP single nucleotide polymorphism
  • SNPs at positions 383, 699 and/or 1332, leading to the amino acid exchanges E with Q, K with Q and F with S, respectively.
  • Sequence identity refers to the degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of for example BLASTP 2.2.5 and in the case of nucleic acids by means of for example BLASTN 2.2.6, wherein the low complexity filter is set on and BLOSUM is 62 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402).
  • Sequence homology refers to the similarity (% positives) of two polypeptide sequences determined by means of for example BLASTP 2.0.1, wherein the filter is set on and BLOSUM is 62 (Altschul et al., 1997, Nucleic Acids Res., 25:3389- 3402).
  • Nucleic acids encoding functional active derivatives can be isolated by using human SEP gene sequences in order to identify homologues with methods known to a person skilled in the art, e.g. through PCR amplification or hybridization under stringent conditions (e.g. 60 0 C in 2.5 x SSC buffer followed by several washing steps at room temperature) with suitable probes derived from e.g. the human SEP sequences according to standard laboratory methods.
  • stringent conditions e.g. 60 0 C in 2.5 x SSC buffer followed by several washing steps at room temperature
  • the same biological activity may also be the ability to compete with membrane bound SEP and therefore to act as an inhibitor of a signal transduced by membrane bound SEP.
  • the term "functional active de- rivative" may refer to the ability to induce the expression of VEGF as shown in Example 8.
  • the sSEP or functional derivative thereof of the invention is devoid of a transmembrane domain of SEP or of functional active variant thereof.
  • sSEP fragments are produced by cleaving at potential protease cleaving sites, more preferably at the following potential cleaving sites:
  • SPRAIPRN amino acids 165 to 172 of SEP as given in SEQ ID NO: 4
  • ARSTPRASRL amino acids 242 to 250 of SEP as given in SEQ ID NO: 4
  • HRPSP amino acids 509 to 513 of SEP as given in SEQ ID NO: 4
  • Cleaving can occur within every amino acid within these sequences, however, a cleaving after the amino acid R is preferred.
  • this includes also that after cleavage with an appropri- ate protease, further amino acids are removed by e.g. carboxypeptidases. Consequently, in a more preferred embodiment, the sSEP or functional derivative thereof of the invention has a C-terminal amino acid corresponding to amino acid 510, 249, 246, 242, 171 or 167 of SEP according to SEQ ID NO: 4 or has a C- terminal amino acid corresponding to the equivalent amino acid of a SEP deriva- tive.
  • an sSEP according to the invention has one of the sequences as shown in Figure 5 (SEQ ID NO: 7-18).
  • this signal peptide may also be cleaved off.
  • the protein depicted in SEQ ID NO: 2, 4 or 6 and soluble variants thereof exhibit an important role in angiogenesis. This enables the use of these proteins in therapy.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising
  • sSEP or derivative thereof of the invention b) SEP as defined in SEQ ID NO: 2, 4 or 6, c) a functional active derivative of the SEP of section b), and/or d) a nucleic acid encoding the proteins of sections a), b) or c) above,
  • the molecules as depicted in sections a) to d) may be provided as defined above.
  • the pharmaceutical composition further comprises VEGF, and/or a functional derivative thereof, preferably in combination with VEGF-A, VEGF-B, VEGF-C, VEGF-D, PLGF, PDGF-A, PDGF-B, PDGF-C, PDGF-D and FGF.
  • VEGF is a well known angiogenic factor.
  • a combination of SEP and VEGF leads to enforced or synergistic effects in the promotion of angiogenesis in mammals.
  • the invention also relates to the sSEP or derivative thereof of the invention or a SEP as defined in SEQ ID NO: 2, 4 or 6 or functional active derivates thereof or of nucleic acids encoding these molecules for use in therapy.
  • composition of the invention may be applied as follows:
  • SEP administration may be effected either as recombinant protein or by gene transfer either as naked DNA or in a vector [Kornowski R,Fuchs S, Leon MB, Epstein SE, Delivery strategies to achieve therapeutic myocardial angiogenesis, Circulation, 2000 101 (4) 454-8; Simons M, Bonow RO, Chronos NA, Cohen DJ 5 Giordano FJ, Hammond FfK 5 et al., Clinical trials in coronary angiogenesis: issues, problems, consensus: An expert panel summary, Circulation, 2000 102 (11) E73-86; and Isner JM, Asahara T, Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization, J Clin Invest, 1999 103 (9) 1231-36].
  • regulatable vectors may be used as described in Ozawa et al, Annu Rev Pharmacol. & Toxicol, 2000 40 295-317.
  • SEP or sSEP can be administered by direct myocardial injection of naked plasmid DNA encoding SEP, sSEP or a functional active derivative thereof during surgery in patients with chronic myocardial ischemia following procedures outlined in Vale, P. R., et al., Left ventricular electromechanical mapping to assess efficacy of phVEGF (165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia, Circulation, 2000 102 965-74.
  • SEP, sSEP or a functional active derivative thereof can also be administered by direct myocardial injection of SEP, sSEP or a functional active derivative thereof protein via a minithoracotomy.
  • it is given as a bolus dose of from 1 pg/kg to 15 mg/kg, preferably between 5 pg/kg and 5 mg/kg, and most preferably between 0.2 and 2 mg/kg.
  • Continuous infusion may also be used, for example, by means of an osmotic minipump as described in Heyman et al., Nat Med, 1999 5 1135-152.
  • the medicament may be infused at a dose between 5 and 20 ⁇ g/(kg-minute), preferably between 1 and 100 ng/(kg-minute), more preferably between 5 and 20 ng/(kg-minute) and most preferably between 7 and 15 pg/(kg-minute).
  • SEP, sSEP or a functional active derivative thereof can be adminis- tered by catheter-based myocardial gene transfer of SEP, sSEP or a functional active derivative thereof.
  • a steerable, deflectable 8F catheter incorporating a 27gauge needle is preferably used and advanced percutaneously to the left ventricular myocardium. For example, a total dose of 200 ⁇ g/kg is administered as 6 injections into the ischemic myocardium (total, 6.0 ml). Injections are guided by e.g.NOGA left ventricular electromechanical mapping. See Vale, P.
  • SEP sSEP
  • Another possibility for SEP, sSEP or a functional active derivative thereof administration is injection of SEP plasmid in e.g. the muscles of an ischemic limb in accordance with procedures described in Simovic, D., et al., Improvement in chronic ischemic neuropathy after intramuscular gene transfer e.g. using phVEGF165 in patients with critical limb ischemia, Arch Neurol, 2001 58 (5) 76168.
  • Still another technique for effective administration is by intra-arterial gene transfer of the gene using adenovirus and replication defective retroviruses as de- scribed for VEGF in Baumgartner I and Isner JM, Somatic gene therapy in the cardiovascular system, Annu. Rev Physiol, 2001 63 427-50.
  • An additional possibility for administering SEP, sSEP or a functional active derivative thereof is by intracoronary and intravenous administration of recombinant SEP, sSEP or a functional active derivative thereof following procedures described in Post, M. J., et al., Therapeutic angiogenesis in cardiology using protein formulations, Cardio- vasc Res, 2001 49 522-31.
  • EPCs ex vivo expanded endothelial progenitor cells
  • SEP sSEP
  • a functional active derivative thereof for myocardial neovascularization as described in Kawamoto, A., et al., Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation, 2001 103 (5) 634-37.
  • a therapeutically effective dose of SEP, sSEP or a functional active derivative thereof is administered by bolus injection of the active substance into e.g. ischemic tissue, e. g. heart or peripheral muscle tissue.
  • the effective dose will vary depending on the weight and condition of the ischemic subject and the nature of the ischemic condition to be treated. It is considered to be within the skill of the art to determine the appropriate dosage for a given subject and condition.
  • the pharmaceutical composition can be administered in further conventional manners, e.g.
  • TTS transdermal therapeutic system
  • SEP, sSEP or a functional active derivative thereof is administered by continuous delivery, e. g., using an osmotic minipump, until the patient is able to self maintain a functional vascular network.
  • SEP, sSEP or a functional active derivative thereof is effectively administered to an ischemic sub- ject by contacting ischemic tissue with a viral vector, e. g. an adenovirus vector, containing a polynucleotide sequence encoding the protein operatively linked to a promoter sequence.
  • a viral vector e. g. an adenovirus vector, containing a polynucleotide sequence encoding the protein operatively linked to a promoter sequence.
  • SEP, sSEP or a functional active derivative thereof may also be effectively admin- istered by implantation of a micropellet impregnated with active substance in the direct vicinity of e.g. the ischemic tissue.
  • the molecules of the present invention are usually formulated with suitable additives or auxiliary substances, such as physiological buffer solution, e.g. sodium chloride solution, demineralized water, stabilizers, such as protease or nuclease inhibitors, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA, gel formulations, such as white Vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.
  • suitable additives or auxiliary substances such as physiological buffer solution, e.g. sodium chloride solution, demineralized water, stabilizers, such as protease or nuclease inhibitors, preferably aprotinin, ⁇ -aminocaproic acid
  • Suitable further additives are, for example, detergents, such as, for example, Triton X-IOO or sodium deoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwit- terionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred.
  • the physiological buffer solution preferably has a pH of approx. 6.0-8.0, expe- cially a pH of approx. 6.8-7.8, in particular a pH of approx. 7.4, and/or an osmo- larity of approx. 200-400 mosmol/1, preferably of approx. 290-310 mosmol/lr.
  • the pH of the medicament is in general adjusted using a suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)pipera- zino]ethanesulphonic acid) or MOPS buffer (3-morpholino-l-propanesulphonic acid).
  • a suitable organic or inorganic buffer such as, for example, preferably using a phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)pipera- zino]ethanesulphonic acid) or MOPS buffer (3-morpholino-l-propanesulphonic acid).
  • a phosphate buffer tris buffer (tris(hydroxymethyl)aminomethane)
  • HEPES buffer [4-(2-hydroxyethyl)pipera- zino]ethanesulphonic acid
  • Injection solutions are in general used if only relatively small amounts of a solu- tion or suspension, for example about 1 to about 20 ml, are to be administered to the body.
  • Infusion solutions are in general used if a larger amount of a solution or suspension, for example one or more liters, are to be administered. Since, in con- trast to the infusion solution, only a few milliliters are administered in the case of injection solutions, small differences from the pH and from the osmotic pressure of the blood or the tissue fluid in the injection do not make themselves noticeable or only make themselves noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according to the invention before use is therefore in general not necessary.
  • the formulation according to the invention should be diluted briefly before administration to such an extent that an at least approximately isotonic solution is obtained.
  • An example of an isotonic solution is a 0.9% strength sodium chloride solution.
  • the dilution can be carried out, for example, using sterile water while the administration can be carried out, for example, via a so-called bypass.
  • subjects which may be treated or diagnosed include animals, preferably mammals and humans, dead or alive. These patients suffer from the diseases as mentioned above.
  • the invention relates to the use of a) the sSEP or derivative thereof of the invention, b) SEP as defined in SEQ ID NO: 2, 4 or 6, c) a functional active derivative of the SEP of section b), and/or d) a nucleic acid encoding the molecules of sections a), b) or c)
  • SEP immobilized to a matrix can be administered directly into the site of fracture to promote the angiogenesis and wound healing.
  • matrices can be used ceramic matrices or bonemeal on which the protein is immobilized.
  • Slow release formulations to have the factor locally enriched can be used as well.
  • neovascularization is an essential requirement for supporting the growing fetus and embryo during pregnancy.
  • vascular development is necessary in the placenta (fetal as well as maternal tissue) as well as in the uterus.
  • Expression analyses which are shown in Figure 3, show the presence of significant levels of VEGF in uterus, reflecting the above described requirement for stimulation of vascular growth in this tissue.
  • the expression levels of VEGF are relatively low in placenta.
  • the limited expression of VEGF in placenta may - by itself - not be sufficient to stimulate sufficient vascularization.
  • SEP in female placenta, as shown in figure 3, provides an explanation for the lower levels of VEGF expression in placenta compared to uterus.
  • SEP is highly expressed in normal placenta.
  • both factors each with defined specificity, are complementing their function to stimulate vascularization.
  • both factors are necessary for sufficient vascularization during ⁇ pregnancy.
  • supplementation of SEP may aid to ameliorate or prevent said dis- orders.
  • inhibition of SEP may be used to prevent angiogenesis in early pregnancies, with the objective to terminate pregnancies in humans (or animals) due to medical indications.
  • the molecules as defined in sections a) to d) induce the formation of vascular vessels.
  • sSEP, SEP or the functional active derivative thereof are able to induce the production of VEGF. Therefore, in a preferred em- bodiment of the use of the present invention, the molecules as defined in sections a) to d) (as defined above) induce the production of VEGF.
  • the molecules as defined in sections a) to d) induce the production of IL-8 and/or RANTES.
  • sSEP, SEP or functional active derivatives thereof are used in combination with VEGF and/or functional active derivatives thereof, preferably in combination with VEGF-A, VEGF-B, VEGF-C, VEGF-D 5 PLGF, PDGF-A, PDGF- B, PDGF-C, PDGF-D and FGF.
  • the invention further includes a method for the treatment of a patient in need of such treatment, wherein an effective amount of SEP, sSEP or a functional active derivative thereof is administered to the patient.
  • Another subject of the present invention is an antibody or fragment thereof which specifically binds an sSEP or derivative thereof of the invention, SEP as defined in SEQ ID NO: 2, 4 or 6 or a functional active derivative of the SEP of the inven- tion.
  • the antibody is a monoclonal or polyclonal antibody.
  • the procedure for preparing an antibody or antibody fragment as described in Example 18 or 20 is effected in accordance with methods which are well known to the skilled person (see below).
  • the term "antibody” is used in the broa- dest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
  • anti- body fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • SEP is especially upregulated in sev- eral tumor diseases. Consequently, SEP, sSEP and functional active derivatives thereof can be used as diagnostic agents.
  • the invention therefore relates to a diagnostic agent comprising
  • sSEP or derivative thereof of the invention b) SEP as defined in SEQ ID NO: 2, 4 or 6, c) a functional active derivative of the SEP of section b), d) a nucleic acid encoding the SEPs of sections a), b) or c), and/or e) means for detection of the molecules of sections a), b), c) or d).
  • This diagnostic agent may be appropriately combined with additional carriers or diluents or other additives which are suitable in this context. With respect to these agents, the same apply as defined above for the pharmaceutical composition of the invention. Furthermore, the invention relates to the sSEP or derivatives thereof of the invention, SEP as defined in SEQ ID NO: 2, 4 or 6, a functional active derivative thereof, a nucleic acid encoding these SEPs or functional active derivatives and/or of means for detecting these SEPs or nucleic acids for use in therapy or diagnosis.
  • the proteins or nucleic acids may be prepared as defined above.
  • means of detecting the proteins of the invention or SEP or functional active derivatives thereof include antibodies or fragments thereof which specifically bind an sSEP or derivative thereof of the invention, SEP as defined in SEQ ID NO: 2, 4 or 6 or a functional active derivative of the SEP.
  • the antibody or fragment thereof can be e.g. a monoclonal or polyclonal antibodies or fragments thereof. It can e.g. be applied in Western Blotting, Immunohistochemistry, ELISA or functional assays for the proteins (Current Protocols, John Wiley & Sons, Inc. (2003)).
  • Means for detecting the nucleic acids as defined above include other nucleic acids being capable of hybridizing with the nucleic acids e.g. in Southern Blots or Northern Blots as well as during In Situ Hybridization (Current Protocols, John Wiley & Sons, Inc. (2003)).
  • the invention relates to the use of a) the sSEP or derivatives of the invention, b) SEP as defined in SEQ ID NO: 2, 4 or 6, c) a functional active derivative of the SEP of section b), d) a nucleic acid encoding the molecules of sections a, b, or c) and/or e) means for detection of the molecules of sections a), b), c) or d) for the diagnosis of tumor or tumor progression.
  • SEP is an important marker of tumor cells (as shown in Fig. 3). Angiogenesis is generally a phenomenon which occurs in later tumor stages. Therefore, SEP represents a marker for later tumor stages, i.e. for tumors which have already achieved a malignant state.
  • sSEP or functional active derivatives thereof may be detected in the serum via antibodies.
  • SEP, sSEP or functional active derivatives thereof may be detected in the tumor tissue via immunohistochemistry.
  • Nucleic acids encoding these molecules, e.g. mRNA, may be detected using quantitative PCR.
  • sSEP expression in the serum may change. Consequently, by measuring serum levels, it can be determined whether a patient is susceptible for an SEP or sSEP mediated tumor therapy. The higher the SEP or sSEP expression, the better a therapeutical success can be predicted.
  • FIG. 1 Proliferation of HUVEC following transfer of supernatants from transfected 293 cells
  • the relative fluorescence units are given as mean value from three independent experiments. Experiments were performed following the manually adapted protocol described above.
  • Vector represents the negative control resulting from transfection of the cloning vector pCMV-Sport ⁇ into 293 cells and meas- urement of Alamar Blue to determine background proliferative effect of the supernatant derived from 293 cells.
  • VEGF and PDGF were derived from the same clone collection to ensure compatibility of expression systems.
  • Figure 2 Proliferation of NHDF (normal human dermal fibroblasts) following transfer of supernatants from transfected HEK 293 cells
  • the relative fluorescence units are given as mean value from three independent experiments. Experiments were performed following the manually adapted protocol described above.
  • Vector represents the negative control resulting from transfection of the cloning vector pCMV-Sport ⁇ into HEK 293 cells and measurement of Alamar Blue to determine background proliferative effect of the supernatant derived from 293 cells.
  • VEGF and PDGF were derived from the same clone collection to ensure compatibility of expression systems.
  • the results shown in Figs. 1 and 2 demonstrate that SEP acts specifically on endothelial but not on fibroblast cells.
  • Figure 3 Increased expression of SEP in tumor vs normal tissue and comparison to VEGF of tumor vs normal specificity
  • Figure 4 shows the putative composition of the domains of hSEP.
  • a globular do- main containing Cysteins at the N-terminus is followed by a Prolin rich domain and two cleavage sites (arrows) for serum proteases / serin proteases, e.g. Thrombin, Plasmin or Urokinase.
  • Repetitive units of similar Prolin containing sequences are followed by a Prolin rich domain and a trans-membrane domain.
  • This Figure shows preferred soluble SEP fragments of the invention.
  • Figure 6 Expression of human SEP in tumors vs. normal tissue by quantitative RT-PCR
  • RNA from mammary gland, and colon tissue was transcribed into cDNA and relative expression of SEP versus 18SrRNA was calculated after quantitative real-time PCR. Absolute expression levels have been analyzed by quantitative real-time PCR for a panel of cDNAs from mammary gland and ovary tissue. Overexpression of SEP was observed in mammary and ovary cancer compared to normal tissue.
  • Figure 7 describes that HEK 293 cells transfected with SEP produce VEGF.
  • the relative fluorescence units (RFU) are given as mean value from three inde- pendent experiments. Experiments were performed following the manually adapted protocol described above.
  • Vector represents the negative control resulting from transfection of the cloning vector pCMV-Sport ⁇ into 293 cells and measurement of Alamar Blue to determine background proliferative effect of the supernatant derived from 293 cells.
  • VEGF was derived from the same clone collec- tion to ensure compatibility of expression systems.
  • Expression of fragment 1-510 (1-510) showed the same activity compared to full length SEP (hSEP). There was also no difference in activity of SEP and the fragment 1-510 when tagged with the HA (hemagglutinin) epitope (hSEP HA; 1-510 HA).
  • Figure 9 Expression of human SEP in tumor vs normal tissue by quantitative RT-PCR
  • RNA from colon, lung, prostate and breast tissue was transcribed into cDNA and relative expression of SEP versus 18SrRNA was calculated after quan- titative real-time PCR. Over-expression of SEP was observed in most colon, lung, prostate and breast cancer compared to normal tissue.
  • Figure 10 Expression of SEP and VEGF in breast tumors versus normal tissue by quantitative RT-PCR
  • RNA from breast tissue was transcribed into cDNA and relative expression of VEGF and SEP versus 18SrRNA was calculated after quan- titative real-time PCR. Over-expression of SEP was observed more frequently in breast cancer versus normal tissue compared to VEGF.
  • Figure 11 Increased expression of SEP in colon cancer versus normal tissues compared to VEGF
  • RNA from colon tissue was transcribed into cDNA and relative expression of SEP and VEGF versus G6PDH was calculated after quantitative real-time PCR.
  • SEP and VEGF a correlation between SEP and VEGF expres- sion in normal colon tissue.
  • colon cancer the tissue where correlation is also found, albeit less pronounced.
  • Expression levels of SEP and VEGF correlated in normal colon tissue and less pronounced in colon cancer tissue.
  • Figure 12 Expression of h SEP in relation to G6PDH under hypoxic conditions bv quantitative RT-PCR
  • Fig. 13 Expression of SEP in colon cancer vs. normal colon tissue
  • This figure shows an example of increased SEP expression in cancer tissue IHC of colon tissue samples.
  • Immunoreactive cells are the malignant tumor cells. Staining for SEP protein was positive in the colon cancer tissue sample compared to normal tissue were staining was negative.
  • Fig. 14 Expression of SEP on cell surface of transfected HEK293
  • HEK 293 transfected with hSEP showed specific staining for SEP protein on the cell surface in FACS analysis compared to control transfections with empty vector.
  • the expression of SEP 1-510 on the cell surface is lower because the protein fragment is secreted.
  • Fig. 15 a Proliferation of HUVEC following transfer of supernatants from transfected 293 cells
  • Fragment 1-167 represents the negative control resulting from transfection of the expression plasmid into 293 cells and measurement of Alamar Blue to determine the non-specific (background) proliferative effect of the supernatant derived from HEK293 cells.
  • the fragment 1-510 showed a similar activity compared to full length SEP (SEP-FuIl).
  • the fragment 1-167 showed no activity.
  • Fig. 15 b Western Blot analysis for SEP of supernatants from transfected HEK293 cells
  • Figure 15 c Proliferation of HUVEC following addition of purified protein (eluates from nickel-agarose column)
  • Fragment 1-167 represents the negative control resulting from expression and purification of the inactive fragment from supernatants of transfected HEK293 cells.
  • 1-167 was derived using the same expression and purification system.
  • PBS represents a negative control to determine the non-specific (background) prolifera- tive effect of the buffer the purified protein was dialysed against.
  • the purified fragment 1-510 showed activity compared to the negative controls.
  • the fragment 1-167 showed no activity.
  • the relative fluorescence units (RFU) are given as mean value from three independent experiments. Experiments were performed following the manually adapted protocol described above.
  • RNA from HEK293 cells transfected with SEP or vector control was transcribed into cDNA and relative expression of IL-8 versus G6PDH was calculated after quantitative real-time PCR. Indicated is the relative induction of IL-8 by SEP compared to empty vector. Induction of IL-8 was observed by overexpression of SEP in HEK293 cells.
  • RNA from HEK293 cells transfected with SEP or vector control was transcribed into cDNA and relative expression of Rantes versus G6PDH was calculated after quantitative real-time PCR. Indicated is the relative induction of Rantes by SEP compared to empty vector. Induction of Rantes was observed by overex- pression of SEP in HEK293 cells.
  • the relative fluorescence units are given as mean value of three independent experiments. Experiments were performed following the manually adapted protocol described above. Supernatants of transfected HEK293 were separated on an Ion exchange column and the fractions were tested for their ability to induce proliferation/survival of HUVEC-cells. To see differences between supernatants of SEP-transfected and control vector transfected cells, AlamarBlue absorption values of cells incubated with a certain fraction of the SEP-supernatants were divided by the corresponding value of cells incubated with fractions of control vector supernatants.
  • 3 activity peaks can be found.
  • the 3 peaks potentially reflect that not only SEP, but also additional factors like IL- 8 and Rantes.have proliferative activity.
  • Figure 18 shows an example of growth inhibition by a rabbit anti-serum specific against SEP.
  • HUVEC cells were incubated with indicated amounts of anti-serum and supernatant of SEP transfected HEK293 cells for 5 days and proliferation of the cells was measured using the AlamarBlue Assay (as described).
  • Anti-SEP anti-serum reduced SEP induced growth of HUVEC cells compared to controls and is therefore suitable for therapeutic intervention of SEP activity.
  • Figure 19 shows an example of growth inhibition by a human F(ab) specific against SEP generated by in vitro selection.
  • HUVEC cells were incubated with 1 ⁇ g/ml of F(ab) and supernatant of SEP transfected HEK293 cells for 5 days and proliferation of the cells was measured using the AlamarBlue Assay.
  • F(ab) 15 reduced SEP induced growth of HUVEC cells to 71% compared to un- treated cells. Antibodies against SEP are therefore suitable for therapeutic intervention of SEP activity.
  • HUVEC cells were incubated with indicated amounts of peptide and supernatant of SEP transfected HEK293 cells for 5 days and proliferation of the cells was measured using the AlamarBlue Assay (as de- scribed).
  • P3061 and pWobble reduced SEP induced proliferation of HUVEC cells compared to untreated cells. Therefore peptides derived from SEP are suitable for therapeutic intervention of SEP activity.
  • Figure 21 a Induction of HUVEC proliferation by stable MCF-7 clones
  • the relative fluorescence units (RFU) are given as mean value of three independent experiments. Experiments were performed following the manually adapted protocol described above. Stably SEP overexpressing clone MCF-7 SEP40 showed induction of proliferative activity on HUVEC compared to a stable con- trol clone MCF-7 Vector 20.
  • RNA from MCF-7 cells stably transfected with SEP or vector control was transcribed into cDNA and relative expression of IL-8 versus G6PDH was calcu- lated after quantitative real-time PCR. Indicated is the relative induction of IL-8.
  • Stably SEP overexpressing clone MCF-7 SEP40 showed increased induction of IL-8 compared to a stable control clone MCF-7 Vector 20.
  • RNA from MCF-7 cells stably transfected with SEP or vector control was transcribed into cDNA and relative expression of Rantes versus G6PDH was cal- culated after quantitative real-time PCR. Indicated is the relative induction of Rantes.
  • Stably SEP overexpressing clone MCF-7 SEP40 showed increased induction of IL-8 compared to a stable control clone MCF-7 Vector 20.
  • the relative fluorescence units (RFU) are given as mean value from three independent experiments. Experiments were performed following the manually adapted protocol described above. The clone PC-3 510-1 stably overexpressing the fragment 1-510 showed induction of proliferative activity on HUVEC compared to a stable control clone PC-3 Vector 7.
  • RNA from PC-3 cells stably transfected with fragment 1-510 or vector control was transcribed into cDNA and relative expression of IL-8 versus G6PDH was calculated after quantitative real-time PCR. Indicated is the relative induction of IL-8.
  • the clone PC-3 510-1 stably overexpressing the fragment 1-510 showed increased induction of IL-8 compared to a stable control clone PC-3 Vector 7.
  • Figure 2 If: Induction of Rantes by stable PC-3 clones
  • RNA from PC-3 cells stably transfected with fragment 1-510 or vector control was transcribed into cDNA and relative expression of Rantes versus G6PDH was calculated after quantitative real-time PCR. Indicated is the relative induction of Rantes.
  • the clone PC-3 510-1 stably overexpressing the fragment 1-510 showed increased induction of Rantes compared to a stable control clone PC-3 Vector 7.
  • Example 1 Isolation of the SEP cDNA by expression screening
  • Plasmid DNAs were prepared on Xantos' proprietary high-throughput robot assembly according to standard Xantos protocols (see WO 03/014346):
  • RNAse containing buffer Pl
  • P2 alkaline buffer
  • P3 acid buffer
  • 2.2x10 4 HEK 293 cells were seeded in 96-well tissue culture plates (Costar) in lOO ⁇ l DMEM medium containing 5% FCS (Invitrogen).
  • Transfection of 18000 cDNAs from a clone collection MMC Clone Collection (IRAK-Collection (,,Mammalian Gene Collection”; RZPD, Berlin) described in Strausberg RL, Feingold EA, Klausner RD, Collins FS. The Mammalian Gene Collection. Science, 1999. 286, 455-457) on 293 cells was performed 24hrs post seeding using calcium phosphate co-precipitation.
  • Precipitates were removed after 4 hours and cells were switched to nutrient deficient DMEM (DMEM, 1.5%FCS, 1% Na- pyruvate, 1% Glutamine, lOO ⁇ g/ml gentamycin, 0.5 ⁇ g/ml amphotericin B).
  • DMEM fetal calf serum
  • supplements Promocell Heidelberg, single quots
  • HUVECS were plated at 2.5 x 10 3 cells /well on day 3.
  • Alamar Blue reagent For each well of a 96well plate, 11 ⁇ l of Alamar Blue reagent were mixed with 9 ⁇ l of ECBM and the resulting 20 ⁇ l were added directly to the HUVEC cells without removal of medium. Incubation was performed at 37 0 C for 4 hours. Alamar Blue fluorescence was measured at 530nm excitation and 590nm emission.
  • Positive control for proliferation of HUVECs was supernatant containing VEGF derived from the clone collection.
  • Negative controls were supernatants from vec- tor-transfected cells and PDGF-transfected 293 cells.
  • SEP Stimulator of Endothelial Proliferation
  • the original SEP clone identified was the IMAGE clone 5123637 derived from a murine liver cDNA library.
  • BLAST searches against the human UniGene database were performed. They revealed the presence of the mRNA sequence of the hypothetical protein KIAA1271 with a low E-value of about le-25. On amino acid level, however, the E-value increases to 5e-125 with an overall homology of 50% between the murine and the human predicted proteins.
  • the assumption that the respective genes may be orthologous is supported by chromosomal localisation studies: the mouse locus of 5123637 is syntenic to the human locus of KIAA1271, 2F2, and 20pl3 respectively.
  • mSEP murine SEP; Xantos clone collection
  • hSEP human SEP; received by RZPD clone services
  • Figure 1 shows the proliferation- inducing activity of mSEP and hSEP in comparison to VEGF.
  • Example 3 Verification of specific expression
  • NHDF normal human dermal fibroblasts
  • Figure 2 demonstrates that mSEP and hSEP were unable to stimulate NHDF proliferation to levels above empty vector controls. However, the cells were clearly responsive to supernatants containing FGF- 2 or PDGF. These results demonstrate that SEP acts specifically on endothelial but not fibroblast cells.
  • Example 4 Expression analysis of hSEP in comparison to VEGF
  • the primary amino acid sequence of SEP forms a protein of 540 amino acids (estimates size 59.4 of kDa), which is anchored to the membrane by a carboxyterminal membrane spanning domain followed by a hydrophilic stop- transfer sequence at the C-terminal end of the molecule. Further details related to the domain structure of SEP are provided in Figure 4. Extracellular domains, which appear to be separated from each other by flexible Gly/Ser rich interdomain linker sequences include repeats which contain 4x multiples of the sequence (L/V)-P-S-K-(L V)-P-T, as well as additional proline rich modules. The amino terminal domain contains multiple cysteins which can form disulfide bonds.
  • N-terminal protein fragments of SEP are to be considered as soluble extracellular proteins and peptides. These products can express their biological function at the site of production (highest extracellular concentration) as well as at nearby and remote locations which are different from their side of production.
  • Example 6 Identification of SEP interacting protein
  • Step 2 Prerequisite: Get an antibody against SEP or fuse SEP with another pro- tein/peptide that could be either a reporter gene (e.g. GFP or enzyme or radioactive label or other chemical compound) or immunoprecipitable by an antibody.
  • a reporter gene e.g. GFP or enzyme or radioactive label or other chemical compound
  • the fusions could be checked for maintained binding properties in the original functional assay.
  • a second transfection screen prepare a cDNA library from a transcriptome compromising the interactor (e.g. the transcriptome of the cell SEP was found functional on). Transfect and over express the cDNA clones individually into an interactor-negative cellular background (this could be checked in advance with the fusion-constructs). Detect labeled cells by visual, enzymatic or physical methods targeted to the fusion-partner of SEP. Gain interactor cDNA from cDNA stock.
  • Extract the whole cellular extract or the appropriate cellular compartment by precipitating the interactor with SEP Precipitation could be performed by immobilization via SEP specific antibodies or immobilization of SEP via a fused protein, peptide or chemical label. [Precipitation of membrane proteins might demand
  • the precipitate could be processed in the following ways: i) Separation on protein gels and blotting (optional: proteolytic cleavage prior to or after electrophoresis). Subsequently mass-spectrometric analysis is performed followed by comparison of peptide data with appropriate mass-spec-databases. In case of no such peptide-map-database entry: sequencing of protein spot or cleavage derived peptides and search in protein and nucleic acid databases (with derived nucleic acid sequences according to the translation code; e.g. search in EST-databases).
  • Step 4 a) A second transfection screen: prepare a cDNA library from a transcriptome compromising the interactor (e.g. the transcriptome of the cell SEP was found functional on). Transfect and over express the cDNA clones individually into a cellular background negative for interactor expression and SEP function (this could be checked in advance with the fusion-constructs and antibodies). Detect SEP function / activation in these cells by monitoring SEP induced phenotype (e.g. induction of VEGF). Gain interactor cDNA from cDNA stock.
  • the interactor e.g. the transcriptome of the cell SEP was found functional on.
  • a supernatant screen prepare a cDNA library from a transcriptome compromising the interactor (e.g. the transcriptome of the cell SEP was found functional on). Transfect and over express the cDNA clones individually into a cellular background potentially negative for interactor expression. Transfer supernatant (containing secreted protein coded by the transfected cDNA) to cells positive for SEP expression. Detect SEP function / activation in these cells by monitoring SEP induced phenotype (e.g. induction of VEGF). Gain interactor cDNA from cDNA stock.
  • Step 1 step 2b, step 3a+b+c, step 4a+b
  • Example 7 Increased expression of SEP in mammary and ovary cancer compared to normal tissue
  • Figure 3 indicates that EST data show high expression of human SEP in cancer versus normal in most tissues.
  • expression levels of SEP in PvNAs and cDNAs from human mammary gland (normal and cancer), ovary (normal and cancer) and colon (normal and cancer) were analysed by quantitative real-time PCR.
  • cDNA was synthesized from 1 ⁇ g of total RNA in a volume of 20 ⁇ l using random hexamers as primer and AMV ReverseTranscriptase (Roche Diagnostics).
  • Real-time PCR was carried out using a LightCycler (Roche Diagnostics).
  • Reac- tions were set up in microcapillary tubes using the following final concentrations: 1 ⁇ M each of SEP sense (TCA GGA GCA GGA CAC AGA AC) and SEP an- tisense (TGG AAG GAG ACA GAT GGA GAC) primers, 3 ⁇ M MgCl 2 , Ix SYBR Greenmaster mix and 0,2 ⁇ l of cDNA. Cycling conditions were as follows: denaturation (95° C for 10 min), amplification and quantitation (95°C for 10 s, 56°C for 10 s and 72°C for 13 s, with a single fluorescence measurement at the end of the 72°C for 13 s segment) repeated 45 times.
  • a melting curve program (55-95°C with a heating rate of 0.1 ° C/s and continuous fluorescence measurement) and a cooling step to 40 0 C followed.
  • the proce- dure was repeated for 18S rRNA as reference gene. Data were analyzed using LightCycler analysis software.
  • Example 8 Induction of VEGF
  • Induction of VEGF by SEP was measured in an ELISA specific for detection of hVEGF.
  • 2x10 4 HEK 293 cells were transfected in parallel with 0.28 ⁇ g of the indicated cDNAs (see Fig. 7) and grown in serum reduced culture medium (1.5% FCS).
  • Concentration of hVEGF in the supernatant was determined 48h after trans- fection according to the manufacturers protocol (PromoKine - Human VEGF ELISA Kit, PromoCell GmbH, Heidelberg, Germany).
  • the empty vector pCMVSport ⁇ was used as negative control.
  • positive control cells were transfected with an expression plasmid for hVEGF. Shown are means of 4 independent experiments.
  • Example 10 Increased expression of SEP in colon, lung, prostate and breast cancer compared to normal tissue
  • Figure 9 indicates higher expression of human SEP in cancer versus normal tissues.
  • expression levels of SEP in PvNAs and cDNAs from human colon (normal and cancer), lung (normal and cancer), prostate (normal and cancer) and breast (normal and cancer) were analysed by quantitative real-time PCR.
  • cDNA was synthesized from 1 ⁇ g of total RNA in a volume of 20 ⁇ l using random hexamers as primer and AMV Reverse Transcriptase (Roche Diagnostics).
  • Realtime PCR was carried out using a LightCycler (Roche Diagnostics).
  • Reactions were set up in microcapillary tubes using the following final concentrations: 1 ⁇ M each of SEP sense (TCA GGA GCA GGA CAC AGA AC) and SEP antisense (TGG AAG GAG ACA GAT GGA GAC) primers, 3 ⁇ M MgCl 2 , Ix SYBR Greenmaster mix and 0,2 ⁇ l of cDNA. Cycling conditions were as follows: dena- turation (95° C for 10 min), amplification and quantitation (95°C for 10 s, 56°C for 10 s and 72°C for 13 s, with a single fluorescence measurement at the end of the 72 0 C for 13 s segment) repeated 45 times.
  • a melting curve program (55-95°C with a heating rate of 0.1 ° C/s and continuous fluorescence measurement) and a cooling step to 40°C followed.
  • a heating rate of 0.1 ° C/s and continuous fluorescence measurement For relative quantification the procedure was re- peated for 18S rRNA as reference gene. Data were analyzed using LightCycler analysis software.
  • Example 11 Increased expression of SEP in breast cancer versus normal tissues compared to VEGF
  • Figure 10 indicates higher expression of human SEP in more breast cancer versus normal tissues compared to VEGF.
  • expression levels of VEGF in RNAs and cDNAs from human breast were analysed by quantitative real-time PCR in the same breast tissue samples as indicated in figure 9.
  • cDNA was synthesized from 1 ⁇ g of total RNA in a volume of 20 ⁇ l using random hexamers as primer and AMV Reverse Transcriptase (Roche Diagnostics).
  • Realtime PCR was carried out using a LightCycler (Roche Diagnostics).
  • Reactions were set up in microcapillary tubes using the following final concentrations: 1 ⁇ M each of VEGF sense (TAC CTC CAC CAT GCC AAG TG) and VEGF antisense (CTA CTA AGA CGG GAG GAG GAA G) primers, 3 ⁇ M MgCl 2 , Ix SYBR Greenmaster mix and 0,2 ⁇ l of cDNA. Cycling conditions were as follows: dena- turation (95° C for 10 min), amplification and quantization (95 0 C for 10 s, 56°C for 10 s and 72°C for 13 s, with a single fluorescence measurement at the end of the 72°C for 13 s segment) repeated 45 times.
  • a melting curve program (55-95°C with a heating rate of 0.1 ° C/s and continuous fluorescence measurement) and a cooling step to 40°C followed.
  • For relative quantification the procedure was repeated for 18S rRNA as reference gene.
  • Data were analyzed using LightCycler analysis software. Relative expression levels of VEGF were compared to relative expression levels of SEP as shown in figure 9.
  • Example 12 Increased expression of SEP in colon cancer versus normal tissues compared to VEGF.
  • Figure 11 indicates correlating expression levels of human SEP and VEGF in normal colon tissue where as correlation is less pronounced in colon cancer.
  • expression levels of VEGF in RNAs and cDNAs from human colon were analyzed by quantitative real-time PCR (as described in figures 9 and 10). Relative expression levels of SEP were compared to relative expression levels of VEGF as shown in Figure 9.
  • Example 13 Increased expression of SEP in HEK 293 cells under hypoxic conditions.
  • Figure 12 indicates higher expression of human SEP in HEK293 cells under hypoxic conditions simulated by incubation with CoCl 2 compared to expression levels of G6PDH.
  • expression levels of SEP in RNAs and cDNAs from HEK 293 cells either untreated or incubated with medium containing 5OmM CoCl 2 for 24 hours were analyzed by quantitative real-time PCR (as described in example 10).
  • Incubation with CoCl 2 is an accepted model for chemical induction of hypoxic conditions in cells.
  • expression levels of VEGF were determined under identical conditions. For relative quantification the procedure was repeated for G6PDH as reference gene. Data were analyzed using LightCy- cler analysis software.
  • tissue samples of patients were stained for SEP protein using immuno histochemistry (IHC) and mRNA levels were measured using quantitative real-time PCR (QPCR) as described in example 7.
  • Table 1 shows expression of SEP in different solid tumors and corresponding normal tissues.
  • Figure 13 shows increased expression of SEP in colon tumor tissue compared to adjacent normal tissue as an example.
  • Indicated tissue samples were either stained for SEP protein by immuno histochemistry using anti-SEP antiserum (described in Example 18) or were analysed for SEP RNA expression by QPCR as described in Example 7.
  • Immunostaining (Applied Phenomics, Estonia) was performed on whole body tissue arrays (core diameter 0.6 and 1.5 mm, paraformaldehyde-fixed and paraffin-embedded material). Manual immunostaining using DAKO secondary reagents (DAKO Duet HRP kit) was performed using standard citrate /microwave pre-treatment. Unspecific binding of secondary reagents was prevented by biotin blocking. The results were evaluated by experts in immunohistochemistry and a pathologist.
  • the immunoreactive cells were the tumor cells while the surrounding stroma was essentially negative. In some instances, sporadic staining was detected in some capillaries of the tumor tissue (not normal), which was in the endothelial cells or smooth muscle cells (Fig. 13). Expression of SEP is increased in tumor tissue of breast, colon, lung and prostate.
  • Normal tissue tested by IHC was found positive for pancreas and salivary gland and negative for brain, peripheral nerve, adrenal gland, ovary, testis, thyroid, bone marrow, spleen, tonsils, myocard, aorta, vena cava, liver, esophagus, stomach, small intestine, kidney, bladder, uterus, cervix, skeletal muscle, skin, lymph node and adipose tissue.
  • Example 15 Expression of SEP on the cell surface of transfected HEK 293
  • HEK 293 cells were transfected with expression plasmids of indicated constructs and expression of SEP was monitored by FACS analysis using anti-SEP antiserum. Expression of SEP was analysed by binding of anti-SEP antibodies (antiserum) to the cell surface of transfected HEK293 and detection by FACS. For this, HEK293 cells were trans- fected with SEP, fragment 1-510 or empty vector as control. 48 h after transfected cells were harvested, washed 3 times with PBS containing 0.1 % BSA (bovine serum albumine) and stained with anti-SEP antiserum (rabbit, 1:1000 in PBS/0.1%BSA, 1 h on ice).
  • BSA bovine serum albumine
  • the secondary antibody (Dianova, FITC labeled anti rabbit, 1:100) was applied for 30 min on ice. Before FACS analysis cells were incubated with propidium iodine (PI) for detection of dead cells. Cells were analysed in a FACSCalibur cytometer (Becton Dickinson) for binding of anti-SEP antiserum to living (PI) negative cells using FACS analysis software. To determine background staining of the secondary antibody cells were incubated without specific anti-SEP antibodies (primary antibody). Specific surface signal was calculated as % positive cells in the presence of primary antibody compared to % positive cells in the absence of primary anti- body.
  • PI propidium iodine
  • HEK 293 transfected with hSEP showed specific staining for SEP protein on the cell surface in FACS analysis compared to control transfections with empty vector.
  • the expression of SEP 1-510 on the cell surface is lower because the protein fragment is secreted (Fig. 14)
  • Example 16 SEP is active as soluble/shedded protein
  • Figure 15a shows the proliferation-inducing activity of purified fragment 1-510 in comparison to PBS and the inactive fragment 1-167.
  • the relative fluorescence units (RFU) are given as mean value from three independent experiments. Experiments were performed following the manually adapted protocol described above.
  • Fragment 1-167 represents the negative control resulting from transfection of the expression plasmid into 293 cells and measurement of Alamar Blue to determine the non-specific (background) proliferative effect of the supernatant derived from HEK293 cells.
  • the fragment 1-510 showed a similar activity compared to full length SEP (SEP-FuIl).
  • the fragment 1-167 showed no activity.
  • SEP was transfected in HEK 203 cells and the supernatant was analysed in Western blot analysis.
  • SEP protein in supernatant of transfected HEK293 was detected in Western Blot analysis using SEP specific antiserum (Fig 15 b).
  • the indicated fragments were secreted into the supernatant of transfected HEK293 cells and could be detected by SEP specific Western Blot Analysis.
  • To analyse the activity of the purified proteins eluates from nickel-agarose column were applied to HUVEC cells and proliferation was monitored using Alamar Blue Assay. The relative fluorescence units (RFU) are given as mean value of three independent experiments. Experiments were performed following the manually adapted protocol described above ( Figure 15c).
  • Fragment 1-167 represents the negative control resulting from expression and purification of the inactive fragment from supernatants of transfected HEK293 cells. 1-167 was derived using the same expression and purification system. PBS represents a negative control to determine the non-specific (background) proliferative effect of the buffer the purified protein was dialysed against. The purified fragment 1-510 showed activity compared to the negative controls. The fragment 1-167 showed no activity.
  • cDNA was synthesized from 1 ⁇ g of total RNA in a volume of 20 ⁇ l using random hexamers as primer and AMV ReverseTranscriptase (Roche Diagnostics).
  • Real-time PCR was carried out using a LightCycler (Roche Diagnostics).
  • Rantes reactions were set up in microcapillary tubes using the following final concentrations: 1 ⁇ M each of Rantes sense (CGC TGT CAT CCT CAT TGC TA; SEQ ID NO: 19) and Rantes antisense (GCA CTT GCC ACT GGT GTA GA; SEQ ID NO: 20) primers, 2.5 ⁇ M MgCl 2 , Ix SYBR Greenmaster mix and 0,2 ⁇ l of cDNA.
  • Cycling conditions were as follows: denaturation (95° C for 10 min), amplification and quantitation (95°C for 10 s, 55°C for 10 s and 72°C for 13 s, with a single fluorescence measurement at the end of the 72 0 C for 13 s segment) repeated 45 times.
  • a melting curve program 55-95°C with a heating rate of 0.1 ° C/s and continuous fluorescence measurement
  • a cooling step to 4°C followed.
  • IL-8 reactions were set up in microcapillary tubes using the following final concentrations: 1 ⁇ M each of IL-8 sense (CTG CGC CAA CAC AGA AAT TA; SEQ ID NO: 21) and IL-8 antisense (TGA ATT CTC AGC CCT CTT CA; SEQ ID NO: 22) primers, 2.5 ⁇ M MgCl 2 , Ix SYBR Greenmaster mix and 0,2 ⁇ l of cDNA.
  • Cycling conditions were as follows: denaturation (95° C for 10 min), amplif ⁇ ca- tion and quantitation(95°C for 10 s, 58°C for 10 s and 72°C for 13 s, with a single fluorescence measurement at the end of the 72 0 C for 13 s segment) repeated 45 times.
  • a melting curve program 55-95°C with a heating rate of 0.1 ° C/s and continuous fluorescence measurement
  • a cooling step to 4 0 C followed.
  • For relative quantification the procedure was repeated for G6PDH RNA as reference gene. Data were analyzed using LightCycler analysis software.
  • Figure 16c shows multiple proliferative activities of supernatants of HEK293 cells transfected with SEP separated by ion exchange chromatography.
  • HEK293 cells were transfected with CaCl 2 in 10 cm plates with SEP full length cDNA or the empty vector as a control. After transfection the medium was exchanged against DMEM containing 0.5% FCS plus supplements. After 48 hours supernatants were collected, centrifuged (5000 g, 10 min) and 15 ml of supernatant was combined with 30 ml of anion exchange loading buffer (20 niM Tris pH8) (load). The protein solution was loaded onto a HiTrap Q FF column (1 ml, Pharmacia) using an AKTA-FPLC system (also Pharmacia).
  • the flowthrough was collected and the protein was eluted by applying a 0 - 400 mM NaCl gradient. 46 fractions were collected and 10 ul of each fraction was applied to a 96 well of HUVEC cells (2,8xlO 3 cells per well, plated 24 hours earlier). A proliferation assay was performed as described above.
  • Figure 16 c shows that 3 activity peaks (starting at 190 mM, 260 mM and 350 mM NaCl) can be found if the supernatant from SEP-transfected cells is compared with the supernatant from control vector transfected cells.
  • Example 18 Generation of antibodies against SEP and detection of SEP pro- tein in Western Blots
  • Antibodies against SEP were produced by immunizing rabbits with peptides PMPVQETQAPESPGENSEQAL (SEQ ID NO: 23) and PADPDGGPRPQADRK (SEQ ID NO: 24). Additionally, rats were immunized with the peptide SKLPINSTRAGM (Eurogentec, Belgium; SEQ ID NO: 25). In vitro selection of human F(ab)s was performed using recombinant SEP (aa 37-510) from E.coli using phage display according to Kretzmar and von Ruden (Curr Opin Biotechnol. 2002 Dec;13(6):598-602). The antibodies were tested in Western Blot and ELISA to show suitability for diagnostic assays. Proliferation assays with HUVEC cells were performed to determine functional activity of the antibodies (growth inhibition). Table 2: Antibodies against SEP
  • Antibodies against SEP recognise SEP protein and are suitable for diagnostic detection of SEP.
  • Antibodies against SEP were produced by immunizing rabbits with peptides PMPVQETQAPESPGENSEQAL (SEQ ID NO: 23) and PADPDGGPRPQADRK (SEQ ID NO: 24). Additionally, rats were immunized with the peptide SKLPINSTRAGM (SEQ ID NO: 25; Eurogentec, Belgium). In vitro selection of human F(ab)s was performed using recombinant SEP (aa 37-510) from E.coli. The antibodies were tested in ELISA to show suitability for diagnostic assays. For this, HEK293 cells were transfected with an expression plasmid for SEP 1-510 tagged with the V5-epitope or with empty vector as control.
  • Antibodies against SEP recognise soluble SEP protein in ELISA and are suitable for diagnostic detection of SEP.
  • Antibodies against SEP were produced by immunizing rabbits with peptides PMPVQETQAPESPGENSEQAL (SEQ ID NO: 23) and PADPDGGPRPQADRK (SEQ ID NO: 24). Additionally, rats were immunized with the peptide SKLPINSTRAGM (Eurogentec, Belgium; SEQ ID NO: 25). In vitro selection of human F(ab)s was performed using recombinant SEP (aa 37-510) from E.coli (Morphosys, Germany). As shown in Examples 18 and 19 the antibodies were tested in Western Blot and ELISA to show suitability for diagnostic assays. Proliferation assays with HUVEC cells were performed to determine functional activ- ity of the antibodies (growth inhibition).
  • Figure 18 shows an example of growth inhibition by a rabbit anti-serum specific against SEP.
  • HUVEC cells were incubated with indicated amounts of anti-serum and supernatant of SEP transfected HEK293 cells for 5 days and proliferation of the cells was measured using the AlamarBlue Assay (as described above).
  • Anti- SEP anti-serum reduced SEP induced growth of HUVEC cells compared to controls and is therefore suitable for therapeutic intervention of SEP activity.
  • FIG. 19 shows an example of growth inhibition by a human F(ab) specific against SEP generated by in vitro selection.
  • HUVEC cells were incubated with 1 ⁇ g/ml of F(ab) and supernatant of SEP transfected HEK293 cells for 5 days and proliferation of the cells was measured using the AlamarBlue Assay (see above).
  • F(ab) 15 reduced SEP induced growth of HUVEC cells to 71% compared to untreated cells. Recombinant antibodies and antibody fragments against SEP are therefore suitable for therapeutic intervention of SEP activity.
  • Example 21 Inhibition of SEP with peptides
  • the peptide pWobble is a mixture of 4 peptides representing the amino acid sequence of 4 repeats within the protein sequence of SEP (N-(L/V)PSK(L/V)PT-C; SEQ ID NO: 26).
  • the peptide p3061 is derived from this sequence (N-LPSKLPT-C; SEQ ID NO: 27).
  • Figure 20 shows an example of growth inhibition by synthetic peptides derived from the extra cellular domain of SEP.
  • HUVEC cells were incubated with indicated amounts of peptide and supernatant of SEP transfected HEK293 cells for 5 days and proliferation of the cells was measured using the AlamarBlue Assay (as described).
  • RNA was extracted from tumor cell lines and transcribed into cDNA. Relative expression levels were analysed by quantitative real-time PCR (QPCR) as described above. Supernatants of these tumor cell lines were transferred to HUVEC and proliferation of HUVEC was monitored using the AlamarBlue assay as described above. Table 3 indicates expression of SEP and proliferative activity of supernatants of different tumor cell lines. The results show that increased SEP expression correlates with increased proliferative activity.
  • QPCR quantitative real-time PCR
  • stably transfected over- expressing tumor cell lines were generated to verify the observed phenotype of inhanced HUVEV proliferation of cell supernatants of SEP overexpressing cells.
  • cells were co-transfected with a plasmid providing Neomycin resistence (pcDNA3.1, Invitrogen) and expression plasmids for SEP, SEP 1-510 or empty vector as control.
  • Stable transfectants were selected with the Neomycin analog Geneticin (Gibco, MCF-7 800 ⁇ g/ml, PC-3 ⁇ g/ml) for 3 weeks and stable clones were isolated. Overexpression of SEP in selected clones was verified by QPCR and Western Blot analysis as described above, Table 3).
  • FIG. 21 shows inhanced induction of HUVEC proliferation and induction of IL- 8 and Rantes of stably transfected MCF-7 (A-C) and PC-3 (D-F) clones compared to clones stably transfected with empty vector as control.
  • Table 3 Expression levels of SEP in selected tumor cell lines and corresponding proliferative activities of supernatants on HUVEC.
  • the indicated stable clones show overexpression of SEP and induction of IL- 8 and Rantes.
  • the clone PC-3 510-1 stably overexpressing the fragment 1-510 showed induction of proliferative activity on HUVEC compared to a stable control clone PC-3 Vector 7.
  • total RNA from PC-3 cells stably transfected with fragment 1-510 or vector con- trol was transcribed into cDNA and relative expression of IL-8 versus G6PDH was calculated after quantitative real-time PCR (Figure 2Ie). Indicated is the relative induction of IL-8.
  • the results of these analyses indicate that the clone PC-3 510-1 stably overexpressing the fragment 1-510 showed increased induction of IL-8 compared to a stable control clone PC-3 Vector 7.

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Abstract

L'invention concerne un nouveau facteur angiogénique, SEP, ainsi que des dérivés solubles de celui-ci, et son utilisation dans des compositions pharmaceutiques ou de diagnostic.
PCT/EP2004/006270 2003-06-10 2004-06-09 Facteur angiogenique et son utilisation medicale WO2004111085A1 (fr)

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WO2007013391A1 (fr) * 2005-07-26 2007-02-01 Japan Science And Technology Agency Molécule ips-1 pouvant induire la production d'un interféron
EP1892528A1 (fr) * 2006-08-25 2008-02-27 Institut Pasteur Utilisation d'un agent modulateur de la production de l'interféron, interagissant avec le pbd de plk

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US11224665B2 (en) * 2016-10-05 2022-01-18 Duke University Mitochondrial antiviral signaling (MAVS) protein compositions and methods of using the same

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007013391A1 (fr) * 2005-07-26 2007-02-01 Japan Science And Technology Agency Molécule ips-1 pouvant induire la production d'un interféron
EP1892528A1 (fr) * 2006-08-25 2008-02-27 Institut Pasteur Utilisation d'un agent modulateur de la production de l'interféron, interagissant avec le pbd de plk
WO2008022805A2 (fr) * 2006-08-25 2008-02-28 Institut Pasteur Utilisation d'un agent modulateur qui interagit avec le pbd de protéines plk pour moduler l'induction ifn
WO2008022805A3 (fr) * 2006-08-25 2008-05-08 Pasteur Institut Utilisation d'un agent modulateur qui interagit avec le pbd de protéines plk pour moduler l'induction ifn

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