WO2000020572A2 - Gewebebindende peptide, ihre identifizierung, herstellung und verwendung - Google Patents
Gewebebindende peptide, ihre identifizierung, herstellung und verwendung Download PDFInfo
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- WO2000020572A2 WO2000020572A2 PCT/EP1999/007296 EP9907296W WO0020572A2 WO 2000020572 A2 WO2000020572 A2 WO 2000020572A2 EP 9907296 W EP9907296 W EP 9907296W WO 0020572 A2 WO0020572 A2 WO 0020572A2
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- the present invention relates to tissue-binding peptides, their identification with the aid of tissues, their production and use as medicaments or diagnostic agents.
- arteriosclerosis The pathogenesis of arteriosclerosis is increasingly attributed to damage to the endothelium, which results in endothelial dysfunction. Endothelial dysfunction is mainly reflected in a changed expression pattern of surface proteins (selectins, adhesion molecules) but also in a change in the synthesis rates of growth hormones. So z. B. in the early stage of arteriosclerosis an increased expression of the E- and P-selectins and the cellular adhesion molecules VCAM and ICAM for the immigration of monocytes into the vessel wall. The conversion of the monocytes into macrophages leads to an altered synthesis of growth factors, which in particular stimulate smooth muscle cells to proliferate and to release mediators which have an inflammatory effect.
- Drug therapy for arteriosclerosis tries to reduce the causes at an early stage, if possible before the manifestation.
- a main goal is to increase the plasma cholesterol level by increasing the excretion of cholesterol metabolites (bile acids) or by reducing cholesterol biosynthesis. Due to the counter-regulation initiated by the organism, drug therapy can only lower the total cholesterol level by approx. 30%. Since this method is also not suitable for inducing regression of an existing arteriosclerosis, percutaneous, transluminal angioplasty (PTA), ie widening of the stenosed section of the vessel using a balloon catheter, is the therapy today of choice.
- PTA percutaneous, transluminal angioplasty
- restenosis ie reclosure
- surgical treatment is therefore carried out by placing a bypass. However, this therapy also causes restenosis of the bypass vessel in about 50% of the patients within 3-6 months after the treatment.
- a number of gene therapy methods have therefore been developed, in which a second catheter is used, via which a vector with a gene therapy effect is applied locally to the affected vessel section (see, for example, WO95 / 27070).
- the transfection efficiency could be increased, for example, by using HVJ liposomes (Morishita, R. et al. (1994) Gene 149, 13).
- Inactivated Sendai viruses hemagglutinating virus of Japan, HVJ
- F and HN protein the viral fusion and binding proteins
- the genetic material can be efficiently introduced directly into the cell's cytoplasm bypassing the lysosomes.
- cell-specific antibodies to liposomal (Vingerhoeds, H. et al. (1994) Immunometh. 4, 259) or viral (Wickha, TJ et al. (1996) J. Virol. 70, 6831) vectors, under the control of a strong viral promoter to be able to target the therapeutic gene only into the desired cell type.
- tissue-specific expression is of great medical importance in particular for the therapy of vascular diseases. This not only opens up opportunities for direct vascular diseases, such as B. to treat arteriosclerosis and its complications (stenosis, restenosis, heart attack), but also all organs and tumors that can be reached via the bloodstream.
- tissue-oriented gene transfer into vascular cells can also be used to express therapeutic proteins which are not or are no longer present in sufficient amounts in the target organism due to pathological or genetic changes, e.g. B. Factor VIII deficiency, insulin deficiency etc.
- An essential goal of somatic gene therapy is therefore to introduce a therapeutic gene into the target cells of the organism as specifically as possible after systemic or local administration and to express the therapeutic gene essentially only in these cells.
- the therapeutic gene should on the one hand be packaged in such a way that it is essentially not degraded by nucleases during transport through the blood vessel system, and on the other hand it should be in a form which enables cell-specific gene expression in the target cells.
- the specific recognition of cells is also referred to as "targeting".
- targeting There are basically two possibilities for this targeting: on the one hand, antibodies against receptors on the Use cell surface, which are integrated into viral or liposomal vector systems (Vingerhoeds, H. et al (1994) supra; Wickham, TJ (1996) supra), and on the other hand peptides with high binding affinity for receptors on the cell surface can be used.
- antibodies for example, certain tumor cells could be recognized and even detected in the intact tissue association using a suitable label.
- a major disadvantage of antibodies is that for technical reasons they are usually produced in the mouse and often lead to serious side effects on patients due to an immune reaction. Even so-called humanized antibodies are not free from this problem. In individual cases, such pronounced immune complexes can occur, which drastically restrict the blood flow to the kidneys and thus lead to fatal kidney failure.
- Peptide presenting libraries that are chemically produced have been developed e.g. B. von Geysen, H.M. et al. (1996) Mol. Immunol. 23, 709 or de Koster, H.S. (1995) J. Immunol. Methods 187, 179-188.
- amino acid mixtures were used on the solid phase, so that the amino acid sequence of the resulting peptides is statistical, i. H. was randomly distributed.
- the peptide banks could also be used in resin-bound form (de Koster, H.S. et al. (1995) supra).
- peptide-presenting banks which are based, for example, on the presentation of the peptides on the surface of phages
- at the N-terminus of the pIII coat protein of filamentous phages see, for example, Kay, BK et al. (1993) Gene 128, 59 or Jellis, CL et al. (1993) Gene 137, 63-68) presented peptides varied from 6 to 38 amino acids were used, among other things, for epitope screening of antibodies (Cortese, R. et al. (1994) Trends Biotechnol.
- peptide-presenting bacterial banks are known.
- An example of this are fusions of a combinatorial peptide library with thioredoxin, which led to the identification of peptide aptamers which recognize the cyclin-dependent kinase 2 (Colas, P. et al. (1996) Nature, 380, 548-550).
- Browsing a combinatorial peptide library was e.g. B. also with fusions to the C-terminus of the alpha subunit of G, (340-350) to identify rhodopsin-binding sequences (Martin, EL et al. (1996) J. Biol. Chem. 271, 361-366 , No. 1).
- Another example is peptide insertions in a thioredoxin-flagellin fusion protein, which is presented on the E. coli cell surface, with which protein-protein interactions can be investigated (Lu, Z. et al. (1995) Bio / Technology 13, 366- 372; U.S. Pat. No. 5,635,182).
- One object of the present invention is therefore a tissue-binding peptide selected from a peptide with the amino acid sequence
- GEGRTVVLSF AWCRGGILGDAM, ' GNLVDLVVGFDD, RVSPPKKSGGGV, GSSKWGLTXKCG, RGGVRQRSRGRR, GEGRTVVCRS or SQRWTALWQWIG and variants thereof.
- variants are understood to mean deletions, additions or substitutions of one or more amino acids, the tissue-specific binding of the peptide essentially not being changed, i. H. the peptide has retained its ability to bind better to one tissue than another.
- deletion preferably no more than 4 amino acids, especially no more than 2 amino acids, are deleted.
- the deletion is preferably carried out at the N- or C-terminus of the peptide, in particular at the N-terminus.
- the term addition means not only the addition of one or more amino acids, for example 1-25 amino acids, but also fusion proteins of the peptide according to the invention with another C- and / or N-terminal peptide or protein.
- a fusion of a tissue-specific peptide with a peptide that contains several histidines leads not only to tissue-specific binding, but also to permeabilization of the cell membrane at a slightly acidic pH (see, e.g., Midoux, P. et al (1998) Bioconjugate Chem. 9, 260-267).
- a fusion protein consists, for example, of the peptide according to the invention and several lysines, for example 16 lysines in succession or the nucleocapsid protein (NCp) 7 derived from HIV-1 or NCp7 Peptides, a nucleic acid, for example a DNA containing a therapeutic gene, can be complexed to the peptide according to the invention in a particularly simple manner (see, for example, Harbottle, RP et al. (1998) Human Gene Therapy 9, 1037-1047 or Bachmann, AS et al. (1998) J. Mol. Med. 76, 126-132).
- a fusion protein which contains a tissue-specific peptide, a peptide which brings about permeabilization of the cell membrane and a nucleic acid-binding peptide, which is particularly advantageous.
- substitutions in which, for example, one or more amino acids with a hydrophobic side chain are replaced by one or more other amino acids with another hydrophobic side chain.
- amino acids with hydrophobic side chains are glycine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline.
- Amino acids with a polar neutral residue can also be interchanged. Examples include serine, threonine, cysteine, tyrosine, asparagine or glutamine.
- amino acids with a polar acidic residue can be interchanged. For example, this includes aspartic acid or glutamic acid.
- Amino acids with a polar basic residue can also be interchanged, such as. B. lysine, arginine or histidine.
- amino acid means not only the amino acids naturally occurring in proteins, but also other amino acids which can be incorporated into a peptide chain, such as, for. As hydroxylysine, hydroxyproline, ornithine, citrulline or homocysteine.
- the individual amino acids of the peptide according to the invention can also be modified.
- the peptide can be marked and / or modified to be protected.
- a suitable label is, for example, the exchange of the hydroxyl group of threonine or serine with the isotope 18 F, which has a half-life of 2 hours and can therefore be used well in clinical practice.
- the peptide can also be labeled with 18 F at the carboxy terminus via a fluoroethyl amide bond .
- the C-terminal fluoramidation takes place, for example, with fluoroethylamine and a urea derivative (e.g. TBU) for activation.
- Another suitable label is the labeling of cysteine or methionine with the sulfur isotope 35 S.
- the isotope 35 S has the advantage over the isotope 18 F that it has a longer half-life.
- the peptide according to the invention can also be labeled with other compounds, for example with ⁇ -galactosidase or biotin or avidin / streptavidin.
- radioactive labeling with in particular short-lived isotopes has the advantage that the structure of the peptide remains essentially unchanged and the distribution of the peptide in the organism can be analyzed in an advantageous manner, for example with the help of positron emission tomography (PET).
- PET positron emission tomography
- the functional side chains of the amino acids are protected beforehand.
- the peptide on the solid phase is removed from the C- to the N-terminus using an acid-labile resin (e.g. SASRIN, a 2-methoxy-4-alkoxybenzyl alcohol resin, Frechet, JMJ et al. (1979) Polymer 20, 675) using the FastMoc ⁇ method (Merrifield, RB (1963) J. Am. Chem. Soc. 95, 1328; Wang, SS (1973) J. Am. Chem. Soc. 95, 1328; Perkin-Elmer Applied Biosystems, Foster City , CA) synthesized.
- an acid-labile resin e.g. SASRIN, a 2-methoxy-4-alkoxybenzyl alcohol resin, Frechet, JMJ et al. (1979) Polymer 20, 675
- Another object of the present invention is a nucleic acid coding for a tissue-binding peptide according to the invention.
- a nucleic acid is particularly preferably selected from a nucleic acid with the nucleotide sequence
- variants is understood in particular to mean variants which code for the same peptide due to the degeneracy of the genetic code.
- variants also means the ribonucleic acids (RNA) corresponding to the deoxyribonucleic acids (DNA) listed.
- RNA ribonucleic acids
- DNA deoxyribonucleic acids
- the DNA or RNA can also be modified to e.g. B. to be better protected against nuclease degradation. Suitable modifications can be found e.g. B. Uhlmann, E. & Peyman A. (1990) Chemical Reviews 90, 543-584 No. 4th
- a vector can, for example, be an expression vector for the production of the peptides according to the invention in prokaryotic or eukaryotic cells.
- prokaryotic expression vectors are for expression in E. coli z.
- Baculovirus vectors as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, and for expression in mammalian cells, for.
- the expression vectors also contain suitable promoters for the respective host cell, e.g. B. the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the ADH2 promoter for expression in yeast (Radorel et al. (1983), J. Biol. Chem. 258, 2674-2682), the baculovirus polyhedrin promoter for expression in insect cells (see e.g. EP-B1-0 127 839) or the early SV40 promoter or LTR promoters e.g. B. from MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232).
- suitable promoters for the respective host cell e.g. B. the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the ADH2 promoter for expression in yeast (Radorel et al. (1983), J. Biol. Chem. 258,
- virus vectors preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, e.g. B. an adeno-associated virus vector, which consists exclusively of two inverted terminal repeat sequences (ITR).
- ITR inverted terminal repeat sequences
- the El gene region is generally replaced by a foreign gene with its own Promoter or replaced by the nucleic acid of the invention.
- a non-replication-capable adenovirus is created. These viruses can then only multiply in a cell line that replaces the missing El genes.
- Replication-deficient adenoviruses are therefore generally formed by homologous recombination in the so-called 293 cell line (human embryonic kidney cell line), which has a copy of the El region stably integrated in the genome.
- the nucleic acid according to the invention is cloned into recombinant adenoviral plasmids under the control of a suitable promoter.
- This is followed by homologous recombination with an El-deficient adenoviral genome, such as, for example, dl327 or dell324 (adenovirus 5), in the Helper cell line 293.
- an El-deficient adenoviral genome such as, for example, dl327 or dell324 (adenovirus 5)
- helper cell line 293 If recombination is successful, viral plaques are harvested.
- the replication-deficient viruses produced in this way are used in high titers (for example 10 ° to 10 "" plaque-forming units or
- the exact insertion site of the nucleic acid according to the invention into the adenoviral genome is not critical. It is e.g. It is also possible to clone the nucleic acid according to the invention in the place of the deleted E3 gene (Karlsson, S. et al. EMBO J. 5, 1986, 2377-2385). However, preferably the El region or parts thereof, e.g. the EIA or EIB region (see e.g. WO 95/00655) is replaced by the nucleic acid according to the invention, especially if the E3 region is also deleted. Also suitable are third generation adenoviral vectors, i. H.
- Vectors which, apart from the ITR and the packaging signal, contain no further sequences coding for viral proteins (Kochanek, S. (1996) Proc. Natl. Acad. Sci. USA 93, 5731).
- the recombination in the helper cell line takes place e.g. B. with the help of a helper plasmid.
- the present invention is not limited to the adenoviral vector system, but adeno-associated virus vectors are also particularly suitable in combination with the nucleic acid according to the invention, since the transfer of the nucleic acid according to the invention into resting, differentiated cells can be carried out by AAV, which is for the treatment of vascular diseases is particularly advantageous.
- AAV vascular diseases
- the two approximately 145 bp long inverted terminal repeat sequences are generally sufficient for the vector functions. They carry the signals necessary for replication, packaging and integration into the host cell genome. The ability to integrate ensures long-lasting gene expression in vivo, which in turn is particularly advantageous.
- AAV AAV vectors or parts thereof by methods known to the person skilled in the art, as described, for example, in WO 95/23867, by Chiorini, JA et al. (1995), Human Gene Therapy 6, 1531-1541 or Kotin, RM (1994), Human Gene Therapy 5, 793-801.
- Vectors with gene therapy effects can also be obtained by complexing the nucleic acid according to the invention with liposomes (neutral or cationic), i.e. the nucleic acid is essentially included in the liposome, since it enables a very high transfection efficiency, in particular of cardiac muscle cells, to be achieved (see, for example, WO95 / 27070) and the nucleic acid is essentially protected against DNases.
- liposomes neutral or cationic
- the nucleic acid is essentially included in the liposome, since it enables a very high transfection efficiency, in particular of cardiac muscle cells, to be achieved (see, for example, WO95 / 27070) and the nucleic acid is essentially protected against DNases.
- Transfection with nucleic acid-liposome complexes with the aid of Sendai viruses in the form of so-called HVJ liposomes (virosomes) is particularly advantageous, since this can further increase the transfection rate.
- small unilamellar vesicles are made from cationic lipids by ultrasound treatment of the liposome suspension.
- the nucleic acid is bound ionically on the surface of the liposomes in such a ratio that a positive net charge remains and the nucleic acid is 100% complexed by the liposomes.
- Feigner et al. Feigner, PL et al. (1987) Proc. Natl. Acad.
- lipid mixtures DOTMA 1,2-dioleyloxypropyl-3-trimefhylammonium bromide
- DOPE dioleoylphosphatidylethanolamine
- new lipid formulations synthesized and tested for the efficiency of transfection of various cell lines (Behr, JP et al. (1989), Proc. Natl. Acad. Sci. USA 86, 6982-6986; Feigner, JH et al. (1994) J. Biol. Chem . 269, 2550-2561; Gao, X. & Huang, L. (1991), Biochim. Biophys. Acta 1189, 195-203).
- the new lipid formulations are DOTAP N- [l- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium ethyl sulfate or DOGS
- nucleic acid-liposome complex contains nucleic acid binding proteins, for example chromosomal proteins, preferably HMG proteins (high mobility group proteins), in particular HMG-1 or HMG-2, or nucleosomal histones such as H2A, H2B, H3 or H4 , since this can increase the expression of the desired nucleic acid at least 3-10 times.
- nucleic acid binding proteins for example chromosomal proteins, preferably HMG proteins (high mobility group proteins), in particular HMG-1 or HMG-2, or nucleosomal histones such as H2A, H2B, H3 or H4 , since this can increase the expression of the desired nucleic acid at least 3-10 times.
- the chromosomal proteins can be isolated, for example, from calf thymus or rat liver by generally known methods or can be produced by genetic engineering.
- Human HMG-1 can be genetically engineered using the human cDNA sequence from Wen, L. et al. (1989) Nucleic Acids Res., 17 (3), 1197-1214.
- Another object of the present invention is therefore also a method for producing a peptide according to the invention, in which the peptide is either chemically synthesized or, as already explained above, produced by genetic engineering.
- the chemical synthesis takes place, for example, with the aid of the generally known Merrifield technique.
- tissue-binding peptides can also be obtained according to the present invention by bringing one or more peptides into contact with a tissue and then isolating the bound peptide (s).
- Another object of the present invention is therefore a method for finding a tissue-binding peptide comprising the following steps:
- non-bound peptides are preferably, for example, by Wash the tissue with a suitable buffer solution.
- peptide is preferably understood to mean peptides with a length of approximately 5 to approximately 40 amino acids, in particular with a length of approximately 5 to approximately 20 amino acids, especially with a length of approximately 10 to approximately 20 , particularly preferably with a length of about 10 to about 15 amino acids.
- tissue is understood to mean cell dressings, in particular cell dressings including their intercellular substance, for example epithelial tissue, connective tissue, supporting tissue, muscle tissue or nerve tissue.
- tissue also includes whole organs or parts thereof and vessels. The organs or vessels are preferably still "living" organs or vessels.
- the intercellular substance is a substance embedded in the intercellular spaces, which contains a structure substance that appears to be without structure and fibrous connective tissue fibers. It was therefore completely surprising that tissue-binding peptides were found by bringing tissue into contact with one or more peptides, which peptides were essentially specific for the tissue used and were not predominantly based on non-specific structures, such as, for example. B. the intercellular substance were bound.
- the specificity of the tissue-binding peptides can preferably be increased by repeatedly bringing the peptides isolated according to step (b) into contact with the tissue one or more times. After their respective isolation, the peptides are preferably brought into contact with the corresponding tissue up to 10 times. In this way, cross-specific peptides, which are specific for several tissue types, can also be found if the peptides are brought into contact with different tissues in succession. If, for example, the peptides are brought into contact in portions with both healthy and diseased tissue, a subtractive comparison can be used to find the peptides required for healthy or diseased tissue are specific. Peptides that are essentially only specific for diseased tissue are particularly advantageous, for example, for diagnosis, for example in the context of a PET examination.
- tissue can be pathologically changed. So change e.g. smooth muscle cells in restenotic vascular areas their entire phenotype and change from the resting, secretory to the proliferative phenotype.
- tissue-binding peptides can consequently also be found which can recognize tissue which is specifically pathologically altered under in vivo conditions. It is also advantageously possible to characterize pathological cell types in the tissue structure, which have not yet been adequately characterized, using the peptides selected according to the method of the invention, and to treat them, for example, with gene therapy.
- a peptide bank for example a combinatorial peptide bank (see eg Colas, P. et al. (1996) supra), is used.
- a peptide bank according to the present invention is understood to be a collection of several different peptides, which are present free or in bound form.
- a peptide bank of free peptides contains a collection of chemically synthesized peptides. (see e.g. Geysen, H.M. et al. (1996) supra). Amino acid mixtures were used on the solid phase in peptide synthesis so that the amino acid sequence of the resulting peptides was statistically distributed. The peptide banks could also be used in resin-bound form (de Koster, H.S. et al. (1995) supra).
- Peptides in bound form are also so-called, for example peptide presenting banks, e.g. B. a peptide-presenting phage bank or preferably a peptide-presenting bacterial bank, as already described in more detail above.
- the peptides are presented in the form of a fusion protein on the surface of, for example, phages or bacteria.
- Preferred fusions are insertions, preferably fusions with thioredoxin, with thioredoxin (TrxA) -flagellin (FliC), with the alpha subunit of G t , as already described in more detail above, or z. B. with LamB (Charbit, A. et al. (1988) Gene 70, 181-189) or Lpp-OmpA (Francisco, JA et al. (1993) Proc. Natl. Acad. Sci. USA 90, 10444-10448 ).
- LamB Charge, A. et al. (19
- peptide-presenting bacterial banks and in particular those with a thioredoxin fusion protein are particularly preferred especially for selection in an in vivo approximate perfusion model of isolated vessels and organs for the following reasons:
- the peptides are presented as fusion proteins together with a protein, for example thioredoxin, the three-dimensional structure of which is generally known.
- the randomized peptides are generally presented in a very accessible domain, which is on the surface of the total protein, such as the so-called "Cys-Cys active site loop" of thioredoxin. Because the peptide is generally sufficiently far from the bacterium and thus is presented in an easily accessible manner, a good interaction with the desired binding partner is generally made possible.
- the peptides can also develop their effect in the form of fusion proteins with a protein having a therapeutic effect without essentially losing their binding capacity.
- Peptides fused to the terminus of a protein and presented in this way in phage libraries generally lose their binding capacity as soon as they are expressed as fusion proteins together with a therapeutically active protein, which is for use in gene therapy is disadvantageous.
- An important reason for this is that the peptides themselves have a free N or C terminus during the presentation which is available for binding. For this reason, it is particularly advantageous if the peptides are presented in the form of inserts into selected surface proteins by a peptide-presenting bank.
- the E.coli thioredoxin is stable against proteases. Consequently, a protease-resistant protein in the form of a fusion protein with the peptide is particularly preferred, for example, for selection in the organ model perfused ex vivo, in particular since proteases can be located on the cells in the vessels. Especially organs, e.g. isolated perfused liver, but also tumors, release a high level of proteases that could cleave the fusion protein in the event of protease sensitivity.
- the term “living” means organs or vessels that are still functional. To maintain functionality over a longer period of time, it is therefore advantageous if the organ or vessel is adequately supplied with oxygen and nutrient substrates
- a nutrient solution which contains Ca 2+ , Mg 2+ and a protein, for example an albumin such as BSA (bovine serum albumin) is preferably suitable for this purpose, preferably the concentration of calcium ions 2.5 mM and magnesium ions 1.1 raM is preferably present in the nutrient solution in a concentration of 1% for the perfusion of isolated
- BSA bovine serum albumin
- the so-called Krebs-Henseleit solution which is composed as follows (data in raM), is particularly suitable for vessels and isolated organs: NaCl 116; KC1 4.6; MgSO 4 1.1; NaHCO 3 24.9; CaCl 2 2.5; KH 2 PO 4 1.2; Glucose 8.3; Pyruvate 2.0 and 1% BSA equilibrated with 95% O 2
- a peptide-presenting bacterial bank was used in an in-vitro vascular perfusion model after a restenosis had previously been induced in a vessel on the whole animal (rat) by denuding the endothelium by means of a balloon catheter.
- This method has the decisive advantages that the changes in the expression patterns of membrane-associated or membrane-bound proteins which occur as a result of pathological vascular processes are retained, the cell polarity does not change and all cells remain in their natural tissue structure.
- the peptides found with the aid of the method according to the invention can, as already described in more detail above, be modified.
- a suitable modification is, for example, a radioactive or non-radioactive label for use in a test system or diagnostic.
- tissue-specific peptides that can be found using the method according to the invention, for example the tissue-specific transfer of substances, in particular pharmaceutically active compounds, especially for tissue-specific gene transfer.
- Tissue-specific gene transfer is used, for example, for diagnosis and / or therapy of vascular and / or organ diseases with the aid of, for example, viral and / or non-viral vectors, preferably with the aid of liposomes, as already described in more detail above.
- the peptide can be physically, chemically or be genetically linked to the desired nucleic acid (see above).
- the peptide can either be combined with a therapeutic protein, e.g. B. nitric oxide synthase (see e.g. WO95 / 27020), insulin (see e.g. EP-B1-0 001 929), erythropoietin (see e.g. EP-B1-0 148 605), or blood coagulation factors , such as B. factor VIII, interferons, cytokines, hormones, growth factors, etc.
- a therapeutic protein e.g. B. nitric oxide synthase (see e.g. WO95 / 27020), insulin (see e.g. EP-B1-0 001 929), erythropoietin (see e.g. EP-B1-0 148 605), or blood coagulation factors , such as B. factor VIII, interferons, cytokines,
- Suitable coupling methods are e.g. B. about lysine residues or nucleocapsid proteins, as already explained in more detail above (see, for example, Harbottle, RP et al. (1998) supra or Bachmann, AS et a 1. (1998) supra). It when one or more tissue-binding peptides via a positively charged domain, for. B. polylysine, are bound to one or more nucleic acids.
- a peptide which increases membrane permeation is also suitable for increasing membrane permeation during gene transfer, e.g. B. a hisitidine-containing polypeptide (see above).
- a so-called polyfection solution containing a nucleic acid with the desired therapeutic gene, a fusion protein of tissue-specific peptide and a DNA-binding part, e.g. B. a positively charged domain, and a peptide that increases membrane permeation.
- a suitable polyfection solution is described, for example, by Midoux, P. (1998) supra.
- a coupling of the peptide to the liposomes via, for example, a C-terminal cysteine introduced to an activated lipid component, e.g. B. via MPB-PE,
- the peptides according to the invention can also be used to change the tropism of viruses, preferably adenoviruses, for example for tissue-specific gene transfer. This is done, for example, by attaching the peptide via a suitable linker to the C-terminus of the Knob domain (see, e.g., Douglas, JT & Curiel, DT (1997) Neuromuscular Disorders 7, 284-298) or by exchanging the region responsible for binding in the knob domain of the fiber protein.
- viruses preferably adenoviruses
- the RGD motif in the hypervariable region of the penton base can be replaced by a cell-specific peptide analogous to the experiments carried out by Wickham with the FLAG epitope, preferably also the natural one Tropism can be switched off (see Wickham et al. (1996) J. Virol. 70, 6831).
- the tropism can be changed, for example, via a shuttle plasmid which codes for the corresponding construct.
- tissue-binding peptides Another possible use of the tissue-binding peptides is the use for the diagnosis of pathologically altered tissue, especially in a pathologically altered vessel, for inflammation, arteriosclerosis, vessels that supply tumor tissue, and tissue with proliferating smooth muscle cells of the vascular system, as well as the representation of different sections of the Vascular system, such as. B. small / large vessels, veins, organ-specific endothelium.
- the diagnosis can either indirectly via z. B. Peptide-recognizing antibodies, for example, in a generally known ELISA test (see, for example, Voller, A. et al. (1976) Bull. World Health Organ., 53, 55-63) or directly by the tissue-binding Peptide is additionally marked.
- the label can be a non-radioactive label, e.g. B. about biotin or avidin / streptavidin, or a radioactive label (see above).
- a radioactive label is particularly useful for a whole-body examination of the patient using imaging methods such as e.g. B. PET (positron emission tomography) or SPECT (single photon emission computer tomography) for the diagnosis of, for example, inflammation, vascular changes or tumors, advantageous.
- imaging methods such as e.g. B. PET (positron emission tomography) or SPECT (single photon emission computer tomography) for the diagnosis of, for example, inflammation, vascular changes or tumors, advantageous.
- the labeled peptide is administered intravenously to the patient and the distribution of the peptide in the organism is analyzed, for example, using “whole-body PET”.
- the present invention therefore furthermore relates to a medicament and / or test system, for example in the form of a diagnostic agent, comprising one or more peptides according to the invention or one or more peptides found by the method according to the invention, or one or more nucleic acids according to the invention, and, if appropriate, suitable ones Auxiliaries and / or additives.
- a diagnostic agent comprising one or more peptides according to the invention or one or more peptides found by the method according to the invention, or one or more nucleic acids according to the invention, and, if appropriate, suitable ones Auxiliaries and / or additives.
- Another object of the present invention also relates to a composition, for example in the form of a transfection or polyfection system, containing one or more peptides according to the invention or one or more peptides found by the method according to the invention, or one or more nucleic acids according to the invention and one further substance, preferably a pharmaceutically active compound, as already described in more detail above, and, if appropriate, suitable auxiliaries and / or additives.
- a composition for example in the form of a transfection or polyfection system, containing one or more peptides according to the invention or one or more peptides found by the method according to the invention, or one or more nucleic acids according to the invention and one further substance, preferably a pharmaceutically active compound, as already described in more detail above, and, if appropriate, suitable auxiliaries and / or additives.
- auxiliaries and / or additives are, for example, generally known protease or nuclease inhibitors.
- Fig. 1 shows the carotid model from Clowes. After ligating the internal and external carotid arteries, a balloon catheter is inserted through the external carotid artery into the common carotid artery, dilated and the vessel denuded over a distance of 1 - 1.5 cm. The catheter is then removed again, the external carotid artery ligated and the internal carotid artery reopened.
- Fig. 2 shows the selection of the peptides in the ex vivo perfused rat carotid.
- 3 shows the relative binding strength of the peptide P36 to a dilated rat carotid, which was removed on the 8th postoperative day, in comparison to the untreated control carotid.
- Figure 4 shows the binding constants of peptide P36 to primary aortic porcine endothelial cells (PAEC), primary porcine smooth muscle cells (VSMC) and monkey kidney cells (COS).
- PAEC primary aortic porcine endothelial cells
- VSMC primary porcine smooth muscle cells
- COS monkey kidney cells
- the endothelial cells were stimulated by LPS and TNF ⁇ (PAEC stimulated).
- Tab. 1 shows the DNA sequences and peptide sequences derived therefrom of the isolated clones.
- IMC medium contains (lxM9 salts, 0.2% (casamino acids, 0.5%
- the bacteria contain an expression cassette which codes for a fusion protein of flagellin and thioredoxin.
- the flagellin serves to anchor the fusion protein in the bacterial membrane, while the thioredoxin presents a foreign 12 amino acid long peptide in an exposed domain, the coding 36 base pair long oligonucleotide of which was cloned at the appropriate position in the coding sequence of the thioredoxin.
- the oligonucleotide used for cloning was synthesized at random, so that the bacterial bank represents a total of 10 9 different peptides.
- the expression cassette is under the control of the tryptophan promoter, so that the expression of the fusion protein can be switched on by the administration of tryptophan at the time of an exponentially growing bacterial culture. Since each bacterium only takes up one plasmid, only one type of peptide with a defined amino acid sequence is expressed from each bacterium and presented on the surface.
- IMC medium 50 ml of IMC medium were mixed with 10 9 bacteria and incubated overnight at +25 ° C for 15 hours. The cell number was then determined by measuring the OD ⁇ , 10 ° cells were incubated in 50 ml IMC medium with 100 ⁇ g / ml ampicillin and the expression of the bacterial bank was induced by adding 100 ⁇ g / ⁇ l tryptophan. The cells were incubated for another 6 hours at 25 ° C before being used for selection.
- Restenosis was induced in an established restenosis model in the rat (see, for example, Clowes, AW et al. (1983) Lab. Invest. 49, 327).
- the common carotid artery in an anesthetized rat was freed from the endothelial cells by introducing a balloon catheter (denudation). This procedure stimulates the smooth vascular muscle cells to proliferate and changes their phenotype from the resting, secretory smooth muscle cell to the proliferating smooth muscle cell.
- the vascular segment marked in FIG. 1 was dissected out and perfused in a special ex vivo perfusion system with a saline medium with the addition of glucose and gassing with oxygen. After an equilibration time of 10 min, which was used to wash out any blood cells and blood components, this vascular segment with a total of 10 10 peptide-presenting bacteria in oxygen-gassed saline medium with a flow of 0.2 ml / min. Recirculated perfused for 60 min. The non-binding bacteria were detached by treatment with saline medium and the specifically binding bacteria were eluted by perfusion for ten minutes under stringent conditions with 0.1 M glycine pH 2.0. The eluted bacteria were cultivated again and subjected to a further five selection cycles (see FIG. 2) in order to increase the specificity.
- Rats weighing approximately 500 g were anesthetized by administering 100 ⁇ l / 100 g body weight Dormicum and 100 ⁇ l / 100 g body weight Hypnorm.
- the carotid artery was then dissected cranially to the bifurcation, the internal carotid artery and all branches branching from it were ligated and the carotid communis was denuded starting from the external carotid about 2 cm along the common carotid with a 2F balloon catheter at 1 bar.
- the internal carotid artery ligature was reopened postoperatively.
- the denuded common carotid artery was dissected and ex vivo with 0.1 ml / min PBS perfused with 0.1 M glucose. Meanwhile, 10 ml of the tryptophan-induced bacterial suspension was centrifuged at 1000 rpm for 3 min, washed in PBS, centrifuged again and the sediment in 6 ml total volume of PBS, 0.1 M glucose, 0.1% BSA, 1 mM CaCl 2 , 10 mM MgCl 2 suspended. The carotid was then perfused recirculating with this suspension with gassing of oxygen at 0.2 ml / min for 1 h.
- the suspension was then washed out by perfusion with 2-3 ml of PBS at 0.2 ml / min before the bound bacteria were eluted by elution with 3 ml of 0.1 M HCl (pH 2.2 adjusted with glycine) and in 50 ml of IMC Medium, 100 ⁇ g / ml ampicillin were grown overnight at +25 ° C.
- bacteria were obtained which express peptides on their surface which bind specifically to proliferating smooth muscle cells.
- the bacteria obtained after six selections were then plated on nutrient medium and cultures were grown from 60 individual colonies. From these cultured bacteria, the plasmid DNA was isolated and sequenced.
- specifically binding peptides could be detected using a method closest to the in vivo situation (organ culture model).
- the sequence of peptide 36 shows homologies to the ⁇ 4 integrin, which is expressed on leukocytes and is responsible for the attachment of the leukocytes to activated vascular cells. The recognition is mediated by the vascular cellular adhesion molecule VCAM-1. The homology is not in the area that is characterized as the binding area of the integrin (RGD-Motif). 6. Attachment studies
- the peptide from clone 36 (peptide 36) used for the binding studies was produced using the Fast-Moc solid-phase synthesis on a fully automatic peptide synthesizer from Applied Biosystems on a 0.1 mmol scale.
- the peptide was radioactively labeled to determine the binding affinities to the desired cell type.
- the N-terminal amino acid (glycine) was used for the radioactive labeling.
- 14 C-gycin was protected with activated N-fluorenyl-methoxycarbonyl-succinimide, purified and coupled covalently N-terminally to the peptide located on the resin and protected in the side chains by means of dicyclohexyl-carbodiimide.
- the peptide was then cleaved from the resin, purified by HPLC and examined for binding to the target cells in the perfused vessel model and in the cell culture.
- the mixture was then left to stand at room temperature overnight, the aqueous phase was acidified by adding 2 M HCl and extracted twice with 10 ml of ethyl acetate each time.
- the combined ethyl acetate phases were washed neutral with water several times, dried with MgSO 4 and the product was precipitated by adding petroleum ether and filtered off.
- the product was dissolved in ethyl acetate and slowly precipitated by adding small amounts of petroleum ether.
- the labeled Fmoc-Glycine was used for the synthesis of a total of 0.4 mmol peptide, so that each peptide was labeled 75%.
- the appropriate amount of the resin / peptide mixture (0.1 mmol) was weighed out and placed in a glass frit with a stopper and a tap. 25 mg of the I4 C-Fmoc glycine (0.075 mmol) were dissolved in 1 ml of NMP, 155 ⁇ l of 0.45M HBTU / HOBt and 750 ⁇ l of 2 M DIEA in NMP were added to the resin / peptide mixture and added for one hour incubated with gentle shaking at room temperature.
- Fmoc-glycine 150 mg were then dissolved in 2 ml of NMP, 2 ml of 0.45M HBTU / HOBt and 1 ml of 2M DIEA were added and the mixture was added to the reaction mixture. The mixture was 30 min. incubated at room temperature, the unreacted Fmoc-glycine filtered off and the mixture washed four times with 6-10 ml of NMP each. The protective group was split off by incubation for 30 minutes with 22% piperidine in NMP with stirring. The piperidine was filtered off, and the resin / peptide mixture was then washed four times with NMP and DCM.
- the mixture was ice-cooled and treated with a solution of 0.75 g phenol, 0.25 ml EDT, 0.5 ml thioanisole, 0.5 ml water and 10 ml of TFA are added with stirring.
- the mixture was then incubated for two hours at room temperature with stirring, the peptide released was filtered off and precipitated by adding five times the volume of ice-cooled diethyl ether. After centrifugation, the peptide in the sediment was washed several times with dieethyl ether and centrifuged again.
- the peptides were chromatographed using an ⁇ kta Purifier 100 over an RPC resource column (3 ml, Pharmacia) with a 20 minute gradient of water, 0.1% TFA to 95% acetonitrile, 0.07% TFA at a flow rate of 2 ml / min cleaned and then lyophilized.
- the vessels or the cells were incubated for different time intervals with different concentrations of the peptides to be examined and the amount of bound peptide per cm vessel or per cell number was determined.
- the peptide 36 binds approximately four times more strongly to the dilated carotid than to the untreated control in the ex vivo perfusion.
- PAEC porcine endothelial cells
- LPS lipopolysaccharides
- TNF ⁇ tumor necrosis factor
- the peptide was able to bind specifically to smooth muscle cells (VSMCs) as well as to activated PAECs. Surprisingly, the peptide binds even more strongly to activated PAECs than to VSMCs. In addition, the binding constants of the peptide differ from activated and non-activated PAECs by almost 3 orders of magnitude (FIG. 4). The peptide practically does not bind to control cells (COS).
- COS control cells
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CA002345214A CA2345214A1 (en) | 1998-10-02 | 1999-10-01 | Tissue binding peptides, identification, production and utilization thereof |
EP99948916A EP1117775A2 (de) | 1998-10-02 | 1999-10-01 | Gewebebindende peptide, ihre identifizierung, herstellung und verwendung |
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DE19845434A DE19845434C1 (de) | 1998-10-02 | 1998-10-02 | Gewebebindende Peptide, ihre Identifizierung, Herstellung und Verwendung |
DE19845434.1 | 1998-10-02 |
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WO1997035022A1 (de) * | 1996-03-15 | 1997-09-25 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin | Exportsysteme für rekombinante proteine |
WO1998040508A1 (en) * | 1997-03-14 | 1998-09-17 | Selective Genetics, Inc. | Adenoviral vectors with modified tropism |
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1998
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1999
- 1999-10-01 CA CA002345214A patent/CA2345214A1/en not_active Abandoned
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Patent Citations (2)
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WO1997035022A1 (de) * | 1996-03-15 | 1997-09-25 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin | Exportsysteme für rekombinante proteine |
WO1998040508A1 (en) * | 1997-03-14 | 1998-09-17 | Selective Genetics, Inc. | Adenoviral vectors with modified tropism |
Non-Patent Citations (6)
Title |
---|
BARRY M.A. ET AL.: "Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries" NATURE MEDICINE, Bd. 2, Nr. 3, M{rz 1996 (1996-03), Seiten 299-305, XP002128875 * |
DATABASE GENBANK [Online] Accession No. AA370946, 21. April 1997 (1997-04-21) ADAMS M.D.: "EST82673; Prostate gland H. sapiens cDNA" XP002129127 * |
MOGFORD J.E. ET AL.: "RGDN peptide interaction with endothelial alpha5-beta1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles." J. CLIN. INVEST., Bd. 100, Nr. 6, September 1997 (1997-09), Seiten 1647-1653, XP002128876 * |
MOGFORD J.E. ET AL.: "Vascular Smooth Muscle alpha5-beta3 integrin mediates artetiolar vasodilation in response to RGD peptides" CIRCULATION RES., [Online] Bd. 79, Nr. 4, 1996, Seiten 821-826, XP002129122 Retrieved from the Internet: <URL:http://circres.ahajournals.org> [retrieved on 2000-01-28] * |
PASQUALINI R. & RUOSLAHTI E.: "Organ targeting in vivo using display peptide libraries" NATURE, Bd. 380, Nr. 6572, 28. M{rz 1996 (1996-03-28), Seiten 364-366, XP002128874 * |
RAJOTTE D. ET AL.: "Molecular heterogeneity of the vacular endothelium revealed by in vivo phage display." J. CLIN. INVEST., Bd. 102, Nr. 2, Juli 1998 (1998-07), Seiten 430-437, XP002128873 * |
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EP1117775A2 (de) | 2001-07-25 |
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WO2000020572A3 (de) | 2000-07-20 |
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