WO2004014951A2 - Molecules de liaison - Google Patents

Molecules de liaison Download PDF

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Publication number
WO2004014951A2
WO2004014951A2 PCT/EP2003/008651 EP0308651W WO2004014951A2 WO 2004014951 A2 WO2004014951 A2 WO 2004014951A2 EP 0308651 W EP0308651 W EP 0308651W WO 2004014951 A2 WO2004014951 A2 WO 2004014951A2
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Prior art keywords
side chain
binding molecule
amino acids
support structure
binding
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PCT/EP2003/008651
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English (en)
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WO2004014951A3 (fr
Inventor
Udo Haberl
Christian Frosch
Hans-Georg Frank
Andreas Rybka
Raimund Wieser
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Aplagen Gmbh
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Priority claimed from EP02017525A external-priority patent/EP1394180B1/fr
Application filed by Aplagen Gmbh filed Critical Aplagen Gmbh
Priority to CA002493019A priority Critical patent/CA2493019A1/fr
Priority to AU2003253381A priority patent/AU2003253381A1/en
Priority to EP03784178A priority patent/EP1527096A2/fr
Priority to JP2004526881A priority patent/JP4667868B2/ja
Publication of WO2004014951A2 publication Critical patent/WO2004014951A2/fr
Publication of WO2004014951A3 publication Critical patent/WO2004014951A3/fr
Priority to IL166361A priority patent/IL166361A/en
Priority to US11/389,667 priority patent/US20070060508A1/en

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Definitions

  • Such biologically active binding molecules may be of the protein family (examples: insulin, growth factors, cytokines, releasing hormones, antibodies, enzymes, etc.), the lipid class (prostaglandins and similar fatty acid derivatives), the steroid class (examples: glucocorticoids, mineral corticoids).
  • cytokine is to be interpreted in a prototypical manner for biologically active binding molecules involved in binding processes or signal transducing processes.
  • Cytokines are biological messengers with high specificity and defined activity.
  • cytokine-specific receptors which are preferably provided on the surface of target cells.
  • the messenger contacts the receptor, in the typical case the latter will trigger an intracellular signal transducing cascade resulting in the biological effect within the cell or tissue.
  • Typical effects which may be triggered by such receptors are, e.g., the entry of a cell into the cell cycle and therewith an increase of the multiplication jate or also the triggering of apoptosis, a cell death form.
  • cytokine-specific receptors which are preferably provided on the surface of target cells.
  • Typical effects which may be triggered by such receptors are, e.g., the entry of a cell into the cell cycle and therewith an increase of the multiplication jate or also the triggering of apoptosis, a cell death form.
  • cytokines play an important role in the homoeostasis of the respective organism.
  • the receptor at the cell surface consists of different proteins which converge due to the binding of the cytokine.
  • many cytokines have specific binding sites which have to bind to two or more receptor proteins at the same time to trigger the effect with high efficiency. This indicates that in the cytokine receptor interaction precisely defined distances and spatial orientations of the various binding regions in the cytokine molecule with respect to each other are critical and necessary for the complete display of the effect.
  • the cytokine as such consist of different proteins which are required to combine in the course of the receptor interaction (C. Aul, W.
  • cytokines Since the cytokines are to be administered regularly, it has to be ensured that they are purified after expression which regularly is performed in bacteria and subsequently are absolutely free from bacterial contaminants to avoid a systemic allergy. This necessitates the use of complex methods which have an enormous impact on the pricing.
  • Proteins prepared by recombination often have tertiary structures deviating from those of native proteins and therefore are recognized as "alien" by the human body.
  • the antibodies induced thereby neutralize the cytokine and result in a loss of activity of the cytokine.
  • Recombinant proteins often exhibit a remarkable lability to proteolytic - i.e. protein degrading - enzymes. This necessitates a frequent administration in relatively high concentrations which on the one hand stresses the patient and on the other hand makes the therapy very costly.
  • cytokine An economic alternative to the recombinant preparation of a cytokine are organic-chemically accessible, completely synthetic mimetics of biologically active proteins such as, e.g., cytokines.
  • Another approach is the computer-assisted design of small, specific binding molecules of, e.g., peptide nature.
  • small specific binding molecules usually have a sequence of less than 100 amino acids. Due to the size thereof, such molecules cannot contribute to the association of receptor subunits which would be required for a complete cytokine activity. Normally, they will also not be designed to be highly affine for two or more binding sites on a large receptor molecule at the same time.
  • peptides can chemically be synthesized by numerous standardized methods quite easily, although an individual synthetic strategy has to be developed for each peptide.
  • the peptides are synthesized on solid phases with the operations of activating an N-terminally protected amino acid, coupling, washing, deblocking, activating being repeated until the desired peptide is finished.
  • Said product is removed from the solid phase, purified by HPLC and subsequently transferred to further investigations such as, e.g., sequence verification and biological tests.
  • the synthesis is the simpler and the more reliable the shorter the peptide is.
  • Peptides have an extremely short in vivo half life, i.e., they are degraded and excreted very fast by endogenous enzymes.
  • Various approaches are applied to metabolically stabilize peptides and proteins.
  • unnatural amino acids which cannot be cleaved are used in synthesis, on the other hand proteins are modified by inert chemical groups to protect them from degradation.
  • PEG intron Schering-Plough
  • interferon modified with polyethylene glycol an interferon modified with polyethylene glycol.
  • the object forming the basis of the invention is attained by a binding molecule consisting of a support structure of at least one cyclic molecular subunit and at least two side chain subunits, wherein the side chain subunits are polypeptide chains synthesized from natural and/or unnatural D- and/or L-amino acids and wherein the side chain subunits are covalently bonded to the support structure.
  • the cyclic molecular subunit is preferably a cyclic polypeptide synthesized from natural or unnatural D- and/or L-amino acids, an aromatic ring, an aromatic ring system, a polylactone, a polylactam, benzenetriamide, trihydroxybenzoic acid, a trilactone or a trilactam.
  • the cyclic support structure can also be structurally minimized and be replaced by a linear symmetrical structure.
  • the amino acids forming the cyclic polypeptide are advantageously linked by peptide bonds.
  • a cyclic molecular subunit within the meaning of the invention is not present if a cyclic structure is formed by the formation of a disulfide bridge.
  • This type of a cyclic peptide structure is a constituent part of many naturally occurring proteins. Due to the lability thereof, it is not suited to be used as the central cyclic molecular subunit of the support structure within the meaning of the invention. Of course, this does not exclude that the binding molecules of the invention also contain disulfide bridges in addition to a more stabile cyclic molecular subunit of the support structure.
  • cyclic molecular subunit is a cyclic polypeptide
  • said at least two side chain subunits of the binding molecule are bonded to the cyclic molecular subunit by amino acid side chain moieties of the amino acids lysine, serine, threonine, glutamate, asparagine and/or aspartate of the cyclic molecular subunit.
  • the cyclic polypeptide preferably consists of 2 to 10, in particular of 2, 3, 4 or 6 amino acids.
  • the cyclic molecular subunit has one of the structures a) benzenetriamide:
  • the molecules comprise at least two side chain subunits which may be identical or also different from each other.
  • said at least two side chain subunits of the binding molecule of the invention are polypeptide chains, they may have the same amino acid sequence or different sequences.
  • the polypeptide chains of the side chain subunits preferably consist of 5 to 60, preferably 10 to 50, in particular 12 to 40 amino acids.
  • the molecular weight of such polypeptide chains is preferably not greater than 10 kDa, in particular below 8 kDa or below 5 kDa.
  • the sterical position of two or more side chains on a cyclic support structure or at the the ends of a linear connecting molecule can be fixed during the synthesis of the whole molecule by introduction of disulfide bonds between two sterically suitably neighbouring residues (e.g. cystein or homocystein) of the respective side chains.
  • residues e.g. cystein or homocystein
  • the amino acid sequences of the side chains can be modified or optimized to contain one or more suitable SH-containing residues at suitable location.
  • binding molecules wherein the side chain subunits are oligo or polynucleic acids consisting of natural and unnatural nucleic acid units. Then, the nucleotides may be connected in a natural manner or they may exist as peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • the molecular weight of such side chain subunits is preferably not greater than 10 kDa, in particular below 8 kDa or below 5 kDa.
  • binding molecules of the invention may be used to bind to DNA-binding proteins or protein complexes with several DNA-binding sites.
  • the side chain subunits are connected with the support structure by spacer molecular subunits (spacers) especially consisting of natural and unnatural carbohydrates or nucleic acids.
  • spacers spacer molecular subunits
  • the free ends of the side chain subunits are covalently bonded to suitable protective groups to prevent exopeptidase degradation.
  • suitable protective groups are D-amino acids, preferably as di- or tripeptide.
  • protective groups usually employed within the scope of solid phase synthesis and monomeric and polymeric carbohydrates may be used.
  • the internal amino acids may also covalently be bonded to protective groups.
  • Preferred protective groups are carbohydrates which preferably are selected from glucose, mannose, N-acetyl glucosamine or N-acetylgalactosamine or sialic acid.
  • the present invention enables the development and preparation of fully synthetical mimetics of physiologic binding molecules which overcome the above-mentioned drawbacks of the state of the art.
  • the present invention designs support structures on the skeleton of which binding molecules are oriented such that the binding properties of a biologically active binding molecule are mimiced.
  • a great advantage of the invention is the simple synthetic preparation of the binding molecules of the invention.
  • the organic- chemical synthesis can be carried out cheaper and controlled better than the recombinant preparation of an analogous protein molecule.
  • unnatural amino acids e.g., D-amino acids or other suitable monomeric units can be used of which a better compatibility or immunologic tolerance is to be expected.
  • the invention described here overcomes the drawbacks of the state of the art in the following items: a) It describes an easily preparable cyclic support structure which usually is easily preparable and available and which can be used for the positioning of specific binding molecules. b) The cyclic structure of the support structure enables the positioning of specific binding molecules in practically any desired spatial orienting by varying the ring size and the distances between the sites the side chain subunits are bonded to and optionally by incorporating suitable spacer molecules. Hence, the binding molecules of the invention distinctly differ from those according to the state of the art where only a random crosslinking of peptides with synthetic molecules was performed and wherein a defined spatial orientation of the molecule components is not possible.
  • the cyclic support structure can be synthesized on the solid phase in a defined sequence and the specific binding molecules can be attached or synthesized as side chain subunits.
  • the support structure is flexible with respect to the number, type and length of the specific binding molecules to be attached as side chain subunits.
  • amino acid sequences oriented in a parallel or antiparallel manner can be coupled with the support structure in one synthesis step in the solid phase and optionally synthesized therewith in one operation.
  • a parallel orientation refers to the side chain subunits all being bonded to the support structure by the N- or C-terminal end in the same direction.
  • An antiparallel orientation refers to at least one side chain subunit being bonded N-terminally and at least one being bonded C- terminally to the support structure.
  • the flexibility with the planned positioning of specific binding molecules may be increased by using unnatural amino acids and D-amino acids.
  • the use of unnatural and D-amino acids or lactones and lactams increases the stability against proteolytic degradation. Additional functionalities increasing stability and half life can be introduced due to the variability of the support structures. This applies to, e.g., glycosylations, acetylations, disulfide-bridging or other chemical derivatizations known to the skilled person from protective group chemistry.
  • the specific binding molecules to be positioned on the support structure of the invention are peptides, they are orientable and synthesizable in a parallel and antiparallel manner on the support skeleton.
  • a preferred embodiment of the support structures of the invention are cyclic peptides. Suitable state-of-the-art protective group strategies are available for cyclic peptides. Cyclic peptides degrade slower in vivo since they can only be cleaved by endopeptidases. Moreover, according to the process of the invention they can be synthesized with an increased proteolysis resistance by incorporating unnatural amino acids such as, e.g., D-amino acids. In case of exactly two functional side chains to be linked the support structure can be minimized to a linear structure.
  • IL2 interleukin-2
  • synthetic binding molecules which - in analogy to interleukin-2 (IL2) - have the capability to bind to IL2 specific receptors with high affinity.
  • IL2 is a cytokine used in tumor therapy. It is a protein consisting of 133 amino acids and having a molecular weight of 15.4 kDa.
  • IL2 binds to specific receptors (IL2R) whereby the IL2- specific intracellular signals are triggered.
  • the highly affine receptor is a receptor complex consisting of the subunits a, b and g (also designated as p55, p75 and p64, resp.). Stimulating the receptor complex consisting of p75 and p64 is sufficient to induce the complete activity of IL2.
  • IL2 has stimulating effects on the growth of T and B lymphocytes, activates cytotoxic and cytolytic NK cells. Thus it has a central significance in the regulation of the immune response. Thus, IL2 is of fundamental importance in the immune response to tumors and inflammatory reactions.
  • LAKs lymphokine activated killer cells
  • the side chain subunits preferably have at least one of the sequences
  • X 1 is selected from I or L
  • X 2 is selected from I or L
  • X 3 is selected from V, L or M,
  • X 4 is selected from E, D or K,
  • X 5 is selected from L or F
  • X 6 is selected from E or D
  • X 7 is selected from A, G or C,
  • X 8 is selected from A or I and in the Ti position a protective group according to the state of the art, preferably a carbohydrate moiety, preferably selected from glucose, mannose, N-acetyl glucosamine or N-acetylgalactosamine, is covalently bonded to threonine in a suitable manner as a protection against proteolytic degradation.
  • a protective group preferably a carbohydrate moiety, preferably selected from glucose, mannose, N-acetyl glucosamine or N-acetylgalactosamine
  • binding molecules wherein the side chain subunits with the stated sequences have sequence homologies of at least 80 %, preferably 90 or 95 % may be used. In this case the sequence deviations especially are conservative amino acid exchanges.
  • Preferred binding molecules have the structure:
  • the IL2R specific binding molecules can overcome the essential drawbacks of the state of the art.
  • the binding molecules can be produced easily and economically. High production costs and the high physiological instability of recombinant molecules do not exist, the insufficient receptor stimulation observed with peptides does not occur since the binding molecules of the invention bind to the receptor complex in a functional cooperative manner in a defined spatial arrangement with the support structure having no affinity to the receptor.
  • the receptor molecules of the invention also offer the possibility to increase the activity and to minimize side effects by a purposive sequence optimization.
  • the above-mentioned synthetic IL2R specific binding molecules are suited for the treatment of diseases of the immunologic system, e.g., inflammations and arthritic processes or of immunodeficiency syndromes of all types and genesis; diseases connected with an increased proliferation of cells, e.g., carcinoses, for example in the form of carcinomas, sarcomas, lymphomas and leukaemias; or infectious processes.
  • diseases of the immunologic system e.g., inflammations and arthritic processes or of immunodeficiency syndromes of all types and genesis
  • diseases connected with an increased proliferation of cells e.g., carcinoses, for example in the form of carcinomas, sarcomas, lymphomas and leukaemias
  • infectious processes e.g., recombinant interleukin 2 in combination with interleukin 12 has positive effects in the tumor therapy.
  • such agents may also be combined with the pharmaceutical of the invention as preferred combination agents triggering apoptosis in target
  • the expected positive effect on the therapeutic success is achieved by a local apoptosis within the tumor and a general immunostimulation by a pharmaceutical of the invention causing a local effectivity increase.
  • TRAIL TNF related apoptosis inducing ligand
  • the respective human sequences can be replaced by their animal counterparts and be combined in a way typical for this invention in order to optimize effects in an animal and thus to enable optimal veterinary use.
  • the Alignment given below shows the sequences relevant for cat and dog, which are homologous to the human sequences. These sequences are characterized for their ability to bind the respective receptors subunits in cat or dog.
  • the invention includes all usual modifications and combinations of these side chains as outlined above for the human sequences. In preferred embodiments these sequences can be modified by introduction of conservative exchanges of amino acids in the sequences, by introduction of SH-containing residues and disulfide bridging as well as by other chemical modifications, which optimize the sterical design and stability of the resulting molecule.
  • Side chains they can be combined on a support structure according to the
  • TRAIL TNF related apoptosis inducing ligand
  • TNF TNF related apoptosis inducing ligand
  • the TRAIL specific binding and effector molecules synthesized here preferably on the basis of suitable cyclic support structures specifically bind to TRAIL receptors expressed by tumor cells and trigger apoptosis. Proteins triggering apoptosis such as the TNF (tumor necrosis factor) or Fas and AOP1 have been known for a longer time. Initially, the cytokine TNF was characterized as a protein having a strong tumoricide effect in mice. However, it could not therapeutically be used in humans due to its strong systemic toxicity.
  • TRAIL was described for the first time in 1999 (Walczak, H. et al. Nature Medicine 5 (1999) 157-163).
  • This protein consists of 281 amino acids and is a type II membrane protein which is converted into a soluble molecule consisting of the amino acids 114 - 281 by cleavage. Subsequent to a spontaneous trimerization it binds to its specific receptors, thereby induces trimerization of the receptors. Thus it triggers the intracellular cell death program mediated by caspase.
  • TRAIL-R1 and 2 both of which being involved in the signal transmission, and the receptors TRAIL R3 and 4, which, however, do not have a so-called "death domain” and hence cannot trigger signals after the binding of TRAIL.
  • Both TRAIL itself and the mentioned receptors are expressed in most of the human tissues and organs; the selective tumoricide effect is probably based on the fact that normal cells have more of the "decoy receptors" 3 and 4 preventing an efficient signal triggering by inhibiting the trimerization of the active receptors.
  • TRAIL In the amino acid sequence of naturally or recombinantly prepared TRAIL there are various domains which are responsible for the binding to the receptor and therefore for biological activity, namely the domains AA131-163, AA201-205, AA214-220, AA237-240, AA258-283. These short domains positioned in sterical neighbourhood are cooperatively responsible for the interaction with the TRAIL receptor.
  • the present invention enables the provision of binding molecules specifically binding to TRAIL receptors with high affinity.
  • binding molecules of the invention the analogous domains mentioned in the above sections are coupled to cyclic support structures in a suitable cooperative arrangement in order to achieve the biological effect.
  • the TRAILR binding molecules of the invention exist in a trimerized form. Due to the covalent binding to a cyclic molecular subunit they have a high biological half life and due to the trimerization the biologically maximum possible activity with minimum side effects at the same time. These peptides are the minimum structure of RAIL required for the biological activity thereof. This achieves the highest specificity with the smallest side effects.
  • the trimers may be prepared either homotrivalent or by a cyclic support structure or by a benzenetriamide linker.
  • the TRAIL peptides are prepared in a saccharide-modified form such that they have a high biological half life. This reduces the administration frequency and amount and with that the stress on the patient.
  • a purposive amino acid exchange the peptides can be converted on the one hand into a form being even more biologically active or a form having an increased biological half life or on the other hand into an antagonistic - i.e. depressant - form.
  • X 3 W or L or R or A or Y
  • X 4 E or N or A or D or H
  • binding molecules wherein the side chain subunits have an sequence homology to the mentioned sequences of at least 80 %, preferably 90 or 95 % .
  • sequence deviations are in particular amino acid exchanges.
  • binding molecules wherein the side chain subunits have a sequence homology to the mentioned sequences of at least 80 %, preferably 90 or 95 % are usable.
  • sequence deviations are in particular conservative amino acid exchanges.
  • Antibodies are substances produced within the body which show a specific binding to substances which are usually foreign to the body and designated as antigens. Antibodies are of importance in the defence against pathogens and for the protection of the organism against infections. In various therapeutic and diagnostic applications antibodies having a relevant binding capacity for an planned indication are used or developed. Thus, antibodies are used to specifically enrich cytostatic agents or other tumor-impairing agents in tumors, or they are used to depict certain parts of the body or diseased area in imaging processes by loading them with a contrast agent. Antibodies may be isolated from the human body as natural antibodies.
  • monoclonal antibodies are antibodies which are grown in laboratory gnawers by immunization and subsequently immortalized by fusion of antibody-producing cells with myeloma cells and monodoned by isolation.
  • antibodies can also be monodoned by recombinant techniques transferring the coding genetic information in suitable, often bacterial or yeast-based expression systems.
  • Recombinantly prepared antibodies may be obtained from all species with an immunoglobulin gene repertoire which is sufficiently sequenced and known.
  • monodonally or recombinantly prepared antibodies will be referred to.
  • Antibodies are relatively large molecules, the specific binding area of which normally contains 6 hypervariable regions (based on the amino acid sequence).
  • the binding properties of an antibody are defined by these so- called CDR ("Complementary Determining Regions").
  • CDR Complementary Determining Regions
  • Both monoclonal antibodies and recombinantly prepared antibodies are therapeutically or diagnostically used in various indications.
  • drawbacks ensue in the therapeutical application of antibodies.
  • the preparation of antibodies by both methods (monodonally and recombinantly) is complicated and very expensive.
  • antibodies themselves are natural molecules the patient's immunologic system reacts to.
  • the therapy can substantially be impeded by the production of the patient's own antibodies (against the therapeutic antibodies) or by anaphylactic reactions.
  • the methods for preparing therapeutic antibodies have to be changed and, e.g., mural monoclonal antibodies have to be introduced into the recombinant production in a molecular-biological, time-consuming and complicated process and at the same time humanized in this manner, i.e. optimized for the therapeutical use by incorporating human sequences.
  • anti- idiotypic substances from libraries, e.g., recombinant peptide libraries. Normally, the peptides isolated this way bind to the antibody only with low affinity and are not or only restrictedly able to competitively prevent the pathologic antibody formation.
  • the present invention solves the above-mentioned problems by providing synthetic binding molecules wherein the binding properties of a therapeutically relevant antibody are mimiced.
  • the support structure of the invention enables to overcome the above-mentioned problems of the state of the art and to develop and design completely synthetic mimetics of the respective antibodies.
  • the organic-chemical synthesis can be carried out more economical and with a better control than the recombinant preparation of an analogous molecule.
  • basically also unnatural amino acids or other suitable monomeric units of which a better compatibility, life time or immunotolerance is to be expected can be used.
  • antibodies may be important not only in therapy but they may also be pathogenetic reagents.
  • antibodies are pathologic if they attack autologous tissue and thereby trigger a so-called autoimmune disease or if they are at least associated therewith.
  • Another variant of the pathologic role of antibodies or other substances of the immunoglobuline superfamily exists in the challenge by allergenic substances in patients being allergic to the respective substance. Also these diseases are false reactions of the immunological apparatus, and the triggering thereof is initiated by the binding of the respective allergen.
  • anti-idiotypic it would be desirable to neutralize the pathogenically relevant antibodies by reacting them with so- called anti-idiotypic substances.
  • anti-idiotypic it has to be understood that the substance specifically binds to the antibody's binding cavity just like the actual antigen. This prevents the pathologic binding to autologous antigens.
  • Another subject of the invention is the process for preparing a binding molecule, said process being performed as a solid phase synthesis process wherein the peptide of the side chain subunit bonded to the solid phase is successively extended by the respective amino acids and optionally a coupling to the support structure follows in a last step.
  • Another subject of the invention are peptides of the sequence
  • X 2 is selected from I or L
  • X 3 is selected from V, L or M,
  • X 4 is selected from E, D or K,
  • X 5 is selected from L or F
  • X 6 is selected from E or D
  • X 7 is selected from A, G or C,
  • X 8 is selected from A or I.
  • Another object of the invention are peptides wherein the side chain subunits have a sequence homology to the stated sequences of at least 80 %, preferably 90 or 95 % and which are not completely homologous to segments of the TRAIL sequence.
  • Another subject of the invention are pharmaceuticals and diagnostic agents containing the binding molecules or peptides of the invention.
  • the binding molecules of the invention are preferably used in the preparation of a pharmaceutical or diagnostic agent for the treatment or identification of diseases of the immunologic system, in particular inflammations, arthritic processes, immunodeficiency syndromes, auto-immune syndromes and immunodeficiency syndromes, fertility disturbances, for the contraception or antiviral prophylaxis, diseases connected with an increased proliferation of cells, in particular tumor diseases, carcinoses, sarcomas, lymphomas and leukaemias; and/or infectious processes with humans or animals.
  • the pharmaceutical of the invention is provided in particular in the form of a micro encapsulation, liposomal preparation or depot preparation containing suitable additives, carriers, adjuvants and/or at least one additional active substance.
  • the pharmaceutical of the invention is provided in combination with a common suitable pharmaceutical carrier.
  • suitable carriers include buffered sodium chloride solutions, water, emulsions, e.g., oil/water emulsions, wetting agents, sterile solutions, etc.
  • the pharmaceutical of the invention may be provided in the form of an injection solution, a tablet, an oinment, a suspension, an emulsion, a suppository, an aerosol, etc. administered in the form of depots (microcapsules, zinc salts, liposome, etc.).
  • the peptides may be microencapsulated due to the small size thereof resulting in depot forms with different durations of effect according to the respective pore size of the capsules.
  • the depot form may be administered locally such that the highest concentration is ensured in the required area (e.g., initial carcinoma) over a long period of time.
  • the mode of administration of the pharmaceutical depends on the form in which the active substance is present; it may be performed orally or parenterally. The skilled person knows several methods. The suitable dosage is determined by the attending physician and depends on several factors, e.g., the age, sex, weight of the patient, the nature and the stage of the disease, the mode of administration, etc.
  • a binding molecule of the invention takes place in particular IL2-specifically by using said molecule for the preparation of human LAK (Lymphokine Activated Killer Cells) ex vivo.
  • LAK Lymphokine Activated Killer Cells
  • cells are taken from the patient, treated with the active substance and activated in a culture dish and subsequently re-infused into the patient.
  • interleukin 12 an interleukin 12 receptor-specific binding and effector molecule, or recombinant IL12 is used as an additional active substance.
  • TRAIL TNF related apoptosis inducing ligand
  • a TRAIL receptor-specific binding and effector molecule, or recombinantly prepared TRAIL is used as an additional active substance.
  • a virostatic in particular a virostatic capable of retarding or blocking the entry of virus particles, in particular HIV particles, into target cells, in particular T-lymphocytes, is added to the pharmaceutical of the invention as an additional active substance.
  • a cyclic peptide is generated and used as the starting point for the solid phase synthesis. Initially, the first peptide strand is synthesized on the solid phase, then the cyclopeptide is coupled thereto and subsequently the second peptide strand is either synthesized on this loaded resin step by step or the previously purified peptide B is coupled thereto.
  • the combined organic phases are extracted by shaking with saturated sodium hydrogencarbonate solution and 5 % of sodium hydrogensulfate solution. After drying over sodium sulfate, the remaining solution is concentrated in a Rotavapor and the residue is suspended in methylene chloride, where the product is obtained in the form of a white solid.
  • a cydohexapeptide of the sequence D-K-D-K-D-K is generated according to the scheme mentioned below.
  • the cydopeptide can be bonded to a polymer support by the side chain of one of the asparaginic acids, optionally by spacer molecules, and then three peptide strands can be synthesized on the resin in parallel or the purified peptides can be coupled to a solid phase. Harz I X
  • the cleavage of Fmoc was performed by a 20 min treatment with 50 % (v/v) of piperidine in DMF.
  • the subsequent cleavage from the resin was performed in the presence of a mixture of 95 % of trifiuoroacetic acid, 2.5 % of water, 2.5 % of diisopropylsilane for 20 min.
  • the cleaved peptides were precipitated by adding tert-butyl methyl ether, centrifuged off and again combined with tert- butyl methyl ether and centrifuged off. Subsequently, the peptides were lyophilized and purified by reversed phase HPLC and characterized by LC/MS (Thermoquest LCQDuo). The purified peptides were checked in a cell culture for the cytotoxicity increasing effect thereof.
  • Sequence 3 APTSSSTKKT QLQLEHFLMK FQMILNGINN were prepared and tested.
  • NK cells which specifically kill tumor cells, as effector cells were incubated with tumor cells as target cells in the presence of the test peptides.
  • the cytotoxicity measurement was performed by measuring the release of lactate dehydrogenase (LDH) according to the standard protocol by a cytotoxicity test kit (Promega, Germany) and in a microwell plate reader.
  • LDH lactate dehydrogenase
  • E NK cells
  • YT cells DSMZ, Germany
  • target cells T
  • Daudi cells DSMZ, Germany
  • YT and Daudi cells were seeded in microwell plates at a ratio of 1:10 and incubated in the presence of 0.3, 3, 30 :M test peptide for 16 h.
  • Human IL-2 (Sigma) in a concentration of 5 nM served as the positive control.
  • Table 1 illustrates that already in a concentration of 0.3 :M the cytotoxicity inducing effect of the described test peptides is equally high as that of human IL-2.
  • the synthesis is carried out on 2-chlorotrityl chloride resin (200-400 mesh) with a substitution rate of 0.2mmol/g.
  • the first seven amino acids are coupled as a fragment. Attachement of the following amino acids is achieved by single, double or triple coupling with lOfold excess of amino acids and PyBOP/HOBT/DIPEA as coupling additives.
  • N-terminal deprotection of the growing peptide chain is achieved by double treatment with piperidine/DMF (1/3). In difficult cases a third treatment is done with DBU/piperidine/DMF (2/2/96). The number of coupling and deprotection cycles used and is shown in the following scheme (Information on difficult couplings/deprotections gained by monitoring of each elongation by HPLC-MS.)
  • Protocol 1 Synthesis of the first peptide fragment
  • the attachment of amino acids 2-7 is achieved by the following procedure: 5mmol of the amino acid, 7.5mmol HOBT and lOmmol DIPEA are dissolved in 15ml DMF. After 5 minutes 5mmol of PyBOP and lOmmol DIPEA are added and this solution is poured onto the resin. After 60 minutes on a vortexer the resin is washed 6 times with 20ml DMF, treated twice with 20ml piperidine/DMF (1:3) for 20/20 minutes and washed 6 times with 20ml DMF. In case of the N-terminal amino acid the resin is not treated with piperidine/DMF.
  • Protocol 2 Cleavage of the peptide fragment
  • the resin After attachement of the last amino acid the resin is washed 6 times with 20ml DMF, twice with 20ml DCM and then allowed to react with 50ml TFE/DCM (2/8) for 60 minutes. The resin is filtered off, the solvents removed in vacuo and the crude peptide fragment used without further purification.
  • Protocol 3 Reattachement of the peptide fragment lmmol of the fragment and 2mmol DIPEA are dissolved in 50ml dry DCM. This solution is added to 5.0g of dry 2-chlorotrityl chloride resin (200-400 mesh) and the mixture allowed to react for 12 hours on a vortexer. At the end of the reaction the resin is allowed to react twice with 50ml of DCM/MeOH/DIPEA for 3 minutes, washed twice with 50ml DCM and twice with 50ml DMF.
  • Protocol 4 Coupling of amino acids 8-57
  • the dried resin is allowed to swell in 50ml piperidine/DMF (1/3) for 30 minutes, treated with 30ml piperidine/DMF (1/3) for 20 minutes, treated with 30ml of DBU/piperidine/DMF (2/2/96) for 20 minutes and washed 6 times with 30ml DMF.
  • lOmmol of the amino acid, 15mmol HOBT and 20mmol DIPEA are dissolved in 30ml DMF. After 5 minutes lOmmol of PyBOP and 20mmol DIPEA are added and this solution is poured onto the resin. After 60 minutes on a vortexer the resin is washed twice with 30ml DMF and the coupling repeated once or (in difficult cases) twice for 60 minutes. The resin is then washed 6 times with 30ml DMF.
  • the resin can directly be used to couple the next amino acid or - after 2 washings with 30ml DCM - dried in vacuum and stored at - 80°C.
  • the N-terminal protecting group is removed by a double treatment with 30ml piperidine/DMF (1/3) for 20 minutes and treatment with DBU/piperidine/DMF (2/2/96) for 20 minutes.
  • the resin is washed 6 times with 30ml DMF, twice with 30ml DCM and treated with 50ml TFE/DCM (2/8) for 180 minutes.
  • the solvent is removed in vacuo and the protecting groups removed by treatment with 30ml TFA/TIS/EDT/water (94/1/2.5/2.5) for 180 minutes under an inert atmosphere.
  • Protocol 6 Refolding Procedure The respective elution fractions were diluted to a final volume of 20ml using exactly the same solvent as being present in the elution fraction. This solution of the purified product was incubated for 30 min. at room temperature with 10ml Ni-NTA Superflow (Qiagen) under slight agitation in a beaker. The Superflow particles were packed into an empty FPLC Column and attached to an FPLC-machine. Using the chromatography programme of this machine, the solvent was exchanged in a 10 min. Gradient by Water, then another 10 min. gradient was used to change the solvent to a mixture of water/trifluorethanol (1/1).
  • this solvent was oxygenized by bubbling oxygen through the reservoir bottle and using oxygen as atmosphere in the bottle in order to close the disulfide bridge.
  • oxygenation a constant flow of lml/min was passed for 24h along the column, while the eluate was constantly recirculated into the reservoir bottle.
  • the binding molecule is able to bind to the beta/gamma Interleukin 2 Receptor Heterodimer, to activate it and to induce signal transduction.

Abstract

L'invention concerne des molécules de liaison constituées d'une structure porteuse d'au moins une sous-unité moléculaire cyclique et d'au moins deux sous-unités à chaînes latérales. Lesdites sous-unités à chaînes latérales sont des chaînes polypeptidiques constituées d'acides D- et/ou L-aminés naturels et/ou non naturels, les sous-unités de chaîne latérale étant liées de manière covalente à la structure de support.
PCT/EP2003/008651 2002-08-06 2003-08-05 Molecules de liaison WO2004014951A2 (fr)

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CA002493019A CA2493019A1 (fr) 2002-08-06 2003-08-05 Molecules de liaison
AU2003253381A AU2003253381A1 (en) 2002-08-06 2003-08-05 Binding molecules
EP03784178A EP1527096A2 (fr) 2002-08-06 2003-08-05 Molecules de liaison
JP2004526881A JP4667868B2 (ja) 2002-08-06 2003-08-05 結合分子
IL166361A IL166361A (en) 2002-08-06 2005-01-18 Binding molecules
US11/389,667 US20070060508A1 (en) 2002-08-06 2006-03-27 Binding molecules

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US40103202P 2002-08-06 2002-08-06
US60/401,032 2002-08-06
EP02017525.3 2002-08-06
EP02017525A EP1394180B1 (fr) 2002-08-06 2002-08-06 Molécules synthétiques mimetiques de molécules de liason physiologiques
EP03010892 2003-05-15
EP03010892.2 2003-05-15

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004104023A2 (fr) * 2003-05-23 2004-12-02 Aplagen Gmbh Synthese de peptides
EP1616957A1 (fr) * 2003-04-18 2006-01-18 Japan as represented by president of National Cardiovascular Center Vecteur
US7589063B2 (en) 2004-12-14 2009-09-15 Aplagen Gmbh Molecules which promote hematopoiesis

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4269422A1 (fr) * 2020-12-25 2023-11-01 Chugai Seiyaku Kabushiki Kaisha Procédé de production d'un composé peptidique contenant un résidu d'acide aminé n-substitué

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021206A1 (fr) * 1992-04-08 1993-10-28 The Scripps Research Institute Polypeptides synthetiques tridimensionnels stabilises
WO1998020036A1 (fr) * 1996-11-06 1998-05-14 Genentech, Inc. Peptide a structure helicoidale contrainte et leurs procedes de fabrication
WO2000004048A1 (fr) * 1998-07-16 2000-01-27 Institut Pasteur Peptides d'yl-2 et leurs derives et leur utilisation comme agents therapeutiques
WO2000024782A2 (fr) * 1998-10-23 2000-05-04 Amgen Inc. Peptides modifies utilises comme agents therapeutiques
US6096710A (en) * 1995-11-17 2000-08-01 The Regents Of The University Of California Collagen-like peptoid residue-containing structures
WO2001011050A1 (fr) * 1999-08-04 2001-02-15 Amgen Inc. Fhm, NOUVEAU MEMBRE DE LA FAMILLE DES SUPERGENES LIGANDS DU TNF__

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5932189A (en) * 1994-07-29 1999-08-03 Diatech, Inc. Cyclic peptide somatostatin analogs

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021206A1 (fr) * 1992-04-08 1993-10-28 The Scripps Research Institute Polypeptides synthetiques tridimensionnels stabilises
US6096710A (en) * 1995-11-17 2000-08-01 The Regents Of The University Of California Collagen-like peptoid residue-containing structures
WO1998020036A1 (fr) * 1996-11-06 1998-05-14 Genentech, Inc. Peptide a structure helicoidale contrainte et leurs procedes de fabrication
WO2000004048A1 (fr) * 1998-07-16 2000-01-27 Institut Pasteur Peptides d'yl-2 et leurs derives et leur utilisation comme agents therapeutiques
WO2000024782A2 (fr) * 1998-10-23 2000-05-04 Amgen Inc. Peptides modifies utilises comme agents therapeutiques
WO2001011050A1 (fr) * 1999-08-04 2001-02-15 Amgen Inc. Fhm, NOUVEAU MEMBRE DE LA FAMILLE DES SUPERGENES LIGANDS DU TNF__

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
APPELHANS ET AL.: "Synthesis and characterization of poly(ether amide) dendrimers containing different core molecules" MACROMOLECULES, vol. 33, 2000, pages 9494-9503, XP002259018 *
CIARDELLI T L ET AL: "Structural studies of interleukin 2" PEPTIDES. STRUCTURE & FUNCTION, PROCEEDINGS OF THE AMERICAN PEPTIDE SYMPOSIUM, ROCKFORD, PIERCE CHEMICAL CO, US, vol. PROC. 9, 1985, pages 75-78, XP002100685 *
FUJII H ET AL: "ANTIMETASTATIC ACTVITIES OF SYNTHETIC ARG-GLY-ASP-SER (RGDS) AND ARG-LEU-ASP-SER (RLDS) PEPTIDE ANALOGUES AND THEIR INHIBITORY MECHANISMS" BIOLOGICAL & PHARMACEUTICAL BULLETIN (OF JAPAN), PHARMACEUTICAL SOCIETY OF JAPAN, JP, vol. 18, no. 12, December 1995 (1995-12), pages 1681-1688, XP001108987 ISSN: 0918-6158 *
RANGANATHAN D ET AL: "DESIGN AND SYNTHESIS OF AB3-TYPE (A=1,3,5-BENZENETRICARBONYL UNIT;B=GLU DIOME OR GLU7 OCTA OME) PEPTIDE DENDRIMERS: CRYSTAL STRUCTURE OF THE FIRST GENERATION" BIOPOLYMERS, NEW YORK, NY, US, vol. 54, no. 4, 2000, pages 289-295, XP001040555 ISSN: 0006-3525 *
See also references of EP1527096A2 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1616957A1 (fr) * 2003-04-18 2006-01-18 Japan as represented by president of National Cardiovascular Center Vecteur
EP1616957A4 (fr) * 2003-04-18 2010-03-10 Japan Government Vecteur
US8298817B2 (en) 2003-04-18 2012-10-30 National Cerebral And Cardiovascular Center Vector
WO2004104023A2 (fr) * 2003-05-23 2004-12-02 Aplagen Gmbh Synthese de peptides
WO2004104023A3 (fr) * 2003-05-23 2005-06-02 Aplagen Gmbh Synthese de peptides
EA008305B1 (ru) * 2003-05-23 2007-04-27 Аплаген Гмбх Синтез пептидов
EP1810977A2 (fr) * 2003-05-23 2007-07-25 AplaGen GmbH Phases solides renfermant des complexes de métal chélates pour la préparation de peptides
EP1810977A3 (fr) * 2003-05-23 2009-05-20 AplaGen GmbH Phases solides renfermant des complexes de métal chélates pour la préparation de peptides
US7589063B2 (en) 2004-12-14 2009-09-15 Aplagen Gmbh Molecules which promote hematopoiesis

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JP4667868B2 (ja) 2011-04-13
CN1675243A (zh) 2005-09-28

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