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WO2002051869A1 - Composes comprenant au moins deux domaines de proteines identiques lies par un peptide possedant une longueur telle que chacun des domaines de proteines peut interagir simultanement avec sa structure de surface cellulaire specifique et bloquer ainsi la fonction de la structure - Google Patents

Composes comprenant au moins deux domaines de proteines identiques lies par un peptide possedant une longueur telle que chacun des domaines de proteines peut interagir simultanement avec sa structure de surface cellulaire specifique et bloquer ainsi la fonction de la structure Download PDF

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Publication number
WO2002051869A1
WO2002051869A1 PCT/SE2001/002892 SE0102892W WO02051869A1 WO 2002051869 A1 WO2002051869 A1 WO 2002051869A1 SE 0102892 W SE0102892 W SE 0102892W WO 02051869 A1 WO02051869 A1 WO 02051869A1
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WIPO (PCT)
Prior art keywords
receptor
protein
cell
peptide
compound
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PCT/SE2001/002892
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English (en)
Inventor
Ann-Christin Koch-Schmidt
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Ann-Christin Koch-Schmidt
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Publication of WO2002051869A1 publication Critical patent/WO2002051869A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to a compound forming a strong comlex with surface structures on cells or pathogens and thereby block the function of this structure. Also contemplated is a method for preparation and its use as well as a pharmaceutical composition.
  • a cell is surrounded by a cell membrane composed of a double lipid layer and globular proteins.
  • a membrane protein normally has an extracellular, a membrane spanning, and an intracellular part.
  • the membrane protein is either composed of a single peptide chain, which can span the membrane once, twice, or several times, or is composed of several chains, so called subunits, which can be identical or different.
  • Membrane proteins and their functions The extracellular part of membrane proteins is normally extensive and often subdivided into domains. This part interacts or communicates with its surroundings. Thus, it is to this part that another membrane bound protein on the neighbouring cell binds to achieve a cell-cell interaction, or a soluble ligand, which often is a polypeptide or a small protein, binds in order to affect the cell. It is also to a specific region or domain of this extra cellular part of a specific membrane protein that a certain virus binds through one of its surface molecules in order to attach to and initiate the infection of the host cell.
  • the extracellular part of such a membrane protein which usually is characterized as a receptor, is often cut off. Then, this soluble domain can bind a free ligand molecule, which thereby is prevented from binding to the membrane receptor.
  • the receptor When the ligand binds to the extracellular part, the receptor undergoes a conformational change, which is transferred to the intracellular part of the receptor. This part can thereby be changed, eg. by phosphorylation, which often results in interactions to other intracellular proteins and thereby initiating of a signalling process or processes.
  • phosphorylations are often found when ligand binding results in aggregation of receptor subunits, like the IL-2 induced aggregation of ⁇ , ⁇ and ⁇ -subunits of the IL-2R.
  • the intracellular domains of the two juxtaposed ⁇ and ⁇ -subunits obtain tyrosine kinase activity and will therefore cross-phosphorylate each other.
  • Such phosphorylations are also found when homodimeric ligands, like erythropoietin, binds to its receptor. In the unbound state, the intracellular domains of such receptors, which usually also are homodimers, are separated from each other. Upon ligand binding, the intracellular domains are forced towards each other and the phophorylation process is initiated (Remy et al., 1999). Human growth hormone, having two receptor binding sites, acts in a similar way by forcing two receptors together resulting in cross-phosphorylation of the juxtaposed intracellular domains (Cunningham et al, 1991). In all these cases, rather long-lived ligand-receptor complexes are formed in order to allow the consecutive reactions to happen so that the intracellular signalling reaches the desired level.
  • ligand-induced ion channels like the acetyl choline receptor NAchR, form very short-lived ligand-receptor complexes.
  • ligand binding results in formation of a channel through which ions are transported.
  • these ligand-receptor complexes exist only for a short time.
  • Membrane proteins can act as receptors for various soluble ligands, such as hormones, growth factors, cytokines, which are destined for affecting the cell, or for nutrients.
  • they can be receptors for stationary components of the extra cellular matrix in order to anchor the cell, or for membrane structures on neighbouring cells in order to form long-lived interactions between the cells.
  • a receptor is exposed on the cell surface only when it is needed there and returns then back to the cytoplasm for storage, or for destruction followed by synthesis of a new receptor/receptor subunit, directly or later.
  • some membrane proteins like ion channels and nutrient receptors, are normally always exposed on the cell surface. Others will be there only during specific periods of the life of the cell, or if an external factor, like a cytokine, a hormone, or a transmitter, binds to the receptor or receptor subunit and thereby initiate other receptor molecules of the same kind to be exposed on the surface (Subtil et al., 1997).
  • receptors like those of the cytokines, are composed of several subunits (Theze et al., 1996). These have to be associated before the receptor can mediate the cell signalling process (Hemar et al., 1995). The association of the subunits seems to be ligand-induced (Subtil et al., 1997). Some of these subunits are found in various cytokine receptors. It seems as if the concentration of the ligand is the factor that determines in what constellation a certain subunit will act for the moment (Yamada et al., 1998).
  • the ligand-receptor complex is internalised.
  • receptors for ligands like nutrients normally turn back to the membrane ready for binding a new ligand.
  • receptors for growth factors normally degrade in order to temporarily diminish the effect of the ligand. If such a receptor contains several subunits, generally one or two of these return back to the surface or are stored, while the others are degraded and must be synthesised again in order to obtain a fully functional receptor (Subtil 1997).
  • ligand binding seems to initiate processes that affect the composition, concentration and position of the receptor and are of greatest importance for a correct function of the receptor. If such a process is inhibited, the receptor mediated cell signalling will be prevented.
  • Endogenous compounds for blockade of receptors on cells, viruses and microorganisms or as analytical tools Today, about 45 % of all drugs are directed to membrane proteins and act as agonists or antagonists. Most of these drugs are foreign substances, purified from various organisms or chemically synthesized. Only a small number are endogenous substances.
  • a foreign compound is seldom receptor-specific and therefore binds to several similar receptors in the host resulting in side effects. Further, non-endogenous compounds can often stimulate antibody production, which results in resistance of the host to the drag. Moreover, synthesized receptor-binding drugs of today show rather high K ⁇ _ values (> 10 "8 M). Therefore, they have to be used in high doses, which can in a negative way influence metabolic processes in the patient.
  • CD4 an endogenous membrane receptor to which HIV-1 binds via its surface protein Gpl20 (Dalgleish et al., 1984), has been used as an antiviral substance.
  • a substance will show resistance not only because of its endogenous nature. It can also develop uninfectious viruses as a mutation in the virus, resulting in decreased affinity to CD4, also decreases the affinity to the host cell.
  • too high doses of CD4 had to be used, due to the high Kp-value of the CD4-Gpl20p complex, to get the desired effect, which resulted in side effects (Schacker et al., 1995).
  • antibiotic drugs of today are specific to enzymes and inhibit processes of microorganisms and thereby their proliferation. By time, such substances loose their effect as microorganisms can exchange genetic material and thus the underlying factors of resistance. Only few antibiotic drugs of today are targeted against surface receptors on microorganisms. Those used are normally rather unspecific, have low affinity, are not endogenous and have to be used in high concentrations, which can result in side effects (Sharon et al., 1981).
  • Kn of ligand-receptor complexes determines the sensitivity of analytical methods based on L-R interactions
  • Analytical/diagnostic methods involving biomolecular interactions like ELISA is based on a ligand-receptor(L-R) complex containing only one ligand and one receptor.
  • the K D of such a complex determines the sensitivity of the method. By decreasing the K D the sensitivity will increase.
  • the first aim of the invention is design of a compound, which forms a very strong complex (K D « 10 "8 M) with surface receptors, specific for the compound, on a cell, a virus, or a pathogenic microorganism, and thereby blocks the biological function of the receptor.
  • the design of the compound includes two or more receptor binding domains of identical or identical couples of endogenous proteins/polypeptides repeated in tandem but interlinked by an endogenous peptide having such a length that each of the various domains of the formed compound/fusion protein can simultaneously bind to a specific surface receptor.
  • the protein/peptide domain(s) of the compound is an endogenous ligand, or part of it, that binds to a specific receptor molecule on an endogenous cell.
  • the endogenous protein domains of the compound consist of two identical human interleukin 2, IL-2, in tandem but interlinked by a peptide containing 8-14 amino acids.
  • the peptide linker between the IL-2 domains consists of 15 amino acids.
  • the endogenous protein domains of the compound consist of two identical human epidermal growth factor, EGF, in tandem but interlinked by a peptide containing 6-10 amino acids.
  • the protein/peptide domains of the compound consist of an extracellular part of an endogenous membrane receptor to which a certain receptor on the surface of a virus or a microorganism specifically binds.
  • the endogenous protein domains of the compound consist of the N-terminal domain of the human coxsackie adeno virus receptor, HCAR, fused in tandem but interlinked by a peptide of 20 amino acids normally found in the HCAR molecule.
  • the compound also contains one or more domains of an endogenous protein, which have the ability to specifically bind to a receptor on a phagocyte or a cytoiytic cell.
  • this additional domain is a Fc ⁇ R binding domain.
  • the compound contains a dimer of the Fc ⁇ R binding domain.
  • the second aim of the invention is that the compound is produced exclusively by gene technological and biotechnical methods.
  • the compound is produced by fusing, in solution and/or in a cloning vector, cDNAs of the involved peptide/protein domain(s) and designed linker DNA, transferring the formed dsDNA to an expression vector, transfer the vector to a suitable host cell, and finally induce the host cell to express the compound.
  • the third aim of the invention is that the compound shall be used as a drug in a pharmaceutical composition.
  • This drug is in one performance used for blocking the biological function of a membrane receptor and thereby the aggregation of receptor subunits and/or the initiation of the receptor mediated intracellular process.
  • the compound composed of tandemly fused IL-2, separated by a peptide linker of 15 amino acids, shall be used for blocking of the biological function of IL- 2R on cells having an abnormal high expression of IL-2R like T-cells in some T-cell lymphoma.
  • the compound composed of two EGFs, separated by a peptide linker, shall be used for blockade of the initiation of the cell signalling process, which normally follows upon binding of EGF to its receptor, in cells having an abnormal high expression of epidermal growth factor receptors like some tumour cells.
  • the compound is used as an antiviral drug by blocking those receptors on the viral surface by which the virus interacts with receptors on its host cell.
  • the compound consisting of tandemly repeated HCAR domains is used for blockade of HCAR binding receptors on some adenoviruses.
  • the compound is used as an antibiotic drug by blocking the specific surface structure(s) by which a microorganism interacts with its host.
  • a fourth aim of the invention is that the compound shall be used as an analytical/diagnostic tool.
  • This analytical/diagnostic tool will in one performance have a detection device in form of an additional protein like an enzyme, a fluorescent protein or a ligand-binding protein, linked to the compound.
  • this detection device consists of avidin to which a biotin labelled ssDNA, for use as the target in a PCR, will bind.
  • a fifth aim of the invention is to perform a procedure for preparation of a compound with the ability to form a strong complex with receptor molecules on endogenous cells or pathogenic microorganisms or viruses, including the following steps: to fuse, in solution and/or in a cloning vector, in tandem but separated by a DNA linker coding for an endogenous peptide, two or more identical cDNAs or identical couples of cDNAs coding for endogenous protein, protein domains or polypeptide(s), alternatively to fuse, in solution and/or in a cloning vector, in tandem but separated by a DNA coding for an endogenous peptide, two or more identical cDNAs or identical couples of cDNA:s coding for endogenous protein, protein domains or polypeptide(s), and an additional cDNA coding for a protein that does not have to be endogenous but will be used in the detection device of the compound, to insert these fused DNA-sequences into an expression vector containing specific tags for simple purification of
  • the cDNA of the cell receptor binding domains is coding for an endogenous, biologically active ligand.
  • the cDNA is coding for an endogenous extracellular domain or extracellular peptide loop of an endogenous cell receptor to which a receptor on the surface of a pathogenic virus or microorganism specifically binds.
  • the DNA, coding for the endogenous peptide that interlink the receptor binding domains shall have such length that the two neighbouring domains of the compound simultaneously bind two juxtaposed receptors on the surface of a cell, a virus or a microorganism.
  • the additional cDNA coding for a ligand, which binds to a receptor on a phagocyte or a cytoiytic cell, is coding for the FcR binding domains of human IgGl.
  • the cDNA coding for a detection device protein unit of the compound, is coding for avidin.
  • a sixth aim of this invention is to produce a pharmaceutical composition involving one of these abovementioned compounds, which can be used as drugs, and one or more pharmaceutically accepted components.
  • Fig. 1 shows a virus that has several identical receptors (Rsv), strategically positioned on its surface, so that the virus simultaneously can interact with several juxtaposed, specific membrane receptors ( R C M) of the host cell and thereby form a long-lived interaction which has to precede the infection process,
  • Rsv identical receptors
  • R C M specific membrane receptors
  • Fig. 2 shows in a-g examples of models of fused cDNA (
  • h-m shows examples of models of fused cDNA, coding for endogenous peptides/proteins, linked to cDNA ( __ ⁇ ) ) coding for foreign proteins like e.g. an enzyme (E), a fluorescent protein (FP) or a protein with very high affinity for a specific ligand (PL) for preparation of analytical/diagnostic tools according to the invention
  • E enzyme
  • FP fluorescent protein
  • PL specific ligand
  • Fig. 3A-B shows models of a compound, based on endogenous peptide/protein and produced according to the invention, with the ability to bind to an extra cellular part of a specific membrane receptor (R C M) and thereby block the subsequent receptor/receptor subunit 7b mediated processes.
  • the ligand (L) is made as a dimer (LD) (or a trimer, a tetramer, etc) resulting in long-lived ligand-receptor complex, which strongly delays recirculation (RC) of the receptor, Fig. 3 A, or of some of the receptor subunits, Fig.
  • LD dimer
  • RC recirculation
  • Fig 3C D shows a dimeric ligand with a tailor-made linker ( ⁇ ) can inhibit the interaction between two juxtaposed intracellular domains (IC) of the same receptor or different receptors, and thereby the receptor mediated signalling process, by simultaneous binding to two juxtaposed receptors (R CM ) forcing these receptors apart,
  • Fig. 3E shows that a drug can be targeted to the correct cell by being fused with one or more ligands having affinity for a membrane receptor specific for that cell (R SCM ),
  • Fig. 4A-C shows models of a compound (L) of tandemly fused endogenous extra cellular domains/peptide loops of host cell membrane receptors and interlinked by a peptide of such a length that the compound simultaneously binds to two or more surface receptors (Rsv) of the virus (V) in fig. 4A, or to couples of surface receptors, Fig. 4.
  • Rsv surface receptors
  • I and Q are extracellular parts of endogenous membrane receptors, and — ⁇ a linker
  • Fig. 5 shows a model of a compound (L) of tandemly fused endogenous extra cellular domains/peptide loops (
  • Fig. 6A-C shows models of compounds for use as an analytical or diagnostic tool to identify surface receptors (Rs) on endogenous cells, virus, microorganisms, or on an artificial surface by containing tandemly fused identical sequences of receptor binding proteins (L) and one or more fused proteins in form of Fig 6 A,
  • Fig. 6A shows an enzyme (E) which converts a substrate (S) to a detectable product (P),
  • Fig. 6B shows a fluorescent protein, FP, which can be detected by fluorescent microscopy
  • Fig. 6C shows a ligand binding protein (LP), to which a ligand (
  • Fig. 7 A shows compounds, prepared according to the invention, for use as drugs containing one cell-targeting ligand,
  • Fig. 7B shows a dimer of the cell-targeting ligand
  • Fig. 8 shows interleukin 2, IL-2, binding normally i.e. with high affinity (K D 10 "11 M) to its receptor, IL-2R, containing three different subunits, ⁇ , ⁇ , ⁇ , which results in cross phosphorylation of the intracellular domains of the ⁇ and ⁇ subunits and thereby initiation of the receptor mediated cell signalling process,
  • Fig. 9 shows IL-2 as a dimer with an interlinking peptide containing 15 amino acids
  • Fig. 10 shows IL-2 as a dimer with an interlinking peptide containing 8 amino acids 7c (L 8aa ), prepared according to the invention, seems to be too short to simultaneously bind two neighbouring IL-2R,
  • Fig. 11 shows IL-2 as a dimer with an interlinking peptide containing 8 amino acids (Ls aa ), prepared according to the invention, seems to bind simultaneously to both antigen binding sites on the anti-IL-2,
  • Fig. 12 shows IL-2 as a dimer with an interlinking peptide containing 15 amino acids (Li 5aa ), prepared according to the invention, seems to be too long to simultaneously bind to both antigen-binding sites on the same anti-IL-2,
  • Fig 13 shows PCR products of various IL-2 inserts, prepared according to the invention, in a cloning vector (pUCl 8) and in an expression vector (pEZZl 8) separated on 1.5 % agarose.
  • Lane 1 IL-2 monomer in pUC;
  • lane 2 IL-2-8L-IL-2 dimer in pUC;
  • lane 3 IL-2-15L-IL-2 dimer in pUC;
  • lane 4 Marker 1 kb (bands from below — 250, 500, 750, 1000, 1500 bp etc);
  • lane 5 IL-2 monomer in pEZZ;
  • lane 6 IL-2-8L-IL-2 dimer in pEZZ;
  • lane 7 IL-2-15L-IL-2 dimer in pEZZ
  • Fig. 14 shows the effect of commercial rIL-2 and of the IL-2 compounds IL-2s
  • IL-2-8L (dimer)
  • IL-2-15L (dimer) prepared according to the invention on proliferation of CTLL-2. Mean values of five proliferation studies are shown. As reference, culture medium without IL-2, was used.
  • the invention consists of a new concept to design receptor binding compounds, which will result in a compound with high specificity and affinity to surface bound receptors, with, due to its endogenous nature, is resistant against the immune defence of the host, and which can be used as an endogenous antagonistic drug on a specific cell membrane receptor and thereby 9 block the subsequent receptor mediated cell signalling process, as an antiviral or antimicrobial drug, and in very low doses due to its high affinity for the surface bound receptors.
  • the compound can be used for analytical and diagnostic purposes.
  • a compound which forms a strong complex with surface structures, specific for the compound, on cells or pathogenic organisms or viruses, comprising two or more identical, or identical couples, of endogenous proteins/protein domains/peptide loops in tandem interlinked by a peptide having such a length that each of the protein domains/peptide loops can interact simultaneously to its specific surface structure and thereby block the function of this structure.
  • the term "two or more”, is intended to mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, identical cDNAs.
  • the cDNAs may also be couples of different cDNAs, such as a heterodimer of cDNA, coding for an endogenous protein, protein domain or peptide loop, here called the ligand, which specifically binds to a certain receptor on an endogenous cell or on the surface of a virus or a microorganism.
  • the fused cDNAs are normally interlinked with a DNA coding for a polypeptide of such a length that all the various receptor binding domains of the formed compound simultaneously bind to their specific receptors. In this way, a very stable and long-lived complex is formed between the compound and the surface receptors as shown in figure 2 a-g and figure 3-6.
  • Further embodiments include a compound, wherein the protein domains consist of an endogenous ligand, which binds to a specific surface receptor on an endogenous cell.
  • the receptor binding ligand of the compound should be composed of the endogenous ligand of the receptor, and the receptor should be able to juxtapose to another identical receptor in the cell membrane.
  • the compound according to the invention may be a compound wherein the specific surface receptor on the endogenous cell is an receptor selected from the group consisting of growth factor receptors, such as EGF binding receptors, and HGF-R;; cytokine receptors, such as IL2-R, TNF- ⁇ -R, IL-12-R, IL-16-R, IL-4-R, and IL-10-R, G-protein coupled receptors (7TM-R), such as D2R, D4R; and ligand induced channel receptors, such as NAchR, GABA-A-R, andNMDA-R.
  • growth factor receptors such as EGF binding receptors, and HGF-R
  • cytokine receptors such as IL2-R, TNF- ⁇ -R, IL-12-R, IL-16-R, IL-4-R, and IL-10-R
  • TNF- ⁇ -R IL-12-R
  • IL-16-R IL-16-R
  • IL-4-R IL-10-R
  • the compound according to the invention may in specific embodiments be a compound, wherein the protein domains/peptide loops are identical to an extracellular domain or peptide loop of that endogenous protein to which a specific virus or microorganism bind by 10 one of its surface structures.
  • the compound according to the invention is a compound, wherein the specific virus or microorganism is selected from the group consisting of Adenoviruses, HIV, Influenza viruses, Epstein Barr virus, Hepatite viruses, Herpes viruses, Papilloma viruses., Streptococcus, Staphylococcus, Plasmodium vivax.
  • the specific virus or microorganism is selected from the group consisting of Adenoviruses, HIV, Influenza viruses, Epstein Barr virus, Hepatite viruses, Herpes viruses, Papilloma viruses., Streptococcus, Staphylococcus, Plasmodium vivax.
  • the compound according to the invention may be produced as a fusion protein by gene technological and biochemical/biological methods.
  • gene technological and biochemical/biological methods are known to the skilled man in the art, and may be methods described in e.g. Maniatis et al. or Current Protocols in Molecular Biology.
  • the compound according to the invention may comprise one or more additional endogenous protein domain(s) with the ability to bind specifically to a receptor on a phagocyte or a cytoiytic cell, figure 3E.
  • the compound may also be a compound, wherein the domain, binding to receptors on a phagocyte or a cytoiytic cell, is a Fc ⁇ R binding domain.
  • Cytoiytic cells may be CTLL (cytotoxic T cell lymphoma line), naturally occurring cytotoxic T cells, NK cells, or phagocytes or macrophages.
  • the binding of the compound to the receptors on the cell membrane should inhibit the normal function of the receptor. This is accomplished by forming such a strong complex with juxtaposed receptors that the recirculation of the receptors or its subunits back to the membrane after internalisation of the complex becomes delayed, or that another type of molecular interaction is inhibited, e.g. cross-phosphorylation of the intracellular domains of two receptors, juxtaposed by ligand binding, and thereby the cell signalling process, figure 3 A-D.
  • the compound is used as an antiviral drug, i.e.
  • the ligand should be identical to that/those extra cellular domain/s of a cell membrane protein to which the virus specifically binds through one of its surface receptors in order to initiate the infection of the host cell.
  • the receptor molecules on the surface of the virus are most often fixed in number and are regularly positioned, i.e. the distance between them is normally rather constant. The reason for this is probably that the virus thereby can bind simultaneously to several receptors on the host cell - and thereby strongly enough for infection - if these cell membrane receptors can be repositioned and juxtaposed in the cell membrane. That this happens is shown in the underlying studies of this invention. Model of virus blocking compounds, prepared according to the invention, are shown in figure 4 A-C.
  • the ligand should consist of tandemly cupled protein domains of that protein domain/unit to which the microorganism binds in order to infect the host.
  • Figure 5 shows design of antimicrobial compounds according to the invention.
  • the compound should also contain one or more of an endogenous ligand specific for a receptor on the target cell, figure 3E and figure 7.
  • the compound according to the invention may be used as an antiviral compound by blocking surface structures of a virus by which the virus interacts with its specific host cell. Even further, the compound according to the invention may be for use as an antimicrobial compound by blocking surface structures of a microorganism by which the microorganism interacts with its host.
  • the compound according to the invention may be a compound, to which one or more protein/protein domains, having fluorescent, enzymatic or ligand binding properties, are further fused, figure 2 h-m.
  • the cDNA for one or more fluorescent proteins is via a linker fused to the DNA of the compound, figures 2 j-k and 6B.
  • cDNA of e.g. avidin, binding biotin labelled with ssDNA is fused via a linker to the 3 'end of the DNA of the compound, figures 2 1-m and 6C.
  • the aim of a compound, prepared according to the invention is that it can be used as a receptor blocking compound acting on an endogenous cell so that the formed complex results in inhibition of the normal function of the receptor and thus of all these processes that follow the normal ligand-receptor interaction.
  • the invention can be applied to most membrane receptors.
  • IL-2 and EGF receptors of growth factors, like IL-2 and EGF, as such receptors are over-expressed on certain tumour cells.
  • IL-2 and EGF were chosen as ligands for preparation of receptor blocking compounds according to the invention.
  • - antiviral substance in order to neutralise a virus and thereby prevent infection of the host cell of the virus.
  • the ligands of the compound are separated by a peptide linker with such a length that all ligands simultaneously bind specific receptor molecules on the surface of the virus. This results in strong complexes showing very low K D . The amount of infectios virus can thereby be decreased to very low levels.
  • the invention can be applied to all 12 pathogenic viruses when enough knowledge is known about the surface structures of the virus and that membrane protein these structures specifically interact with.
  • the N- terminal domain of HCAR human coxsackie adenovirus receptor
  • HCAR human coxsackie adenovirus receptor
  • antimicrobial sustance in order to neutralize the micro- organism and prevent it from infecting the host.
  • the invention could be applied to such microorganism that interact with specific extra cellular protein/protein domains of the host and for which these interacting molecules are known. - sensitive analytical or diagnostic tools for quantification and/or identification of those cells, viruses and microorganisms, which contain the specific receptor molecule to which the prepared fusion protein binds.
  • a compound according to the invention can be used intra- and/or extra corporal, figure 7. From the blood the compound will be directed to the correct target cell through the dimer of a ligand, which interacts with a specific receptor on the target cell. The formed complex can then, via e.g. a FcR binding domain of the compound, bind a cell having phagocytic or lytic properties, figure 3E and 7.
  • the compound may be a compound wherein the endogenous protein domains consist of human IL-2 interlinked by a peptide containing 2-20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23,24, 25, 30, or 40 amino acids.
  • inventions may be a compound, wherein the endogenous protein domains consist of human IL-2 interlinked by a peptide containing 5-14 amino acids.
  • human IL-2 may be interlinked by a peptide containing 15 amino acids.
  • the compound according to the invention may also be for medical use.
  • the compound may be a compound wherein the medical use is blocking receptor dynamics and/or a receptor mediated process.
  • the method according to the invention is a method for preparation of a compound according to the invention, with the property to form a strong complex with specific structures on the surface of a cell or a pathogenic virus or microorganism, comprising the steps of: 13 fusing in tandem, with gene technological methods in in a cloning vector, or in soulution, identical cDNA, or couples of identical cDNAs, interlinked by DNA coding for an endogenous peptide linker, coding for at least one endogenous protein/protein domain/peptide loop, - transferring the fused DNA sequence to an expression vector, transfering the expression vector to a host cell, and producing the desired fusion protein in a host cell.
  • cDNA coding for the above mentioned protein/protein domain/peptide loop
  • the cDNA coding for the above mentioned protein/protein domain/peptide loop, is coding for an endogenous extracellular domain/peptide loop.
  • the method according to the invention may be a method, wherein the DNA, coding for the peptide linker between the protein domains/peptide loops of the fusion protein, is coding for a peptide, the length of which is adapted to the size of the mentioned surface structures and the distance between them when juxtaposed on a endogenous cell or a pathogenic virus or microorganism.
  • the method according to the invention comprises one further cDNA, coding for at least one the protein domains/peptide loops with the ability to bind to a specific receptor on phagocytes or cytoiytic cells, that is fused.
  • cDNA is coding for one or more of a Fc ⁇ R binding ligand.
  • a pharmaceutical composition comprising at least one purified compound, according to the invention, and eventually one or more pharmaceutically acceptable components.
  • a compound may be for analytical or diagnostic purposes. It may be, e.g. to analyse the amount of bacteria or viruses in a biological sample, such as blood, urine, tissue sample or a clinical sample.
  • the diagnostic purpose may be to identify the presence of specific pathogens, such as viruses and microorganisms, in blood, tissue, urine or a clinical sample.
  • ligand-receptor complexes where the ligand is a paracrine or an endocrine cytokine, show K D between 10 "n and 10 "8 M. Such low K D are requisited for the ligand- receptor complex to be formed as the cytokine normally exists in very low concentrations at its target cell. By increasing the receptor concentration, by receptor aggregation, in a certain area of the cell membrane, where the cytokine is to be found, enough ligand-receptor complexes will be formed and an intracellular answer obtained.
  • the K D is theoretically decreased to K D 2 if no steric hindrances exist compared to the L-R formation (Jencks, 1981; Mammen et al, 1998). A trimerization would likewise result in K D 3 .
  • a dimeric or trimeric ligand results then in very strong and long-lived complexes, i.e. almost irreversible complexes.
  • the additive affinity which is obtained by forming several ligand-receptor complexes, is called avididty (Mammen et al., 1984).
  • the blocking capacity of the compounds of this invention is based on the avidity that is obtained when endogenous ligands are coupled in tandem but interlinked by a peptide having such a length that all ligands of the compound simultaneously can interact with a receptor on the surface of a cell, a virus or a microorganism and thereby form very stable complexes. The subsequent receptor mediated processes are then inhibited.
  • the compound according to the invention will be used as a drug acting as an antagonist/blocking agent of a specific receptor on an endogenous cell or a pathogen it should consist of endogenous receptor binding ligands in order to diminish side effects and development of host resistance against the drug. This is made possible by using recombinant techniques with human cDNA for the ligands and the linkers.
  • the human cDNA of interest is initially amplified by PCR and then inserted as a monomer or dimer into a cloning ir directly into an expression vector, e.g. a plasmid or a phage.
  • the expression vector is transferred into a host cell which produces the desired human protein. If the formed protein has to be glycosylated, eukaryotic host cells should be used.
  • the procedures used for preparation of recombinant human insulin resulting in a pharmaceutical composition can be applied for compounds prepared according to this invention. 15
  • Prerequisites for preparation of compounds acting as receptor antagonists on endogenous cells are that - the endogenous ligand of the receptor is known as well as its cDNA or amino acid sequence,
  • the effective dose of the compound can be lowered thousands of times down to the Ko-level.
  • Such low concentrations of the effective dose increase the stability against proteolytic degradation, and increases the resistance of the compound against the immune system of the host.
  • tandem-coupled ligands interlinked with a tailor-made polypeptide
  • the ligands can interact with juxtaposed receptors on a surface and complexes with a much lower K D are formed compared to the LR complex.
  • the sensitivity of the method increases correspondingly.
  • - are endogenous and interact specifically with its natural receptor on the cell or a receptor on a certain pathogen, - are endogenous and do not activate the immune defence system,
  • the complex After binding a ligand, like a nutrient, to its receptor, the complex is usualy internalised. Normally the ligand-receptor complex dissociates in the cell and the receptor turns back to the membrane. If such receptors are juxtaposed on the surface, a compound consisting of dimers or trimers of the ligand will form a very stable complex, which is more slowly dissociated.
  • a ligand like a nutrient
  • Blockade of the recirculation of subunits of a multimer receptor internalised as a ligand- receptor complex Blockade of the recirculation of subunits of a multimer receptor internalised as a ligand- receptor complex.
  • a compound, according to the invention forms a complex with two or more juxtaposed multimeric receptors, which is more slowly dissociated in the cell after internalisation. The recirculation of some of the subunits is therefore delayed. If such a subunit also is used by another receptor, the function of this receptor is also inhibited.
  • IL-2R ligand-receptor complex compared to the natural ligand-receptor complex - the ligand IL-2 and its receptor, IL-2R were chosen as: 1.
  • IL-2 is a monomer protein for which the primary sequence of the 133 amino acid long peptide chain as well as the three dimensional structure is known (Bazan and McKay, 1992).
  • Recombinant human IL-2 has the same biological activity as the endogenous one (Landgraf et al., 1992).
  • IL-2R is common on T-cells, which are simple to culture (Rottenberg et al., 1993).
  • IL-2R is composed of three subunits, alpha, beta, and gamma, which have to be associated in order to obtain a fully functional receptor (Taniguchi and Minami, 1993), figure 8.
  • IL-2R The structure of IL-2R is well known and a good example of a very dynamic receptor 18
  • rIL-2 does not have to be glycosylated for its biological activity (Landgraf et al, 1992).
  • IL-2R blocking drugs are therapeutically interesting in treatment of some T-cell lymphoma. Such tumour cells proliferate strongly due to high expression of IL-2R. By blocking these receptors, the proliferation should be diminished or inhibited. Moreover, the blockade should also promote apoptosis of the tumour cells due to decreased recirculation of the gamma subunit of the receptor (Yamada et al., 1998).
  • IL-2 which is categorized as a cytokine, contains 133 amino acids and is produced by activated T-cells.
  • the effect of IL-2 is a general activation and intensification of the immune defence.
  • IL-2R is composed of three different subunits, which are associated upon IL-2 binding. Normally, only two of these subunits, beta and gamma, are expressed on the surface (Hemar et al., 1995). Probably, IL-2 initially binds to the gamma subunit and then fuse with the gamma subunit. Subsequently, the alpha subunit, which can be stored in the cell (Subtil et al., 1997), is associated and a complete IL-2-IL-2R is formed, figure 8. The intracellular domains of the beta and gamma subunits cross-phosphorylate, and intracellular signalling pathways, mentioned above, are initiated, figure 8.
  • I1-2-IL-2R is quickly internalised (Chang et al., 1996).
  • the beta subunit is degraded like the IL-2, while the gamma subunit normally turns back to the membrane.
  • the alpha subunit can be degraded, turn back directly to the membrane, or be stored for a while in the cell (Hemar et al., 1995).
  • IL-2R By binding IL-2 to IL-2R, proliferation of T-cells, cytoiytic activity of NK-cells, and development of antibodies from B-cells, are promoted.
  • tumour T-cells are characterised by too high expression of IL-2R. Binding of IL- 2 results in proliferation, i.e. the amount of tumour cells increases. Blocking such receptors would inhibit the proliferation, which is shown in this application.
  • the distance between two binding sites for IL-2 of juxtaposed IL-2R was calculated to 19 be about 40 A.
  • the length of the peptide linker should therefore be about 50 A (15 amino acids) so that both IL-2-domains of the formed fusion protein will bind to the juxtaposed IL- 2R, figure 9.
  • a compound with a shorter linker (28 A), containing 8 amino acids between the two IL-2 was chosen. This compound should not bind simultaneously to juxtaposed IL-2R, figure 10.
  • such a dimer was supposed to bind to fixed receptor sites, comparable to those of a virus.
  • the antigen binding sites of antiIL-2 bound to a well in an ELISA plate was chosen, figure 11.
  • a compound containing a 15 amino acid long peptide linker between the two IL-2 domains should be too long, figure 12.
  • the recombinant protein was initially tagged with IgG binding ZZ-domains by inserting the DNA of the compound into a pEZZ plasmid in order to simplify the following purification process.
  • the ZZ-tag was cut off by factor Xa, as a Xa cleaving peptide was inserted between the ZZ and the first IL-2 domain.
  • forward primer 1 : 5'- ACAACGGATCCCGCACCTACTTCAAGTTCTACAAAG-3' containing a BamHI site
  • reverse primer 2: 5 OTTGAGTCGACGGCCTGATATGTTTTAAGTGGGAAGCAC-3 ' containing two stop codons and a SAL-I site
  • reverse primer 3: 5'-GCTAAGTCGACAGTCAGTGTTGAGATGATGCTTTGAC-3' containing the 3'endo of IL-2 without stop codon.
  • dNTP and Taq-polymerase was bought from Boehringer-Mannheim, Germany).
  • the annealing temperature of the PCR was 58 °C.
  • PCR-products containing amplified cDNA for IL-2 and the pUC 18 plasmid was cleaved by BamHI and Sal I (Boehringer- Mannheim) and the cleaved products were separated by agarose (1,5 % and 0.8 % respectively) electrophoresis and cleaned by GenecleanTM (Bio 101, Ca, USA).
  • the cDNA products formed by primer 1 + 2 or primer 1 +3 were then ligated by T4-ligase (Pharmacia- Upjohn) with the cleaved pUC plasmid.
  • plasmids Two different plasmids were obtained - one with an IL2 with stop codons (pUC IL-2s) and one with IL-2 without stop codons (pUC IL-2).
  • the plasmids were transferred to E.coli JM 109, and positive transformants were selected as white colonies using ampicillin containing (70 mg/ml) agar plates, IPTG (isopropyl-beta-D- thiogalactopyranoside, 20 ug/ml) as inducer, and X-gal (5-bromo-4-chIoro-3-indoyl-beta-D- galactoside, 40 ug/ml) (all Pharmacia-Upjohn) for detection.
  • ampicillin containing 70 mg/ml
  • IPTG isopropyl-beta-D- thiogalactopyranoside, 20 ug/ml
  • X-gal 5-bromo-4-chIoro-3-ind
  • 5'AATTCTATCGAAGGTCGT -3 'and 5'-GATCACGACCTTCGATAG-3' were hybridized to a DNA sequence containing ATCGAAGGTCGT, coding for ile-glu-gly-arg, which is the cleavage site for factor Xa.
  • This sequence was ligated to the 5 'end of the IL-2 gene by the T4-ligase in pUC-IL2- plasmids and pUC-IL2s, which first had been purified (by Wizard Plus Midiprep, SDS- Promega), cleaved with EcoRI and BamHI (Boehringer-Mannheim) and purified by agarose electrophoresis and GenecleanTM.
  • the pUC-X-IL2s and pUC-X-IL2 were formed.
  • the DNA for the Xa sequence can be incorporated into the 5 'forward primer of IL-2 together with an EcoRI site instead of a BamHI site.
  • the oligonucleotides 5 '-TCGACCAGGTACCGCCTGGTCAGAGCTCTA-3 ' and 5'- AGCTTAGAGCTCTGACCAGGCGGTACCTGG-3 ' were hybridized.
  • the formed DNA contains a Sad and a Kpnl site.
  • the DNA was inserted into a pUC-x-IL2, which had been cleaved with Sal I and Hind III, and a pUC-X-IL2-L plasmid was formed.
  • pUC-x-IL2-L The insert from pUC-x-IL2-L was inserted into cleaved and purified pUC-IL2s.
  • pUC-X-II2-L(15)-IL2s was formed.
  • two cDNA of IL-2 can be interlinked by preparing by PCR cDNA with complementary DNA overhangs in the 5 'and 3 'ends. The overhangs (24 - 45 bp) code for the peptide linkers.
  • the two prepared cDNA:s, one with an overhang in the 5 'end and the other in the 3 'end, are mixed with Taq enzyme and dNTP but no primers and a PCR is performed during 20 cycles using an annealing temperature of about 55 °C
  • a primer 5 'forward and a primer 3 'reverse(containing an EcoRI site and a Hind III site, respectively, but no IL2cDNA sequences) are added and another 35 cycles are performed.
  • the obtained IL-2 dimers are purified by agarose electrophoresis and Geneclean as described above before ckeavage with EcoRI and Hind III. 21 Transfer of IL-2 inserts to an expression plasmid, pEZZ.
  • pEZZ Pulsa-Upjohn- Amersham
  • pUC containing various IL-2 inserts were cleaved by EcoRI and Hind III. Purified IL-2 insert was ligated into purified pEZZ. The plasmids were then transformed to E.coli HB101 and positive transformants were selected.
  • the sequence of the insert was verified by Taq DyeDeoxi-Terminator cycle sequencing kit (Applied Biosystems, Foster city, CA)using the Applied Biosystem model 373 DNA Sequencing system.
  • rIL-2 and various IL-2 dimers were produced from positive transformants, containing the desired IL-2 inserts, cultured over night in LB-medium containing 0.1 % glucose.
  • IL-2 containing fractions identified by IL-2 ELISA (Qiagen) were applied to IgG Sepharose 6FF (Pharmacia-Biotech, Uppsala, Sweden). The gel was washed according to the manufacturers recommendations. The ZZ-IL2 proteins were eluted by
  • Xa was applied to the column, and cleaved IL-2 products were eluted. All IL-2 preparations were sieved through a 0.22 um filter before use.
  • the products were analysed by ELISA and Western blot.
  • CTLL-2 (ATCC) were cultured to the manufacturers recommendations, but the concentration of human IL-2 in the culture medium was 10 "9 M.
  • the cells were washed twice with medium without IL-2. About 10 cells (100 ⁇ l) were transferred to each well in a 96 wells culture plate. After adding culture medium rIL-2, IL-2 dimers or control (eluate from HBlOl-pEZZ plasmid without insert) was added. After 12 hours incubation at 37 °C,
  • rIL-2 as a monomer or as a dimer was obtained from pUC-IL-2 in HB 101 22 induced by IPTG. All of the produced IL-2 was found in the cytoplasmic fraction. As the expressed protein from the pEZZ insert contains the signal sequence for protein A, the produced protein should appear in the culture media. However, most of the produced protein was found in lysed bacteria. Studies of the interaction of the various IL-2 compounds to anti-IL-2 showed that the monomer IL-2 acts in the same was as commercial IL-2. the dimer rIL-2-8L give a false picture- about 1000 times too high value of the IL-2 concentration. the dimer rIL-2- 15L shows about 10 times the vlue of momomer IL-2.
  • IL-2 in dimeric form having a 8 amino acid long peptide shows about 3080 cpm.
  • IL-2 in dimeric form having a 15 amino acid long peptide shows a total stop in proliferaton with only 180 cpm. This value is even lower than that of the control but which can contain some remaining rIL-2. The same stop was found for the preparation even if dimeric IL-2-15L was diluted 100 times. Moreover, these cells seem to have initiated apoptosis.
  • the cell becomes apoptotic.
  • the ⁇ -subunit of IL-2R is decreased on the surface as its presence there inhibits the apoptosis.
  • the results of the IL-28-L dimers which show a two fold proliferation compared to monomeric IL2, can be explained by fact that only one of the IL-2 domains of the compound binds to juxtaposed IL-2 receptors, but that the concentration of IL2 in the vicinity of the IL- 2R is doubled.
  • Presence of the ZZ domains does not affect the binding to the receptor.
  • Studies of the interaction between anti-IL-2 and the various IL-2 compounds are also interesting by showing that a dimeric compound gives a too high value of the antigen if binding simultaneously to both antigen binding sites and thereby lowering the K D .
  • the dimeric IL-2-8L seems to be perfect in binding to both antigen binding sites on the antibody, while dimeric IL-2-15L seems to be too long.
  • the tenfold increase in concentration is probably an effect of that IL-2-15L sometimes can bind two juxtaposted receptors but usually not.
  • a dimer of IL-2 interlinked by a peptide which is tailor-made according to the IL-2R, can simultaneously bind to juxtaposed receptors in the membrane of CTLL-2 and thereby from very strong complexes.
  • receptors like the IL-2R, which normally are not aggregated, can be juxtaposed on a membrane that a dimer of IL-2, interlinked with a peptide of such a length that the IL-2 domains of the dimer can bind to each of the antigen binding sites on the antiIL-2 antibody.
  • Such a binding kan be comparable to the binding of a dimer to a surface structure of a virus.
  • the binding to the membrane receptor causes the intracellular domains of the receptor to interact and the cross- phosphorylation and thereby the cell signalling process to occur.
  • the cross-phorylation process can be inhibited.
  • Dimeric EGF prepared according to the invention has shown such blocking effects.
  • Antiviral compounds according to the invention can be prepared if that membrane protein, by which the virus interact on the host cell, is known and some of its amino acid sequene or cDNA has been identified. Dimeric HCAR, prepared according to the invention against various adenoviruses show blocking effects. 24 Example 5 Antibiotic compounds according to the invention
  • Antibiotic compounds can be prepared to pathogenic microorganisms, which interact with a protein in the host. Parts of the amino acid sequence or cDN A of this protein must be known.
  • the receptor can be a component of the surface of a cell, a virus, a mircoorganism, or can be bound to an artificial surface.
  • the compound contains two or more receptor binding domains. Moreover, the compound consists of one of the following alternatives : one or two domains of an active enzyme showing a high turn over one or two domains of a ligand binding protein one or two domains of Green fluorescent protein, GFP
  • One application contains a compound containing two human IL-2 domains interlinked with a peptide of 15 amino acids in order to identify and quantify CTLL-2 by studying an enzyme coupled analysis, figure 6A by using fluorescence microscopy, figure 6B by PCR /Real time PCR using biotin, which binds strongly to an avidin monomer linked to the compound, and which have a bound nucleotide (40bp) as target for the PCR, figure 6C.
  • EGF epidermal growth factor
  • CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus, Nature, 312, 763-66.
  • HIV-1 entry cofactor functional cDNA cloning of a seven-transmembrane G protein-coupled receptor , Science,
  • Interleukin-15 can replace the IL-2 signal in IL-2 dependent adult T-cell leukemia (ATL) cell lines: Expression of IL-15 receptor alfa on ATL cells, Blood, 91, 4265-72.

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Abstract

Sur la surface d'une cellule, il existe différentes molécules de surface différentes, appelées récepteurs. Ces récepteurs fixent des ligands spécifiques, ce qui permet à la cellule d'être affectée par son environnement. On trouve également des molécules réceptrices sur la surface de virus et d'autres agents pathogènes. En revanche, de tels récepteurs sont utilisés pour la fixation de l'agent pathogène à une protéine spécifique chez l'hôte et par conséquent pour initier le processus d'infection. Si les récepteurs membranaires de la cellule sont bloqués, la cellule ne peut pas être affectée par un ligand spécifique, et si les récepteurs de surface d'un agent pathogène sont bloqués, l'agent pathogène ne peut pas devenir infectieux. Cette invention concerne des composés, qui forment des complexes très forts avec les structures de surface spécifiques de composés et par conséquent inhibent la fonction biologique de structures de surface spécifiques de composés (récepteurs) sur les cellules, les microorganismes pathogènes ou les virus. Cette invention concerne également la conception et la préparation de tels composés destinés à être utilisés en tant que médicaments ou en tant qu'outils à des fins analytiques et diagnostiques. Ces composés comprennent deux ou plusieurs domaines de protéines identiques liés par un peptide possédant une longueur telle que chacun des domaines de protéine peut interagir simultanément avec sa structure de surface spécifique et par conséquent bloquer la fonction de sa structure. Les domaines de protéines peuvent être l'interleukine 2 (IL-2) humaine.
PCT/SE2001/002892 2000-12-22 2001-12-21 Composes comprenant au moins deux domaines de proteines identiques lies par un peptide possedant une longueur telle que chacun des domaines de proteines peut interagir simultanement avec sa structure de surface cellulaire specifique et bloquer ainsi la fonction de la structure WO2002051869A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2010033249A2 (fr) * 2008-09-22 2010-03-25 Massachusetts Institute Of Technology Compositions à base de ligands dimériques et procédés associés
WO2015056713A1 (fr) * 2013-10-15 2015-04-23 国立大学法人東京大学 Agoniste de la protéine c-met
US9029328B2 (en) 2010-03-24 2015-05-12 The Brigham And Women's Hospital, Inc. Methods for cardioprotection and cardioregeneration with dimers of EGF family ligands

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WO1993023550A2 (fr) * 1992-05-18 1993-11-25 Genentech, Inc. Activation de recepteurs d'oligomerisation grace a des ligands de recepteurs fusionnes
EP0816510A1 (fr) * 1995-12-22 1998-01-07 Toray Industries, Inc. Procede de production de proteines fusionnees biologiquement actives
WO1999002710A1 (fr) * 1997-07-10 1999-01-21 Beth Israel Deaconess Medical Center Production et utilisation de multimeres de proteines recombinees a activite biologique modifiee
WO1999063936A2 (fr) * 1998-06-08 1999-12-16 Advanced Medicine, Inc. Agents therapeutiques modulant les recepteurs de l'endotheline

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WO1993023550A2 (fr) * 1992-05-18 1993-11-25 Genentech, Inc. Activation de recepteurs d'oligomerisation grace a des ligands de recepteurs fusionnes
EP0816510A1 (fr) * 1995-12-22 1998-01-07 Toray Industries, Inc. Procede de production de proteines fusionnees biologiquement actives
WO1999002710A1 (fr) * 1997-07-10 1999-01-21 Beth Israel Deaconess Medical Center Production et utilisation de multimeres de proteines recombinees a activite biologique modifiee
WO1999063936A2 (fr) * 1998-06-08 1999-12-16 Advanced Medicine, Inc. Agents therapeutiques modulant les recepteurs de l'endotheline

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010033249A2 (fr) * 2008-09-22 2010-03-25 Massachusetts Institute Of Technology Compositions à base de ligands dimériques et procédés associés
WO2010033249A3 (fr) * 2008-09-22 2010-08-19 Massachusetts Institute Of Technology Compositions à base de ligands dimériques et procédés associés
US9198952B2 (en) 2008-09-22 2015-12-01 The Brigham And Women's Hospital, Inc. Compositions of and methods of using ligand dimers
US9029328B2 (en) 2010-03-24 2015-05-12 The Brigham And Women's Hospital, Inc. Methods for cardioprotection and cardioregeneration with dimers of EGF family ligands
WO2015056713A1 (fr) * 2013-10-15 2015-04-23 国立大学法人東京大学 Agoniste de la protéine c-met
JPWO2015056713A1 (ja) * 2013-10-15 2017-03-09 国立大学法人 東京大学 ｃ−Ｍｅｔタンパク質アゴニスト
US9994616B2 (en) 2013-10-15 2018-06-12 The University Of Tokyo c-Met protein agonist

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