WO2001078785A2 - Wirkstoff-konjugate mit intrazellulär wirksamen liganden - Google Patents
Wirkstoff-konjugate mit intrazellulär wirksamen liganden Download PDFInfo
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- WO2001078785A2 WO2001078785A2 PCT/DE2001/001495 DE0101495W WO0178785A2 WO 2001078785 A2 WO2001078785 A2 WO 2001078785A2 DE 0101495 W DE0101495 W DE 0101495W WO 0178785 A2 WO0178785 A2 WO 0178785A2
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- active ingredient
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- 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
- A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6875—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
- A61K47/6879—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
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- 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
- A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
- A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
Definitions
- the invention relates to active substance conjugates with an intracellularly active ligand, a targeting domain and usually also a localization domain that ensures entry into the cytoplasm of the target cell, as well as their use as diagnostic agents and medicaments, also in various indications and methods for their production.
- An average cell contains approximately 30,000 proteins that participate in intracellular signal transduction, regulation of transcription, cell-cell communication and intracellular transport.
- the functional analysis or therapeutic influence of these processes in the context of the living cell is hindered by the fact that the plasma membrane only allows a selective exchange of substances and that various highly specific binding molecules (eg antibodies) cannot penetrate the plasma membrane.
- micro-injection or pore-forming substances have so far been used for corresponding analyzes, which, however, place an extraordinary strain on the cell and cannot be used in a therapeutic approach.
- Microinjection into individual cells in particular is not suitable for therapy. For this reason, genetic methods have been developed in recent years to remove the complex network of functional relationships of the above. To study proteins.
- RNA antisense approaches are complex to produce because they usually only show significant effects in homozygous animals. They are completely unsuitable for therapy since they would require germline gene therapy.
- the second previously used method of dominant negative mutants by overexpression of proteins with mutations that have destroyed the desired function is often hampered by side effects of the strong overexpression required for this and also requires precise information about the mode of action of the corresponding proteins in order to produce the desired mutants.
- RNA antisense approaches In addition, these two approaches also require a genetic transformation that is not suitable for therapeutic uses.
- the object of the invention was therefore to provide suitable agendas with which a better, in particular more specific and effective, investigation and influencing of living cells is possible.
- an active substance conjugate containing at least one ligand and at least one targeting domain which binds specifically to a surface structure of a target cell of a human or non-human mammal, the ligand modulating an intracellular signal cascade within the target cell.
- the invention differs from previously described couplings of antibodies or ligands with intracellular effectors in that the intracellular effectors were previously substances (diphtheria toxin, RNases, Pseudomonas exotoxin domains, etc.) whose intracellular action is primarily toxic (immunotoxins).
- the intracellular effectors were previously substances (diphtheria toxin, RNases, Pseudomonas exotoxin domains, etc.) whose intracellular action is primarily toxic (immunotoxins).
- an effect that kills the cells is not the primary effect.
- the inactivation or even killing of the cell can take place in the further course of the signal cascade (for example, by apoptosis induction), but this effect differs from the immunotoxins not only in that they are not primarily those described in the invention Agents is caused, but also because it depends on the status of the signal transduction cascade in the respective cell, which allows an additional specificity.
- Hsp70 a primarily cytoplasmic protein
- Many virus proteins also have translocation domains, e.g. B the tat gene product of HIV or a protein of the herpes virus. Synthetic peptides have also been described which can efficiently penetrate biomembranes and thereby transport other peptides coupled to them.
- agents described in this invention have a decoupling of binding and transport, which makes it possible to achieve specific binding via suitable domains, for example antibody fragments.
- This decoupling can be achieved in this invention in that a different molecular domain is responsible for the binding to the cell than for the transition into the cytoplasm.
- the active substance conjugate also contains a separate localization domain, which ensures the passage of the active substance conjugate into the cytoplasm of the target cell.
- an active substance conjugate containing at least one targeting domain that binds specifically to a surface structure of a target cell of a human or non-human mammal, at least one separate localization domain that allows the active substance conjugate to pass into the cytoplasm of the target cell guaranteed, and at least one Li acting within the target cell found, solved.
- this active substance conjugate it is also preferred if the ligand of the active substance conjugate modulates an intracellular signal cascade within the target cell.
- the targeting and localization domains are part of a targeting component composed of several domains.
- intracellular receptor signaling pathways are to be modulated by intracellular ligands in order to generate immunological tolerance, to inhibit inflammation, to treat tumors, or to influence the differentiation or growth status of cells.
- intracellular ligands are coupled with targeting components which ensure a cell / tissue-specific effect of the ligands by allowing them to be transferred into the cytoplasm of these specific cells ("ligand sneaking") ,
- the ligand modulates the intracellular signaling pathways of interleukin 1, interleukin 6, the tumor necrosis factor alpha, CD28, the T cell receptor or the ras / raf signal transduction pathway. It is advantageous if the ligand:
- STAT3 a member of the STAT protein family, in particular STAT3,
- NIK a member of the NF-kB / rel protein family, preferably NIK
- IkB proteins preferably IkB proteins, IkB kinases alpha and beta or AKT,
- the ligand contained in the drug conjugate according to the invention is a dominant negative mutant an IkB protein, an IkB kinase or the NIK, preferably a mutant which is not subject to any signal-induced degradation,
- ras protein preferably a constitutively active or inactive mutant
- the ligand comprises or consists of - preferably recombinant - antibody or an antibody fragment, preferably an intracellular antibody, preferably against units of an intracellular signal transduction pathway,
- the ligand comprises or consists of a natural binding partner of an intracellular element of a mammalian target cell or a mutant of such a natural binding partner,
- the ligand comprises or consists of a natural or synthetic peptide or protein, a carbohydrate, a nucleic acid, an aptamer, a hormone, a neurotransmitter, a lipid, a synthetic organic compound or a derivative of these molecules.
- intracellular ligands can also be antibodies in addition to the natural ligands or their mutants. What is new in this case is that these antibodies do not need to be produced in the cytoplasm, which is extremely unfavorable for their folding, as in the case of intracellular antibodies (intrabodies), but can nevertheless effectively achieve their cytoplasmic targets.
- the invention overcomes the difficulties that arise with the use of the higher molecular weight agents described above for modulating intracellular signal cascades by intracellular antibodies ("intrabodies”) or overexpression of mutants of the natural ligand ("dominant negative mutations").
- Intracellular antibodies have already been expressed in various cell compartments.
- the ras gene product p21 was inactivated by intracellular cytoplasmic antibodies.
- the impairment of the transactivation function of p53 by cytoplasmic antibody fragments has also been described.
- the internal disulphide bridges of cytoplasmic expressed antibody fragments are not properly oxidized due to the reducing environment of the cytoplasm.
- antibodies have been described recently which do not require intracellular disulphide bridges and which may therefore be better suited for function in the cytoplasm, they have shown reduced stability. In addition, this approach cannot be generalized for all antibodies.
- the chaperones required for antibody folding are also not available in the cytoplasm.
- the proposed ligands and conjugates enable the key sites of signal cascades to be modulated, which have a costimulatory effect, for example, in the case of T lymphocyte activation via CD28 or via cytokine receptors for TNFa, IL-1 or IL-6.
- TNFa and IL-1 are also central mediators for acute and chronic inflammatory reactions.
- the blocking of these signaling pathways should be used to dampen inflammatory processes and / or support the induction of immunological tolerance by blocking the second (costimulatory) signal.
- the conjugates are also of universal importance for immunology, oncology and inflammation research, since it makes it possible to selectively stimulate or inhibit cell functions and to use them therapeutically.
- Either natural ligands or their mutations (eg dominant negative mutants), or recombinant antibodies against various elements of intracellular signal transduction cascades are obtained as modulators of the intracellular signaling pathways, or artificial or synthetic ligands, e.g. B. selected because of their special resistance to degradation, manufactured and used, which recognize target epitopes in the elements of the signaling pathways with sufficient affinity.
- Elements of the binding region (paratopes) of a suitable antibody molecule can be genetically introduced into the selected ligand basic structure. The specificity of the modulation can be controlled by the point of attack in the signal path. Particularly attractive as the target structure (antigen) in the model system described are factors (here: kinases) in which several signaling pathways of different receptors converge.
- NFkB-inducing kinase NIK
- IKK IkB kinases
- IKK-a and IKK-b deficient mice die perinatally or still embryonic the targeted inhibition in certain cells such as T cells would be here or monocytes / macrophages using a cell-specific "targeting component" essential for the therapeutic use of highly potent IKK inhibitors.
- NF-kB inhibitors first have to be introduced as a gene into the target cells by means of transfections or else by transduction using viruses (gene therapy), which is relatively unspecific, because the antigenicity of viruses can hardly be carried out repeatedly and is still quite problematic overall.
- viruses gene therapy
- What has been described here overall allows the targeted introduction of a recombinant fusion protein that can be produced in large quantities using biotechnology for the efficient modification of intracellular signaling pathways. Since, in particular, fusion protein, composed of a "targeting" component and a ligand or effector component, preferably consists of human components, an immune response is not to be expected, which permits repeated application.
- the ras / raf signal transduction path plays an important role in growth regulation, among other things.
- constitutively active ras Mutants represent an essential step in oncogenesis.
- the method described here should enable inhibiting ligands of ras proteins or constituents of the ras signal transduction chain to be specifically introduced into tumor cells and thus inhibit the unrestrained cell proliferation.
- Stat3 is an interesting target for the IL-6 receptor, because it is not only involved in the IL-6 signaling pathway, but also plays an important role in the cellular transformation and growth control of tumor cells.
- the intracellular signaling pathways use a ligand which binds to members of the ras superfamily of monomeric GTPases (with the subfamilies of the Ras, Rho, Rac, Rab subfamilies) or a constitutively active or inactive mutant of a ras Proteins are modulated.
- intracellular signaling pathways are modulated with the aid of a ligand binding to members of the MAP kinase signaling pathway or a dominant negative mutant of a component of the MAP kinase signaling pathway.
- intracellular signaling pathways are modified with the aid of a ligand binding to the PI-3 kinase or phosholipase Cg or a dominant negative mutant of these enzymes.
- the targeting domain comprises or consists of an antibody - preferably recombinant -, an antibody fragment or a "single-chain" antibody,
- the targeting domain comprises or consists of a natural ligand of the cell surface structure or a mutant of such a natural ligand,
- the targeting domain comprises or consists of a natural or synthetic peptide or protein, a carbohydrate, a nucleic acid, an aptamer, a hormone, a neurotransmitter, a lipid, a synthetic organic compound or a derivative of these molecules.
- a synthetic or genetically produced ligand can be used, e.g. B. generated by firstly the selection of a suitable basic structure (for example related resistance, degradability, similarity to antibodies etc.) and secondly the change by introducing antigen binding sequences (paratopes) from known antibodies or by controlled mutation by means of genetic methods and optimization of the binding properties, e.g. , B. Affinity. In this way, targeting on the cell surface (resistance blood plasma / serum) or a functional domain in the cell interior (cytoplasm) can be addressed.
- the targeting domain binds to a protein accessible on the cell surface of the target cell, in particular to CD4, CD5, CD71, CD95, CDla, CD19, CD14, CD7, CD20, CD22, CD69, CD25, MHCII, CD21, CD45RA, CD45RO, NCAM, ICAM, the EGF receptor or integrins.
- the localization domain determines the intracellular localization of the molecule by the transition from the endocytotic vesicle pathway into the cytoplasm, preferably a protein with an "endosome escape" signal, in particular for example that Endosome escape domain of Pseudomonas aemginosa exotoxin A or a deletion mutant of diphtheria toxin. It is further preferred if the localization domain of the active substance conjugate according to the invention comprises or consists of a fragment of a biological toxin or an RNase and / or binds to integrin beta5 or integrin beta3.
- the localization domain comprises or consists of at least one fragment of a virus protein, preferably a surface protein or virus capsid protein, preferably selected from surface proteins of the adeno-associated vime (AAV), the adenovime, the hepatitis viruses; of influenza vime, especially the amino terminal peptide of influenza virus hemagglutinin and its acidic variants; of polyoma vims, rabbit parvovirus, rabies vims, semliki forest vims, human rhinovirus, herpes viruses or encephalitis viruses.
- AAV adeno-associated vime
- influenza vime especially the amino terminal peptide of influenza virus hemagglutinin and its acidic variants
- polyoma vims rabbit parvovirus, rabies vims, semliki forest vims, human rhinovirus, herpes viruses or encephalitis viruses.
- the localization domain of the active substance conjugate according to the invention acidifies the biochemical milieu in the endosomes or endocytotic vesicles, preferably contains or consists of adjuvant proteins or their fragments.
- the transition into the cytoplasm can be achieved with a large number of different molecular domains described, for example bacterial toxins, virus capsid proteins, but also by parts of RNases. Coupling to non-peptide molecules, such as cationic lipids, can also promote this transition.
- a particularly preferred embodiment of the drug conjugate according to the invention is when the drug conjugate contains an intracellular antibody against a component of an intracellular signaling pathway or a dominant negative mutant of a component of an intracellular signaling pathway as part of the ligand, an antibody against or ligand for one Surface structure, preferably an internalizing receptor, as part of the targeting domain and preferably an endosome escape sequence as part of the localization domain.
- the active substance conjugate (bispecific antibody fusion protein) contains an intracellular and a surface structure-specific antibody, in particular as two scFv antibodies, in the form of diabodies, triabodies, V-bodies, minibodies, dsFv-dsFv ', tandem -scFv or two chemically conjugated antibodies or antibody fragments, and optionally contains an endosome escape sequence.
- ligands For the cell type-specific delivery of ligands, these are combined in the form of bispecific proteins with a recombinant antibody fragment or the natural ligand of the surface structure, which ideally bind to an internalizing cell surface receptor.
- a protein domain with an endosome escape signal will still be required in some cases.
- cationic lipids are added to the active substance conjugate according to the invention, in particular the localization domain, in particular 3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, N- (1 -2,3-dioleyloxypropyl) - N, N, N-triethylammonium (DOTMA), dioleylphosphatidylethanolamm (DOPE), N- [l- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N- (2-hydroxyethyl) ammonium bromide (DMRIE ) or cholesterol.
- the localization domain in particular 3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, N- (1 -2,3-dioleyloxypropyl) - N, N, N-triethylammonium (DOTMA), dioleyl
- the modulators are genetically introduced into the target cells.
- the genetic information for the modulators is conveyed through tissue-specific somatic gene therapy.
- the surface of the adenoviruses (or AAV) used as gene therapy vectors is modified by inserting tissue-specific peptides or antibody fragments against T-cell surface proteins in order to limit their infectivity to the target cells.
- a special embodiment of the active substance conjugate is therefore present if the ligand contains nucleic acid with section (s) coding for a ligand that modulates an intracellular signal cascade within the target cell, preferably a ligand already described above. It is particularly preferred if the active substance conjugate contains an adenovism vector, an AAV vector, a retroviral vector, an NDV vector, a JCV vector or other DNA molecule complexes. This transmission unit can be modified, preferably contains a targeting domain.
- another object of the invention is an active ingredient conjugate containing nucleic acid with a section (s) coding for an intracellular signal cascade within the target cell, preferably coding already described above, and at least one transmission unit selected from the adenovime -Vector, the AAV vector, a retroviral vector, an NDV vector, a JCV vector or other DNA molecule complexes, wherein the transfer unit can be modified and / or the drug conjugate preferably also contains a targeting domain already described above , In this respect, a specifically modulated ligand can be generated in this way.
- An overall preferred form of all active substance conjugates according to the invention is an embodiment in which the ligand, the targeting domain and possibly other parts of the active substance conjugate, such as the localization domain or the targeting component, are connected to one another via covalent bonds.
- the ligand, the targeting domain and, if appropriate, other parts of the active substance conjugate present are particularly preferred if, in the case of an active substance conjugate according to the invention.
- the application also relates to a method for introducing ligands into living cells, in which an active substance conjugate according to the invention is brought into contact with the cells.
- cationic lipids are added in the process according to the invention, in particular 3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, N- (1 -2,3-dioleyloxy ⁇ ropyl) - N, N, N-triethylammonium (DOTMA), dioleylphosphatidylethanolamm (DOPE), N- [l- (2,3-dimyristyloxy) ⁇ ropyl] -N, N-dimethyl-N- (2-hydroxyethyl) ammonium bromide (DMRIE) or cholesterol.
- DOTMA dioleylphosphatidylethanolamm
- DOPE dioleylphosphatidylethanolamm
- DMRIE N-dimethyl-N- (2-hydroxyethyl) ammonium bromide
- DMRIE N-dimethyl-N- (2-hydroxyethyl) ammonium bromide
- the drug conjugates according to the invention are therapeutically highly effective, specific and toxicologically harmless. They are therefore suitable and intended to be used as therapeutic agents.
- Another The invention therefore relates to medicaments which contain at least one active substance conjugate according to the invention and, if appropriate, suitable additives and / or auxiliaries and optionally further active substances.
- the medicinal products according to the invention can be administered as liquid medicinal forms, in particular as injection solutions, but depending on the area of application also in the form of aerosols, drops or juices or as semi-solid medicinal forms in the form of granules, tablets, pellets or capsules.
- Suitable additives and / or auxiliaries are, for example, solvents or diluents, stabilizers, suspension mediators, buffer substances, preservatives, and also dyes, fillers and / or binders.
- excipients and the amounts to be used depend on whether the medicament is to be administered, for example, by inhalation, orally, orally, parenterally, intravascularly, intravenously, intraperitoneally, rectally, subcutaneously, percutaneously or intramuscularly.
- Preparations in the form of tablets, dragees, capsules, granules or suspensions such as drops, juices and syrups are suitable for oral applications, suspensions and (injection) solutions for intravascular applications.
- Another object of the invention is a diagnostic agent containing at least one active substance conjugate according to the invention and, if appropriate, suitable additives and / or auxiliaries.
- the agents and conjugates described can be used universally. After exchanging the specific elements (i.e. the targeting domain / component and / or the modulating ligand), they can be used to elucidate the functional meaning of individual signal transduction steps by selective inhibition in cellular systems. However, these conjugates can be used in particular for the treatment of infections, autoimmune diseases, degenerative diseases and cancer.
- Another object of the invention is therefore the use of an active ingredient conjugate according to the invention for the manufacture of a medicament for the treatment of diseases in which certain cell types or cell populations differ from the normal physiological function or behavior, in particular for the treatment of cancer, local and systemic inflammation, cellular storage diseases such as amyloidosis, hemochromatosis or other thesaurismosis and M. Alzheimer's; Autoimmune diseases, infections or degenerative diseases or to influence the differentiation or growth status of cells.
- Another object of the invention is the use of an active ingredient conjugate according to the invention for elucidating the functional importance of individual signal transduction steps in cellular systems.
- the use of an active ingredient conjugate according to the invention for introducing ligands into living cells is a further subject of the invention.
- the invention also relates to a method for producing an active substance conjugate according to the invention
- the active substance conjugate is produced from the ligand, targeting domain and / or possibly other parts of the active substance conjugate, such as, in particular, the localization domain or the targeting component as a fusion protein, suitable cells having a correspondingly coding nucleic acid, preferably in an expression vector, are transfected and the fusion protein is expressed and then isolated and purified from the cells,
- ligand, targeting domain and / or possibly other contained parts of the active substance conjugate, such as in particular the localization domain or the targeting component, are chemically covalently conjugated
- the drug conjugate is produced in any sequence and combination of the processes according to a) and b).
- the preparation is carried out by molecular combinatorics, the ligand, the targeting domain and / or possibly other parts of the active ingredient conjugate, such as, in particular, the localization domain or the target, at least in part.
- Geting component are isolated from molecular libraries, preferably using the phage display, ribosomal displays or the "two-hybrid" system.
- the therapeutic agent can benefit in particular from the methods of molecular combinatorics, since parts of the ligand or the targeting component can be isolated from molecular libraries, in particular with the aid of the phage display, ribosomal display or the two-hybrid system. Production can be facilitated by producing the targeting component and the ligand as a coherent fusion protein. If other substances than proteins are used as ligands (e.g. carbohydrates, aptamers, nucleic acids or other biomolecules), chemical conjugation is also possible.
- ligands e.g. carbohydrates, aptamers, nucleic acids or other biomolecules
- Another object of the invention is the treatment of a person or animal who needs this treatment with or using the active substance conjugates according to the invention.
- This treatment is particularly suitable for the aforementioned indications and types of use.
- Figure 1 shows the principle of "ligand sneaking".
- a fusion protein from an endosome escape domain e.g. a peptide from amino acids 252 - 366 of Pseudomonas Exotoxins A ("translocation domain")
- a bispecific scFv (single chain) antibody is shown. Molecules and fusion fractions are not shown to scale.
- Fig. 1 gives a general overview of a principle underlying the objects of the invention.
- Figure 2 is a schematic representation of the fusion protein for immunosuppression according to Example 1. The individual fusion parts are not shown to scale.
- Figure 3 is a schematic representation of a bispecific antibody fusion protein for immunosuppression according to Example 2. The individual fusion parts are not shown to scale.
- the His tag near the carboxy terminus is removed, instead it is inserted at the amino terminus of the expressed fusion protein.
- KDEL a sequence "KDEL" is coded, which causes localization in the endoplasmic reticulum.
- a nucleic acid coding for IkB alpha as an intracellular modulator (ligand) is cloned in, in particular for a mutant, which is no longer degraded in a signal-induced manner by a deletion at the amino terminus, and thus ensures an increased action of the therapeutic agent.
- Soluble fusion protein is produced by the method of Schmiedl, Breitling & Dübel, Protein Engineering 13 (2000), pp. 725-734 and purified using the His tag and with ion exchange chromatography.
- the therapeutic agent can be used for the immunosuppressive treatment of inflammatory diseases with significant involvement of CD4-positive inflammatory cells (T helper cells, monocytes, macrophages, astrocytes).
- T helper cells monocytes, macrophages, astrocytes.
- the expression vector is introduced into CHO cells and the expression product is produced and purified according to the prior art.
- a His tag or another sequence can be cloned into the construct using conventional methods, which facilitates or permits the purification of the protein by affinity chromatography.
- the agent can be used for the immunosuppressive treatment of cytokine-driven inflammatory diseases and especially for the treatment of IL6-dependent immune overreactions.
- the bispecific antibody components can alternatively also be in the form of diabodies, triabodies, V-bodies, minibodies, dsFv-dsFv ', tandem scFv (as defined in Dübel & Konntermann Re- combinant antibodies. (2001) In: Antibody Engineering, ed: R. Kontermann & S. Dübel, Springer Verlag, Heidelberg / New York) or by two chemically conjugated antibodies or antibody fragments.
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Abstract
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DE10191398T DE10191398D2 (de) | 2000-04-18 | 2001-04-18 | Wirkstoff-Konjugate mit Intrazellulär wirksamen Liganden |
AU73844/01A AU7384401A (en) | 2000-04-18 | 2001-04-18 | Active substance conjugates with intracellularly active ligands |
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DE10019157A DE10019157A1 (de) | 2000-04-18 | 2000-04-18 | Verfahren zum Einbringen von Liganden in lebende Zellen |
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Cited By (7)
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EP1345627A1 (de) * | 2000-11-13 | 2003-09-24 | Centre National De La Recherche Scientifique | Gezielte modifikation intrazellulärer verbindungen |
WO2007101202A1 (en) * | 2006-02-27 | 2007-09-07 | Research Development Foundation | Cell-targeted ikb and methods for the use thereof |
DE102007041834A1 (de) * | 2007-09-03 | 2009-03-05 | Siemens Ag | Arzneimittel zur Behandlung eines Karzinoms |
WO2012101235A1 (en) * | 2011-01-26 | 2012-08-02 | Cenix Bioscience Gmbh | Delivery system and conjugates for compound delivery via naturally occurring intracellular transport routes |
US9546211B2 (en) | 2014-10-23 | 2017-01-17 | Singh Molecular Medicine, Llc | Single domain antibodies directed against TNF-alpha |
US9850321B2 (en) | 2014-10-23 | 2017-12-26 | Singh Molecular Medicine, Llc | Single domain antibodies directed against intracellular antigens |
US10195277B2 (en) | 2015-11-02 | 2019-02-05 | Singh Biotechnology, Llc | Single domain antibodies directed against human immunodeficiency virus |
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WO1996004305A1 (en) * | 1994-08-04 | 1996-02-15 | Menarini Ricerche S.P.A. | Hybrid molecules useful in the treatment of tumors, their preparation and use in pharmaceutical compositions |
EP0706799A2 (de) * | 1994-09-16 | 1996-04-17 | MERCK PATENT GmbH | Immunokonjugate |
WO1998042876A1 (en) * | 1997-03-26 | 1998-10-01 | Board Of Regents, The University Of Texas System | Methods and compositions for using membrane-penetrating proteins to carry materials across cell membranes |
WO2000045850A2 (en) * | 1999-02-06 | 2000-08-10 | Aurx Inc. | Drug delivery vehicle |
WO2001053336A1 (en) * | 2000-01-19 | 2001-07-26 | Allergan Sales, Inc. | Clostridial toxin derivatives and methods for treating pain |
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2000
- 2000-04-18 DE DE10019157A patent/DE10019157A1/de not_active Withdrawn
-
2001
- 2001-04-18 DE DE10191398T patent/DE10191398D2/de not_active Ceased
- 2001-04-18 AU AU73844/01A patent/AU7384401A/en not_active Abandoned
- 2001-04-18 WO PCT/DE2001/001495 patent/WO2001078785A2/de active Application Filing
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WO1998042876A1 (en) * | 1997-03-26 | 1998-10-01 | Board Of Regents, The University Of Texas System | Methods and compositions for using membrane-penetrating proteins to carry materials across cell membranes |
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SCHMIDT W ET AL: "Generation of effective cancer vaccines genetically engineered to secrete cytokines using adenovirus-enhanced transferrinfection (AVET)" GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, Bd. 190, Nr. 1, 29. April 1997 (1997-04-29), Seiten 211-216, XP004064404 ISSN: 0378-1119 * |
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1345627A1 (de) * | 2000-11-13 | 2003-09-24 | Centre National De La Recherche Scientifique | Gezielte modifikation intrazellulärer verbindungen |
WO2007101202A1 (en) * | 2006-02-27 | 2007-09-07 | Research Development Foundation | Cell-targeted ikb and methods for the use thereof |
DE102007041834A1 (de) * | 2007-09-03 | 2009-03-05 | Siemens Ag | Arzneimittel zur Behandlung eines Karzinoms |
WO2012101235A1 (en) * | 2011-01-26 | 2012-08-02 | Cenix Bioscience Gmbh | Delivery system and conjugates for compound delivery via naturally occurring intracellular transport routes |
JP2014505064A (ja) * | 2011-01-26 | 2014-02-27 | セニックス バイオサイエンス ゲーエムベーハー | 自然に存在する細胞内輸送経路を介して化合物を送達するための送達システム及びコンジュゲート |
US9546211B2 (en) | 2014-10-23 | 2017-01-17 | Singh Molecular Medicine, Llc | Single domain antibodies directed against TNF-alpha |
US9663570B2 (en) | 2014-10-23 | 2017-05-30 | Singh Molecular Medicine, Llc | Single domain antibodies directed against KRAS |
US9695234B2 (en) | 2014-10-23 | 2017-07-04 | Singh Molecular Medicine, Llc | Single domain antibodies directed against STAT3 |
US9850321B2 (en) | 2014-10-23 | 2017-12-26 | Singh Molecular Medicine, Llc | Single domain antibodies directed against intracellular antigens |
US10195277B2 (en) | 2015-11-02 | 2019-02-05 | Singh Biotechnology, Llc | Single domain antibodies directed against human immunodeficiency virus |
US10369223B2 (en) | 2015-11-02 | 2019-08-06 | Singh Biotechnology, Llc | Single domain antibodies directed against ebola virus VP24 |
Also Published As
Publication number | Publication date |
---|---|
AU7384401A (en) | 2001-10-30 |
DE10019157A1 (de) | 2001-11-15 |
WO2001078785A3 (de) | 2002-05-10 |
DE10191398D2 (de) | 2003-04-30 |
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