DE10019157A1 - Method for introducing ligands into living cells - Google Patents

Abstract

Intracellular receptor signaling pathways are to be modulated by intracellular ligands to generate immunological tolerance, to inhibit inflammation, to treat tumors or to influence the differentiation or growth status of cells. What is new here is that intracellular ligands are coupled to 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"). In addition to the natural ligands or their mutants, intracellular ligands can also be antibodies. What is new in this case is that the antibodies are not produced in the cytoplasm, which is extremely unfavorable for their folding, as in the case of intracellular antibodies (intrabodies), but can nevertheless reach their cytoplasmic targets effectively.

Description

An average cell contains approximately 10,000 proteins that are attached to it intracellular signal transduction, regulation of transcription, cell-cell Participate in communication and intracellular transport. The functional analysis or therapeutic influence of these processes in the context of the living cell is hampered by the fact that highly specific binding molecules (e.g. antibodies) the plasma membrane cannot penetrate. Therefore, for appropriate analyzes previously used microinjection or pore-forming substances that but mean an extraordinary strain on the cell and in one therapeutic approach are not usable. Therefore, in the In recent years, genetic methods have been developed to deal with the complex Network of functional relationships of the above. To study proteins. This are above all: genetic knockouts, dominant negative mutants and Antisense RNA expression. Genetic knockouts are expensive their production, since they are usually only found in homozygous animals show significant effects. They are completely unsuitable for therapy, because they would require germline gene therapy. The second so far used method of dominant negative mutants due to overexpression of proteins with mutations that destroy the desired function are often caused by side effects of the strong ones required for this Overexpression hinders and also requires accurate information about the mode of action of the corresponding proteins for the production of  desired mutants. The same applies to RNA antisense approaches. In addition, these two approaches also require a genetic one Transformation that is not suitable for the desired therapeutic approaches is.

In contrast, the agents described in this patent are functional modulating directly on the target molecule to be examined, without doing so to primarily influence its regulation, production or quantity. Experiments using microinjection of monoclonal antibodies into individual cells or through the intracellular expression of Antibodies or "dominant negative" ligands prove the principle Feasibility of this aspect of the invention. Microinjection into single However, cells are also unsuitable for therapy. The invention overcomes the difficulties that have arisen with the use of so far described higher molecular agents for modulating intracellular Signal cascades through intracellular antibodies ("intrabodies") or Overexpression of mutants of the natural ligand ("dominant negative mutations "). Intracellular antibodies have already been found in expressed in various cell compartments. Inactivation succeeded of the ras gene product p21 by intracellular cytoplasmic Antibody. The impairment of the transactivation function of p53 by cytoplasmic antibody fragments has been described. However, it was also shown that the internal disulphide bridges cytoplasmic expressed antibody fragments because of the reducing milieu of the cytoplasm can not be oxidized correctly.  

Antibodies without intracellular Disulphide bridges and therefore possibly for one Function in the cytoplasm might be more appropriate, but these showed reduced stability. In addition, this approach is not general for everyone Generalize antibodies. They are not in the cytoplasm either Chaperones are available that are necessary for antibody folding. A comparative study using the same scFv antibody different signal sequences in different cell compartments was shown to show significant antigen binding activity only in ER and on the membrane could be detected, but not in Nucleus or cytoplasm. Another disadvantage of intrabodies are one in that they have to be made inside the cell for Therapy without the use of gene transfer is therefore not available. "Dominant negative" mutation products also have the Disadvantage, not available in the target cell without the use of gene transfer to be. The invention circumvents both obstacles by Fusion proteins outside the cells to be treated in one suitable production system.

The invention differs from previously described couplings of antibodies or ligands with intracellular effectors, that the intracellular effectors previously substances (diphtheria toxin, RNases, Pseudomonas exotoxin domains and a.) were their intracellular The effect is primarily toxic (immunotoxins). The invention is against a cell killing effect is not the primary effect. In some  Application examples can be seen in the further course of the signal cascade the cell is inactivated or even killed (e.g. by Induction of apoptosis), this effect differs from that Immunotoxins not only because they are not primarily from those in the Agents described invention is caused, but also by that it depends on the status of the signal transduction cascade in the respective cell depends on what enables additional specificity.

The ability to make the intracellular effector cell / tissue specific Contributing is another advantage of the class of Agents. They differ from them in particular various other transport domains described recently and their conjugates, which are the intakes of peptides or proteins ensure from the outside through the cell membrane. Hsp70, a priumary cytoplasmic protein, can be noncovalently bound peptides of Feed MHC loading machinery if it is outside of the cells is added. Many virus proteins also have translocations domains, e.g. B. the tat gene product of HIV or a protein of Herpes viruses. Synthetic peptides have also been described Can penetrate biomembranes efficiently while being coupled to them transport other peptides. All of these approaches are common, that they don't have the membrane penetration with the necessary specificity carry out. The agents described in this invention exhibit on the other hand, there is a decoupling of binding and transport that enables via suitable domains, e.g. B. antibody fragments, a  to achieve specific attachment. This decoupling can be done in this Invention can be achieved in that for binding to the cell different molecular domain is responsible than for the transition into the cytoplasm. The latter can be described with a variety of different ones Molecular domains can be achieved, e.g. B. bacterial toxins, Virus capsid proteins, but also through parts of RNases. Also Coupling to non-peptide molecules, such as cationic lipids, can favor this transfer.

The therapeutic agent can differ from the methods of molecular Combinatorics benefit in particular since parts of the ligand or the Targeting component can be isolated from molecular libraries especially with the help of the phage display, ribosomal display or the Two hybrid systems. Production can be facilitated that the targeting component and the ligand as a coherent Fusion protein can be produced. In the case of other ligands Substances are used as proteins (e.g. carbohydrates, aptamers, Nucleic acids or other biomolecules), is also a chemical Conjugation possible.

The tolerance for the patient is ensured by a the largest possible proportion of the therapeutic agent, in particular parts of the ligand or the targeting component or the coding thereof Genes are of human origin.  

Local production of the agent near the target cells is complete appropriate targeted gene transfer (through "targeted gene deivery" or targeted perfusion of individual physiological compartments) as well possible.

The method described can be used universally. After exchanging the specific elements (ie the targeting component and / or the modulating ligand), it can be used to elucidate the functional meaning of individual signal transduction steps by selective inhibition in cellular systems. However, this method can be used in particular for the treatment of infections, autoimmune diseases, degenerative diseases and cancer. An overview of the general principle of action is given in Fig. 1.

The proposed methods enable the modulation of the Key points of signal cascades that e.g. B. in T-lymphocyte Activation via CD28 or via cytokine receptors for TNFa, IL-1 or IL-6 costimulatory. TNFa and IL-1 are also central Mediators for acute and chronic inflammatory reactions. The Blockage of these signaling pathways is said to dampen inflammation processes are used and / or induction immunological Tolerance by blocking the second (costimulatory) signal support. However, the process also has a universal meaning for immunology, oncology and inflammation research since it  enables selective stimulation or inhibition of cell functions and to use this therapeutically.

Together, all constructs are optimized for the most extensive Avoiding the immune response against the therapeutic construct. Therefore, human molecules are preferred.

As modulators of the intracellular signaling pathways, either natural ligands or their mutations (e.g. dominant negative Mutants), or recombinant antibodies against various elements obtained from intracellular signal transduction cascades. The specificity the modulation can by the point of attack in the signal path to be controlled. As target structure (antigen) in the described Model system are particularly attractive factors (here: kinases) which converge several signaling pathways of different receptors. The signaling pathways of both TNFα and IL-1 receptors meet for example in NFkB-inducing kinase (NIK). Almost all NF κB-activating signaling pathways are likely at the level of the IB kinases (IKK) α and β meet, which makes them an ideal target for a makes potent inhibition of NF-κB activation, e.g. B. using dominant negative mutants. However, since at least a complete or extensive inhibition of the IKK's in the entire organism considerable side effects (IKK-α and IKK-β deficient Mice die perinatally or still embryonic), would be the targeted one Inhibition in certain cells such as T cells or  Monocytes / macrophages using a cell-specific "targeting Component "essential for a therapeutic use of highly potent IKK inhibitors. Also IkB proteins, especially mutants that none subject to signal-induced degradation, inhibit NF-κB activity extremely effective. Currently, however, these NF-κB inhibitors have to be used first can be introduced as a gene into the target cells using transfections or by transduction using viruses (gene therapy) what is relatively unspecific, hardly repeated due to the antigenicity of viruses can be carried out and is currently still quite problematic is. The method described here allows the targeted introduction of a recombinant biotechnologically producible Fusion protein for the efficient modification of intracellular signaling pathways. Because this fusion protein is made up of a "targeting" and a Ligand or effector component, primarily from human Components, an immune response is not expected, which is a repeated application allowed.

The ras / raf signal transduction path plays an important role other in growth regulation. In this way, constitutively active ras Mutants represent an essential step in oncogenesis. Also here the method described here should enable inhibitory Ligands of ras proteins or components of the ras Targeted signal transduction chain in tumor cells and thus inhibiting the unchecked cell proliferation.  

With the IL-6 receptor, Stat3 is an interesting target because it is not only involved in the IL-6 signaling pathway, but also plays an important role cellular transformation and growth control of tumor cells.

For the cell type-specific delivery of ligands, these are in the form bispecific proteins with a recombinant antibody fragment or combined with the natural ligand of the surface structure, more ideal Way of an internalizing cell surface receptor. To one Converts from the endocytose vesicles to the site of action of the modulators, the To ensure cytoplasm, in some cases a protein Domain with an endosome escape signal may be required.

Alternatively, the modulators are genetically engineered into the target cells smuggled in. For a potential therapeutic approach, the gen Etical information for the modulators through tissue-specific somatic gene therapy mediated. To do this, the surface of the as Gene therapy vectors used adenovirus (or AAV) by Insertion of tissue-specific peptides or antibody fragments against T- Cell surface proteins modified to their infectivity on the Restrict target cells.

Further applications result from the fact that intracellular Signaling pathways using an IκB protein or the IκB kinase α or β (IKK-α / β) or AKT binding ligands or dominant negative Mutants of the IKK's are modulated.  

Further applications result from the fact that the intracellular Signaling pathways using a monomeric to members of the ras superfamily GTPases (with the subfamilies of the Ras, Rho, Rac, Rab subfamilies) binding ligand or a constitutively active or inactive mutant of a ras protein can be modulated.

Further applications result from the fact that the intracellular Signaling pathways to members of the MAP kinase Signal transduction pathway binding ligand or a dominant negative mutant of a component of the MAP kinase Signal transduction path are modulated.

Further applications result from the fact that intracellular Signaling pathways using a to the PI-3 kinase or phosholipase C-γ binding ligands or a dominant negative mutant of these Enzymes are modified.

Further applications result from the fact that the intracellular Signaling pathways of the T cell receptor are modulated.

Other applications arise for the treatment of diseases, which are characterized in that certain cell types or Cell populations from their normal physiological function and Behavior deviate by z. B. hyper (over) are activated or on the contrary, can no longer be activated or can only be activated inadequately.  

These conditions occur e.g. B. in local and systemic inflammation and at Autoimmune diseases. In these cases, through targeted intervention the normal state is restored in signal cascades of these cells become. It is sufficient to add a sufficiently large number of cells to reach. It is not necessary to return every cell to normal to postpone as self-healing mechanisms the healing can support. The same applies analogously to tumor cells, at which often have physiological signal transmission paths permanent are overactivated. The principle of treatment is also applicable for cellular storage diseases in which misdirected syntheses for harmful deposition of molecules (e.g. amyloidosis, Hemochromatosis or other thesaurismoses, u. a. also M. Alzheimer).

Claims (12)

1. A method for introducing ligands into living cells, characterized in that the ligand is bound to a molecule (hereinafter referred to as the targeting component) which ensures specific binding to a surface structure of the cell and passage into the cytoplasm of the target cell, and characterized in that the ligand modulates an intracellular signaling cascade within the cell. 2. The method according to claim 1, characterized in that
the targeting component contains a peptide or polypeptide, in particular an antibody or antibody fragment,
the targeting component contains a natural ligand of the cell surface structure or a mutant of such a natural ligand,
the targeting component contains a carbohydrate, a nucleic acid, an aptamer, a synthetic peptide, a hormone, a neuro transmitter, a lipid, a synthetic orange compound or derivatives of the molecules mentioned. 3. The method according to any one of claims 1 to 2, characterized in that
the ligand contains a protein, in particular an antibody or antibody fragment,
that the ligand contains a natural binding partner or a mutant of such a natural binding partner,
the ligand contains a carbohydrate, a nucleic acid, an aptamer, a synthetic peptide, a hormone, a neurotransmitter, a lipid, a synthetic orange compound or derivatives of the molecules mentioned. 4. The method according to any one of claims 1 to 3, characterized characterized that the intracellular signaling pathways of interleukin 1, interleukin 6, tumor necrosis factor alpha or CD28 be modulated. 5. The method according to any one of claims 1 to 4, characterized characterized that the intracellular signaling pathways with the help of a at the cytoplasmic accessible part of the receptors for the in Claim 4 designated signaling pathways ligands, or Help of a binding to a member of the STAT protein family Ligands, or with the help of a to a member of NF-kB / rel- Protein family or on the NFkB signal transduction pathway Molecule binding ligands or a dominant negative Mutant can be modulated by NIK.   6. The method according to any one of claims 1 to 5, characterized characterized in that the targeting component is at a on the Protein accessible to the cell surface binds, in particular CD4, CD5, CD71, CD95, CD1a, CD19, CD14, CD7, CD20, CD22, CD69, CD25, MHCII, CD21, CD45RA, CD45RO, NCAM, ICAM, EGF- Receptor, integrins. 7. The method according to any one of claims 1 to 6, characterized characterized in that the targeting component consists of several Domains, one of which is the specific binding to the Ensures cell surface and another the intracellular Localization of the molecule determined, in particular by the Passes from the endocytotic vesicle pathway into the cytoplasm. 8. The method according to any one of claims 1 to 7, characterized characterized that the domain which is the intracellular Localization of the molecule ensures a fragment of a biological toxin or an RNase and / or integrin beta5 or integrin beta3 binds. 9. The method according to any one of claims 1 to 8, characterized characterized that the domain which is the intracellular Localization of the molecule determines a fragment of a Virus protein is, especially from surface proteins of the adeno-associated virus (AAV), adenovirus, hepatitis viruses, of the influenza virus (especially the amino terminal peptide of the  Influenza virus hemagglutinins and its acidic variants), des Polyoma virus, rabbit parvovirus, rabies virus, Semliki Forest virus, the human rhinovirus, from herpes viruses or encephalitis viruses. 10. The method according to any one of claims 1 to 9, characterized characterized that the intracellular localization of the molecule is improved by adding cationic lipids, in particular by 3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, N- (1-2,3-dioleyloxypropyl) -N, N, N-triethylammonium (DOTMA), dioleylphosphatidylethanolamine (DOPE), N- [1- (2,3-dimyristyl oxy) propyl] -N, N-dimethyl-N- (2-hydroxyethyl) ammonium bromide (DMRIE) or cholesterol. 11. The method according to any one of claims 1 to 10, characterized characterized that the domain which is the intracellular Localization determines this by acidifying the biochemical Causes milieus in the endosomes or endocytotic vesicles, in particular by adenoviral proteins or their fragments caused acidification. 12. The method according to any one of claims 1 to 8, characterized characterized that the ligand is encoded by a gene which with the help of the targeting component into the cell is introduced, in particular by that for the ligand coding gene by an adenovirus vector, an AAV  Vector, a retroviral vector, an NDV vector, a JCV Vector or with the help of other DNA-molecular complexes in the Cell cells is introduced, the surface of which Transmission units is modified so that a targeting Component a specific uptake into certain cells enables.