SI23508A - Fusion polypeptides comprising tir and dimerization domain for modulation of tlr/innate immunity signaling - Google Patents

Abstract

The invention represents the TLR/IL-1R signaling pathway inhibitory fusion polypeptide comprising TIR and dimerization domain which is composed of TIR domain and dimerization domain wherein TIR domain and dimerization domain are connected by a linker peptide and wherein the polypeptide optionally also contains a segment enabling plasma membrane localization. Addition of the dimerization domain to the TIR domain significantly improves the inhibitory potency of TIR domain. The invention also represents the application of the nucleic acid encoding fusion polypeptide comprising TIR and dimerization domain or the application of the recombinantly produced fusion polypeptide comprising TIR and dimerization domain, especially of the polypeptide additionally containing protein transduction domain, for the inhibition of cellular TLR/IL-1R signaling or for the manufacturing a drug used for the treatment of chronic or acute inflammatory conditions, such as infectious and autoimmune diseases, sepsis, septic shock, cancer and others resulting from improper and/or excessive TLR/IL-1R signaling.

Description

A fusion polypeptide composed of a TIR domain and a dimerization domain to modulate TLR / natural immunity signaling

FIELD OF THE INVENTION

FIELD OF THE INVENTION The fusion polypeptide is comprised of a TIR domain and a dimerization domain that are linked to a binding peptide, the fusion polypeptide being an improved inhibitor of TLR / IL-1R signaling. Field of the invention is a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, said nucleic acid being expressed in the host cells. Field of the Invention is a nucleic acid encoding a fusion protein composed of a TIR domain and a dimerization domain or a recombinantly produced fusion polypeptide consisting of a TIR domain and a dimerization domain that can be used to prepare a drug for introduction into target cells / organisms where said fusion polypeptide operates as an inhibitor of inflammatory conditions resulting from excessive and / or inadequate TLR / IL-1R signaling, such as autoimmune diseases, sepsis, septic shock, and others. FIELD OF THE INVENTION The use of nucleic acids encoding a fusion polypeptide composed of a TIR domain and a dimerization domain or a recombinantly produced fusion polypeptide composed of a TIR domain and a dimerization domain to inhibit cellular TLR / IL-1R signaling in human or animal cells.

The state of the art

Natural immunity is the first line of defense against pathogenic microorganisms. An important component of natural immunity are Toll-like receptors that recognize conserved microbial patterns (so-called pathogen-associated molecular pattems). Ligand binding to TLRs triggers its activation and subsequent binding of adapter proteins to the receptor complex. Through TIR: TIR interactions, TLR adapters are linked to proteins in a further cascade, involving mostly different kinases and ultimately leading to the activation of transcription factors such as NF-κΒ and IRFs, leading to the synthesis of inflammatory cytokines and interferons.

On the other hand, excessive or a deregulated response to the bacterial endotoxin recognized by TLR4 and its MD-2 coreceptor leads to the development of a systemic inflammatory response that often develops into high-mortality-sepsis disease. The development of some chronic inflammatory and autoimmune diseases is also associated with the recognition of endogenous ligands by TLR, which leads • · · • ·

to the development of sterile inflammation. TLR involvement has already been demonstrated in diseases such as arthritis, gouty, chronic inflammatory bowel disease, psoriasis, multiple sclerosis, ulcerative colitis, Chron's disease, neuropathic pain, alcohol-induced inflammation, cancer and others.

Selective intra-cellular inhibition of downstream TLR signaling pathways may be used to limit the inflammatory response. Some approaches have already been described for the selective inhibition of individual TLR adapters, e.g. the use of antisense oligonucleotides, siRNAs, adapter-specific antibodies or adapter inhibitors that reduce the expression / activity of them and thereby inhibit the TLR signaling pathway corresponding to these adapters, e.g. of the TRAM (U.S. Pat. Appl. Pub. 2005/0158799 Al) or TIRAP (U.S. Pat. 7285535 B2) adapters. The anti-inflammatory effect can also be achieved by affecting the expression or activity of endogenous negative TLR signaling pathway regulators such as MyD88s (U.S. Pat. 7122656 B2) or IRAK-M (U.S. Pat. Appl. Pub. 2003/0157539 Al).

Selective inhibition of TLR can also be achieved by involvement in protein-protein interactions in the TLR signaling cascade, where the TIR receptor and adapter domains and often the BB loop within these domains play an important role. Peptides and peptidomimetics of BB loops can occupy the binding site of the adapter at its target, thereby preventing the binding of the native adapter, thereby disrupting the structure of the functional receptor complex. Such is the case with peptides and peptidomimetics that mimic a specific portion of MyD88 and, through interactions with its TIR domain, inhibit the homodimerization of MyD88 and thus can be used to inhibit MyD88 dependent pathways (U.S. Pat. Appl. Pub. 2008/0064643 Al). However, loop peptides of BB MyD88, TRAM, TIRAP and TRIF can selectively inhibit TLR4 signaling (Intl. Pat. Appl. Pub. WO 2006/113530 A2). However, the ability to inhibit the peptides and peptidomimetics described is relatively weak, since high concentrations of these are required for inhibition. In addition, their use is limited to specific TLR signaling pathways, e.g. the peptides of BB MyD88, TRAM, TIRAP and TRIF loops only affect the TLR4 signaling pathway (Intl. Pat. Appl. Pub. WO 2006/113530 A2).

Summary of the Invention

The problem of low activity of peptides interacting with proteins in the TLR signaling pathway of natural immunity was solved by the inventors using fusion polypeptides composed of the TIR domain and the dimerization domain. The inventors have surprisingly discovered that the overactivation of TLR / IL1-R signaling pathways can be limited by the use of fusion polypeptides composed of the TIR domain and dimerization domain that interfere with TIR: TIR interactions between receptors and adapters that both contain the TIR domain. In addition, said fusion polypeptides also comprise a dimerization domain in addition to the TIR domain, the two being linked to a binding peptide. The invention discloses fusion polypeptides composed of the TIR domain and dimerization domains that have significantly better inhibitory potential for TLR / IL-1R signaling than monomic TIR domain inhibitors or peptide inhibitors and peptidomimetics of BB loops. An improved domain inhibitory potential is responsible for the dimerization of TIR domains. Fusion polypeptides composed of the TIR domain and the dimerization domain are further enhanced by an optionally added segment for membrane localization, which enhances their inhibitory potential. In addition, novel fusion polypeptides composed of the TIR domain and the dimerization domain specific for TLR / IL-1R signaling act by directly engaging in the interactions of TIR: TIR signaling complexes and without affecting the expression or activity of individual adapters. Furthermore, the TIR domain in fusion polypeptides composed of the TIR domain and the dimerization domain can be selected from different homologous animal, human or viral TIR domains of TLR signal adapters, TLRs, receptors from the IL-1R family, and TIR domain-containing viral proteins, allowing selective inhibition of TLR / IL-1R signaling pathways in contrast to said MyD88 homodimerization inhibitors that inhibit only the MyD88 dependent pathway (US Pat. Appl. Pub. 2008/0064643 Al) or loop loop peptides that inhibit only the TLR4 signaling pathway (Intl. Pat. Appl. Pub. WO 2006/113530 A2). The proposed fusion polypeptides composed of the TIR domain and the dimerization domain can be used to inhibit TLR / IL-1R cell signaling and / or to prepare a medicament for the treatment of chronic and acute diseases that develop due to excessive or inadequate TLR / IL-1R signaling.

Nucleic acids encoding fusion polypeptides composed of a TIR domain and a dimerization domain can be systemically or locally introduced by various methods of administration such as lipofection, microinjection, electroporation, viral uptake, etc. Instead of nucleic acids that can be used as a medicament, the improvement of the present invention is the use of recombinantly produced fusion polypeptides composed of a TIR domain and a dimerization domain, thereby avoiding the need for gene therapy. Of particular importance in this respect are the fusion polypeptides composed of the TIR domain and the dimerization domain, which further comprise the protein transduction domain, which enables efficient and rapid translocation of the proposed drugs into target cells.

The invention relates to a fusion polypeptide composed of a TIR domain and a dimerization domain consisting of a) a native or mutated homologous animal, human or viral TIR domain of TLR signal adapters, TLRs, IL-1R family receptors, viral proteins containing the domain TIR, b) dimerization domains and a) and b) are linked to a binding peptide containing flexible and hydrophilic amino acids. Optionally, the fusion polypeptide composed of the TIR domain and the dimerization domain comprises a peptide marker and / or a membrane localization segment. The invention relates to inhibitors of a TLR signaling pathway fusion polypeptide composed of a TIR domain and a dimerization domain, wherein the TIR domain of a fusion polypeptide consisting of a TIR domain and a dimerization domain refers to a native or mutated homologous animal, human or viral TIR domain of TLR signaling adapters MyD88, MAL , TRIF, TRAM and SARM, TLRs, receptors from the IL-1R family, viral proteins containing the TIR domain, preferably relates to the TIR domain of the MyD88 signaling adapter, preferably to SEQ ID NO: 12. The invention relates to inhibitory signaling fusion polypeptide of the TLR pathway consists of a TIR domain and a dimerization domain, wherein the dimerization domain of the fusion polypeptide composed of the TIR domain and the dimerization domain is a protein domain that allows spontaneous dimerization of said fusion polypeptide or protein domain that allows dimerization of said fusion polypeptide after addition of a chemistry fusion polypeptide dimerization by binding to this prot ein domain. The dimerization domain allowing spontaneous dimerization of said fusion polypeptide is preferably, but not limited to, the helix-forming segment, preferably the homodimeric helix-forming segment, 3 to 12 heptad long, preferably GCN4-p1, preferably SEQ ID NO: 16 The invention relates to an inhibitory fusion polypeptide consisting of a TIR domain and a dimerization domain, wherein the TIR domain and the dimerization domain in said fusion polypeptide are linked by a binding peptide. The binding peptide is from 3 to 40 amino acids long, preferably from 15 to 30 amino acids, preferably 25 amino acids, preferably SEQ ID NO: 18. Amino acids are preferably, but not limited to, selected from amino acids serine, glycine, threonine, proline , valine, alanine, glutamine, asparagine, glutamate and aspartate.

The invention also relates to a fusion polypeptide composed of a TIR domain and a dimerization domain which, in addition to a) native or mutated homologous animal, human or viral TIR domains of TLR signal adapters, TLRs, receptors of the IL-1R family, viral proteins containing

the TIR domain, b) the dimerization domains, and wherein a) and b) are linked to a binding peptide containing flexible and hydrophilic amino acids, and optionally also containing a peptide marker or / and a membrane localization segment, containing a protein transduction domain for transduction fusion polypeptide into eukaryotic target cells. The protein transduction domain (PTD) is selected from cationic peptide sequences such as the PTD HIV-1 TAT protein, VP22 DNA-binding protein herpes simplex virus 1 (HSV-1) and Antennapedia (Antp) homeotic transcription factor of the wine fly, preferably PTD HIV-1 TAT protein, preferably SEQ ID NO: 22.

The invention relates to nucleic acids encoding a fusion polypeptide composed of a TIR domain and a dimerization domain described above wherein the nucleic acid is operatively linked to a regulatory element, promoter and terminator that allows expression of a TIR domain fusion polypeptide and a dimerization domain in host cells / organisms.

The invention also relates to a host cell / organism expressing the nucleic acid described above encoding a fusion polypeptide composed of a TIR domain and a dimerization domain and to a target cell / organism where a fusion polypeptide composed of a TIR domain and a dimerization domain inhibits the TLR signaling pathway / IL-1R.

The invention also relates to the production of a fusion polypeptide composed of a TIR domain and a dimerization domain and includes the introduction of a nucleic acid encoding a fusion polypeptide composed of the TIR domain and a dimerization domain into a host organism, culturing the host organism under conditions appropriate for protein expression and isolation, and protein purification.

The invention relates to a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain that can be used to inhibit TLR / IL-1R cell signaling for research on natural immunity and / or for the preparation of a medicament for the treatment of inflammatory processes such as inflammatory and autoimmune diseases, sepsis and septic shock, cancer and others resulting from inappropriate and / or overexpression of TLRs in target cells / organisms by methods of administration including but not limited to lipofection, microinjection, biolistics, immunolipofection, electroporation, genetic bombing, virus entry.

The invention relates to a fusion polypeptide consisting of a TIR domain and a dimerization domain that is produced, isolated and purified from host cells / organisms and can be used to inhibit TLR / IL-1R cell signaling for natural immunity research and / or for the preparation of a medicament for treatment of inflammatory processes such as inflammatory and autoimmune diseases, sepsis and septic shock, cancer and others resulting from inappropriate and / or excessive TLR signaling in target cells / organisms by methods of administration including but not limited to electroporation, microinjection, and liposome entry.

The invention also relates to a fusion polypeptide composed of a TIR domain and a dimerization domain which, in addition to a) native or mutated homologous animal, human or viral TIR domains, of TLR signal adapters, TLRs, receptors of the IL-1R family, viral proteins containing the TIR domain , b) dimerization domains and wherein a) and b) are linked to a binding peptide containing flexible and hydrophilic amino acids, and optionally also containing a peptide marker or / and a membrane localization segment, containing a protein transduction domain for fusion transduction polypeptide into eukaryotic target cells and is produced, purified and isolated from host cells / organisms and can be used to inhibit TLR / IL-1R cell signaling for research on natural immunity and / or for the preparation of a medicament for the treatment of inflammatory processes such as inflammatory and autoimmune diseases , sepsis and septic shock, cancer and others resulting from inappropriate and / or excessive signaling TLRs in target cells / organisms by introduction into target cells / organisms by protein transduction.

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Description of the pictures

Figure 1. Schematic representation of a fusion polypeptide composed of a TIR domain and a dimerization domain. The dimerization of the fusion polypeptide of the present invention may be spontaneous (a) or ligand-induced (b).

Figure 2. Constitutive activity measured via activation of the NF-κΒ promoter in HEK293 cells, fusion polypeptides composed of the TIR domain and dimerization domain: ccs (TcpB) -TIR MyD88 and GCN-lin-TIR MyD88, each consisting of segment for the formation of the coiled helix, the binding peptide and the TIR domain (a). Enhanced agonist inhibition of TLR4 (b), TLR5 (c) and TLR9 (d) MyD88-dependent signaling pathways by fusion polypeptides composed of the TIR domain and the dimerization domain: ccs (TcpB) -TIR MyD88 and / or GCN-lin-TIR MyD88 versus the TIR MyD88 monomo domain.

Figure 3. Constitutive activity measured via IFNP promoter activation in HEK293 cells, fusion polypeptides composed of the TIR domain and dimerization domain: ccs (TcpB) -TIR MyD88 and GCN-lin-TIR MyD88 (a). Enhanced agonist inhibition of the TLR3-induced signaling pathway by the GCN-lin-TIR MyD88 fusion polypeptide compared to the TIR MyD88 monomo domain (b).

Figure 4. The binding peptide inserted between the coil-forming segment and the TIR domain is required for effective inhibition by fusion polypeptides composed of the TIR domain and dimerization domain, as shown by enhanced inhibition by the agonist-induced TLR4 / MD-2 signaling by GCN-lin-TIR MyD88 compared to GCN-TIR MyD88, which does not contain a binding peptide.

Figure 5. Enhanced agonist inhibition-induced MyD88-dependent IL-1R signaling by GCN-lin-TIR MyD88 compared to the monomeric TIR MyD88 domain measured via NF-κΒ promoter activation in HEK293 cells.

Figure 6. ccs (TcpB) -TIR MyD88 and GCN-lin-TIR MyD88 do not affect the TNFaR signaling pathway.

Figure 7. Effect of added segment for membrane localization of myr-TAT to GCN-lin-TIR MyD88 on its constitutive activity (a) and inhibitory potential of TLR4 / MD-2 (b) agonist-induced signaling measured via NF-κΒ promoter activity in HEK293 cells.

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Figure 8. SDS-PAGE of recombinant TAT-GCN-lin-TIR MyD88 polypeptide (a). Inhibition of agonist-induced TLR5 signaling in HEK293 cells by transducing recombinant TAT-GCN-lin-TIR MyD88 (b).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel inhibitors of the natural immunity signaling pathways of Tollu-like receptors (TLRs) / receptors from the IL-1R (interleukin-1 receptor) family. The invention is based on the discovery that TLR / IL-1R signaling can be inhibited by the use of fusion polypeptides composed of a TIR domain and a dimerization domain, with the addition of domains allowing dimerization of the fusion polypeptides described to improve their inhibition.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly known to those skilled in the art. The terminology used to describe the invention is intended to explain a particular segment of the invention and is not intended to limit the invention. All publications mentioned in the description of the invention are cited as references. In the description of the invention and the claims, the description is in the singular but also includes the plural, which is not specifically emphasized in the description for ease of understanding.

A fusion polypeptide composed of a TIR domain and a dimerization domain

The invention relates to a fusion polypeptide composed of a TIR domain and a dimerization domain. In the field of the invention, the TIR domains of TLR signaling adapters, such as MyD88, are known to act inhibitory to TLR signaling (Medzhitov et al. 1998, Mol Celi 2, 253-258). Possible mechanisms for their inhibition of the TLR signaling pathway include their ability to bind to TLRs, since they may prevent the binding of native adapters and / or the TIR domain and may also bind to other adapters (Fitzgerald et al. 2001, Nature 413, 78-83) .

The inventors have discovered that the addition of a dimerization domain to the TIR domain, with the dimerization domain and the TIR domain associated with the binding peptide, greatly improves the inhibitory potential of the TIR domain and, consequently, the dimers of the TIR domain inhibit the TLR signaling pathway more effectively as monomemes.

The term fusion polypeptide composed of the TIR domain and dimerization domain refers to a fusion polypeptide composed of a) native or mutated homologous animal, human or viral TIR domain of TLR signal adapters, TLRs, IL-1R family receptors, viral proteins, which contain the TIR domain, b) the dimerization domains and a) and b) are linked to a binding peptide containing flexible and hydrophilic amino acids. Optionally, the fusion polypeptide composed of the TIR domain and the dimerization domain comprises a peptide marker and / or a membrane localization segment. A fusion polypeptide composed of a TIR domain and a dimerization domain inhibits the TLR signaling pathway by interfering with TIR: TIR interactions between receptors and TLR signal adapters, thereby impeding signal transduction.

Figure 1 is a schematic illustration of a fusion polypeptide composed of a TIR domain and a dimerization domain that can dimerize spontaneously or after the addition of a chemical ligand allowing dimerization, as described below. In Figure 1, the dimerization domain is at the Infinite portion of the TIR domain, the two being linked to the binding peptide. Likewise, our results clearly show that the addition of a dimerization domain to the N-terminal portion of the TIR domain significantly enhances the inhibitory potential of the monomeric TIR domains on agonist-induced TLR signaling (Figures 2 b), c), d), 3 b) and 5), which was determined by the dual reporter system described in the embodiments below. Improved inhibitory potential of the addition of a dimerization domain to the C-terminal portion of the TIR domain could be determined by the same method, except using constructs having a dimerization domain at the C-terminal rather than the N-terminal portion of the TIR domain.

The term TIR domain in the description refers to a native or mutated homologous animal, human or viral TIR domain of TLR signal adapters, TLRs, receptors of the IL1-R family, viral proteins containing a TIR domain that are different in length and have a sequence conservation of 20% and are composed of five central β bands surrounded by five α helices and containing three conserved essential signaling sequences. In addition to TLR signaling adapters, the TIR domain also contains TLR / IL-1R and is essential for signaling because it connects receptors to proteins in a further cascade through adapters.

The term homologous refers to amino acid sequences of proteins that originate from the same or different organism and are well conserved. The term homologous also refers to mutant protein segments in which mutations minimally alter the structure and function of the polypeptide.

The term native refers to a fragment that can be obtained from an organism without prior genetic manipulation, and wherein a protein or protein fragment is encoded in the genome of that organism.

The term mutant refers to a fragment different from the native protein / protein fragment in at least one amino acid.

The term TLR signaling adapters refers to cytosolic adapter proteins that participate in the TLR signaling pathway, namely MyD88, MAL, TRIF, TRAM, and SARM. The adapters contain a TIR domain that can bind to the TIR TLR domain, thus linking activated TLRs to proteins in a further cascade via the TIR domain. MyD88 participates in all TLR signaling pathways except TLR3, while TRIL participates in TLR3 and TLR4 signaling pathways. TIRAP and TRAM are auxiliary adapters, with TIRAP binding MyD88 to associated receptors in the TLR4 and TLR2 signaling pathways, and TRAM connecting the TRIF to the receptor in the TLR4 signaling pathway.

The term Tollu-like receptor (TLR) refers to any animal or human TLR receptor. TLRs are members of a type I transmembrane receptor group consisting of leucine-rich repeats at the N-terminus, a transmembrane region and a cytosolic domain containing the TIR domain. The term refers to native receptors capable of activating cells or immune responses after the addition of appropriate ligands. Tollu-like receptors are located on the cell surface (TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, TLR11) or within cell vesicles (TLR3, TLR7, TLR8, TLR9). TLR activation is triggered by the binding of TLR-activating molecules (agonists). Agonist binding results in the merger of cytosolic TIR domains. Upon merging TIR domains, adapters such as MyD88 and TRIF bind to them, leading to activation of the signaling cascade and activation of transcription factors and transcription of genes involved in the immune response. Different TLRs trigger different responses along the MyD88-dependent or TRIF-dependent pathway, leading to the formation of inflammatory cytokines such as IL-1, IL-6, TNF-α or interferons.

The term IL-1R family of receptors refers to receptors from a receptor subfamily with an intracellular TIR domain and an immunoglobulin (Ig) -like extracellular domain. They include the receptor for type I IL-1 (IL-1RI) and its accessory protein (IL-IRAcP), the IL-18 receptor (IL-18Ra and IL-18Rb), the T1 / ST2 receptor regulators, and the IL-1R-related molecule ( And its orphan receptors (TIGIRR-1, TIGIRR-2, and IL-1Rrp2).

The term viral proteins containing the TIR domain refers to viral proteins containing the TIR domain, which is similar in amino acid sequence to mammalian TIR domains. Examples are the A46R and A52R proteins from the vaccine virus, which are antagonists of IL-1 and TLR signaling hosts.

The dimerization of the fusion protein composed of the TIR domain and the dimerization domain of the present invention can be initiated due to the dimerization domain that is part of the fusion polypeptide. The dimerization may be spontaneous or may be initiated by the addition of a chemical ligand. The dimerization domain that results in spontaneous dimerization is preferably, but is not limited to, a homodimeric parallel segment for the formation of helixes of 3 to 12 heptads. The dimerization of the fusion polypeptide described above composed of the TIR domain and the dimerization domain can also be initiated by the addition of a small chemical ligand that binds to two dimerization domains, thereby causing the dimerization of the fusion polypeptide described above composed of the TIR domain and dimerization domain. A small chemical ligand may be, but is not limited to e.g. FK506 that triggers dimerization of FKBP (FK506 binding protein) or cyclosporin that triggers dimerization of cyclophilin.

The term dimerization domain refers to a protein domain that allows spontaneous dimerization of a fusion polypeptide composed of a TIR domain and a dimerization domain, or to a protein domain that allows dimerization of a fusion polypeptide composed of a TIR domain and a dimerization domain after addition of a dimerization-binding chemical ligand these protein domains. A dimerization domain that allows spontaneous dimerization is preferred, but is not limited to the segment for forming coiled helices. Examples of dimerizations e triggered by the addition of a chemical ligand include, but are not limited to, FK506 that initiates dimerization of FKBP (FK506 binding protein) or cyclosporine that initiates dimerization of cyclophilin.

The term spontaneous dimerization refers to the dimerization of a fusion polypeptide composed of a TIR domain and a dimerization domain, wherein the dimerization domain can dimerize by itself, thereby triggering the dimerization of an entire fusion polypeptide composed of the TIR domain and the dimerization domain.

The term ligand-induced dimerization refers to the dimerization of a fusion polypeptide composed of a TIR domain and a dimerization domain triggered by the addition of a chemical ligand that binds to two dimerization domains of a fusion polypeptide, thereby causing its dimerization.

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The term coil-forming segment has a general meaning and refers to a structural protein motif composed of a coil capable of homodimerization with the coil-forming segment of a fusion polypeptide composed of a TIR domain and a dimerization domain, thereby causing its dimerization. The segments for forming coiled helixes are wrapped around each other to form an additional coiled helix. They contain a heptad repeat designated by (abcdefg) n, which is repeated every two turns of the helix, a and d usually represent non-polar, hydrophobic amino acid residues occurring on the interface of two helices, e ing the solvent exposed to amino acid residues, which form electrostatic interactions with each other, b, c and f 'being hydrophilic and exposed to the solvent. The various amino acids at sites a-g determine the oligomerization state, specificity, helix orientation, and stability. Specifically, the helix-forming segment in the description refers to natural or designed helix-forming segments, consisting of 3 to 12 heptads, are in parallel orientation and may form homodimers. Segments for coiled helix formation may be natural or engineered protein structural motifs for coiled helix formation, preferably natural, preferably GCN4-p1, preferably SEQ ID NO: 16.

The term homodimerization has a general meaning in the description of the invention and refers to the process of formation of dimers composed of one type of monomers.

The term monomer refers above to one segment for the formation of coiled helices, which may interact with another segment to form coiled helices by wrapping the two around each other.

The term parallel orientation has a general meaning and refers to two helixes, both extending from N-terminal to C-terminal side by side in the same direction.

It is also an object of the invention that, for enhanced inhibition by polypeptides composed of the TIR domain and the dimerization domain, an important binding peptide of 3 to 40 amino acids is located between the dimerization domain and the TIR domain. Optionally, the fusion polypeptide composed of the TIR domain and the dimerization domain comprises a peptide marker and / or a membrane localization segment. The signaling complex, which includes TLRs and adapters, is formed on the membrane, often in special membrane microdomains, such as e.g. lipid rafts. The localization of fusion polypeptides composed of the TIR domain and the dimerization domain to the membrane would therefore contribute to more successful inhibition, since such fusion polypeptides, by involvement in the interactions of TIR: TIR receptors and adapters, can disrupt the formation of a membrane-forming signaling complex.

The term binding peptide refers to a shorter amino acid sequence located between the dimerization domain and the TIR domain, separating them appropriately and providing an optimal arrangement of the TIR domains. The binding peptide is typically 3 to 40 amino acids long, preferably 15 to 30 amino acids, preferably 25 amino acids long. Preferably, the amino acids are selected from, but not limited to, serine, glycine, threonine, proline, valine, alanine, glutamine, asparagine, glutamate, aspartate, which allow flexibility.

The term marker refers to short amino acid sequences added to a fusion polypeptide to facilitate purification / isolation / detection of an isolated protein.

The term segment for membrane localization has a general meaning and refers to an amino acid sequence that allows the fusion polypeptide composed of a TIR domain and a dimerization domain to be directed to a membrane. The membrane localization segment may be an amino acid sequence that can be covalently modified by attached lipid groups that contribute to the affinity of the fusion protein for membrane binding. An example is the myristoylation sequence. In addition, the membrane localization segment may also be a collection of basic amino acids that forms electrostatic interactions with acidic phospholipid heads in the membrane.

A fusion polypeptide composed of a TIR domain and a dimerization domain is encoded by a nucleic acid inserted into an expression vector. An expression vector with an inserted nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain is used to inhibit TLR / IL-1R cell signaling in human or animal cells and / or to prepare a medicament for treating diseases resulting from inappropriate and / or excessive TLR / IL-1R signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer, and others. An expression vector with an inserted nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, or a prepared drug containing an expression vector with an inserted nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain is introduced into the host cell a cell that then expresses a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain. A fusion polypeptide consisting of a TIR domain and a dimerization domain, which is then synthesized in a host cell / target cell, is used in this cell as an inhibitor of TLR / IL-1R signaling for the treatment of diseases resulting from inappropriate and / or excessive TLR / signaling IL-1R, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and more. In this context, to treat diseases resulting from inadequate and / or excessive TLR / IL-IR signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, a nucleic acid encoding a fusion polypeptide consisting of TIR domains and dimerization domains and is inserted into the expression vector and introduced into the host cell / target cell by gene therapy, which then expresses a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain.

In another context, however, a fusion polypeptide composed of a TIR domain and a dimerization domain encoded by a nucleic acid is recombinantly produced, isolated and purified from a host cell / organism. The recombinantly produced fusion polypeptide composed of the TIR domain and the dimerization domain is then used to inhibit TLR / IL-IR cell signaling in human or animal cells or / and to prepare a drug that is subsequently introduced into target cells / organisms where it acts as an inhibitor TLR / IL-IR signaling pathways for the treatment of diseases resulting from inadequate and / or excessive TLR / IL-IR signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, to avoid the need for gene therapy with nucleic acids encoding the fusion polypeptide of the present invention. In this context, a fusion polypeptide consisting of a TIR domain and a dimerization domain preferably in addition to a) a native or mutated homologous animal, human or viral TIR domain of TLR signal adapters, TLRs, IL-1R family receptors, TIR domain-containing viral proteins, b) dimerization domains and wherein a) and b) are linked to a binding peptide containing flexible and hydrophilic amino acids and optionally also containing a peptide marker or / and a membrane localization segment also containing a protein transduction domain for the transduction of the fusion polypeptide of the present invention into eukaryotic target cells.

Figure 8 b) represents an example of inhibition of flagellin-induced cellular activation via TLR5 by the recombinantly produced fusion polypeptide of the present invention with the addition of a protein transduction domain, the inhibition determined by the double reporter system described in embodiments below. In this case, the transduction domain is the end portion of the dimerization domain of the fusion polypeptide of the present invention. The inhibitory potential of the recombinantly produced fusion polypeptide of the present invention with the addition of a transduction domain to the C-terminal portion of the TIR domain can be determined by the same method except using the fusion polypeptide of this invention having a transduction domain at the C-terminal portion of the TIR domain instead of the N-terminal part of the dimerization domain.

The term protein transduction domain (PTD) has a general meaning and refers to cationic peptide sequences that allow peptides and proteins to be introduced into a cell. The fusion of basic PTD with the inhibitory fusion polypeptide of the present invention enables the transport of the fusion polypeptide across the cell membrane into the cell. Examples of PTD include, but are not limited to, the PTD of the HIV-1 TAT protein, the VP22 DNA-binding protein of the herpes simplex virus 1 (HSV-1), and the Antennapedia (Antp) homeotic transcription factor of the wine fly. The advantage of using the fusion polypeptide of the present invention with the addition of a protein transduction domain is the rapid, concentration-dependent entry of such a fusion polypeptide into a wide variety of cells and low toxicity of the protein transduction domains.

DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain

The invention also relates to DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain consisting of a) a native or mutated homologous animal, human or viral TIR domain of TLR signal adapters, TLRs, receptors from the IL-1R family, viral proteins containing the TIR domain, b) the dimerization domains and a) and b) being linked to a binding peptide containing flexible and hydrophilic amino acids. Optionally, the fusion polypeptide composed of the TIR domain and the dimerization domain comprises a peptide marker and / or a membrane localization segment. The invention also relates to DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, preferably in addition to a) native or mutated homologous animal, human or viral TIR domains of TLR signal adapters, TLRs, IL-1R family receptors, viral proteins containing the TIR domain, b) dimerization domains and a) and b) linked to a binding peptide containing flexible and hydrophilic amino acids and optionally also containing a peptide marker or / and a membrane localization segment also containing a protein transduction domain.

The term DNA / nucleic acid in the description refers to an open reading sequence of nucleotides encoding a protein of the present invention and operatively linked to regulatory elements, a promoter and a terminator, to ensure protein expression in host cells. The length of the DNA sequence can be highly variable depending on the individual protein.

The term regulatory elements has a general meaning in the description and refers to regions of DNA in the expression vector where regulatory proteins, such as transcription factors that control gene expression, bind.

The term promoter has a general meaning in the description and refers to DNA regions in the expression vector that promote transcription of target genes. The type of promoter depends on the host organism in which the gene will be expressed. In the case of expression in prokaryotes, e.g. E. coli, promoters such as e.g. Salary, PT5 and preferably PT7. In the case of expression in eukaryotes, e.g. mammalian cells are promoters of mostly viral origin, such as e.g. PCMV or PSV40.

The term terminator has a general meaning in the description and refers to the sequence that indicates the end of a transcription gene. The type of terminator depends on the host organism in which the gene will be expressed. In the case of expression in prokaryotes, e.g. E. coli, the bacteriophage T7 terminator can be used.

Unless otherwise stated, molecular biology methods have been used in the invention, such as: gene cloning, polymerase amplification, nucleic acid detection, preparation of fusion constructs, expression of peptides, proteins in host cells and the like. The methods are generally known to those skilled in the art and are described in detail in the following manuals (see: Sambrook et al. 1989 Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor, NY; Ausubel et al. Current Protocols and Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

The fusion polypeptide composed of the TIR domain and the dimerization domain described above is encoded by DNA inserted into the expression vector (viral or non-viral). A fusion polypeptide composed of a TIR domain and a dimerization domain encoded by a nucleic acid of the invention can be produced in host cells / organisms expressing a nucleic acid encoding a fusion polypeptide from the expression vector of the invention. A synthesized fusion polypeptide composed of a TIR domain and a dimerization domain is used in target cells / organisms to inhibit the TLR / IL-1R signaling pathway and / or to treat diseases resulting from inadequate and / or over-signaling, including inflammatory and auto immune diseases, sepsis, septic shock, cancer and more.

Suitable vectors for expressing DNA encoding a TIR fusion polypeptide and dimerization domains include, but are not limited to: plasmids, viral vectors, and • · others. Expression vectors, which are compatible with host cell organisms, are well known to those skilled in the art and contain appropriate control elements for transcription and translation of nucleic acid. Typically, the expression vector includes an expression cassette that includes a 5 'to 3' promoter, a coding sequence for the fusion protein operatively linked to the promoter, and a terminator including a stop codon for RNA polymerase and a polyadenylation signal for polyadenylase.

In the context of using DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, to inhibit cellular TLR / IL-IR signaling in human or animal cells and / or to prepare a drug to be used for gene therapy for treatment diseases resulting from inappropriate and / or over-signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer, and others, the expression vector should be ready for expression in eukaryotic cells. According to the invention, prokaryotic cells are used to prepare sufficient amounts of nucleic acid. Eukaryotic expression vectors, however, are used to express DNA in eukaryotes.

In a context where, instead of DNA encoding a fusion polypeptide composed of the TIR domain and a dimerization domain, a recombinantly produced fusion polypeptide consisting of a TIR domain and a dimerization domain is used to inhibit TLR / IL-1R cell signaling in human or animal cells and / or for the preparation of a medicament for the treatment of diseases resulting from inadequate and / or excessive signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, the expression vector should be ready for expression in prokaryotic or eukaryotic cells. Eukaryotic vectors are used to express DNA in eukaryotes, prokaryotic vectors are used to express DNA in prokaryotes.

The expression vector generally contains operably linked control elements operatively linked to DNA encoding a fusion polypeptide composed of the TIR domain and the dimerization domain of the invention. The control elements are selected in such a way that they correspond to the amount of expression and the tissue-specific expression. The promoter may be constitutive or inducible according to the desired pattern of expression. The promoter may be native or of foreign origin (not represented in the cells where it is used) and may be natural or synthetic. The promoter is selected to function in host / target cells. In addition, initiation signals for efficient translation of the fusion protein are included, which includes ATG and associated sequences. In case the vector used in the invention includes two or more reading frames, the reading frames should be operatively linked to the control elements independently and the control elements should be the same or different, depending on the desired protein production.

Examples of bacterial expression vectors include, but are not limited to: pET vectors, pRSET vectors, and others. In cases where vectors are used in bacterial cells, the control elements are of bacterial origin.

Examples of mammalian mammalian expression vectors include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others. When vectors are used in mammalian cells, the controls are mostly viral in origin, for example: adenovirus 2, cytomegalovirus, simian virus 40.

Host cell / organism / Target cell / organism

The invention also relates to a host cell / organism expressing the DNA described above encoding the fusion polypeptide of the invention and to a target cell / organism where the fusion polypeptide composed of the TIR domain and the dimerization domain inhibits TLR / IL-1R signaling pathways.

The term host cell / organism refers to the cell / organism into which the DNA encoding the fusion polypeptide composed of the TIR domain and the dimerization domain has been inserted to express it in that cell / organism. Expression of DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain leads to the synthesis of this fusion polypeptide in the host cell / organism.

In a context in which DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain and / or for the preparation of a medicament to be used for gene therapy for treatment will be used to inhibit cellular TLR / IL-1R signaling in human or animal cells diseases resulting from inadequate and / or over-signaling, including inflammatory and auto-immune diseases, sepsis, septic shock, cancer, and others, the host cell / organism is also a target cell / organism since the fusion polypeptide will be composed of the TIR domain and not only synthesize the dimerization domain but will also inhibit TLR / IL-1R signaling pathways in the host cell / organism. In this context, host cells / target cells are animal, preferably vertebrate, preferably mammalian or human cells or organisms. The methods of inserting DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain into host / target cells are described in more detail below.

In a context where for the inhibition of cellular TLR / IL-1R signaling in human or animal cells or for the preparation of a medicament to be used for the treatment of diseases resulting from inappropriate and / or excessive signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, instead of DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, uses a recombinantly produced fusion polypeptide composed of a TIR domain and a dimerization domain, the host cell / organism may be prokaryotic / prokaryotic or eukaryotic eukaryotic. Eukaryotic cells suitable for expressing the fusion polypeptide of the present invention are not restricted as long as the cell lines are compatible with expression vector propagation methods and expression of the fusion protein. Preferably, eukaryotic cells include, but are not limited to, yeast, fungi, plant cells, and mammalian cells such as: mouse, rat, monkey, or human fibroblast. Any bacterial host of the invention may be used to express DNA. Bacteria or yeasts are preferably used to express the fusion polypeptide of the present invention. The polypeptide can be expressed in E. coli or B. subtilis or in the yeast S. cerevisiae or P. pastoris. The bacterium E. coli is preferably used. The invention relates to the expression of a polypeptide in bacteria or yeasts. The invention relates to bacteria or yeasts that express a polypeptide, preferably E. coli or P. pastoris.

The insertion of expression vector with DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain into a host cell / organism is by conventional methods known to those skilled in the art, and these methods relate to transformation, transfection and involving chemical uptake, electroporation, microinjection, DNA lipofection, particle bombardment, viral DNA uptake and more.

The term target cell / organism refers to a cell / organism in which TLR / IL-1R signaling is inhibited as a result of the uptake and expression of a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain or introduction of a recombinantly produced fusion polypeptide consisting of a TIR domain and a dimerization domain into a target cell / organism for research into natural immunity or for treating diseases resulting from inappropriate or / and excessive TLR signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others. Target cells / organisms are animal, preferably vertebrate, preferably mammalian cells, or human cells or organisms.

In the context where we use DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain to inhibit TLR / IL-1R signaling or / and to prepare a drug that

• · will be intended for gene therapy for the treatment of diseases resulting from inappropriate or / and excessive TLR signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, DNA encoding a TIR fusion polypeptide and dimerization domains, inserts and expressions in the target cell, and a synthesized fusion polypeptide composed of the TIR domain and the dimerization domain inhibits TLR / IL-1R signaling pathways.

In another context, instead of DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, a recombinantly produced fusion polypeptide composed of a TIR domain and a dimerization domain is produced, isolated and purified from the respective host cells / organisms described above and used for the inhibition of TLR / IL-1R signaling or / and the preparation of a medicament for treating diseases in target cells / organisms resulting from inadequate or / and excessive TLR signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others.

Methods for introducing DNA encoding a fusion polypeptide composed of the TIR domain and dimerization domain, and methods for introducing a recombinantly produced fusion polypeptide composed of the TIR domain and dimerization domain into target cells / organisms are described below.

Recombinant production of a fusion polypeptide composed of a TIR domain and a dimerization domain

The invention also relates to recombinant production of a fusion polypeptide composed of a TIR domain and a dimerization domain, which includes the insertion of DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain into the host organism, cultivation of the host organism under conditions suitable for protein expression and isolation, and protein purification. According to the invention, a fusion polypeptide composed of a TIR domain and a dimerization domain can be produced, isolated and purified from a host organism that expresses DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain. A fusion polypeptide comprised of the TIR domain and the dimerization domain of the invention is used to inhibit TLR / IL-1R signaling in human or animal cells and / or to prepare a drug to be used on target cells / organisms for the treatment of resulting diseases inadequate or / and excessive TLR signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer, and others.

In general, DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain is included in the expression vector (viral or non-viral), as described above. The invention also includes host cells and organisms containing the DNA of the invention (transient or stably introduced) encoding a fusion polypeptide composed of a TIR domain and a dimerization domain. Suitable host organisms are known to those skilled in the art of molecular biology and include prokaryotic and eukaryotic cells as described above.

The introduction of vectors into host cells is accomplished by conventional methods known in the art and described above.

DNA uptake can be transient or stable. Transient expression refers to the introduction of vector DNA that is not incorporated into the host cell genome of the invention. With stable uptake, the DNA of the invention is incorporated into the host genome. The uptake of DNA according to the invention, especially when DNA is incorporated into the host cell, can be controlled by the presence of selection markers. DNA encoding selection markers, e.g. refers to antibiotic resistance, and may be included in the vector of the invention or a separate vector.

A fusion polypeptide composed of a TIR domain and a dimerization domain is expressed in a suitable host organism. Expression of higher amounts of fusion polypeptide can be achieved in bacteria such as E. coli in P. pastoris yeast, or mammalian cultures can be used to produce smaller amounts of protein, if posttranslational modifications are necessary for proper protein twist and function. Protein can be expressed in the mammalian cells of the following organisms: humans, rodents, cattle, pigs, poultry, rabbits and the like. The host cells may be primary cell lines or immortalized cell lines. Expression of a fusion polypeptide composed of a TIR domain and a dimerization domain may be constitutive or inducible, e.g. protein expression can be induced by the addition of an IPTG inducer, which, by binding to the lac repressor, triggers the expression of the desired protein. A fusion polypeptide composed of a TIR domain and a dimerization domain can be expressed in a soluble or insoluble fraction.

Protein purification techniques including gel filtration, ion chromatography, reverse phase chromatography, affinity chromatography are well known to those skilled in the art.

Methods of introduction into target cells / organisms

The invention relates to the inhibition of the TLR / IL-1R signaling pathway in the target cell / organism, by introducing into the cell an inhibitory concentration of an expression vector with inserted DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain. In this context of the invention, DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain inserted into an expression vector described above for in vitro or in vivo transfection of target cells via the interaction of the vector and the target cells. The fusion polypeptide composed of the TIR domain and the dimerization domain is then expressed from the DNA encoding the fusion polypeptide.

Methods of introducing a fusion encoding a fusion polypeptide of the present invention into target cells / organisms include lipofection, microinjection, biolisting, immunolipofection, electroporation, gene bombardment, or uptake, whereby the nucleic acid is inserted into DNA or RNA viruses, which infect the target cells. After viral insertion into the target cell, the nucleic acid can remain free and reproduce as episomes autonomously or the nucleic acid may be incorporated into the target cell genome. Viral systems include retroviral, lentiviral, adenovirus, and herpes simplex virus vectors for gene transfer. The viral vectors used herein are generated using a production cell line that packs the nucleic acid vector into the viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent incorporation into the target cell and expression cassette for the protein to be expressed. Viral vectors may be modified to be specific to a particular cell type by expressing the ligand as a fusion protein with a viral coat protein, with the ligand having affinity for the receptor of the desired cell type. For all of the methods of introduction described above, the fusion polypeptide composed of the TIR domain and the dimerization domain is produced from the nucleic acid it encodes in the target cells, where it then acts as an inhibitor of the TLR signaling pathway.

Nucleic acid uptake can be in vivo or ex vivo. Ex vivo entry involves first collecting a sample of individual cells, e.g. immune cells, endothelial cells or any other cells for which treatment is beneficial, culturing the collected cells ex vivo, followed by nucleic acid uptake by one of the methods described above. The cells can then be inserted back into the individual for prophylaxis or therapy. In vivo therapy, however, involves the introduction of a nucleic acid directly into an individual by systemic administration (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously) or topically, with the complex being introduced directly to a target site in the body. The nucleic acids are introduced by any suitable route, preferably with a pharmaceutically acceptable carrier. DNA insertion can be temporary or stable. Temporary DNA insertion takes place with a vector that prevents DNA from being incorporated into the target cell genome. However, stable DNA uptake is achieved by incorporating DNA into the target cell genome, e.g. using viruses (e.g., retro viruses, virus bands, and adeno virus-related viruses) to ensure the incorporation of a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain into the target cell genome.

An overview of gene therapy is given in more detail in Chapter 20 of the book "Gene Therapy and Other Genetics-Based Therapeutic Approaches in Human Molecular Genetics, by T. Strachan and A. P. Read, BIOS Scientific Publishers Ltd (1996)."

In another context, for the inhibition of TLR / IL-1R signaling and / or for the preparation of a medicament for use on target cells / organisms for the treatment of diseases resulting from inadequate and / or excessive TLR / IL-1R signaling, including inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, use a fusion protein composed of a TIR domain and a dimerization domain that is produced, isolated and purified from the host cells / organisms described above and preferably also contains a PTD that enables the uptake of the fusion polypeptide consisting of a TIR domain and a dimerization domain into a target cell. A fusion polypeptide composed of a TIR domain and a PTD-free dimerization domain can be introduced into target cells / organisms by methods including but not limited to electroporation, microinjection, and liposome uptake.

A fusion polypeptide consisting of a TIR domain and a dimerization domain for inhibiting TLR / IL-1R cell signaling and for preparing a medicament for the treatment of disease

The invention relates to a fusion polypeptide composed of a TIR domain and a dimerization domain, which can be used to inhibit TLR / IL-1R cell signaling for the study of natural immunity and / or for the preparation of a medicament for the treatment of inflammatory conditions by the administration of a therapeutically prepared composition an effective dose of a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, or a recombinantly produced fusion polypeptide composed of a TIR domain and a dimerization domain that is produced, isolated and purified from host cells / organisms in combination with a pharmaceutically acceptable carrier target cells / organisms described above. The fusion polypeptide of the invention inhibits the TLR / IL-1R signaling pathway by interfering with the TIR: TIR signaling complex of the TLR signaling complex, thus producing anti-inflammatory and immunoinhibitory effects in the target cells / organisms. The reconstituted drug can be used to treat inflammatory conditions such as inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, which are more fully described below.

The term drug preparation refers to the process of preparing the pharmaceutical compositions described below. The term drug preparation refers to a pharmaceutical composition described below consisting of a therapeutically effective dose of a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, or a recombinantly produced fusion polypeptide composed of a TIR domain and a dimerization domain in combination with a carrier .

The invention relates to both prophylactic and therapeutic methods for treating a target organism that is susceptible to disease or has a disease associated with TLR over-signaling. The term treatment refers to the administration of a preparation as described above to a target organism having a disease, a symptom of a disease or a predisposition to a disease for the purpose of treating, alleviating or ameliorating a disease, a symptom of a disease or a predisposition to a disease. A therapeutically effective dose of DNA encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, or a therapeutically effective dose of the recombinantly produced fusion polypeptide of this invention in combination with a pharmaceutically acceptable carrier is administered to the target cell / organism by the methods of administration described above with the pharmacist described below .

The invention relates to methods for the use of a medicament described above for the treatment or amelioration of conditions curable by inhibiting an individual's TLR / IL-1R signaling.

The term status refers to the physiological states that result from TLR overexpression and include inflammation. More specifically, the invention relates to the treatment of target organisms with a medicament of the invention for the treatment and prevention of inflammatory and autoimmune diseases, sepsis, septic shock, cancer and others, which are described in more detail below.

The formulation of the invention described above can be used to treat many immune diseases resulting from excessive or inadequate TLR / IL-1R signaling. Examples include inflammatory conditions such as bacterial, viral, fungal infectious and infectious diseases, sepsis and septic shock, autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Chron's disease, ulcerative colitis, insulin resistance, cancer, neuropathic pain, neurological inflammation, alcohol-induced inflammation, ischemia, hypoxia, hemorrhage, atherosclerosis, asthma, allergy, graft rejection, ankylosing spondylitis, putica.

The formulation of the invention described above can be used to treat or prevent inflammatory responses caused by germs (or microbial toxins), e.g. bacteria (eg Gram positive or negative bacteria) such as E. coli, Salmonella typhimurium or other Salmonella 'species, Pseudomonas species or Gram positive bacteria such as Staphylococcus, Streptococcus, Pneumococcus. Other examples of microbial infections that can be treated with the manufactured drug of the invention described above also include viruses, e.g. Ebola virus, West Nile virus and hepatitis a, b, c; and fungi, e.g. Candida albicans, Cryptococcus neoformans, and primers, e.g. Plasmodium falciparum and other malaria-causing Plasmodium species.

The preparation described above can be used to treat or prevent inflammatory reactions caused by microbial toxins that cause inflammatory responses, e.g. lipopolysaccharide or any superantigen produced by microbes, e.g. staphylococcal enterotoxin A or B.

The preparation described above can be used to treat or prevent any inflammatory reactions that can be treated by the methods of the invention, including plant toxins, nickel, latex, environmental toxins or allergens that stimulate an immune response upon skin contact or inhalation.

The preparation described above can be used to treat both systemic and local inflammatory responses. The methods and the medicament of the invention can be used for

treating or preventing inflammatory responses that affect the function of specific organs or organ systems, e.g. liver, intestines, kidneys, joints, skin, central nervous system, bladder or reproductive organs. The methods and the drug can also be used to treat chronic or acute inflammatory diseases and skin conditions, e.g. psoriasis, eczema or contact dermatitis.

The invention relates to the medical or veterinary application of a medicament. The target organisms involved in the healing process are vertebrates, preferably mammals, including but not limited to humans, primates, dogs, cats, rabbits, goats, ungulates and others.

The invention also relates to a method of inhibiting the immune response by administering a formulation of the present invention by inhalation, oral, intravenous, transdermal, parenteral, subcutaneous, intradermal, intrapleural, intracerebral, intraarterial, or by injection directly into an organ or tissue.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain, or a recombinantly produced fusion polypeptide consisting of a TIR domain and a dimerization domain in combination with a pharmaceutically acceptable carrier, which may include a salt, buffer, dextrose, water, glycerol, stabilizers, solvents, lubricants, osmostabilizers and others. The term pharmaceutically acceptable refers to a material that is not toxic to the target cell / organism. The pharmaceutical composition must be compatible with the route of administration, which may be parenteral (intravenous, intradermal, subcutaneous), oral, topical, transmucosal or administered by inhalation.

In the context of the use of a nucleic acid encoding a fusion polypeptide consisting of a TIR domain and a dimerization domain for the treatment of autoimmune, chronic inflammatory diseases, sepsis, septic shock, cancer and other gene therapy, the pharmaceutical carrier is preferably formulated for viral entry or for introduction with any other method of inserting DNA into the target cells / organisms described above.

In the context of the use of a recombinantly produced fusion polypeptide consisting of a TIR domain and a dimerization domain that is produced, isolated and purified from host cells / organisms for the treatment of autoimmune, chronic inflammatory diseases, sepsis,

septic shock, cancer, and the like, the pharmaceutical composition is preferably formulated for, but not limited to, transduction uptake, since the fusion polypeptide consisting of the TIR domain and the dimerization domain may contain PTD that allows efficient and rapid translocation into target cells / organisms.

In the context of the invention, a nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain or a recombinantly produced fusion polypeptide composed of a TIR domain and a dimerization domain in combination with a pharmaceutically acceptable carrier is introduced into target cells / organisms in a therapeutically effective amount for the purpose of preventing treatment or amelioration of disorders associated with excessive or inadequate TLR / IL1R signaling, wherein the term therapeutically effective amount refers to the amount of nucleic acid or recombinantly produced fusion polypeptide of the invention described above that is sufficient to inhibit the TLR / IL-1R signaling pathway, and improving the symptoms of the disorder. Optionally, the dosage amount is sufficient when the improvement of symptoms of the disease resulting from excessive or inadequate TLR / IL-1R signaling begins. Inhibition may not be complete and permanent, it is sufficient that the benefit of administration of the pharmaceutical composition outweighs the undesirable effects. The therapeutically effective amount depends on the route of administration, on the nucleic acid encoding the fusion polypeptide composed of the TIR domain and the dimerization domain, or on the recombinantly produced fusion polypeptide of the invention, on the efficiency of protein expression in the case of gene therapy, and on the target organism. Effective amounts are determined in the manner known in the art.

The following are embodiments intended to illustrate the invention. The description of embodiments is not intended to limit the invention but should be construed as demonstrating the operation of the invention.

Implementation examples

Example 1: Preparation of DNA constructs

The constructs listed below (Table 1, Table 2) were prepared in order to illustrate the operation of the invention. Fusion polypeptides composed of the TIR domain and the dimerization domain of the present invention were designed by combining the TIR domain, dimerization domains, binding peptide and membrane localization segment. The amino acid sequence of the TIR domain of the MyD88 adapter was selected as the sequence for the TIR domain (SEQ ID NO: 12). For

the GCN4 peptide (SEQ ID NO: 16) was selected for the dimerization of the segments, which forms a homodimeric parallel segment for the formation of helical coils and the N-terminal segment (ak 1-117) of B. melitensis TcpB protein, which predictably includes a dimerization domain consisting of homodimeric segment for helix formation (ccs (TcpB); SEQ ID NO: 14). A sequence of 29 flexible and hydrophilic amino acids (SEQ ID NO: 18) was selected for the binding peptide. However, the myristoylation sequence of the TRAM adapter (SEQ ID NO: 20) and the basic TAT segment (SEQ ID NO: 22) were selected for the membrane localization segment. Fusion proteins may contain a signal sequence that directs the protein to a specific site in the cell and a peptide marker for the detection and purification of the protein.

DNA constructs and associated fusion proteins are described in Table 1. A more detailed description and function of individual DNA or proteins is described in Table 2. DNA sequences for the GCN, lin, and TAT segments were synthesized by GeneArt (http: //www.geneart .com /). DNA constructs were folded into the pcDNA3 vector (http://www.lablife.org/p?a=vdb_view&id=g2.9Qyh n9D3As.29eaY9eZRAcswvk-) for expression in mammalian cells, the TAT-GCN-lin-TIR MyD88 construct was bent into the pET3a vector for expression in E. coli. The pDeNy-hMyD88 vector with the TIR gene MyD88 was purchased from Invivogen (http://www.invivogen.com/PDF/pDeNv-hMvD88_TDS.pdf).

DNA constructs were prepared by molecular biology methods described in the book: Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular cloning: A laboratory manual. 2nd ed. New York, Cold Spring Harbor Laboratory Press: 1659 p. Plasmids and constructs were transformed into E. coli DH5a. Plasmids for transfection into the HEK293 cell line were isolated with the PureLink HiPure Plasmid Kit (InVitrogen), which provides plasma isolation without endotoxin.

The final constructs prepared by the methods described above are given in Table 1. The nucleotide sequence of the constructs was confirmed by sequencing. The individual parts of the DNA-encoded fusion proteins are given in Table 2, which also contains intermediate sequences between the individual genes.

• ·

Table 1: The fusion polypeptides used to illustrate the operation of the invention

No. Name The composition of the construct Nucleotide / amino acid sequence Plasmid Framework 1 ccs (TcpB) -TIR MyD88 FLAG-ccs (TcpB) -TIR MyD88 SEQ ID NO: 1 / SEQ ID NO: 2 pcDNA3 2 GCN-TIR MyD88 GCN-TIR MyD88 SEQ ID NO: 3 / SEQ ID NO: 4 pcDNA3 3 GCN-lin-TIR MyD88 GCN-lin-TIR MyD88 SEQ ID NO: 5 / SEQ ID NO: 6 pcDNA3 4 myr-TAT-GCN-lin-TIR MyD88 myr-TAT-GCN-lin-TIR MyD88 SEQ ID NO: 6 / SEQ ID NO: 8 pcDNA3 5 TAT-GCN-lin-TIR MyD88 His-GG-TAT-GG-GCN-lin-TIR MyD88 SEQ ID NO: 9 / SEQID: 10 pET3a

Table 2: Genes, Intermediate Sequences, Their Function and Amino Acid / Nucleotide Sequence

The name of the gene Nucleotide sequence Amino acid sequence Function HIS tag CATCATCATCATCATCA T HHHHHH a marker FLAG _tag GACTACAAAGACGAT GACGACAAG DYKDDDDK a marker GG GGTGGT GG binding dipeptide TIR MyD88 SEQ ID NO: 11 SEQ ID NO: 12 the TIR adapter domain MyD88 ccs (TcpB) SEQID: 13 SEQ ID NO: 14 N-terminal portion of TcpB, which is predicted to form a homodimeric segment for the formation of coiled helices GCN SEQID: 15 SEQ ID NO: 16 GCN4-pl forming a homodimeric parallel segment for the formation of helixes lin SEQID: 17 SEQ ID NO: 18 binding peptide myr SEQID: 19 SEQ ID NO: 20 sequence for myristoylation of the TRAM adapter TAT SEQ ID NO: 21 SEQ ID NO: 22 basic segment for protein transduction (PTD)

Example 2: Effect of fusion polypeptides composed of a TIR domain and a dimerization domain on TLR / IL-IR and TNFα-R signaling

The effect of fusion polypeptides composed of the TIR domain and the dimerization domain of the invention was illustrated by transfection of nucleic acids encoding the fusion polypeptides of this invention into the HEK293 cell line and activation of a reporter system reflecting activation of the NF-κΒ or IFNP promoter.

• ·

The methods and techniques of cell culture cultivation are well known to those skilled in the art and are therefore only briefly described to illustrate an embodiment. HEK293 cell lines were grown at 37 ° C and 5% CO2. DMEM medium with 10% FBS containing all the necessary nutrients and growth factors was used for cultivation. When the cell culture was sufficiently dense, the cells were glued to a new bottle and / or diluted. For the use of cells in the experiments, cell counts were determined with a hemocytometer and grafted 2.5 * 10 ⁴ per well into a 96-well microtiter plate 24 hours before transfection. The inoculated plates were incubated at 37 ° C and 5% CChdok ler cells were not 50-70% confluently grown for transfection with JetPei transfection reagent. The transfection was performed according to the manufacturer's instructions, adapted for a 96-well microtiter plate.

Dual luciferase assay

Cell activation and inhibition by the fusion polypeptides of the invention were monitored by a double luciferase assay based on reporter plasmids. A test with two reporter proteins was used to determine luciferase activity: (a) godfather luciferase (Fluc) and (b) Renilla luciferase (Rluc). The godfather luciferase gene, which encodes a godfather luciferase that uses luciferin as a substrate, is functionally linked to the NF-κΒ (NF-κB -Fluc) or IFNP (IFNβfluc) promoter and thus detects their activation. Activation of TLR leads to activation of NF-κB and / or IFN3 transcription factors, which can be detected by measuring the activity of godfather luciferase. The plasmid with the Rluc reporter is transfected into cells simultaneously with the pFluc plasmid and plasmids encoding fusion polypeptides composed of the TIR domain and the dimerization domain and serve as a transfection success reporter. This reporter bears the record for Renilla luciferase, which uses coelentrazine as a substrate. Rluc is expressed constitutively and is used to normalize transfection.

Plasmids with DNA encoding the fusion polypeptides of the invention listed in Table 1, different TLRs and two reporters were used for transfection into HEK293 cell line: Rluc and NF-κΒ Fluc / IFNβ-Fluc at the following concentrations:

Figure 2.

a) To determine the constitutive activity of fusion polypeptides composed of the TIR domain and the dimerization domain on the activity of the NF-κB promoter: 10 ng Rluc; 50 ng NF-κΒ -Fluc; 140 ng of the plasmid examined

To determine the inhibitory effect of fusion polypeptides composed of the TIR domain and the dimerization domain on ligand-triggered cellular activation of TLRs compared to the TIRMyD88 domain:

b) to TLR4 / MD2 signaling: 1 ng TLR4, 1 ng MD2, 10 ng Rluc; 50 ng NF-κΒ -Fluc; 1 ng, 10 ng and 20 ng of the plasmid examined

c) TLR5 signaling: 30 ng TLR5; 10 ng Rluc; 50 ng NF-κΒ -Fluc, 1 ng, 10 ng and 20 ng of the plasmid examined

d) TLR9 signaling: 40 ng TLR9; 5 ng Unc93Bl, 10 ng Rluc, 100 ng NF-κΒ -Fluc; 1 ng, 10 ng and 20 ng of the plasmid examined

Figure 3.

a) to determine the constitutive activity of fusion polypeptides composed of the TIR domain and the dimerization domain to IFNP-Fluc promoter activity: 10 ng Rluc; 40 ng ΙΡΝβ-fluc; 100 ng of the plasmid examined

b) for determining the inhibitory effect of fusion polypeptides composed of the TIR domain and the dimerization domain on ligand-induced cellular activation of TLR3 compared to the TIR domain of MyD88: TLR3 20 ng; 10 ng Rluc; 40 ng ΙΡΝβ-fluc; Ing, 10 ng and 20 ng of the plasmid examined

Figure 4. To compare inhibition of fusion polypeptides composed of TIR domain and dimerization domain with or without binding peptide with ligand-triggered signaling via TLR4 / MD-2: 1 ng TLR4, 1 ng MD2, 10 ng Rluc; 50 ng NF-κΒ -Fluc; 1 ng, 10 ng and 20 ng of the plasmid examined

Figure 5. To determine the inhibitory effect of fusion polypeptides composed of the TIR domain and the dimerization domain compared to the TIR MyD88 domain on ligand-induced IL-1R signaling: 10 ng Rluc, 50 ng NF-κΒ-Fluc; 5 ng, 75 ng, 145 ng of the plasmid examined

Figure 6. To determine the effect of fusion polypeptides composed of the TIR domain and the dimerization domain versus the TIR MyD88 domain on ligand-induced TNFα-R signaling: 10 ng Rluc, 50 ng NF-κΒ -Fluc; 5 ng, 35 ng, 75 ng of plasmid examined »9

Figure 7. To determine the effect of an added segment for membrane localization of myr-TAT to GCN-lin-TIR MyD88:

a) on its constitutive activity: 10 ng Rluc; 50 ng NF-κΒ -Fluc; 5 ng of the plasmid examined

b) the inhibitory potential of the ligand-induced TLR4 / MD-2 signaling: 1 ng TLR4; 1 ng MD2; 10 ng Rluc; 50 ng NF-κΒ -Fluc; 1 ng, 10 ng, 20 ng of the plasmid examined

The medium was replaced with cells 6 hours after transfection, except when monitoring the effect of fusion polypeptides composed of the TIR domain and the dimerization domain on the ligand induced TLR9, IL-1R and TNFα-R signaling. In these cases, the medium was replaced with cells 24 hours after transfection.

After changing the medium, cells were stimulated with appropriate TLR agonists, as shown in Figures 1-7. Cells were stimulated for 16 h, except when the influence of fusion polypeptides composed of the TIR domain and dimerization domain on ligand-induced TLR9, IL-1R, and TNFα-R signaling was monitored. In these cases, the cells were stimulated for 24 h.

In the experiments where the constitutive activity of the fusion polypeptides of the present invention was determined, the medium was replaced with cells 6 hours after transfection, after which the cells were incubated for a further 16 hours.

Cells were then lysed with buffer according to the manufacturer's instructions (Promega). We first measured the activity of firefighter luciferase (Fluc) and then the activity of Renilla luciferase (Rluc; phRL-TK http://www.promega.com/vectors/prltk.txf). The dual luciferase assay is described in the manufacturer's instructions (Promega). Rluc activity reflects the proportion of successfully transfected cells, while Fluc activity reflects activation of NF-κΒ or IFNP promoters. The Fluc / Rluc ratio (RLU relative luciferase units) is the normalized value of activated cells that have been transfected.

The results shown in Figure 2 support the inventors' idea that constructs containing a TIR domain with an added dimerization domain, where these two are linked to a binding peptide, inhibit TLR signaling significantly better than monomemes of the TIR domain. Furthermore, the GCN-lin-TIR construct of MyD88 can also inhibit the intracellular TLR3 and TLR9 receptors as shown in Figures 2 and 3, potentially useful for the treatment of diseases involving inadequate / over-activation of endogenous TLRs, e.g. TLR9, which is involved in the development of SLE. The importance of inserting a binding peptide between the dimerization domain and the TIR domain is illustrated by · ·

Figure 4. Fusion polypeptides composed of the TIR domain and the dimerization domain are specific for both TLR and IL-1R signaling (Figure 5) and are therefore potentially useful for the treatment of inflammatory diseases involving not only TLR but also IL-1R signaling ; e.g. putika. Fusion polypeptides composed of the TIR domain and the dimerization domain have no effect on TNFα-R signaling, confirming their specificity for TLR / IL-1R signaling. The effect of an optionally added membrane localization segment on the fusion polypeptide of the present invention is shown in Figure 7.

Example 3: Recombinant production of a fusion polypeptide composed of a TIR domain and a dimerization domain with the added transduction domain for introduction into target cells

In another context of the present invention, a fusion polypeptide consisting of a TIR domain and a dimerization domain is used which is produced, isolated and purified as a recombinant target cell insertion protein, where it acts as a TLR / IL-1R signaling pathway inhibitor. In this context, a fusion polypeptide composed of a TIR domain and a dimerization domain, which further comprises a protein transduction domain, is preferably used for rapid and efficient transduction into target cells. To illustrate the invention, a recombinant TAT-GCN-lin-TIR MyD88 polypeptide (SEQ ID NO: 10) was produced. The plasmid encoding the open reading frame of TAT-GCN-lin-TIR MyD88 was transformed by chemical transformation into competent cells of E. coli BL21 (DE3) pLysS. The selected bacterial colony grown on the LB plate with the selected antibiotic (ampicillin) was inoculated into 10 mL of liquid LB medium with the appropriate antibiotic. After several hours of growth at 37 ° C, 10-100 pL of culture were inoculated into 100 mL of the selected medium and the bacteria were allowed to grow overnight by shaking 37 ° C. The next day, the overnight culture was diluted 20-50 times, reaching OD600 of the diluted culture between 0.1 and 0.2. 500 ml diluted culture flasks were shaken at 37 ° C until OD600 was reached between 0.6-0.8. At that time, protein expression was induced by the addition of an IPTG inducer (1 mM). Four hours after induction, the culture was centrifuged and the bacterial cells were resuspended in lysis buffer (Tris pH 8.0 0.1% deoxycholate with protease inhibitory addition) and frozen at -80 ° C overnight. The thawed cell suspension was then further lysed by sonication and centrifuged. The presence of the expressed TAT-GCN-lin-TIR MyD88 protein in the insoluble fraction and in the supernatant by SDS-PAGE and Westem transfer with primary antibodies against the histidine marker were verified. TAT-GCN-lin-TIR MyD88 was isolated from the insoluble cell fraction, which was washed twice, then dissolved in 6 M GdnHCl, 10 mM β-mercaptoethanol, 10 mM TRIS, 100 mM sodium phosphate, pH 8. After centrifugation dissolved The fractions were applied to the supernatant on a reversed phase C5 column from which TAT-GCN-lin-TIR MyD88 was isolated. The isolated protein was dialyzed against MQ and concentrated. Protein purity was verified by SDS polyacrylamide gel electrophoresis (Figure 8 a). The purified protein can be effectively transduced into target cells where it acts as an inhibitor of the TLR signaling pathway.

Example 4: Effect of a recombinant fusion polypeptide composed of a TIR domain and a dimerization domain with an added protein transduction domain on TLR5 signaling on HEK293 cells

To determine the effect of a recombinant fusion polypeptide composed of the TIR domain and the TAT-GCN-lin-TIR MyD88 dimerization domain on TLR signaling, HEK293 cells were grafted onto a 96-well microtiter plate and transfected with 20 ng TLR5, 10 ng Rluc the next day ng Fluc. At 24 h after transfection, the cells were replaced with medium and recombinant TAT-GCN-lin-TIR MyD88 was added at a final concentration of 50 μΜ. After 3 hours of incubation, cells were stimulated with flagellin at a final concentration of 10 ng / mL and incubated for a further 16 hours. The cells were then lysed and luminescence was measured. A more detailed description of the double luciferase assay is provided above. The results in Figure 8 b) show the inhibitory effect of the recombinant TAT-GCN-lin-TIR MyD88 protein, which has an inhibitory effect on TLR5 signaling on HEK293 cells. The TAT-GCN-lin-TIR MyD88 can be used to prepare a medicament for the treatment of inflammatory conditions resulting from inadequate and / or excessive TLR signaling, whereby drug administration is via protein transduction.

LIST OF ORDERS <110> Institute of Chemistry, Ljubljana, Slovenia Jerala, Roman Fekonja, Ota <120> Fusion polypeptides composed of TIR domain and dimerization domain for modulation of TLR / natural immunity signaling <130> <160> 22 <170> Patent version 3.5 < 210> 1 <211> 801 <212> DNA <213> artificial sequence <220>

<223> ccs (TcpB) -TIR MyD88 <220>

<221> CDS <222> (1) .. (801) <400> 1

atg Met 1 gac Asp tac Tyr aaa Lys gac Asp 5 gat Asp gac Asp gac Asp aag Lys atg Met 10 tet Sir aaa Lys gag Glu aaa Lys caa Gin 15 gcc Ala 48 caa tca aaa gcc cac aaa gcc caa caa gct ate agt tca gca aaa tca 96 Gin Sir Lys Al a 20 His Lys Al a Gin Gin 25 Al a Ile Sir Sir Ala 30 Lys Sir ctc tcc aca cag aaa agc aaa atg tca gag ctt gag ege gcc acg agg 144 Leu Sir Thr 35 Gin Lys Sir Lys Met 40 Sir Glu Leu Glu Arg 45 Ala Thr Arg gat ggt gcc gcg att ggc aag aag ego gcg gat ate gcc aaa aaa att 192 Asp Gly 50 Al a Al a Ile Gly Lys 55 Lys Arg Al a Asp Ile 60 Ala Lys Lys Ile gcc gat aag gca aaa cag tta agc tcc Dad cag gct aag caa ttc aaa 240 Al a 65 Asp Lys Al a Lys Gin 70 Leu Sir Sir Tyr Gin 75 Al a Lys Gin Phe Lys 80 gct gat gag cag gct gtt aaa aag gtc gcg cag gag caa aag cgt tta 288 Al a Asp Glu Gin Al a 85 Val Lys Lys Val Al a 90 Gin Glu Gin Lys Arg 95 Leu tca gat gag ego acc aag cat gaa gct ttc ate aaa caa tca ttg agc 336 Sir Asp Glu Ar g 100 Thr Lys His Glu Al a 105 Phe Ile Lys Gin Sir 110 Leu Sir tcc atg ego aca act gcg agc gcg act atg gaa gca gaa gaa gaa gct 384 Sir Met Ar g 115 Thr Thr Al a Sir Al a 120 Thr Met Glu Ala Glu 125 Glu Glu Ala

agc Sir gca Al a 130 cgt Arg ttc Phe gat Asp gcc Ala ttc Phe 135 ate Ile tgc Cys Dad Tyr tgc Cys ccc Pro 140 agc Sir gac Asp ate Ile cag Gin ttt gtg cag gag atg ate cgg caa ctg gaa cag aca aac Dad ego ctg Phe Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu 145 150 155 160 aag ttg tgt gtg tet gac ege gat gtc ctg cct ggc acc tgt gtc tgg Lys Leu Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp 165 170 175 tet att get agt gag ete ate gaa aag agg tgc ege cgg atg gtg gtg Sir Ile Al a Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val 180 185 190 gtt gtc tet gat gat tac ctg cag agc aag gaa tgt gac ttc cag acc Val Val Sir Asp Asp Tyr Leu Gin Sir Lys Glu Cys Asp Phe Gin Thr 195 200 205 aaa ttt gca ete agc ete tet approx ggt gcc cat cag aag ego ctg ate Lys Phe Al a Leu Sir Leu Sir Pro Gly Ala His Gin Lys Arg Leu Ile 210 215 220 ccc ate aag tac aag gca atg aag aaa gag ttc ccc agc ate ctg agg Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu Arg 225 230 235 240 ttc ate act gtc tgc gac tac acc aac ccc tgc acc aaa tet tgg ttc Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp Phe 245 250 255 tgg act ege ctt gcc aag gcc ttg tcc ctg ccc Trp Thr Arg Leu Al a Lys Ala Leu Sir Leu Pro

260 265 <210> 2 <211> 267 <212> PRT <213> artificial sequence <220>

<223>, synthetic construct <400> 2 Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Sir Lys Glu Lys Gin Ala 1 5 10 15 Gin Sir Lys Ala His Lys Ala Gin Gin Ala Ile Sir Sir Ala Lys Sir 20 25 30 Leu Sir Thr Gin Lys Sir Lys Met Sir Glu Leu Glu Arg Ala Thr Arg 35 40 45 Asp Gly Ala Ala Ile Gly Lys Lys Arg Ala Asp Ile Ala Lys Lys Ile 50 55 60 Ala Asp Lys Ala Lys Gin Leu Sir Sir Tyr Gin Ala Lys Gin Phe Lys

• · ·

65 70 75 80 Ala Asp Glu Gin Ala Val Lys Lys Val Ala Gin Glu Gin Lys Arg Leu 85 90 95 Sir Asp Glu Arg Thr Lys His Glu Ala Phe Ile Lys Gin Ser Leu Ser 100 105 110 Sir Met Arg Thr Thr Ala Sir Ala Thr Met Glu Ala Glu Glu Alu 115 120 125 Sir Ala Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gin 130 135 140 Phe Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu 145 150 155 160 Lys Leu Cys Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp 165 170 175 Sir Ile Ala Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val 180 185 190 Val Val Sir Asp Asp Tyr Leu Gin Ser Lys Glu Cys Asp Phe Gin Thr 195 200 205 Lys Phe Ala Leu Ser Leu Ser Pro Gly Ala His Gin Lys Arg Leu Ile 210 215 220 Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg 225 230 235 240 Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe 245 250 255 Trp Thr Arg Leu Ala Lys Ala Leu Ser Leu Pro 260 265 <210> 3 <211> 516 <212> DNA <213> artificial sequence <220> <223> GCN-TIR MyD88 <220> <221> CDS <222> (1) · (516) <400> 3 atg agg atg aag cag ctg gaa gat aag gtg gag gaa ctg ctg tcc aag Met Arg Met Lys Gin Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys 1 5 10 15 aac tac cac ctg gaa aac gag gtg gcc cgg ctg aag aaa ctg gtc ggc Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly

25 30 gag aga gca cgt ttc gat gcc ttc ate tgc tat tgc ccc agc gac ate

144 • ·

Glu Arg Al a 35 Arg Phe Asp Ala Phe 40 Ile Cys Tyr Cys Pro 45 Sir Asp Ile cag ttt gtg cag gag atg ate cgg caa ctg gaa cag aca aac Dad ego 192 Gin Phe Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg 50 55 60 ctg aag ttg tgt gtg tet gac ege gat gtc ctg cct ggc acc tgt gtc 240 Leu Lys Leu Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val 65 70 75 80 tgg tet att get agt gag ete ate gaa aag agg tgc ege cgg atg gtg 288 Trp Sir Ile Al a Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val 85 90 95 gtg gtt gtc tet gat gat tac ctg cag agc aag gaa tgt gac ttc cag 336 Val Val Val Sir Asp Asp Tyr Leu Gin Sir Lys Glu Cys Asp Phe Gin 100 105 110 acc aaa ttt gca ete agc ete tet approx ggt gcc cat cag aag ego ctg 384 Thr Lys Phe Al a Leu Sir Leu Sir Pro Gly Ala His Gin Lys Arg Leu 115 120 125 ate ccc ate aag tac aag gca atg aag aaa gag ttc ccc agc ate ctg 432 Ile Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu 130 135 140 agg ttc ate act gtc tgc gac tac acc aac ccc tgc acc aaa tet tgg 480 Arg Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp 145 150 155 160 ttc tgg act ege ctt gcc aag gcc ttg tcc ctg ccc 516 Phe Trp Thr Arg Leu Ala Lys Ala Leu Sir Leu Pro

165 170 <210> 4 <211> 172 <212> PRT <213> artificial sequence <220>

<223> synthetic construct <400> 4

Met 1 Arg Met Lys Gin 5 Leu Glu Asp Lys Val 10 Glu Glu Leu Leu Sir 15 Lys Asn Tyr His Leu 20 Glu Asn Glu Val Ala 25 Arg Leu Lys Lys Leu 30 Val Gly Glu Arg Ala 35 Arg Phe Asp Ala Phe 40 Ile Cys Tyr Cys Pro 45 Sir Asp Ile Gin Phe 50 Val Gin Glu Met Ile 55 Arg Gin Leu Glu Gin 60 Thr Asn Tyr Arg Leu Lys Leu Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val

70 75 80

Trp Ser Ile Ala Ser Glu Leu Ile 85

Val Val Val Ser Asp Asp Tyr Leu 100

Thr Lys Phe Ala Leu Ser Leu Ser 115 120

Ile Pro Ile Lys Tyr Lys Ala Met 130 135

Arg Phe Ile Thr Val Cys Asp Tyr 145 150

Phe Trp Thr Arg Leu Ala Lys Ala 165 <210> 5 <211> 603 <212> DNA <213> artificial sequence <220>

<223> GCN-lin-TIR MyD88 <220>

<221> CDS <222> (1) .. (603) <400> 5 atg agg atg aag cag ctg gaa gat

Met Arg Met Lys Gin Leu Glu Asp

5 aac tac cac ctg gaa aac gag gtg

Asn Tyr His Leu Glu Asn Glu Val gag aga ggt acc ggc tcc gag gga

Glu Arg Gly Thr Gly Ser Glu Gly

40 tcc aag gtg acc gac tet ggc tcc

Ser Lys Val Thr Asp Ser Gly Ser

55 cgt ttc gat gcc ttc ate tgc tat

Arg Phe Asp Ala Phe Ile Cys Tyr

70 cag gag atg ate cgg caa ctg gaa

Gin Glu Met Ile Arg Gin Leu Glu tgt gtg tet gac ege gat gtc ctg

Cys Val Ser Asp Arg Asp Val Leu

100 get agt gag ete ate gaa aag agg

Glu Lys Arg Cys Arg Arg Met Val 90 95

Gin Ser Lys Glu Cys Asp Phe Gin 105 110

Pro Gly Ala His Gin Lys Arg Leu 125

Lys Lys Glu Phe Pro Ser Ile Leu 140

Thr Asn Pro Cys Thr Lys Ser Trp 155 160

Leu Ser Leu Pro 170 aag gtg gag gaa ctg ctg tcc aag Lys Val Glu Glu Leu Leu Ser Lys

15 gcc cgg ctg aag aaa ctg gtc ggc

Ala Arg Leu Lys Lys Leu Val Gly

30 aag tcc tet ggc tcc gga tet gag

Lys Ser Ser Gly Ser Gly Ser Glu gag acc ggc tcc tet gga tcc gca

Glu Thr Gly Ser Ser Gly Ser Ala tgc ccc agc gac ate cag ttt gtg

Cys Pro Ser Asp Ile Gin Phe Val

80 cag aca aac tat ega ctg aag ttg

Gin Thr Asn Tyr Arg Leu Lys Leu

95 cct ggc acc tgt gtc tgg tet att

Pro Gly Thr Cys Val Trp Ser Ile

105 110 tgc ege cgg atg gtg gtg gtt gtc

144

192

240

288

336

384 • ·

Al a Sir Glu 115 Leu Ile Glu Lys Arg 120 Cys Arg Arg Met Val 125 Val Val Val tet gat gat tac ctg cag agc aag gaa tgt gac ttc cag acc aaa ttt 432 Sir Asp Asp Tyr Leu Gin Sir Lys Glu Cys Asp Phe Gin Thr Lys Phe 130 135 140 gca ete agc ete tet approx ggt gcc cat cag aag ego ctg ate ccc ate 480 Ala Leu Sir Leu Sir Pro Gly Ala His Gin Lys Arg Leu Ile Pro Ile 145 150 155 160 aag tac aag gca atg aag aaa gag ttc ccc agc ate ctg agg ttc ate 528 Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu Arg Phe Ile 165 170 175 act gtc tgc gac tac acc aac ccc tgc acc aaa tet tgg ttc tgg act 576 Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp Phe Trp Thr 180 185 190 ege ctt gcc aag gcc ttg tcc ctg ccc 603 Arg Leu Ala Lys Ala Leu Sir Leu Pro

195 200 <210> 6 <211> 201 <212> PRT <213> artificial sequence <220>

<223>: synthetic construct <400> 6 Met Arg Met Lys Gin Leu Glu Asp Lys Val Glu Glu Leu Leu Sir Lys 1 5 10 15 Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly 20 25 30 Glu Arg Gly Thr Gly Sir Glu Gly Lys Sir Sir Gly Sir Gly Sir Glu 35 40 45 Sir Lys Val Thr Asp Sir Gly Sir Glu Thr Gly Sir Sir Gly Sir Ala 50 55 60 Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Sir Asp Ile Gin Phe Val 65 70 75 80 Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu Lys Leu 85 90 95 Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Sir Ile 100 105 110 Ala Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val 115 120 125

Ser Asp Asp Tyr Leu Gin Ser Lys Glu Cys Asp Phe Gin Thr Lys Phe 130 135 140 ·· · ·

Ala 145 Leu Sir Leu Sir Pro 150 Gly Ala His Gin Lys 155 Arg Leu Ile Pro Ile 160 Lys Tyr Lys Ala Met 165 Lys Lys Glu Phe Pro 170 Sir Ile Leu Arg Phe 175 Ile Thr Val Cys Asp 180 Tyr Thr As n Pro Cys 185 Thr Lys Sir Trp Phe 190 Trp Thr Arg Leu Ala Lys Ala Leu Sir Leu Pro

195 200 <210> 7 <211> 654 <212> DNA <213> artificial sequence <220>

<223> myr-TAT-GCN-lin-TIR MyD88 <220>

<221> CDS <222> (1). . (654) <400> 7

atg Met 1 ggc Gly ate Ile ggc Gly aag Lys 5 tcc Sir aag Lys Dad Tyr ggt Gly cgt Arg 10 aaa Lys aaa Lys cgt Arg cgt Arg cag Gin 15 cgt Arg 48 cgt cgt agg atg aag cag ctg gaa gat aag gtg gag gaa ctg ctg tcc 96 Arg Arg Arg Met Lys Gin Leu Glu Asp Lys Val Glu Glu Leu Leu Sir 20 25 30 aag aac tac cac ctg gaa aac gag gtg gcc cgg ctg aag aaa ctg gtc 144 Lys Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val 35 40 45 ggc gag aga ggt acc ggc tcc gag gga aag tcc tet ggc tcc gga tet 192 Gly Glu Arg Gly Thr Gly Sir Glu Gly Lys Sir Sir Gly Sir Gly Sir 50 55 60 gag tcc aag gtg acc gac tet ggc tcc gag acc ggc tcc tet gga tcc 240 Glu Sir Lys Val Thr Asp Sir Gly Sir Glu Thr Gly Sir Sir Gly Sir 65 70 75 80 gca cgt ttc gat gcc ttc ate tgc Dad tgc ccc agc gac ate cag ttt 288 Ala Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Sir Asp Ile Gin Phe 85 90 95 gtg cag gag atg ate cgg caa ctg gaa cag aca aac Dad ego ctg aag 336 Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu Lys 100 105 110 ttg tgt gtg tet gac ege gat gtc ctg cct ggc acc tgt gtc tgg tet 384 Leu Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Sir 115 120 125 att gct agt gag ete ate gaa aag agg tgc ege cgg atg gtg gtg gtt 432 Ile Ala Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val 130 135 140

gtc Val 145 tet Sir gat Asp gat Asp tac Tyr ctg Leu 150 cag Gin agc Sir aag Lys gaa Glu tgt Cys 155 gac Asp ttc Phe cag Gin acc Thr aaa Lys 160 480 ttt gca ete agc ete tet approx ggt gcc cat cag aag ego ctg ate ccc 528 Phe Ala Leu Sir Leu Sir Pro Gly Ala His Gin Lys Arg Leu Ile Pro 165 170 175 ate aag tac aag gca atg aag aaa gag ttc ccc agc ate ctg agg ttc 576 Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu Arg Phe 180 185 190 ate act gtc tgc gac tac acc aac ccc tgc acc aaa tet tgg ttc tgg 624 Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp Phe Trp 195 200 205 act ege ctt gcc aag gcc ttg tcc ctg ccc 654 Thr Arg Leu Ala Lys Ala Leu Sir Leu Pro 210 215

<210> <211> <212> <213> 8 218 PRT artificial sequence <220> <223> synthetic construct <400> 8 Met Gly Ile Gly Lys Ser Lys 1 5 Tyr Gly Arg Lys 10 Lys Arg Arg Gin Arg 15

Arg Arg Arg Met Lys Gin Leu Glu Asp Lys Val Glu Glu Leu Leu Ser 20 25 30

Lys Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val 35 40 45

Gly Glu Arg Gly Thr Gly Ser Glu Gly Lys Ser Ser Gly Ser Gly Ser 50 55 60

Glu Ser Lys Val Thr Asp Ser Gly Ser Glu Thr Gly Ser Ser Gly Ser

70 75 80

Ala Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gin Phe

90 95

Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu Lys 100 105 110

Leu Cys Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser 115 120 125

Ile Ala Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val 130 135 140

Val Ser Asp Asp Tyr Leu Gin Ser Lys Glu Cys Asp Phe Gin Thr Lys 145 150 155 160 • · · ·

Phe Ala Leu Sir Leu 165 Sir Pro Gly Ala His 170 Gin Lys Arg Leu Ile 175 Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu Arg Phe 180 185 190 Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp Phe Trp 195 200 205 Thr Arg Leu Ala Lys Ala Leu Sir Leu Pro

210 215 <210> 9 <211> 666 <212> DNA <213> artificial sequence <220>

<223> TAT-GCN-lin-TIR MyD88 <220>

<221> CDS <222> (1). . (666) <400> 9 atg cat cat cat cat cat cat cat ggt ggt tat ggt cgt aaa aaa cgt cgt 48

Met His His His His His His Gly Gly Tyr Gly Arg Lys Lys Arg Arg 15 10 15 cag cgt cgt cgt ggt ggt agg atg aag cag ctg gaa gat aag gtg gag 96

Gin Arg Arg Arg Gly Gly Arg Met Lys Gin Leu Glu Asp Lys Val Glu

25 30 gaa ctg ctg tcc aag aac tac cac ctg gaa aac gag gtg gcc cgg ctg 144

Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu

40 45 aag aaa ctg gtc ggc gag aga ggt acc ggc tcc gag gga aag tcc tet 192

Lys Lys Leu Val Gly Glu Arg Gly Thr Gly Ser Glu Gly Lys Ser Ser

55 60 ggc tcc gga tet gag tcc aag gtg acc gac tet ggc tcc gag acc ggc 240

Gly Ser Gly Ser Glu Ser Lys Val Thr Asp Ser Gly Ser Glu Thr Gly

70 75 80 tcc tet gga tcc gca cgt ttc gat gcc ttc ate tgc tat tgc ccc agc 288

Ser Ser Gly Ser Ala Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser

90 95 gac ate cag ttt gtg cag gag atg ate cgg caa ctg gaa cag aca aac 336

Asp Ile Gin Phe Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn

100 105 110 tat ega ctg aag ttg tgt gtg tet gac ege gat gtc ctg cct ggc acc 384

Tyr Arg Leu Lys Leu Cys Val Ser Asp Arg Asp Val Leu Pro Gly Thr

115 120 125 tgt gtc tgg tet att get agt gag ete ate gaa aag agg tgc ege cgg 432

Cys Val Trp Ser Ile Ala Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg

130 135 140 atg gtg gtg gtt gtc tet gat gat tac ctg cag agc aag gaa tgt gac 480 Met Val Val Val Val Sir Asp Asp Tyr Leu Gin Sir Lys Glu Cys Asp 145 150 155 160 ttc cag acc aaa ttt gca ete agc ete tet approx ggt gcc cat cag aag 528 Phe Gin Thr Lys Phe Ala Leu Sir Leu Sir Pro Gly Ala His Gin Lys 165 170 175 ego ctg ate ccc ate aag tac aag gca atg aag aaa gag ttc ccc agc 576 Ar g Leu Ile Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir 180 185 190 ate ctg agg ttc ate act gtc tgc gac tac acc aac ccc tgc acc aaa 624 Ile Leu Arg Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys 195 200 205 tet tgg ttc tgg act ege ctt gcc aag gcc ttg tcc ctg ccc 666 Sir Trp Phe Trp Thr Arg Leu Ala Lys Ala Leu Sir Leu Pro

210 215 220 <210> 10 <211> 222 <212> PRT <213> artificial sequence <220>

<223> synthetic construct <400> 10

Met 1 His His His His 5 His His Gly Gly Tyr 10 Gly Arg Lys Lys Arg 15 Arg Gin Arg Arg Arg Gly Gly Arg Met Lys Gin Leu Glu Asp Lys Val Glu 20 25 30 Glu Leu Leu Sir Lys Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu 35 40 45 Lys Lys Leu Val Gly Glu Arg Gly Thr Gly Sir Glu Gly Lys Sir Sir 50 55 60 Gly Sir Gly Sir Glu Sir Lys Val Thr Asp Sir Gly Sir Glu Thr Gly 65 70 75 80 Sir Sir Gly Sir Ala Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Sir 85 90 95 Asp Ile Gin Phe Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn 100 105 110 Tyr Arg Leu Lys Leu Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr 115 120 125 Cys Val Trp Sir Ile Ala Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg

130 135 140

Met Val Val Val Val Asp Asp Tyr Leu Gin Ser Lys Glu Cys Asp

·· ·

145 150 155 160 Phe Gin Thr Lys Phe 165 Ala Leu Sir Leu Sir 170 Pro Gly Ala His Gin 175 Lys Ar g Leu Ile Pro 180 Ile Lys Tyr Lys Ala 185 Met Lys Lys Glu Phe 190 Pro Sir Ile Leu Ar g 195 Phe Ile Thr Val Cys 200 Asp Tyr Thr Asn Pro 205 Cys Thr Lys Sir Trp Phe Trp Thr Ar g Leu Ala Lys Ala Leu Sir Leu Pro

210 215 220 <210> 11 <211> 417 <212> DNA <213> Homo sapiens <220>

<221> CDS <222> (1) .. (417) <400> 11

atg Met 1 gca Ala cgt Ar g ttc Phe gat Asp 5 gcc Ala ttc Phe ate Ile tgc Cys Dad Tyr 10 tgc Cys ccc Pro agc Sir gac Asp ate Ile 15 cag Gin 48 ttt gtg cag gag atg ate cgg caa ctg gaa cag aca aac Dad ego ctg 96 Phe Val Gin Glu Met Ile Ar g Gin Leu Glu Gin Thr Asn Tyr Arg Leu 20 25 30 aag ttg tgt gtg tet gac ege gat gtc ctg cct ggc acc tgt gtc tgg 144 Lys Leu Cys Val Sir Asp Ar g Asp Val Leu Pro Gly Thr Cys Val Trp 35 40 45 tet att get agt gag ete ate gaa aag agg tgc ege cgg atg gtg gtg 192 Sir Ile Ala Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val 50 55 60 gtt gtc tet gat gat tac ctg cag agc aag gaa tgt gac ttc cag acc 240 Val Val Sir Asp Asp Tyr Leu Gin Sir Lys Glu Cys Asp Phe Gin Thr 65 70 75 80 aaa ttt gca ete agc ete tet approx ggt gcc cat cag aag ego ctg ate 288 Lys Phe Ala Leu Sir Leu Sir Pro Gly Ala His Gin Lys Arg Leu Ile 85 90 95 ccc ate aag tac aag gca atg aag aaa gag ttc ccc agc ate ctg agg 336 Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu Arg 100 105 110 ttc ate act gtc tgc gac tac acc aac ccc tgc acc aaa tet tgg ttc 384 Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp Phe 115 120 125 tgg act ege ctt gcc aag gcc ttg tcc ctg ccc 417 Trp Thr Ar g Leu Ala Lys Ala Leu Sir Leu Pro

130 135

<210> 12 <211> 139 <212> PRT <213> Homo sapiens <400> 12

Met 1 Ala Arg Phe Asp 5 Ala Phe Ile Cys Tyr 10 Cys Pro Sir Asp Ile 15 Gin Phe Val Gin Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu 20 25 30 Lys Leu Cys Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp 35 40 45 Sir Ile Ala Sir Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val 50 55 60 Val Val Sir Asp Asp Tyr Leu Gin Sir Lys Glu Cys Asp Phe Gin Thr 65 70 75 80 Lys Phe Ala Leu Sir Leu Sir Pro Gly Ala His Gin Lys Arg Leu Ile 85 90 95 Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Sir Ile Leu Arg 100 105 110 Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Sir Trp Phe 115 120 125 Trp Thr Arg Leu Ala Lys Ala Leu Sir Leu Pro 130 135 <210> 13 <211> 354 <212> DNA <213> Brucella melitensis <220> <221> CDS <222> d) · (354) <400> 13 atg tet aaa gag aaa caa gcc caa tca aaa gcc cac aaa gcc caa caa 48 Met Sir Lys Glu Lys Gin Ala Gin Sir Lys Ala His Lys Ala Gin Gin 1 5 10 15 gct ate agt tca gca aaa tca ete tcc aca cag aaa agc aaa atg tca 96 Ala Ile Sir Sir Ala Lys Sir Leu Sir Thr Gin Lys Sir Lys Met Sir 20 25 30 gag ctt gag ege gcc acg agg gat ggt gcc gcg att ggc aag aag ego 144 Glu Leu Glu Arg Ala Thr Arg Asp Gly Ala Ala Ile Gly Lys Lys Arg 35 40 45 gcg gat ate gcc aaa aaa att gcc gat aag gca aaa cag tta agc tcc 192 Ala Asp Ile Ala Lys Lys Ile Ala Asp Lys Ala Lys Gin Leu Sir Sir 50 55 60

tat cag get aag caa ttc aaa get gat gag cag get gtt aaa aag gtc Tyr Gin Ala Lys Gin Phe Lys Ala Asp Glu Gin Ala Val Lys Lys Val 65 70 75 80 gcg cag gag caa aag cgt tta tca gat gag ego acc aag cat gaa get Ala Gin Glu Gin Lys Arg Leu Sir Asp Glu Arg Thr Lys His Glu Ala 85 90 95 ttc ate aaa caa tca ttg agc tcc atg ego aca act gcg agc gcg act Phe Ile Lys Gin Sir Leu Sir Sir Met Arg Thr Thr Ala Sir Ala Thr 100 105 110 atg gaa gca gaa gaa gaa Met Glu Ala Glu Glu Glu 115 <210> 14 <211> 118 <212> PRT <213> Brucella melitensis <400> 14 Met Ser Lys Glu Lys Gin Ala Gin Sir Lys Ala His Lys Ala Gin Gin 1 5 10 15 Ala Ile Sir Sir Ala Lys Sir Leu Sir Thr Gin Lys Sir Lys Met Sir 20 25 30 Glu Leu Glu Arg Ala Thr Arg Asp Gly Ala Ala Ile Gly Lys Lys Arg 35 40 45 Ala Asp Ile Ala Lys Lys Ile Ala Asp Lys Ala Lys Gin Leu Sir Sir 50 55 60 Tyr Gin Ala Lys Gin Phe Lys Ala Asp Glu Gin Ala Val Lys Lys Val 65 70 75 80 Ala Gin Glu Gin Lys Arg Leu Sir Asp Glu Arg Thr Lys His Glu Ala 85 90 95 Phe Ile Lys Gin Sir Leu Sir Sir Met Arg Thr Thr Ala Sir Ala Thr 100 105 110 Met Glu Ala Glu Glu Glu 115 <210> 15 <211> 99 <212> DNA <213> synthetic sequence <220> <221> CDS <222> (D.  (99) <400> 15 agg atg aag cag ctg gaa gat aag gtg gag gaa ctg ctg tcc aag aac Arg Met Lys Gin Leu Glu Asp Lys Val Glu Glu Leu Leu Sir Lys Asn 1 5 10 15

• ·

tac cac ctg Tyr His Leu aga Arg gaa Glu 20 aac Asn gag Glu gtg Val gcc Ala cgg Arg 25 ctg aag aaa Lys ctg Leu gtc Val 30 ggc Gly gag Glu 96 99 Leu Lys <210> 16 <211> 33 <212> PRT <213> synthetically arrests <400> 16 Arg Met Lys 1 Gin Leu 5 Glu Asp Lys Val Glu 10 Glu Leu Leu Sir Lys 15 Asn Tyr His Leu Arg Glu 20 Asn Glu Val Ala Arg 25 Leu Lys Lys Leu Val 30 Gly Glu

<210> 17 <211> 75 <212> DNA <213> synthetic sequence <220> <221> CDS <222> d) ·. (75) <400> 17 ggc tcc gag gga aag tcc tet ggc tcc gga tet gag tcc aag gtg acc 48 Gly Ser Glu Gly Lys Sir Ser Gly Sir Gly Sir Glu Sir Lys Val Thr 1 5 10 15 gac tet ggc tcc gag acc ggc tcc tet 75 Asp Ser Gly Sir Glu Thr Gly Sir Sir 20 25 <210> 18 <211> 25 <212> PRT <213> synthetic sequence <400> 18 Gly Ser  Glu Gly Lys Sir Ser Gly Sir Gly Sir Glu Sir Lys Val Thr 1 5 10 15

Asp Ser Gly Ser Glu Thr Gly Ser Ser 20 25 <210> 19 <211> 21 <212> DNA <213> Homo sapiens

<220> <221> <222> CDS (D · · (21) <400> 19 atg ggc ate ggc aag tcc aag Met Gly Ile Gly Lys Ser Lys 1 5 <210> 20 <211> 7 <212> PRT <213> Homo sapiens <400> 20 Met Gly  Ile Gly Lys Ser Lys

5 <210> 21 <211> 33 <212> DNA <213> synthetic sequence <220>

<221> CDS <222> (1) .. (33) <400> 21 tat ggt cgt aaa aaa cgt cgt cag cgt cgt cgt Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg 15 10 <210> 22 <211> 11 <212> PRT <213> synthetic sequence <400> 22

Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Arg 15 10 • ·

Claims (17)

A fusion polypeptide consisting of a TIR domain and a dimerization domain, characterized in that the TIR domain and the dimerization domain are linked to a binding peptide. A fusion polypeptide according to claim 1, characterized in that the dimerization domain is selected from the dimerization domains that allow spontaneous dimerization. Fusion polypeptide according to claim 2, characterized in that the dimerization domain is selected from segments for the formation of coiled helices, consisting of 3 to 12 heptads, are in parallel orientation and may form homodimers, preferably GCN4-pl, in particular SEQID: 16. A fusion polypeptide according to claim 1, characterized in that the dimerization domain is selected from the dimerization domains that allow ligand-induced dimerization. A fusion polypeptide according to any one of claims 1 to 4, characterized in that the TIR domain is selected from homologous animal, human or viral domains of Toll / Interleukin-1 receptor TLR receptors, Tollu-like receptors, receptors of the Interleukin-1 family receptor, viral proteins containing the TIR domain. 6. The fusion polypeptide of claim 5, wherein the TIR domain is selected from the TIR MyD88, TRIF, TRAM, TIRAP and SARM domains, preferably the TIR MyD88 domain, in particular SEQ ID NO: 12. A fusion polypeptide according to any one of claims 1 to 6, characterized in that the binding peptide is typically from 3 to 40 amino acids long, preferably from 15 to 30 amino acids, most preferably 25 amino acids and amino acids are preferably selected from , but not limited to serine, glycine, threonine, proline, valine, alanine, glutamine, asparagine, glutamate, aspartate. A fusion polypeptide consisting of a TIR domain and a dimerization domain according to any one of claims 1 to 7, characterized in that the dimerization segment is on the N-terminal portion of the TIR domain. • · · · A fusion polypeptide composed of a TIR domain and a dimerization domain according to any one of claims 1 to 8, characterized in that the fusion polypeptide composed of the TIR domain and the dimerization domain comprises a membrane localization segment. A fusion polypeptide composed of a TIR domain and a dimerization domain according to any one of claims 1 to 9, characterized in that the fusion polypeptide comprised of the TIR domain and the dimerization domain optionally contains a protein transduction domain selected from cationic peptide sequences, such as PTD of HIV-1 TAT protein, VP22 DNA-binding protein of herpes simplex virus 1 (HSV-1) and Antennapedia (Antp) homeotic transcription factor of fly wine, preferably PTD of HIV-1 TAT protein, in particular SEQ ID NO: 22. A nucleic acid encoding a fusion polypeptide composed of a TIR domain and a dimerization domain according to any one of claims 1 to 10, characterized in that the DNA is operatively linked to regulatory elements, a promoter and a terminator that provide expression of the fusion polypeptide composed of the domain TIRs and dimerization domains in host cells / organisms. 12. Nucleic acid-containing host cells / organisms expressing a fusion polypeptide composed of a TIR domain and a dimerization domain according to any one of claims 1 to 10. A fusion polypeptide according to any one of claims 1 to 10, characterized in that the fusion polypeptide consisting of the TIR domain and the dimerization domain is produced, isolated and purified from host cells / organisms according to claim 12. 14. The fusion polypeptide of claim 13 for use in the manufacture of a medicament for the treatment of inflammatory conditions such as bacterial, viral, fungal infectious diseases and infectious diseases caused by yeast, sepsis and septic shock, autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Chron's disease, ulcerative colitis, insulin resistance, cancer, neuropathic pain, neurological inflammation, alcohol-induced inflammation, ischemia, hypoxia, hemorrhage, atherosclerosis, asthma, allergy, transplant rejection, ankylosing spondylitis or non-emergent ankylosing spondylitis overexpressing TLRs or treating a pathway whose development involves IL-1R / MyD88 signaling. • * A nucleic acid according to claim 11, which encodes a fusion polypeptide consisting of a TIR domain and a dimerization domain according to any one of claims 1 to 9, which is used for the preparation of a medicament for the treatment of inflammatory conditions such as bacterial, viral, fungal infectious diseases and infectious diseases caused by yeasts, sepsis and septic shock, autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Chron's disease, ulcerative colitis, insulin resistance, cancer, neuropathic pain, neurological inflammation, alcohol-induced hemorrhage, ischemia, ischemia , atherosclerosis, asthma, allergy, graft rejection, ankylosing spondylitis resulting from inappropriate and / or excessive TLR signaling, or for treatment of a pathway whose development involves targeting IL-1R / MyD88 signaling, including but not limited to not limited to lipofection, microinjection, biolistics, immunolipofection, electroporation, gene bombardment m, viral input. The fusion polypeptide of claim 13 for inhibiting cellular TLR / IL-1R signaling in human or animal cells. The nucleic acid of claim 11 for inhibiting cellular TLR / IL-1R signaling in human or animal cells.