Bimer or an oligomer of a dimer, trimer, quatromer or pentamer of recombinant fusion proteins

Abstract

The invention relates to oligomers of a dimer, trimer, quatromer or pentamer of recombinant fusion proteins. Said oligomers are characterized in that the recombinant fusion proteins have at least one component A and at least one component B, whereby component A contains a protein or a protein segment with a biological function, in particular with a ligand function for antibodies, for soluble or membranous signal molecules, for receptors or an antibody, or an antibody segment, and component B contains a protein or a protein segment which dimerizes or oligomerizes the dimer, trimer, quatromer or pentamer of the recombinant fusion protein, without the action of third-party molecules. The invention also relates to the use of dimers or oligomers of this type for producing a medicament, to the fusion proteins which cluster in dimers or oligomers and to their DNA sequence and expression vectors or host cells comprising this DNA sequence.

1 . A bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins characterized by the recombinant fusion proteins having at least one component A and at least one component B, wherein component A comprises a protein or a protein segment with biological function, in particular with ligand function for antibodies, for soluble or membrane-bound signal molecules or for receptors or an antibody or segment of an antibody and component B comprises a protein or a protein segment selected from the group consisting of the family of C1q proteins and the collecting, which bimerizes or oligomerizes the dimer, trimer, quadromer or pentamer of the recombinant fusion protein without the effect of third molecules. 
     
     
         2 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component A of the recombinant fusion proteins in the dimer, trimer, quadromer or pentamer being identical or different. 
     
     
         3 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component A of the recombinant fusion proteins being a peptide hormone, a growth factor, a cytokine, an interleukin, a segment thereof or a functional derivative of the above-mentioned sequences. 
     
     
         4 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component A of the recombinant fusion proteins being a cytokine from the family of TNF cytokines, a segment of such a TNF cytokine or a functional derivative of the above-mentioned sequences. 
     
     
         5 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 4  characterized by the component A of the recombinant fusion proteins being a TNF cytokine or a segment of a TNF cytokine selected from the group consisting of CD40L, FasL, TRAIL, TNF-a, CD30L, OX40L, RANKL, TWEAK, Lta, Ltab2, LIGHT, CD27L, 41-BB, GITRL, APRIL, EDA, VEGI and BAFF, or a functional derivative of the above-mentioned sequences. 
     
     
         6 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component A of the recombinant fusion proteins being an antigen or a segment of an antigen of viral, bacterial or protozoological pathogens. 
     
     
         7 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 6  characterized by the component A of the recombinant fusion proteins being a surface antigen or a segment of a surface antigen of a viral, bacterial or protozoological pathogen. 
     
     
         8 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component A of the recombinant fusion proteins being an amino acid sequence coupled to a receptor agonist or to a receptor antagonist. 
     
     
         9 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component B of the recombinant fusion proteins containing a multimerizing and oligomerizing segment or a functional derivative of such a segment of a protein, selected from the group consisting of C1q, MBP, SP-A, SP-D, BC, CL43 and ACRP30, or containing the collagen domain of the EDA protein or a functional derivative thereof. 
     
     
         10 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 9  characterized by the component B containing an amino acid sequence, as shown in  FIG. 6A  for the sequence of amino acids 18 to 111 of mACRP30 or a functional derivative thereof, or containing an amino acid sequence, as shown in  FIG. 6B  for the sequence of amino acids 18 to 108 of hACRP30 or a functional derivative thereof, whereby  FIGS. 6A and 6B , respectively is part of the claim. 
     
     
         11 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the component B containing a sequence of 8 to 20 amino acids which bimerizes or oligomerizes dimers or multimers of recombinant fusion proteins. 
     
     
         12 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 11  characterized by the component B forming one or more disulfide bridges. 
     
     
         13 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 12  characterized by the component B containing the amino acid sequence LEGSTSNGRQCAGIRL or a functional derivative of this sequence. 
     
     
         14 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 11  characterized by the component B containing the sequence LEGSTSNGRQSAGIRL or a functional derivative of this sequence. 
     
     
         15 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the multimerized and oligomerized fusion proteins having antigenic segments for detecting and crosslinking by antibodies. 
     
     
         16 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the recombinant fusion proteins having a linker sequence between component A and component B. 
     
     
         17 . The bimer or oligomer of dimers, trimers, quadromers or pentamers of recombinant fusion proteins according to  claim 1  characterized by the recombinant fusion proteins having two or more different components B. 
     
     
         18 . A medicament comprising a pharmaceutical composition of a bimer or oligomer according to  claim 1 . 
     
     
         19 . Method of treatment of a disorder or disease comprising administration of a bimer or oligomer according to claim wherein the disorder or disease is a hyperinflammatory disorders, autoimmune diseases, diseases based on hyperapoptotic or hypoapoptotic disorders, infectious diseases, viral infectious diseases, tumors, in particular tumors of the lymphatic system, and/or endocrinological disorders. 
     
     
         20 . A vaccine comprising a bimer or oligomer according to  claim 1  wherein the vaccine is for active or passive immunization against infectious diseases. 
     
     
         21 . A vaccine according to  claim 20  for vaccination against German measles, measles, poliomyelitis, rabies, tetanus, diphtheria, BCG, malaria, yellow fever, HIV or influenza. 
     
     
         22 . A vaccine according to  claim 20  which is formulated for parenteral or oral administration. 
     
     
         23 . A method for in-vitro diagnosis comprising applying a bimer or oligomer according to  claim 1  to an in-vitro protein-based derivative. 
     
     
         24 . A fusion protein characterized by the recombinant fusion protein containing a component A and a component B, whereby component A contains a protein or a protein segment with biological function, in particular ligand function for antibodies or receptors or an antibody or segment of an antibody or for soluble or membrane-bound signal molecules, and component B contains a multimerizing and oligomerizing segment or a functional derivative of such a segment of a protein, selected from the group consisting of the family of the C1q proteins and the collecting. 
     
     
         25 . The fusion protein according to  claim 24  characterized by the component B of the fusion protein containing a multimerizing and oligomerizing segment of a protein or a functional derivative of the same, selected from the group consisting of C1q, MBP, SP-A, SP-D, BC, CL43 and ACRP30, or containing the collagen domain of the EDA protein or a functional derivative thereof. 
     
     
         26 . A DNA sequence characterized by the DNA sequence encoding the fusion protein according to  claim 24 . 
     
     
         27 . An expression vector characterized by the expression vector containing the DNA sequence according to  claim 26 . 
     
     
         28 . A host cell characterized by the host cell being transfected with an expression vector according to  claim 27 . 
     
     
         29 . A soluble multimeric polypeptide of at least two trimer units, wherein each trimer unit comprises a fusion protein trimer strand consisting of: a first polypeptide comprising the first about 100 to 250 N-terminus residues of a collectin family scaffold protein, wherein the first polypeptide comprises a hub and a body region of the collectin family scaffold protein; and a second polypeptide comprising the last about 100 to 250 C-terminus residues of a tumor necrosis factor superfamily (TNFSF) ligand, wherein the second polypeptide comprises an extracellular domain (ECD) of the TNFSF ligand, wherein the carboxy-terminal residue of the first polypeptide is operably linked to the amino-terminal residue of the second polypeptide via: i) deletion of a carbohydrate recognition domain (CRD) of the collectin family scaffold protein and ii) replacement of the CDR with the ECD of the TNFSF ligand, whereby a single trimer strand spontaneously trimerizes with two additional trimer strands to form a trimer unit and the trimer unit binds at the hub to form the multimeric polypeptide. 
     
     
         30 . The multimeric polypeptide of  claim 29 , wherein the TNFSF ligand is selected from lymphotoxin-A (LTA), lymphotoxin-B (LTB), tumor necrosis factor (TNF), or any of TNFSF4-15 and TNFSF18. 
     
     
         31 . The multimeric polypeptide of  claim 29 , wherein the collectin family scaffold protein is selected from complement factor 1 (C1q), mannose binding protein, mannose-binding lectin type 1 (MBL1), mannose-binding lectin type 2 (MBL2), pulmonary surfactant protein A (SPA), pulmonary surfactant protein D (SPD), conglutinin, collectin 43, C-type lectin L1 (CL-L1), adipocyte complement related protein of 30 kDa (ACRP30), or hibernation specific protein 27 (Hib27). 
     
     
         32 . The multimeric polypeptide of  claim 29 , wherein the trimer unit comprises homomeric trimer strands. 
     
     
         33 . The multimeric polypeptide of  claim 29 , wherein the trimer unit comprises heteromeric trimer strands. 
     
     
         34 . The multimeric polypeptide of  claim 29 , wherein the collectin family scaffold protein is surfactant protein D. 
     
     
         35 . The multimeric polypeptide of  claim 29 , wherein the TNFSF ligand is CD40L. 
     
     
         36 . The multimeric polypeptide of  claim 29 , wherein the trimer strand is SPD-CD40L. 
     
     
         37 . The multimeric polypeptide of  claim 29 , wherein the TNFSF ligand is receptor activator of NF-kappaB ligand (RANKL). 
     
     
         38 . The multimeric polypeptide of  claim 29 , wherein the trimer strand is SPD-RANKL. 
     
     
         39 . The multimeric polypeptide of  claim 29 , wherein the TNFSF ligand is CD27L/CD70. 
     
     
         40 . The multimeric polypeptide of  claim 29 , wherein the trimer strand is SPD-CD27L/CD70. 
     
     
         41 . The multimeric polypeptide of  claim 29 , wherein amino acid residues comprising the trimerized strands which are susceptible to proteolytic degradation are removed from the multimeric polypeptide. 
     
     
         42 . The multimeric polypeptide of  claim 29 , wherein the multimer is a dimer of trimer units. 
     
     
         43 . A soluble multimeric polypeptide of at least two trimer units, wherein each trimer unit comprises a fusion protein trimer strand comprising a first polypeptide comprising a multimerizing and oligomerizing segment of an extracellular matrix protein having a carbohydrate recognition domain (CRD) and wherein the protein is selected from the group consisting of the family of C1q proteins and collectin family proteins; and a second polypeptide of a membrane-bound signal protein comprising an extracellular domain (ECD), wherein the carboxy-terminal residue of the first polypeptide is operably linked to the amino-terminal residue of the second polypeptide via: i) deletion of a carbohydrate recognition domain (CRD) of the first polypeptide and ii) replacement of the CRD with the ECD of the second polypeptide, whereby a single trimer strand spontaneously trimerizes with two additional trimer strands to form a trimer unit and the trimer unit binds to form the multimeric polypeptide. 
     
     
         44 . The multimeric polypeptide of  claim 43  wherein the extracellular matrix protein is cartilage oligomeric matrix protein (COMP) or cartilage matrix protein (CMP), C1q, collagen α 1 (X), collagen α2(VII), ACRP30, the internal ear structure protein, cerebelin, multimerin, Lung surfactant protein (SP-A), mannose binding protein (MBP), lung surfactant protein (SP-D), bovine serum conglutinin (BC), or bovine collectin-43 (CL43). 
     
     
         45 . The multimeric polypeptide of  claim 43  wherein the membrane-bound signal protein comprising an extracellular domain (ECD) is a TNF cytokine. 
     
     
         46 . The multimeric polypeptide of  claim 45  wherein the TNF cytokine is CD40L, FasL, TRAIL, TNF, CD30L, OX40L, RANKL, TWEAK, Lta, Ltab2, LIGHT, CD27L, 41-BB, GITRL, APRIL, EDA, BEGI or BAFF. 
     
     
         47 . The multimeric polypeptide of  claim 45  further comprising a peptide linker sequence between the first and second polypeptide.