Tumor necrosis factor receptor (TNFR) binding protein complex with improved binding and bioactivity

1. A tumor necrosis factor receptor (TNFR) binding protein complex comprising 12 or more protein ligands (PLs) that specifically bind to the extracellular part of the same TNFR.

2. The TNFR binding protein complex of claim 1 , comprising between 2 to 18 PLs.

3. The TNFR binding protein complex of claim 1 , wherein between 2 to 6 PLs, form a protein ligand group (PLG) with the following structure:

PL 1 -L1-PL 2 -L1-PL 3 -L1-PL 4 -L1-PL 5 -L1-PL 6 ,

wherein

any of PL 4 to PL 6 , and/or L1 may be absent or present, L1 in each case independently means a peptide linker.

4. The TNFR binding protein complex of claim 3 , comprising between 2 to 6 PLGs and each PLG comprising between 2 to 6 PLs.

5. The TNFR binding protein complex of claim 3 , wherein the PLGs are linked to each other through a peptide linker 2 (L2) to form a PLG-multimer.

6. The TNFR binding protein complex of claim 3 , further comprising two or more polymerization domains (PD).

7. The TNFR binding protein complex of claim 6 , wherein the two or more PD are selected from the group consisting of dimerization domains, trimerization domains or tetramerization domains.

8. The TNFR binding protein complex of claim 1 , wherein each PL is independently of each other selected from the group consisting of a TNF homology domain of a TNF-ligand family member protein (THD), a scaffold-protein and a peptidomimetic.

9. The TNFR binding protein complex of claim 1 , wherein, when at least one of the PLs is a TNF-ligand family member protein (THD), the TNF-ligand family member protein is TNF or LTA.

10. The TNFR binding protein complex of claim 8 , wherein, when at least one of the PLs is a TNF homology domain of a TNF-ligand family member protein (THD) the C-terminus of a first THD, which is defined by a C-terminal consensus sequence

(SEQ ID NO: 1)

-S/T/V-F/Y/S-F-G-A/L/V/I-X 1 ,

is linked to the N-terminus of a second THD, which is defined by an N-terminal consensus sequence

(SEQ ID NO: 2)

X 2 -V/A/F-A-H-V/L/I/Y 

or

(SEQ ID NO: 3)

X 3 -V/W/F/C-A/L-E/Y/Q/H-L,

through L1, which has a length of 2 to 20 amino acids;

wherein X 1 is a non-polar/hydrophobic or polar/neutral amino acid;

wherein X 2 is selected from the group consisting of P, K, V, I, and A; and

wherein X 3 is selected from the group consisting of D, S, M, and I;

optionally further comprising one to four further THDs each consecutively linked to each other in the same way as the first and second THD.

11. The TNFR binding protein complex of claim 1 , wherein the TNFR is TNFR 2.

12. The TNFR binding protein complex of claim 6 , wherein the two or more polymerization domains (PD) are linked via their N- and/or C-terminus to a PLG or a PLG-multimer through a peptide linker 3 (L3).

13. The TNFR binding protein complex of claim 7 , wherein

(i) when the two or more PD are dimerization domains, the dimerization domains are selected from the group consisting of heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), immunoglobulin Fc region, heavy chain domain 3 (CH3) of IgG or IgA, heavy chain domain 4 (CH4) of IgM or IgE, Fab, Fab2, leucine zipper motifs, barnase-barstar dimers, miniantibodies, and ZIP miniantibodies;

(ii) when the two or more PDs are trimerization domains, the trimerization domains are selected from the group consisting of tenascin C (TNC), the trimerization region of the C-terminal noncollagenous domain (NC1) of collagen XVIII, fab3-like molecules, and TriBi-minibodies; or

(iii) when the two or more PD are tetramerization domains, the tetramerization domains are selected from the group consisting of the tetramerization domain of p53, the tetramerization domain of the general control protein 4 (GCN4), the tetramerization domain of VASP (vasodilator stimulated phosphoprotein), tandem diabodies, and di-diabodies.

14. The TNFR binding protein complex of claim 8 , wherein, when at least one of the PL is a TNF homology domain of a TNF-ligand family member protein (THD), the TNF-ligand family member protein is selected from the group consisting of TNF, TNF-related apoptosis inducing ligand (TRAIL or TNFSF10, tumor necrosis factor superfamily member), CD40L (TNFSF5), CD27L (TNFSF7), CD30L (TNFSF8), FasL (TNFSF6), 4-1BBL (TNFSF9), OX40L (TNFSF4), EDAM, LTA (TNFSF1), LTB (TNF SF3), CD153 (TNF SF 8), RANKL (TNF SF 11), TWEAK (TNF SF 12), APRIL (TNFSF13), BAFF (TNFSF13B), LIGHT (TNFSF14), VEGI (TNFSF15), and GITRL (TNFSF18).

15. The TNFR binding protein complex of claim 9 , wherein, when at least one of the PLs is TNF, the TNF comprises a sequence according to SEQ ID NO: 43 which comprises one or more TNFR2 specific mutations selected from the group consisting of D143Y, D143F, D143E, D143N, E146Q, E146H, E146K A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R//S147T, E146D/S147D, E146N/5147, K65W, D143N, D143E, D143F, D143W, D143Y, D143V, D143V/F144L/A145S, D143N/A145R, D143V/A145S, A145R, A145H, A145K, A145F, and A145W.

16. The TNFR binding protein complex of claim 10 , wherein L1 has a length of 2 to 15 amino acids, and wherein X 1 is selected from the group consisting of F, V, Q, A, I, L, and Y.

17. A pharmaceutical composition comprising as an active agent the TNFR binding protein complex according to claim 1 .

18. A nucleic acid encoding the TNFR binding protein complex according to claim 1 or a PLG comprised therein.

19. A vector comprising the nucleic acid according to claim 18 .