Granted Patent B2

US 12,735,472 · App. 17/617,137 · Granted Sep 15, 2026

Inventors: Roman Fischer (Nuremberg, DE); Martin Siegemund (Stuttgart, DE); Klaus Pfizenmaier (Tiefenbronn, DE); Roland Kontermann (Nurtingen, DE)

Assignee: UNIVERSITÄT STUTTGART

C07K14/70575A61K38/191C07K14/525C07K19/00A61K38/00C07K2319/30C07K2319/70C07K2319/74

Abstract

Claims (46)

1 . A polypeptide, comprising a binding domain consisting of three peptide tumor necrosis factor (TNF) homology domains of TNF-ligand family member proteins (THD) that specifically bind to the extracellular part of TNF receptor 2 (TNFR2), wherein the C-terminus of the first and second THD, respectively, which is in each case defined by the C-terminal sequence

(SEQ ID NO: 3)

V-Y-F-G-I-I,

is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal sequence

(SEQ ID NO: 4)

P-V-A-H-V

through a peptide X a , which is in each case independently selected and has a length of 9 to 10 amino acids, wherein the peptide X a consists of

X C -X L -X N

wherein

X C is A-L;

X L is absent or is selected from the group consisting of G, G-G, G-G-G, and G-G-G-G (SEQ ID NO: 16);

X N is selected from the group consisting of P-S-D-K (SEQ ID NO: 6), T-P-S-D-K (SEQ ID NO: 7), R-T-P-S-D-K (SEQ ID NO: 8), and S-R-T-P-S-D-K (SEQ ID NO: 9), and

wherein the THD comprises a contiguous amino acid sequence consisting of amino acids 88 to 231 of full length human TNF alpha of SEQ ID NO: 5, but comprising the mutations D143N and A145R of the ectodomain of human TNF alpha corresponding to D219N and A221R, respectively, relative to SEQ ID NO: 5.

2 . The polypeptide according to claim 1 , wherein the three THDs are identical.

3 . The polypeptide according to claim 2 , wherein:

(i) X L is absent and X N is S-R-T-P-S-D-K (SEQ ID NO: 9);

(ii) X L is G-G-G-G (SEQ ID NO: 16) and X N is P-S-D-K (SEQ ID NO: 6);

(iii) X L is G and X N is selected from R-T-P-S-D-K (SEQ ID NO: 8), and S-R-T-P-S-D-K (SEQ ID NO: 9);

(iv) X L is G-G and X N is selected from T-P-S-D-K (SEQ ID NO: 7), and R-T-P-S-D-K (SEQ ID NO: 8); or

(v) X L is G-G-G and X N is selected from P-S-D-K (SEQ ID NO: 6), and T-P-S-D-K (SEQ ID NO: 7).

4 . The polypeptide according to claim 1 , wherein the polypeptide has an onset of aggregation temperature (T m ) of more than 62° C. as determined by dynamic light scattering.

5 . A polypeptide multimer comprising at least two polypeptides according to claim 1 that are

(a) linked together; or

(b) linked to a protein selected from the group consisting of: a multimerization domain, a serum protein, a cytokine, a targeting moiety and a toxin.

6 . The polypeptide multimer according to claim 5 , wherein:

A. the polypeptide multimer has at least one of the following properties:

(i) an onset of aggregation temperature (T m ) of at least 72° C.;

(ii) an EC 50 for binding to TNFR2 in HeLa-TNF-R2 cells that is not decreased by more than 15%, 12%, or 10%, after 8 days of incubation in human plasma at 37° C.;

(iii) an EC 50 for binding to TNFR2 expressed on MEFs of less than 100 pM;

(iv) an EC 50 for binding to TNFR2 on Kym-1 cells of less than 200 pM;

(v) an EC 50 for activation of NF-κB in HeLa-TNF-R2 cells of less than 30 pM; and/or

B. the polypeptide multimer further comprises a ligand specific for an organ, tissue or cell-type.

7 . The polypeptide multimer according to claim 5 , wherein said polypeptides are:

(a) linked together by an amino acid linker that has a length of between 1 to 30 amino acids or between 7 to 15 amino acids; or

(b) linked to said protein by an amino acid linker that has a length of between 1 to 30 amino acids.

8 . The polypeptide multimer according to claim 5 , wherein the at least two polypeptides are linked to a protein that is a multimerization domain, and wherein the multimerization domain is a dimerization domain, a trimerization domain or a tetramerization domain.

9 . The polypeptide multimer according to claim 8 , wherein the dimerization domain is selected from the group consisting of an antibody, an antibody heavy chain, immunoglobulin Fc region, heavy chain domain 2 (CH2) of IgM (MHD2), heavy chain domain 2 (CH2) of IgE (EHD2), heavy chain domain 3 (CH3) of IgG, heavy chain domain 3 (CH3) of IgA, heavy chain domain 3 (CH3) of IgD, heavy chain domain 4 (CH4) of IgM, heavy chain domain 4 (CH4) of IgE, Fab, Fab 2 , leucine zipper motifs, barnase-barstar dimers, miniantibodies, and ZIP miniantibodies.

10 . The polypeptide multimer according to claim 8 , wherein the

(i) the trimerization domain is 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

(ii) the tetramerization domain is 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.

11 . The polypeptide multimer according to claim 8 , wherein the dimerization domain is selected from the group consisting of FcΔab, LALA, LALA-GP, IgG2, IgG2σ, aglycosylated IgG1, IgG1 L234F/L235E/P331S, IgG2m4, and IgG4 ProAlaAla.

12 . A nucleic acid molecule encoding the polypeptide according to claim 1 .

13 . A vector comprising the nucleic acid molecule according to claim 12 .

14 . A pharmaceutical composition comprising as an active agent a polypeptide according to claim 1 .

15 . A method of treating, hyperproliferative disorders, inflammatory disorders, neurodegenerative disorders, neuropathic diseases, neurological insults autoimmune disorders, or metabolic disorders in a subject, the method comprising: administering to the subject in need thereof an effective amount of a polypeptide according to claim 1 or a nucleic acid molecule encoding the polypeptide according to claim 1 .

16 . The method of claim 15 , wherein the hyperproliferative disorders are cancer or malignancies of the hematologic system, or the metabolic disorders are metabolic syndromes or cardiovascular diseases.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (166)