Internalized bound molecules

Abstract

The present invention provides a conjugation molecule comprising at least two single variable antibody domains that target receptors present on fibrogenic effector cells, including activated myofibroblasts and similar and / or related profibrosis cells and / or contractile cells. [Solution] The present invention relates to an internalized binding molecule for cell-specific targeted delivery of anti-fibrotic substances. The present invention also relates to a binding molecule comprising at least two single variable antibody domains, each targeting a receptor on fibrillating effector cells. The present invention further relates to nucleic acids encoding such binding molecules, host cells for the expression of such binding molecules, and methods for preparing such binding molecules. The present invention further relates to pharmaceutical compositions comprising such binding molecules, and the use of such binding molecules and / or pharmaceutical compositions, in particular, for preventive, therapeutic, or diagnostic purposes.

Claims

1. A conjugation molecule comprising at least two monovariable antibody domains and at least one diagnostic or therapeutic molecule, wherein the at least two monovariable antibody domains can specifically bind to transmembrane receptors expressed on fibrogenic effector cells, and the fibrogenic effector cells are characterized by the expression of alpha smooth muscle actin and / or stress fibers that can be identified by immunofluorescence microscopy, for example, one or more of the following precursor cells of activated: portal vein fibroblasts, hepatic stellate cells, pericytes, alveolar smooth muscle cells, alveolar fibroblasts, stromal fibroblasts, mesangial cells, cutaneous fibroblasts, mucosal smooth muscle cells, intestinal (muscle) fibroblasts, Cajal's stromal cells, or tumor stromal fibroblasts in cancer, preferably activated: alveolar smooth muscle cells in the lung A binding molecule comprising: a fibrogenic effector cell selected from one or more of the following precursor cells: alveolar fibroblasts and / or pericytes in the lungs; stromal fibroblasts in the kidneys; pericytes and / or mesangial cells in the kidneys; dermal fibroblasts and / or pericytes in the skin; mucosal smooth muscle cells in the intestines; intestinal (muscle) fibroblasts in the intestines; Cajal's stromal cells in the intestines; or pericytes in the intestines; or tumor stromal fibroblasts in cancer, wherein the at least two single variable antibody domains are variable domains (VHH) of heavy chain-only antibodies, and the transmembrane receptor is either an insulin-like growth factor 2 receptor (IGF2R) or a platelet-derived growth factor β receptor (PDGFRB).

2. The binding molecule according to claim 1, wherein the transmembrane receptor is insulin-like growth factor 2 receptor (IGF2R).

3. The binding molecule according to claim 1 or 2, comprising a first VHH capable of specifically binding to IGF2R, and a second VHH capable of specifically binding to IGF2R.

4. The binding molecule according to claim 2 or 3, comprising at least two single variable antibody domains, preferably two single variable antibody domains, which can independently and specifically bind to the transmembrane receptor IGF2R expressed on the fibrinoplastic effector cells.

5. A multiparatopic binding molecule, preferably a biparatopic binding molecule, wherein the binding of a first VHH contained in the binding molecule to IGF2R preferably enhances the binding of a second VHH contained in the binding molecule to IGF2R, preferably the reverse, and / or preferably the binding of the first VHH and the second VHH to IGF2R enhances the internalization of the binding molecule compared to the internalization of a second binding molecule containing only the first VHH or the second VHH.

6. A multiparatopic binding molecule, preferably a biparatopic binding molecule, wherein the binding of the first VHH to IGF2R expressed on cells of a first species and to IGF2R expressed on cells of a second species enhances the binding of the second VHH to IGF2R expressed on cells of a first species and to IGF2R expressed on cells of a second species, preferably the reverse as well, according to any one of claims 3 to 5.

7. The biparatopic binding molecule according to any one of claims 3 to 6, wherein at least one, preferably both, of the first VHH and the second VHH can compete for binding to IGF2R with one or more single-domain antibodies having sequences of 13A8 having SEQ ID NO: 257, 13A12 having SEQ ID NO: 289, 13C11 having SEQ ID NO: 297, 13E8 having SEQ ID NO: 249, 13F11 having SEQ ID NO: 273, or 13G10 having SEQ ID NO: 305, and preferably both the first VHH and the second VHH do not compete with the same single-domain antibody having sequences of 13A8, 13A12, 13C11, 13E8, 13F11, or 13G10.

8. A first VHH capable of specifically binding to IGF2R, wherein the first VHH comprises the CDR1 sequence described in SEQ ID NO: 251, the CDR2 sequence described in SEQ ID NO: 253, and the CDR3 sequence described in SEQ ID NO: 255, and a second VHH capable of specifically binding to IGF2R, wherein the second VHH comprises the CDR1 sequence described in SEQ ID NO: 275, the CDR2 sequence described in SEQ ID NO: 277, and the CDR3 sequence described in SEQ ID NO: 279; or the CDR1 sequence described in SEQ ID NO: 259, the CDR2 sequence described in SEQ ID NO: 261, and the CDR3 sequence described in SEQ ID NO: 263; or the CDR1 sequence described in SEQ ID NO: 291, the CDR2 sequence described in SEQ ID NO: 293, and the CDR3 sequence described in SEQ ID NO: 295; or the CDR1 sequence described in SEQ ID NO: 299 A binding molecule according to any one of claims 3 to 7, comprising: the CDR2 sequence described in SEQ ID NO: 301 and the CDR3 sequence described in SEQ ID NO: 303; or the CDR1 sequence described in SEQ ID NO: 307, the CDR2 sequence described in SEQ ID NO: 309 and the CDR3 sequence described in SEQ ID NO: 311; or any combination of CDR1, CDR2 and CDR3 sequences in which at least four amino acids, preferably at least three, more preferably at least two, and most preferably at least one amino acid are conservatively substituted independently; or any one combination of CDR1, CDR2 and CDR3 sequences in which 85 to 99% of amino acids, preferably 90 to 99%, more preferably 95 to 99%, and most preferably 97 to 99% of amino acids are conservatively substituted independently.

9. The present invention comprises a first VHH capable of specifically binding to IGF2R, wherein the first VHH comprises the CDR1 sequence described in SEQ ID NO: 251, the CDR2 sequence described in SEQ ID NO: 253, and the CDR3 sequence described in SEQ ID NO: 255, and a second VHH capable of specifically binding to IGF2R, wherein the second VHH comprises the CDR1 sequence described in SEQ ID NO: 275, the CDR2 sequence described in SEQ ID NO: 277, and the CDR3 sequence described in SEQ ID NO: 279, or independently, one or two CDR1 sequences and one or two CDR2 sequences. The binding molecule according to any one of claims 3 to 7, wherein at least four amino acids, preferably at most three, more preferably at most two, and most preferably at most one, in the sequence and / or one or two CDR3 sequences are conservatively substituted, or independently, 85 to 99% of the amino acids, preferably 90 to 99%, more preferably 95 to 99%, and most preferably 97 to 99%, in one or two CDR1 sequences, one or two CDR2 sequences and / or one or two CDR3 sequences are conservatively substituted.

10. A binding molecule according to any one of claims 3 to 8, comprising a first VHH capable of specifically binding to IGF2R, wherein the first VHH has the sequence 13E8 having SEQ ID NO: 249, and a second VHH capable of specifically binding to IGF2R, wherein the second VHH has the sequence of any one of 13A8 having SEQ ID NO: 257, 13A12 having SEQ ID NO: 289, 13C11 having SEQ ID NO: 297, 13F11 having SEQ ID NO: 273, and 13G10 having SEQ ID NO: 305, preferably comprising a first VHH having the sequence 13E8 having SEQ ID NO: 249 and a second VHH having the sequence 13F11 having SEQ ID NO:

273.

11. The binding molecule according to claim 1, wherein the transmembrane receptor is platelet-derived growth factor beta receptor (PDGFRB).

12. The binding molecule according to claim 1 or 11, comprising a first VHH capable of specifically binding to PDGFRB, and a second VHH capable of specifically binding to PDGFRB.

13. The binding molecule according to claim 11 or 12, comprising at least two single variable antibody domains, preferably two single variable antibody domains, which can independently and specifically bind to the transmembrane receptor PDGFRB expressed on the fibrinoplastic effector cells.

14. A multiparatopic binding molecule, preferably a biparatopic binding molecule, wherein the binding of a first VHH contained in the binding molecule to PDGFRB preferably enhances the binding of a second VHH contained in the binding molecule to PDGFRB, preferably the reverse, and / or preferably the binding of the first VHH and the second VHH to PDGFRB enhances the internalization of the binding molecule compared to the internalization of a second binding molecule containing only the first VHH or the second VHH.

15. A multiparatopic binding molecule, preferably a biparatopic binding molecule, wherein the binding of the first VHH to PDGFRB expressed on cells of a first species and to PDGFRB expressed on cells of a second species enhances the binding of the second VHH to PDGFRB expressed on cells of a first species and to PDGFRB expressed on cells of a second species, preferably the reverse as well, according to any one of claims 12 to 14.

16. The biparatopic conjugation molecule is such that at least one, preferably both, of the first VHH and the second VHH have SEQ ID NO: 1 (SP01P), 9 (SP02P), 25 (SP05P), 33 (SP07P), 41 (SP08P), 49 (SP10P), 65 (SP12P), 73 (SP13P), 81 (SP14P), 113 (SP20P), and 153 (SP153). The binding molecule according to any one of claims 12 to 15, which can compete for binding to PDGFRB with one or more single-domain antibodies having the sequence SP26P or SP28P having sequence number 169, preferably the first VHH and the second VHH do not compete with the same single-domain antibody having the sequence SP01P, SP02P, SP05P, SP07P, SP08P, SP10P, SP12P, SP13P, SP14P, SP20P, SP26P or SP28P.

17. A first VHH capable of specifically binding to PDGFRB, wherein the first VHH comprises the CDR1 sequence described in SEQ ID NO: 155, the CDR2 sequence described in SEQ ID NO: 157, and the CDR3 sequence described in SEQ ID NO: 159, and a second VHH capable of specifically binding to PDGFRB, wherein the second VHH comprises the CDR1 sequence described in SEQ ID NO: 3, the CDR2 sequence described in SEQ ID NO: 5, and the CDR3 sequence described in SEQ ID NO: 7; or the CDR1 sequence described in SEQ ID NO: 11, the CDR2 sequence described in SEQ ID NO: 13, and the CDR3 sequence described in SEQ ID NO: 15; or the CDR1 sequence described in SEQ ID NO: 35, the CDR2 sequence described in SEQ ID NO: 37, and the CDR3 sequence described in SEQ ID NO: 39 A binding molecule according to any one of claims 12 to 16, comprising: 3 sequences; or the CDR1 sequence described in SEQ ID NO: 75, the CDR2 sequence described in SEQ ID NO: 77, and the CDR3 sequence described in SEQ ID NO: 79; or any combination of CDR1, CDR2, and CDR3 sequences in which at least 4 amino acids, preferably at least 3, more preferably at least 2, and most preferably at least 1 amino acid are conservatively substituted independently; or any one combination of CDR1, CDR2, and CDR3 sequences in which 85 to 99% of amino acids, preferably 90 to 99%, more preferably 95 to 99%, and most preferably 97 to 99% of amino acids are conservatively substituted independently.

18. The first VHH comprises a first VHH capable of specifically binding to PDGFRB, wherein the first VHH comprises the CDR1 sequence described in SEQ ID NO: 155, the CDR2 sequence described in SEQ ID NO: 157, and the CDR3 sequence described in SEQ ID NO: 159, and the second VHH capable of specifically binding to PDGFRB, wherein the second VHH comprises the CDR1 sequence described in SEQ ID NO: 11, the CDR2 sequence described in SEQ ID NO: 13, and the CDR3 sequence described in SEQ ID NO: 15, or independently, one or two CDR1 sequences and one or two CDR2 sequences. The binding molecule according to any one of claims 12 to 16, wherein at least four amino acids, preferably at most three, more preferably at most two, and most preferably at most one, are conservatively substituted in the column and / or one or two CDR3 sequences, or independently, 85 to 99% of the amino acids, preferably 90 to 99%, more preferably 95 to 99%, and most preferably 97 to 99%, in one or two CDR1 sequences, one or two CDR2 sequences, and / or one or two CDR3 sequences are conservatively substituted.

19. A binding molecule according to any one of claims 12 to 17, comprising a first VHH capable of specifically binding to PDGFRB, wherein the first VHH has a sequence of SP26P having SEQ ID NO: 153, and a second VHH capable of specifically binding to PDGFRB, wherein the second VHH has a sequence of any one of SP01P having SEQ ID NO: 1, SP02P having SEQ ID NO: 9, SP07P having SEQ ID NO: 33, and SP13P having SEQ ID NO: 73, preferably comprising a first VHH having a sequence of SP26P having SEQ ID NO: 153 and a second VHH having a sequence of SP02P having SEQ ID NO:

9.

20. The binding molecule according to any one of claims 1 to 19, comprising at least two single variable antibody domains, wherein the at least two single variable antibody domains are separated by a linker amino acid sequence.

21. The binding molecule according to any one of claims 1 to 20, comprising an N-terminal or C-terminal cysteine ​​or histidine residue, preferably an N-terminal or C-terminal cysteine ​​residue.

22. A binding molecule according to any one of claims 1 to 21, comprising a half-life extender.

23. The binding molecule according to claim 22, wherein the half-life extender comprises or consists of one or more groups, residues, parts or binding units that provide the binding molecule having an increased half-life compared to a binding molecule without the half-life extender.

24. The binding molecule according to claim 23, wherein the group, residue, part or binding unit that provides the binding molecule having an increased half-life is an albumin-binding domain or an albumin-binding VHH or a fragment thereof.

25. The binding molecule according to any one of claims 1 to 24, wherein the binding of the binding molecule to the receptor enables the induction of ligand internalization or antibody internalization.

26. The binding molecule according to claim 25, wherein the binding of one of the single variable antibody domains to the antigen modulates the binding of the other single variable antibody domain to the antigen.

27. The binding molecule according to any one of claims 1 to 26, wherein the therapeutic molecule can inhibit one or more of the contractile activity, fibrogenic activity, chemotactic activity, and pro-inflammatory activity of the effector cell.

28. The binding molecule according to any one of claims 1 to 27, wherein the therapeutic molecule is a kinase inhibitor, preferably a kinase inhibitor selected from Rho kinase inhibitors, JAK-2 inhibitors, or neprilysin inhibitors, or an angiotensin II receptor antagonist such as losartan, and preferably the therapeutic molecule is a Rho kinase inhibitor or a JAK-2 inhibitor.

29. A nucleic acid encoding at least some amino acid residues of the binding molecule according to any one of claims 1 to 28, preferably an amino acid residue of the binding molecule.

30. A host cell for the expression of at least some amino acid residues of a binding molecule according to any one of claims 1 to 28, comprising the nucleic acid according to claim 29, preferably the amino acid residues of the binding molecule.

31. A method for producing a binding molecule according to any one of claims 1 to 28, comprising the steps of: culturing a host cell according to claim 30; enabling the expression of at least a portion of amino acid residues of the binding molecule, preferably amino acid residues of the binding molecule; recovering at least a portion of the binding molecule or the expressed amino acid residues of the binding molecule; and optionally binding the therapeutic molecule or the diagnostic molecule to a portion of the binding molecule or the amino acid residues of the binding molecule via a linker.

32. A binding molecule according to any one of claims 1 to 28, for use as a pharmaceutical product.

33. A conjugation molecule according to any one of claims 1 to 28, for use in a method for the prevention and / or treatment of a disease or disorder related to or characterized by the upregulation of PDGFRB and / or IGF2R expression.

34. The binding molecule for use according to claim 33, wherein the disease or disorder is one or more of the following: hepatic fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and cancers associated with (sclerosing) malignancies such as breast cancer, lung cancer, colon cancer, and prostate cancer, preferably one or more of the following: pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and cancers associated with (sclerosing) malignancies such as breast cancer, lung cancer, colon cancer, and prostate cancer.

35. The binding molecule for use according to claim 33 or 34, wherein the prevention and / or treatment is the prevention and / or treatment of one or more (secondary) clinical symptoms resulting from hepatic fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and cancers associated with (sclerosing) malignancies such as breast cancer, lung cancer, colon cancer, and prostate cancer, for example, portal hypertension in hepatic fibrosis and / or cirrhosis, and pulmonary hypertension such as pulmonary arterial hypertension and / or pulmonary venous hypertension in pulmonary fibrosis.

36. The binding molecule for use according to claim 35, wherein the prevention and / or treatment of the (secondary) clinical symptoms is the prevention and / or treatment of one or more acute complications of such (secondary) clinical symptoms, such as esophageal bleeding in portal hypertension and / or cardiovascular failure, and / or pulmonary failure in pulmonary hypertension such as pulmonary arterial hypertension and / or pulmonary vein hypertension.

37. A pharmaceutical composition comprising at least one binding molecule and at least one pharmaceutically acceptable excipient as described in any one of claims 1 to 28.

38. A pharmaceutical composition according to claim 37, for use as a pharmaceutical product.

39. The pharmaceutical composition according to claim 37, for use in a method for the prevention and / or treatment of a disease or disorder related to or characterized by the upregulation of PDGFRB and / or IGF2R expression.

40. The pharmaceutical composition for use according to claim 39, wherein the disease or disorder is one or more of the following: hepatic fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and cancers associated with (sclerosing) malignant tumors such as breast cancer, lung cancer, colon cancer, and prostate cancer, preferably one or more of the following: pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and cancers associated with (sclerosing) malignant tumors such as breast cancer, lung cancer, colon cancer, and prostate cancer.

41. The pharmaceutical composition for use according to claim 39 or 40, wherein the prevention and / or treatment is the prevention and / or treatment of one or more (secondary) clinical symptoms resulting from liver fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and cancers associated with (sclerosing) malignant tumors such as breast cancer, lung cancer, colon cancer, and prostate cancer, such as portal hypertension in liver fibrosis and / or cirrhosis, and pulmonary hypertension such as pulmonary arterial hypertension and / or pulmonary venous hypertension in pulmonary fibrosis.

42. The pharmaceutical composition for use according to claim 41, wherein the prevention and / or treatment of the (secondary) clinical symptoms is the prevention and / or treatment of one or more acute complications of such (secondary) clinical symptoms, such as esophageal bleeding in portal hypertension and / or cardiovascular failure, and / or pulmonary failure in pulmonary hypertension such as pulmonary arterial hypertension and / or pulmonary vein hypertension.

43. A pharmaceutical composition comprising at least one binding molecule according to any one of claims 1 to 28 and at least one pharmaceutically acceptable excipient, further comprising at least one other compound useful for the treatment of one or more clinical conditions of hepatic fibrosis, pulmonary fibrosis, renal fibrosis, systemic sclerosis / scleroderma, inflammatory bowel disease, and (sclerosing) malignant tumors.

44. The pharmaceutical composition according to claim 43, for use as an adjuvant treatment for secondary hemodynamic symptoms of fibrotic diseases such as portal hypertension and pulmonary hypertension.

45. The pharmaceutical composition according to claim 44, for use in the treatment of acute symptoms of portal hypertension such as acute esophageal bleeding or pulmonary hypertension such as acute cardiac decompensation.

46. A binding molecule according to any one of claims 1 to 28, wherein the diagnostic molecule is a contrast agent.

47. A diagnostic composition comprising at least one binding molecule according to claim 46 and a diluent and / or excipient.

48. Use of the binding molecule according to claim 46 or the diagnostic composition according to claim 47 in a method for diagnosing a disease or disorder related to or characterized by the upregulation of PDGFRB and / or IGF2R expression.

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