Tetravalent FZD and WNT co-receptor binding antibody molecules and uses thereof

Abstract

Described herein are tetravalent binding antibody molecules comprising a FZD receptor binding domain and an LRP5 / 6 coreceptor binding domain on opposite ends of an Fc domain that activate the Wnt β-catenin signaling pathway, and methods of use thereof.

Claims

1. (a) an Fc domain; (b) a bivalent low density lipoprotein receptor-related protein 5 (LRP5) binding domain; (c) a bivalent Frizzled4 (FZD4) binding domain; and A tetravalent binding antibody molecule comprising: An LRP5 binding domain is attached to one end of an Fc domain, and a bivalent FZD4 binding domain is attached to the other end of the Fc domain; The bivalent LRP5 binding domain comprises a bivalent diabody that binds to LRP5, and the FZD binding domain comprises two Fabs that bind to FZD4; Tetravalent binding antibody molecules (a) a dimer of a first heavy chain monomer and a second heavy chain monomer, each comprising a single polypeptide chain comprising from N-terminus to C-terminus: (1) a first heavy chain variable domain (VH) and a light chain variable domain (VL); (2) an Fc region comprising a constant heavy chain domain 3 (CH3); (3) a second VH, and (4) constant heavy chain domain 1 (CH1); Where: (5) the first VH of the first heavy chain monomer comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 528, a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 553, and a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 586; (6) the first VH of the second heavy chain monomer comprises CDR-H1 of SEQ ID NO:536, CDR-H2 of SEQ ID NO:566, and CDR-H3 of SEQ ID NO:603; (7) The VL of the first heavy chain monomer comprises a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 491, and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 510; (8) the VL of the second heavy chain monomer comprises CDR-L1 of SEQ ID NO:1, CDR-L2 of SEQ ID NO:2, and CDR-L3 of SEQ ID NO:493; and (9) The second VH of both the first heavy chain monomer and the second heavy chain monomer comprises CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 61, and CDR-H3 of SEQ ID NO:

90. and (b) a first light chain monomer and a second light chain monomer comprising a VL and a constant light chain domain 1 (CL1), each of which comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 12, The first heavy chain monomer and the second heavy chain monomer dimerize via their Fc regions to form an Fc domain, and the VL and the first VH of the first heavy chain monomer pair with the first VH and VL of the second heavy chain monomer to form a bivalent diabody that binds to LRP5; a first light chain monomer and a second light chain monomer pair with a first heavy chain monomer and a second heavy chain monomer, respectively, to form two Fabs that bind to FZD4; A bivalent diabody that binds to LRP5 forms a bivalent LRP5 binding domain, and two Fabs that bind to FZD4 form a bivalent FZD4 binding domain; Tetravalent binding antibody molecule.

2. The tetravalent binding antibody molecule described in claim 1, wherein the Fc regions of the first heavy chain monomer and the second heavy chain monomer dimerize via a knob-in-hole arrangement.

3. The tetravalent binding antibody molecule of claim 2, wherein the Fc region of the first heavy chain monomer comprises the mutations T366S, L368A and Y407V, and the Fc region of the second heavy chain monomer comprises the mutation T366W, according to EU numbering.

4. The tetravalent binding antibody molecule of claim 3, wherein the Fc region of the first heavy chain monomer further comprises the mutations S354I and E357L, and the Fc region of the second heavy chain monomer further comprises the mutations Q347M, Y349F, T350D and L368M, according to EU numbering.

5. The tetravalent linked antibody molecule of claim 3, wherein an additional disulfide bond is introduced between the Fc region of the first heavy chain monomer and the Fc region of the second heavy chain monomer, and wherein, according to EU numbering, the first heavy chain monomer comprises the mutation Y349C and the second heavy chain monomer comprises the mutation S354C.

6. The tetravalent binding antibody molecule of any one of claims 1 to 5, wherein the Fc region has reduced or no effector function.

7. The tetravalent binding antibody molecule described in claim 6, wherein the Fc region of the first heavy chain monomer and / or the Fc region of the second heavy chain monomer comprises the amino acid mutations L234A, L235A and P331S (LALAPS).

8. The tetravalent binding antibody molecule of any one of claims 1 to 7, wherein the LRP5 binding domain and the bivalent FZD binding domain are each linked to the Fc domain by one or more linkers.

9. The tetravalent binding antibody molecule described in claim 8, wherein the one or more linkers comprise 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 10, 1 to 6 amino acids, 1 to 5 amino acids, or 2 to 4 amino acids.

10. A tetravalent binding antibody molecule as described in claim 8 or 9, wherein the VL of the first heavy chain monomer and the VL of the second heavy chain monomer are each fused to the Fc region of the first heavy chain monomer and the second heavy chain monomer, respectively, via a linker comprising SEQ ID NO: 892 (GGGGSGGGGGSEPKSSDKTHT).

11. A tetravalent binding antibody molecule described in any one of claims 8 to 10, wherein the second VH of the first heavy chain monomer and the second VH of the second heavy chain monomer are each fused to the Fc region of the first heavy chain monomer and the second heavy chain monomer, respectively, via a linker comprising SEQ ID NO: 891 (GGGSGGGSGGGSGGGSGSTG).

12. A tetravalent binding antibody molecule described in any one of claims 1 to 11, wherein the first VH of the first heavy chain monomer is linked to the VL of the first heavy chain monomer, and the first VH of the second heavy chain monomer is linked to the VL of the second heavy chain monomer, each via a linker comprising SEQ ID NO:886 (GGGGS).

13. A tetravalent binding antibody molecule, The first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:908, the second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:907, and the first light chain monomer and the second light chain monomer each comprise the amino acid sequence of SEQ ID NO:

909. A tetravalent binding antibody molecule as described in claim 1.

14. A tetravalent binding antibody molecule, (a) a first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:908 or SEQ ID NO:940, a second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:944 or SEQ ID NO:948, and a first light chain monomer and a second light chain monomer comprise the amino acid sequence of SEQ ID NO:909 or SEQ ID NO:952, respectively; or (b) the first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:937 or SEQ ID NO:941, the second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:945 or SEQ ID NO:949, and the first light chain monomer and the second light chain monomer comprise the amino acid sequence of SEQ ID NO:909 or SEQ ID NO:952, respectively; or (c) the first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:938 or SEQ ID NO:942, the second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:946 or SEQ ID NO:950, and the first light chain monomer and the second light chain monomer comprise the amino acid sequence of SEQ ID NO:909 or SEQ ID NO:952, respectively; or (d) the first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:939 or SEQ ID NO:943, the second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:947 or SEQ ID NO:951, and the first light chain monomer and the second light chain monomer comprise the amino acid sequence of SEQ ID NO:909 or SEQ ID NO:952, respectively. A tetravalent binding antibody molecule as described in claim 1.

15. The tetravalent linked antibody molecule of claim 1, wherein the first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:941, the second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:949, and the first light chain monomer and the second light chain monomer each comprise the amino acid sequence of SEQ ID NO:

952.

16. A tetravalent binding antibody molecule comprising: (a) a first low density lipoprotein receptor-related protein 5 (LRP5) binding domain comprising a heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO:528, CDR-H2 having the amino acid sequence of SEQ ID NO:553, CDR-H3 having the amino acid sequence of SEQ ID NO:586, and a light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:1, CDR-L2 having the amino acid sequence of SEQ ID NO:2, and CDR-L3 having the amino acid sequence of SEQ ID NO:493; (b) a second LRP5 binding domain comprising a heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 536, CDR-H2 having the amino acid sequence of SEQ ID NO: 566, CDR-H3 having the amino acid sequence of SEQ ID NO: 603, and a light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 1, CDR-L2 having the amino acid sequence of SEQ ID NO: 491, and CDR-L3 having the amino acid sequence of SEQ ID NO: 510; and (c) a first Frizzled 4 (FZD4) binding domain and a second FZD4 binding domain, each of which comprises a VH comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 24, a CDR-H2 having the amino acid sequence of SEQ ID NO: 61, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 90, and a VL comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 1, a CDR-L2 having the amino acid sequence of SEQ ID NO: 2, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 12; Tetravalent binding antibody molecule.

17. The tetravalent binding antibody molecule of claim 16, wherein the first heavy chain monomer comprises a VH of a first LRP5 binding domain, a VL of a second LRP5 binding domain, and a VH of a first FZD4 binding domain; the second heavy chain monomer comprises a VH of a second LRP5 binding domain, a VL of a first LRP5 binding domain, and a VH of a second FZD4 binding domain; the first light chain monomer comprises a VL of a first FZD4 binding domain; and the second light chain monomer comprises a VL of a second FZD4 binding domain.

18. The tetravalent binding antibody molecule of claim 17, wherein the first heavy chain monomer and the second heavy chain monomer each comprise an Fc region comprising a CH2 and a CH3 domain, and each Fc region comprises an alanine at position 234, an alanine at position 235, a serine at position 331, or any combination thereof, wherein each position is according to EU numbering.

19. The tetravalent binding antibody molecule of claim 18, wherein the VL of the first heavy chain monomer and the VL of the second heavy chain monomer are each linked to the N-terminus of the Fc region of the first heavy chain monomer and the second heavy chain monomer, respectively, via a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 892 (GGGGSGGGGGSEPKSSDKTHT).

20. The tetravalent binding antibody molecule of claim 18 or 19, wherein the VH of the first FZD4 binding domain and the VH of the second FZD4 binding domain are linked to the C-terminus of the Fc region of the first heavy chain monomer and the second heavy chain monomer, respectively, via a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 891 (GGGSGGGSGGGSGGGSGSTG).

21. The tetravalent binding antibody molecule of any one of claims 18 to 20, wherein the VH of the first LRP5 binding domain of the first heavy chain monomer is linked to the VL of the first heavy chain monomer by a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 886 (GGGGS), and the VH of the second LRP5 binding domain of the second heavy chain monomer is linked to the VL of the second heavy chain monomer by a polypeptide linker comprising the amino acid sequence of SEQ ID NO: 886 (GGGGS).

22. The tetravalent binding antibody molecule of any one of claims 17 to 21, wherein the first heavy chain monomer comprises the amino acid sequence of SEQ ID NO:941, the second heavy chain monomer comprises the amino acid sequence of SEQ ID NO:949, and the first light chain monomer and the second light chain monomer each comprise the amino acid sequence of SEQ ID NO:

952.

23. A tetravalent linked antibody molecule comprising a first heavy chain monomer having the amino acid sequence of SEQ ID NO:941, a second heavy chain monomer having the amino acid sequence of SEQ ID NO:949, and a first light chain monomer and a second light chain monomer, each having the amino acid sequence of SEQ ID NO:

952.

24. A pharmaceutical composition comprising a tetravalent binding antibody molecule according to any one of claims 1 to 23 and a pharma- ceutically acceptable carrier.

25. 25. A pharmaceutical composition according to claim 24 for use as a medicament.

26. 25. A pharmaceutical composition as described in claim 24 for use in the treatment or prevention of disorders or conditions characterized by defective retinal or cerebral vascularization and / or characterized by reduced endothelial cell barrier function and / or vascular leakage.

27. 27. The pharmaceutical composition for use according to claim 26, wherein the disorder is selected from the group consisting of diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema, pediatric vitreous retinopathy, Alzheimer's disease, epilepsy, multiple sclerosis, stroke and ischemia.

28. The pharmaceutical composition of claim 24 for use in the treatment or prevention of a retinal or macular disorder selected from the group consisting of diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema and pediatric vitreous retinopathy, or in the treatment or prevention of a disorder selected from the group consisting of Alzheimer's disease, epilepsy, multiple sclerosis, stroke and ischemia.

29. A nucleic acid molecule encoding SEQ ID NO:941, SEQ ID NO:949, and / or SEQ ID NO:

952.

30. A nucleic acid molecule encoding a tetravalent binding antibody molecule according to any one of claims 1 to 23.

31. 31. One or more vectors comprising the nucleic acid molecule of claim 29 or claim 30.

32. 32. The one or more vectors of claim 31 , wherein the vector is a plasmid; an animal virus, including a virus selected from the group consisting of reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus, varicella virus, baculovirus, papilloma virus, and papovavirus.

33. A host cell expressing one or more vectors of claim 31 or claim 32, wherein one or more vectors encode sequence numbers 941, 949, and 952.

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