Methods of treating cancer with low density lipoprotein-related protein 6 (LRP6)—half life extender constructs

Abstract

The present invention relates to LRP6 constructs that bind to LRP6 receptor. The LRP6 constructs comprise at least one LRP6 binding moiety and a half-life extender molecule such that the LRP6 construct inhibit the Wnt signaling pathway without potentiation of the Wnt signal. The LRP6 constructs also have an increased half-life to provide more time for the therapeutic benefit.

We claim: 
     
       1. A method of treating a disease mediated by a canonical Wnt signaling pathway cancer, comprising the step of administering to a subject in need thereof an effective amount of a composition comprising a low density lipoprotein-related protein 6(LRP6) construct, wherein said LRP6 construct comprises:
 (a) first and second LRP6 single chain Fv molecules (scFvs) or anti-LRP6 antibody fragments thereof that bind to LRP6; and 
 (b) a half-life extender molecule chosen from a human serum albumin, or a protein that binds to human serum albumin; 
 
       wherein the first and second scFv molecules or fragments thereof are linked to the N- and C-termini of the human serum albumin, wherein the LRP6 construct inhibits a canonical Wnt signal transduction pathway, and wherein the the LRP6 construct displays no significant potentiation of a Wnt signal in the presence of an LRP6 binding protein, and wherein the disease is a cancer; and 
       wherein:
 (i) the first anti-LRP6 antibody fragment is linked to the N-terminus of the half-life extender molecule and binds to the Propeller 1 region of LRP6, and the second anti-LRP6 antibody fragment is linked to the C-terminus of the half-life extender molecule and binds to the Propeller 3 region of LRP6; or 
 (ii) the first anti-LRP6 antibody fragment is linked to the C-terminus of the half-life extender molecule and binds to the Propeller 1 region of LRP6, and the second anti-LRP6 antibody fragment is linked to the N-terminus of the half-life extender molecule and binds to the Propeller 3 region of LRP6. 
 
     
     
       2. The method of  claim 1 , wherein the cancer is selected from the group consisting of breast cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, and melanoma. 
     
     
       3. The method of  claim 1 , wherein the first LRP6 scFv is linked to the N-terminal of the human serum albumin and binds to the Propeller 1 region of LRP6, and the second LRP6 scFv is linked to the C-terminal of the human serum albumin and binds to the Propeller 3 region of LRP6. 
     
     
       4. The method of  claim 1 , wherein the first LRP6 scFv is linked to the C-terminal of the human serum albumin and binds to the Propeller 1 region of LRP6, and the second LRP6 scFv is linked to the N-terminal of the human serum albumin and binds to the Propeller 3 region of LRP6. 
     
     
       5. The method of  claim 1 , wherein the LRP6 binding protein is a Wnt binding protein selected from the group consisting of Wnt1, Wnt3, and Wnt3a. 
     
     
       6. The method of  claim 1 , wherein the first and second LRP6 scFvs anti-LRP6 antibody fragments are indirectly linked to the N- and C-termini of the human serum albumin half-life extender molecule via an attachment linker selected from the group consisting of an Fc linker, a hinge linker, a Gly-Ser linker, an Ala linker, and a KTHT linker. 
     
     
       7. The method of  claim 1 , wherein the first and second LRP6 scFvs are directly linked to the N- and C-termini of the human serum albumin by direct fusion to the human serum albumin. 
     
     
       8. The method of  claim 1 , wherein the human serum albumin is selected from the group consisting of a mutant human serum albumin, or a fragment of a human serum albumin. 
     
     
       9. The method of  claim 8 , wherein the mutant human serum albumin comprises mutations C34S and N503Q. 
     
     
       10. The method of  claim 8 , wherein the fragment of human serum albumin comprises at least one domain of human serum albumin selected from the group consisting of DI, DII, DIII, and DIV. 
     
     
       11. The method of  claim 1 , wherein the first or second scFv fragment comprises at least one amino acid mutation that improves stability of the scFv compared with the unmutated scFv fragment, wherein the amino acid mutation is selected from:
 VH:G34V, VH:I37F, VH:V85E, and VH:M95F of scFv06475 VH (SEQ ID NO: 82) using the Kabat numbering system;   VL:D93N of scFv06475 VL (SEQ ID NO: 81) using the Kabat numbering system;   VH:V33N, VH:I34M, VH:I34F, VH:V48I, VH:S49A, VH:G50S, VH:W52aG, and VH:H58Y of scFv08168 VH (SEQ ID NO: 14) using the Kabat numbering system; and   VL:S22T, VL:V47L, VL:G64V, and VL:T78V of scFv08168 VL (SEQ ID NO: 13) using the Kabat numbering system.   
     
     
       12. The method of  claim 11 , wherein the scFv fragment binds to the Propeller 1 region of LRP6 and comprises at least one amino acid mutation that improves stability of the scFv compared with the unmutated scFv fragment, wherein the amino acid mutation is selected from the group consisting of I34M and S49A. 
     
     
       13. The method of  claim 11 , wherein the scFv fragment binds to the Propeller 3 region of LRP6 and comprises at least one amino acid mutation that improves stability of the scFv compared with the unmutated scFv fragment, wherein the amino acid mutation is M95F. 
     
     
       14. The method of  claim 11 , wherein the first scFv fragment binds to the Propeller 1 region of LRP6 and comprises at least one amino acid mutation that improves stability of the first scFv compared with the unmutated first scFv fragment, wherein the amino acid mutation is selected from the group consisting of I34M and S49A; and a second scFv fragment that binds to the Propeller 3 region of LRP6 and comprises at least one amino acid mutation that improves stability of the second scFv compared with the unmutated second scFv fragment, wherein the amino acid mutation is M95F. 
     
     
       15. The method of  claim 1 , wherein the construct inhibits phosphorylation of LRP6 as assessed by a Wnt ligand induced phosphorylation assay. 
     
     
       16. The method of  claim 1 , wherein the construct has the functional activity of depleting a cell population, inhibiting or reducing proliferation of a cell population, inhibiting or reducing secretion of inflammatory mediators from a cell population, or inhibiting or reducing secretion of cytoplasmic granules from a cell population, wherein the cell population is selected from the group consisting of tumor cells and Wnt dependent cells. 
     
     
       17. The method of  claim 16 , wherein the low density lipoprotein-related protein 6 (LRP6) construct inhibits or reduces proliferation of a cell population. 
     
     
       18. The method of  claim 1 , wherein the construct shows increased half-life of about 5 hours compared with the first or second LRP6 single chain Fv molecule without a half-life extender. 
     
     
       19. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises SEQ ID NO: 293 or an amino acid sequence comprising at least 95% sequence identity with SEQ ID NO: 293. 
     
     
       20. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises SEQ ID NO: 295 or an amino acid sequence comprising at least 95% sequence identity with SEQ ID NO: 295. 
     
     
       21. The method of  claim 1 , wherein the first or the second LRP6 scFv is indirectly linked to the N- and C-termini of the human serum albumin via an attachment linker selected from the group consisting of an Fc linker, a hinge linker, a Gly-Ser linker, an Ala linker, and a KTHT linker. 
     
     
       22. The method of  claim 1 , wherein the human serum albumin is a mutant human serum albumin. 
     
     
       23. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises six CDRs according to SEQ ID NOS: 1-6. 
     
     
       24. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises six CDRs according to SEQ ID NOS: 7-12. 
     
     
       25. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises six CDRs according to SEQ ID NOS: 69-74. 
     
     
       26. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises six CDRs according to SEQ ID NOS: 75-80. 
     
     
       27. The method of  claim 1 , wherein Wnt1 and Wnt3a signaling is inhibited. 
     
     
       28. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct has a half-life of at least 18 hours in serum. 
     
     
       29. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises an amino acid sequence that comprises at least 90% sequence identity with SEQ ID NO: 293. 
     
     
       30. The method of  claim 1 , wherein the low density lipoprotein-related protein 6 (LRP6) construct comprises an amino acid sequence that comprises at least 90% sequence identity with SEQ ID NO: 295. 
     
     
       31. A method of treating a cancer, comprising administering to a subject in need thereof an effective amount of a composition comprising a low density lipoprotein-related protein 6 (LRP6) construct, wherein said LRP6 construct comprises a first antibody or antibody fragment thereof that binds to the Propeller 1 region of LRP6 and a second LRP6 antibody or antibody fragment thereof that binds to the Propeller 3 region of LRP6, said LRP6 construct further comprising a protein that binds to human serum albumin, wherein the LRP6 construct inhibits a canonical Wnt signal transduction pathway, and displays no significant potentiation of a Wnt signal in the presence of an LRP6 binding protein, and 
       wherein:
 (i) the first anti-LRP6 antibody or antibody fragment (a) is linked to the N-terminus of the protein that binds to human serum albumin and (b) binds to the Propeller 1 region of LRP6, and the second anti-LRP6 antibody or antibody fragment (c) is linked to the C-terminus of the protein that binds to human serum albumin and (d) binds to the Propeller 3 region of LRP6; or 
 (ii) the first anti-LRP6 antibody or antibody fragment (a) is linked to the C-terminus of the protein that binds to human serum albumin and (b) binds to the Propeller 1 region of LRP6, and the second anti-LRP6 antibody or antibody fragment (c) is linked to the N-terminus of the protein that binds to human serum albumin and (d) binds to the Propeller 3 region of LRP6. 
 
     
     
       32. The method of claim 1, wherein the first anti-LRP6 antibody fragment and the second anti-LRP6 antibody fragment are linked to the N-terminus and the C-terminus, respectively, of the protein that binds to human serum albumin. 
     
     
       33. The method of claim 1, wherein the second anti-LRP6 antibody fragment and the first anti-LRP6 antibody fragment are linked to the N-terminus and the C-terminus, respectively, of the protein that binds to human serum albumin. 
     
     
       34. The method of claim 1, wherein the first anti-LRP6 antibody fragment and the second anti-LRP6 antibody fragment are linked to the N-terminus and the C-terminus, respectively, of the human serum albumin. 
     
     
       35. The method of claim 1, wherein the first anti-LRP6 antibody fragment and the second-anti-LRP6 antibody fragment are linked to the C-terminus and the N-terminus, respectively, of the human serum albumin. 
     
     
       36. The method of claim 1, wherein the LRP6 construct inhibits both Wnt1- and Wnt3-mediated Wnt signal transduction pathways. 
     
     
       37. The method of claim 1, wherein the first anti-LRP6 antibody fragment and the second anti-LRP6 antibody fragment are independently chosen from a monovalent fragment consisting of the light chain variable domain, a heavy chain variable domain, and CL and CH1 domains (Fab); a Fab fragment, a bivalent fragment comprising two Fab fragments (F(ab′) 2 ); a single chain Fv (scFv); a scFab consisting of light chain variable domain, a heavy chain variable domain, and a CL and a CH1 domains; a dAb including a light chain variable domain and a heavy chain variable domain; a dAb fragment consisting of a light chain variable domain or a heavy chain variable domain; a SMIP; a domain antibody; a diabody; a minibody; an affibody; or a nanobody. 
     
     
       38. The method of claim 1, wherein the first anti-LRP6 antibody fragment and the second anti-LRP6 antibody fragment is independently chosen from a single-chain Fv (scFv), a Fab or a Fab fragment, a single-chain Fab (scFabs), a diabody, a domain antibody, or a nanobody. 
     
     
       39. The method of claim 1, wherein the LRP6 construct comprises a bispecific or a biparatopic antibody fragment. 
     
     
       40. The method of claim 1, wherein the first anti-LRP6 antibody fragment or the second anti-LRP6 antibody fragment comprises a first or a second single domain antibody, or a first or a second nanobody. 
     
     
       41. The method of claim 1, wherein the first anti-LRP6 antibody fragment and the second anti-LRP6 antibody fragment comprise a first and a second domain antibody fragments. 
     
     
       42. The method of claim 41, wherein the first and second domain antibody fragments, each one consists of a heavy chain variable domain or a light chain variable domain; or each one consists of a single variable domain and a CH2 domain and a CH3 domain. 
     
     
       43. The method of claim 1, wherein the protein that binds human serum albumin is an antibody, a nanobody, a Fab, a DARPin, an avimer, an affibody, or an anticalin. 
     
     
       44. The method of claim 1, wherein the human serum albumin or the protein that binds to human serum albumin is fused or chemically linked to the first anti-LRP6 antibody fragment and second anti-LRP6 antibody fragment. 
     
     
       45. The method of claim 1, wherein the human serum albumin or the protein that binds to human serum albumin is linked to the first anti-LRP6 antibody fragment and the second anti-LRP6 antibody fragment by an amino acid attachment linker. 
     
     
       46. The method of claim 45, wherein the attachment linker comprises alanine, glycine, or serine residues used alone or in combination. 
     
     
       47. The method of claim 45, wherein the attachment linker is a Gly/Ser linker. 
     
     
       48. The method of claim 2, wherein the LRP6 construct is administered in combination with another therapeutic or cytotoxic agent. 
     
     
       49. The method of claim 2, wherein the LRP6 construct is administered parenterally. 
     
     
       50. The method of claim 1, wherein the LRP6 construct shows increased half-life of at least 5 hours or at least 10 hours compared to an LRP6 construct without the half-life extender. 
     
     
       51. A method of treating a cancer, comprising administering to a subject in need thereof an effective amount of a composition comprising a low density lipoprotein-related protein 6 (LRP6) construct, wherein said LRP6 construct comprises:
 (a) a biparatopic antibody fragment comprising a first anti-LRP6 antibody fragment and second anti-LRP6 antibody fragment; and   (b) a protein that binds to human serum albumin, wherein the protein that binds to human serum albumin is linked to each of the first antibody fragment and the second antibody fragment by an amino acid attachment linker that comprises alanine, glycine, or serine residues used alone or in combination,   wherein the LRP6 construct inhibits both Wnt1- and Wnt3-mediated Wnt signal transduction pathways, and displays no significant potentiation of a Wnt signal in the presence of an LRP6 binding protein; and   
       wherein:
 (i) the first anti-LRP6 antibody fragment is linked to the N-terminus of the protein that binds to human serum albumin and binds to the Propeller 1 region of LRP6, and the second anti-LRP6 antibody fragment is linked to the C-terminus of the protein that binds to human serum albumin and binds to the Propeller 3 region of LRP6; or 
 (ii) the first anti-LRP6 antibody fragment is linked to the C-terminus of protein that binds to human serum albumin and binds to the Propeller 1 region of LRP6, and the second anti-LRP6 antibody fragment is linked to the N-terminus of the protein that binds to human serum albumin and binds to the Propeller 3 region of LRP6. 
 
     
     
       52. The method of claim 51, wherein the first and second domain antibody fragment is chosen from:
 (i) a first and a second nanobody;   (ii) a first and a second dAb fragment;   (iii) a first and a second domain antibody fragment, each one consisting of a heavy chain variable domain or a light chain variable domain; or   (iv) a first and a second domain antibody fragment, each one consists of a single variable domain and a CH2 domain and a CH3 domain.