Biparatopic polypeptides antagonizing Wnt signaling in tumor cells
The invention provides novel biparatopic LRP5/LRP6 cross-reactive binding polypeptides, and more specifically novel biparatopic LRP5/LRP6 cross-reactive immunoglobulin single variable domain constructs which can inhibit Wnt signaling pathways. The invention also relates to specific sequences of such polypeptides, methods of their production, and methods of using them, including methods of treatment of diseases such as cancer.
1. A method of treatment of tumors expressing human LRP5 and/or human LRP6 by inhibition of the Wnt-1 and/or Wnt-3a signaling pathways in a cancer patient comprising administering to said patient a cross-reactive biparatopic polypeptide which specifically binds to an epitope of the human low-density receptor-like protein 5 (LRP5) as well as an epitope of the human low-density receptor-like protein 6 (LRP6) in an amount effective to reduce said patient's tumor burden and/or tumor size wherein said cross-reactive biparatopic polypeptide comprises:
(a) a first immunoglobulin single variable domain, wherein said immunoglobulin single variable domain is a humanized VHH domain capable of inhibiting the Wnt1 signaling pathway comprising the CDR sequences:
(SEQ ID NO: 4)
CDR1: SYAMG
(SEQ ID NO: 5)
CDR2: AIRRSGRRTYYADSVKG
and
(SEQ ID NO: 6)
CDR3: ARRVRSSTRYNTGTWWWEY;
and
(b) a second immunoglobulin single variable domain wherein said second immunoglobulin single variable domain is a humanized VHH domain capable of inhibiting the Wnt3a signaling pathway comprising the following CDR sequences:
(SEQ ID NO: 13)
CDR1: SYAMG
(SEQ ID NO: 14)
CDR2: AISWRSGSTYYADSVKG
and
(SEQ ID NO: 15)
CDR3: DPRGYGVAYVSAYYEY.
wherein said first and said second immunoglobulin single variable domains are covalently linked by a linker peptide, wherein said linker peptide comprises a third immunoglobulin single variable domain which binds to albumin comprising SEQ ID NO:24.
2. The method of claim 1 , wherein the cross-reactive biparatopic polypeptide comprises
a first immunoglobulin single variable domain comprising the amino acid sequence SEQ ID NO: 20 and
a second immunoglobulin single variable domain comprising the amino acid sequence SEQ ID NO:23.
3. The method of claim 2 , wherein the cross-reactive biparatopic polypeptide comprises SEQ ID NO: 26.