Polypeptides antagonizing Wnt signaling in tumor cells
The invention provides novel LRP5-binding polypeptides, and more specifically novel LRP5-binding 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 biparatopic polypeptide wherein each paratope binds to an epitope of the human low-density receptor-like protein 5-(LRP5), comprising a first LRP5 epitope-binding immunoglobulin single variable domain (ISVD) and a second LRP5 epitope-binding immunoglobulin single variable domain (ISVD)
wherein the first LRP5 epitope-binding domain binds a Wnt3a binding region of LRP5, thereby inhibiting Wnt3a ligand binding and the Wnt3a signaling pathway, and wherein said first LRP5 epitope-binding domain is distinct from the second LRP5 epitope-binding domain that binds a Wnt1 binding region of LRP5, thereby inhibiting Wnt1 ligand binding and the Wnt1 signaling pathway
wherein said first LRP5 epitope-binding ISVD comprises the following CDR sequences:
(SEQ ID NO: 1)
CDR1: TYVMG,
(SEQ ID NO: 2)
CDR2: AISWSGGSTYYADSVKG,
(SEQ ID NO: 3)
CDR3: SRGTSTPSRASGVSRYDY,
and
wherein said second LRP5 epitope-binding ISVD comprises the following CDR sequences:
(SEQ ID NO: 7)
CDR1: IGAMG,
(SEQ ID NO: 8)
CDR2: AVSSGGSTYYVDSVKG,
(SEQ ID NO: 9)
CDR3: ETGPYGPPKRDY.
2. The biparatopic polypeptide of claim 1 , wherein said ISVDs are VHH domains.
3. The biparatopic polypeptide according to claim 1 , wherein
said first LRP5 epitope binding ISVD comprises the sequence of SEQ ID NO:11 or the sequence of SEQ ID NO:23, and
wherein said second LRP5 epitope-binding ISVD comprises the sequence of SEQ ID NO:13 or the sequence of SEQ ID NO:22.
4. The biparatopic polypeptide according to claim 1 comprising a sequence of SEQ ID NO: 20.
5. A pharmaceutical composition comprising (i) as the active ingredient, the biparatopic polypeptide according to claim 1 , and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
6. A An isolated nucleic acid molecule encoding the biparatopic polypeptide according to claim 1 .
7. An expression vector comprising the nucleic acid of claim 6 .
8. A host cell comprising an expression vector according to claim 7 .
9. A method of manufacturing a biparatopic polypeptide comprising the steps of
a. culturing a host cell according to claim 8 under conditions that allow expression of the biparatopic polypeptide; and
b. recovering the biparatopic polypeptide.