Lipocalin muteins with binding-affinity for glypican-3 (GPC-3) and use of lipocalin muteins for target-specific delivery to cells expressing GPC-3

1. A human tear lipocalin mutein comprising a mutated amino acid residue at two or more positions corresponding to positions 26-34, 55-61, 64, 79, 101, 104-106, 108, 111, 114 and 153 of the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9), wherein the mutein is capable of binding glypican-3 (GPC-3) with a K D of about 10 nM or lower, wherein the amino acid sequence of the mutein has at least 70% sequence identity to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9), and wherein the amino acid sequence of the mutein comprises the following amino acid substitution corresponding to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9): Arg 60→Tyr, Asp, Thr, Trp, Ile, Pro, Glu, Gln, Val, Ser, or Gly.

2. The mutein of claim 1 , wherein the mutein is capable of competing for binding to GPC-3 in a competition assay with an IC50 or EC50 value of about 1 nM or lower.

3. The mutein of claim 1 , wherein the mutein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 mutated amino acid residues at the sequence positions 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 60, 61, 64, 79, 101, 104, 105, 106, 108, 111, 114 and 153 of the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9).

4. The mutein of claim 1 , wherein the amino acid sequence of the mutein further comprises at least one of the following amino acid substitutions: Arg 26→Asp, Thr, Ser, Gly, Phe, Tyr, Val or Glu, Glu 27→Thr, Asn, Asp, Arg, Leu, Phe or Val, Pro 29→Arg, Lys, Ser, Glu, Leu or Phe, Glu 30→Gly, Lys, Phe, Trp or Asn, Met 31→Ala, His, Leu, Trp, Gly, Ser or Arg, Asn 32→Tyr, Trp, Gln, His, Leu, Lys, Phe or Arg, Leu 33→Gln, His, Gly, Val, Glu or Phe, Glu 34→Gly, Asn, Pro, Trp, Arg or His, Met 55→Gln, Asn, Ile, Thr, Ser or Leu, Leu 56→Pro, Ser, Phe, Trp, Arg, Asn, Ala, Val, Asp, Gln, Glu or Thr, Ile 57→Leu or Ser, Ser 58→Asp, Trp, Phe, Ala, Glu, His, Asn, Pro or Val, Cys 61→Arg, Ser, Gly, Ala, Trp, Lys, Tyr, Asp, Val, Ile, Thr, Phe, Asn, Leu, Gln or Glu, Glu 104→Trp, Thr, Ser, His, Ala or Asp, and His 106→Ala, Tyr, Phe, Pro, Thr or Glu, and Lys 108→Leu, Ser, Phe or Trp.

5. The mutein of claim 4 , wherein the amino acid sequence of the mutein further comprises one of the following sets of amino acid substitutions:

(1) Met 31→Ala, Asn 32→Arg, Glu 34→His, Leu 56→Trp, Ser 58→Asn, Arg 60→Trp; Cys 61→Arg; His 106→Glu; and Lys 108→Trp; or

(2) Met 31→Arg, Asn 32→Phe, Glu 34→Gly, Leu 56→Thr, Ser 58→Asp, Arg 60→Glu; Cys 61→Tyr; His 106→Ala; and Lys 108→Leu.

6. The mutein of claim 4 , wherein the amino acid sequence of the mutein further comprises one of the following sets of amino acid substitutions:

(a) Arg 26→Thr; Glu 27→Leu; Phe 28→Cys; Met 31→Ala; Glu 34→Gly; Leu 56→Thr; Cys 61→Tyr; Ala 79→Val; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;

(b) Arg 26→Thr; Glu 27→Leu; Phe 28→Cys; Asn 32→Phe; Leu 33→Glu; Met 55→Asn; Cys 61→Tyr; Ala 79→Val; Cys 101→Ser; Leu 105→Cys; Lys 108→Leu; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;

(c) Arg 26→Thr; Glu 27→Leu; Phe 28→Cys; Glu 30→Gly; Leu 33→Glu; Glu 34→Gly; Leu 56→Thr; Ser 58→Asp; Cys 101→Ser; His 106→Ala; Lys 108→Leu; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;

(d) Arg 26→Thr; Phe 28→Cys; Glu 29→Arg; Asn 32→Tyr; Leu 56→Trp; Ile 57→Leu; Arg 60→Trp; Cys 61→Arg; Cys 101→Ser; Leu 105→Cys; Lys 108→Trp; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;

(e) Arg 26→Thr; Phe 28→Cys; Met 31→Ala; Asn 32→Tyr; Glu 34→His; Leu 56→Trp; Ser 58→Asn; Arg 60→Trp; Cys 101→Ser; Leu 105→Cys; His 106→Glu; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser; or

(f) Arg 26→Thr; Phe 28→Cys; Glu 29→Arg; Met 31→Ala; Leu 33→His; Ile 57→Leu; Ser 58→Asn; Cys 61→Arg; Cys 101→Ser; Glu 104→Asp; Lys 108→Trp; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser.

7. The mutein of claim 1 , wherein the amino acid sequence of the mutein further comprises at least one of the following amino acid substitutions: Arg 26→Thr; Glu 27→Leu; Phe 28→Cys; Pro 29→Phe; Glu 30→Gly; Leu 33→Glu; Met 55→Asn; Ile 57→Leu; Val 64→Trp; Val 64→Leu; Val 64→Asp; Val 64→Ala; Ala 79→Val; Cys 101→Ser; Glu 104→Asp; Leu 105→Cys; Arg 111→Pro; Lys 114→Trp and Cys 153→Ser.

8. The mutein of claim 1 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-8 and 11-43 or at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-8 and 11-43.

9. The mutein of claim 1 , wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein or fused at its N-terminus and/or its C-terminus to a fusion partner.

10. The mutein of claim 1 , wherein the amino acid sequence of the mutein further comprises at least one of the following amino acid substitutions corresponding to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9): Glu 30→Gly, Lys, Phe, Trp or Asn, and Leu 33→Gln, His, Gly, Val, Glu or Phe.

11. The mutein of claim 1 , wherein the amino acid sequence of the mutein has at least 80% sequence identity to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9), and wherein the amino acid sequence of the mutein comprises at least 9 of the following amino acid substitutions corresponding to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9): Arg 26→Asp, Thr, Ser, Gly, Phe, Tyr, Val or Glu, Glu 27→Thr, Asn, Asp, Arg, Leu, Phe or Val, Pro 29→Arg, Lys, Ser, Glu, Leu or Phe, Glu 30→Gly, Lys, Phe, Trp or Asn, Met 31→Ala, His, Leu, Trp, Gly, Ser or Arg, Asn 32→Tyr, Trp, Gln, His, Leu, Lys, Phe or Arg, Leu 33→Gln, His, Gly, Val, Glu or Phe, Glu 34→Gly, Asn, Pro, Trp, Arg or His, Met 55→Gln, Asn, Ile, Thr, Ser or Leu, Leu 56→Pro, Ser, Phe, Trp, Arg, Asn, Ala, Val, Asp, Gln, Glu or Thr, Ile 57→Leu or Ser, Ser 58→Asp, Trp, Phe, Ala, Glu, His, Asn, Pro or Val, Arg 60→Tyr, Asp, Thr, Trp, Ile, Pro, Glu, Gln, Val, Ser, or Gly, Cys 61→Arg, Ser, Gly, Ala, Trp, Lys, Tyr, Asp, Val, Ile, Thr, Phe, Asn, Leu, Gln or Glu, Glu 104→Trp, Thr, Ser, His, Ala or Asp, and His 106→Ala, Tyr, Phe, Pro, Thr or Glu, and Lys 108→Leu, Ser, Phe or Trp.

12. The mutein of claim 1 , wherein the amino acid sequence of the mutein has at least 85% sequence identity to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9), and wherein the amino acid sequence of the mutein comprises at least 12 of the following amino acid substitutions corresponding to the amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 9): Arg 26→Asp, Thr, Ser, Gly, Phe, Tyr, Val or Glu, Glu 27→Thr, Asn, Asp, Arg, Leu, Phe or Val, Pro 29→Arg, Lys, Ser, Glu, Leu or Phe, Glu 30→Gly, Lys, Phe, Trp or Asn, Met 31→Ala, His, Leu, Trp, Gly, Ser or Arg, Asn 32→Tyr, Trp, Gln, His, Leu, Lys, Phe or Arg, Leu 33→Gln, His, Gly, Val, Glu or Phe, Glu 34→Gly, Asn, Pro, Trp, Arg or His, Met 55→Gln, Asn, Ile, Thr, Ser or Leu, Leu 56→Pro, Ser, Phe, Trp, Arg, Asn, Ala, Val, Asp, Gln, Glu or Thr, Ile 57→Leu or Ser, Ser 58→Asp, Trp, Phe, Ala, Glu, His, Asn, Pro or Val, Arg 60→Tyr, Asp, Thr, Trp, Ile, Pro, Glu, Gln, Val, Ser, or Gly, Cys 61→Arg, Ser, Gly, Ala, Trp, Lys, Tyr, Asp, Val, Ile, Thr, Phe, Asn, Leu, Gln or Glu, Glu 104→Trp, Thr, Ser, His, Ala or Asp, and His 106→Ala, Tyr, Phe, Pro, Thr or Glu, and Lys 108→Leu, Ser, Phe or Trp.

13. A pharmaceutical composition comprising the mutein of claim 1 and a pharmaceutically acceptable excipient.

14. A diagnostic or analytical kit comprising the mutein of claim 1 .

15. A nucleic acid molecule comprising a nucleotide sequence encoding the mutein of claim 1 .

16. A host cell containing a nucleic acid molecule of claim 15 .

17. A method of producing the mutein of claim 1 , wherein the mutein is produced in a cultured host cell comprising a nucleic acid molecule encoding for the mutein of claim 1 .