Specific binding agents of human angiopoietin-2
Disclosed are peptides that bind to Ang-2. Also disclosed are peptibodies comprising the peptides, methods of making such peptides and peptibodies, and methods of treatment using such peptides and peptibodies.
1. An isolated polynucleotide encoding a polypeptide capable of binding Ang-2, wherein said polypeptide comprises the amino acid sequence
Cz 2 WDPWT
(SEQ ID NO: 67)
wherein z 2 is an acidic or neutral polar amino acid residue, and physiologically acceptable salts thereof.
2. An isolated polynucleotide according to claim 1 , wherein said polypeptide further comprises the amino acid sequence
Cz 2 WDPWTC
(SEQ ID NO: 68)
wherein z 2 is an acidic or neutral polar amino acid residue, and physiologically acceptable salts thereof.
3. An isolated polynucleotide encoding a polypeptide capable of binding Ang-2 comprising an amino acid sequence of the formula:
a 1 a 2 a 3 Ca 5 WDPWTCa 12 a 13 a 14
(SEQ ID NO: 69)
wherein:
a 1 , a 2 , and a 3 are each independently amino acid residues;
a 5 is an amino acid residue;
a 12 is absent or an amino acid residue;
a 13 is absent or a neutral hydrophobic, neutral polar, or a basic amino acid residue;
a 14 is a neutral hydrophobic or neutral polar amino acid residue;
and physiologically acceptable salts thereof.
4. An isolated polynucleotide encoding the polypeptide according to claim 3 wherein:
a 1 is V, I, P, W, G, S, Q, N, E, K, R, or H;
a 2 is V, P, M, G, S, Q, D, E, K, R, or H;
a 3 is A, V, P, M, F, T, G, D, E, K, or H;
a 8 is A, V, G, Q, N, D, or E;
a 12 is S, Q, N, D, E, K, or R;
a 13 is L, T, or H;
a 14 is V, L, I, W, or M;
and physiologically acceptable salts thereof.
5. An isolated polynucleotide encoding the polypeptide according to claim 3 wherein:
a 1 is Q;
a 2 is E;
a 3 is E;
a 5 is D or E;
a 12 is D or E;
a 13 is H; and
a 14 is M;
and physiologically acceptable salts thereof.
6. An isolated polynucleotide encoding a polypeptide capable of binding Ang-2 comprising an amino acid sequence of the formula:
(SEQ ID NO: 70)
b 1 b 2 b 3 b 4 b 5 b 6 Cb 8 WDPWTCb 15 b 16 b 17 b 18 b 19 b 20
wherein:
b 1 is absent or an amino acid residue;
b 2 is absent or a neutral hydrophobic, neutral polar, or a basic amino acid residue;
b 3 , b 4 , b 5 , and b 6 are each independently absent or amino acid residues;
b 8 is an amino acid residue;
b 15 is absent or an amino acid residue;
b 16 is absent or a neutral hydrophobic, neutral polar, or a basic amino acid residue;
b 17 is absent or a neutral hydrophobic or neutral polar amino acid residue;
b 18 , b 19 , and b 20 are each independently absent or amino acid residues;
and physiologically acceptable salts thereof.
7. An isolated polynucleotide encoding the polypeptide according to claim 6 wherein:
b 1 is absent, or A, V, L, P, W, F, T, G, S, Q, N, K, R, or H;
b 2 is absent, or A, V, L, I, P, W, M, T, G, S, Y, N, K, R, or H;
b 3 is absent, or A, L, I, P, W, M, T, G, S, Q, N, E, R, or H;
b 4 is V, I, P, W, G, S, Q, N, E, K, R, or H;
b 5 is V, P, M, G, S, Q, D, E, K, R, or H;
b 6 is A, V, P, M, F, T, G, D, E, K, or H;
b 8 is A, V, G, Q, N, D, or E;
b 15 is S, Q, N, D, E, K, or R;
b 16 is L, T, or H;
b 17 is V, L, I, W, or M;
b 18 is absent, or A, V, L, P, W, F, T, G, Y, Q, D, E, or R;
b 19 is absent, or V, L, I, P, T, G, S, Y, Q, N, D, E, or R; and
b 20 is absent, or V, L, P, W, M, T, G, S, Y, Q, N, D, K, or R;
and physiologically acceptable salts thereof.
8. An isolated polynucleotide encoding the polypeptide according to claim 6 wherein:
b 1 is absent, or P, or T;
b 2 is absent, or I, or N;
b 3 is absent, or R, or I;
b 4 Q;
b 5 is E;
b 6 is E;
b 8 is D or E;
b 15 is D or E;
b 16 is H;
b 17 is M;
b 18 is absent, or W, or P;
b 19 is absent, or G, or E; and
b 20 is absent, or V, or K;
and physiologically acceptable salts thereof.
9. An isolated polynucleotide encoding a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4, and SEQ ID NO: 76 to SEQ ID NO: 118, inclusive, wherein said polypeptide is capable of binding to Ang-2, and physiologically acceptable salts thereof;
PEPTIDE
SEQ ID NO.
PEPTIDE SEQUENCE
Con4-44
76
PIRQEECDWDPWTCEHMWEV
Con4-40
77
TNIQEECEWDPWTCDHMPGK
Con4-4
78
WYEQDACEWDPWTCEHMAEV
Con4-31
79
NRLQEVCEWDPWTCEHMENV
Con4-C5
80
AATQEECEWDPWTCEHMPRS
Con4-42
81
LRHQEGCEWDPWTCEHMFDW
Con4-35
82
VPRQKDCEWDPWTCEHMYVG
Con4-43
83
SISHEECEWDPWTCEHMQVG
Con4-49
84
WAAQEECEWDPWTCEHMGRM
Con4-27
85
TWPQDKCEWDPWTCEHMGST
Con4-48
86
GHSQEECGWDPWTCEHMGTS
Con4-46
87
QHWQEECEWDPWTCDHMPSK
Con4-41
88
NVRQEKCEWDPWTCEHMPVR
Con4-36
89
KSGQVECNWDPWTCEHMPRN
Con4-34
90
VKTQEHCDWDPWTCEHMREW
Con4-28
91
AWGQEGCDWDPWTCEHMLPM
Con4-39
92
PVNQEDCEWDPWTCEHMPPM
Con4-25
93
RAPQEDCEWDPWTCAHMDIK
Con4-50
94
HGQNMECEWDPWTCEHMFRY
Con4-38
95
PRLQEECVWDPWTCEHMPLR
Con4-29
96
RTTQEKCEWDPWTCEHMESQ
Con4-47
97
QTSQEDCVWDPWTCDHMVSS
Con4-20
98
QVIGRPCEWDPWTCEHLEGL
Con4-45
99
WAQQEECAWDPWTCDHMVGL
Con4-37
100
LPGQEDCEWDPWTCEHMVRS
Con4-33
101
PMNQVECDWDPWTCEHMPRS
AC2-Con4
102
FGWSHGCEWDPWTCEHMGST
Con4-32
103
KSTQDDCDWDPWTCEHMVGP
Con4-17
104
GPRISTCQWDPWTCEHMDQL
Con4-8
105
STIGDMCEWDPWTCAHMQVD
AC4-Con4
106
VLGGQGCEWDPWTCRLLQGW
Con4-1
107
VLGGQGCQWDPWTCSHLEDG
Con4-C1
108
TTIGSMCEWDPWTCAHMQGG
Con4-21
109
TKGKSVCQWDPWTCSHMQSG
Con4-C2
110
TTIGSMCQWDPWTCAHMQGG
Con4-18
111
WVNEVVCEWDPWTCNHWDTP
Con4-19
112
VVQVGMCQWDPWTCKHMRLQ
Con4-16
113
AVGSQTCEWDPWTCAHLVEV
Con4-11
114
QGMKMFCEWDPWTCAHIVYR
Con4-C4
115
TTIGSMCQWDPWTCEHMQGG
Con4-23
116
TSQRVGCEWDPWTCQHLTYT
Con4-15
117
QWSWPPCEWDPWTCQTVWPS
Con4-9
118
GTSPSFCQWDPWTCSHMVQG
TN8-Con4
4
QEECEWDPWTCEHM.
10. An isolated polynucleotide encoding a Fc-fusion polypeptide capable of binding Ang-2, said Fc-fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 25.
11. The polynucleotide of claim 10 , wherein said Fc in SEQ ID NO:25 is a human IgG1 Fc.
12. The polynucleotide of claim 10 , wherein said Fc in SEQ ID NO:25 has the amino acid sequence set forth in SEQ ID NO:60.
13. An isolated polynucleotide having a nucleic acid sequence as set forth in SEQ ID NO:46.
14. An isolated polynucleotide encoding 2xCon4(C)1K peptibody as set forth in SEQ ID NO:25.
15. An isolated polynucleotide encoding a Fc-fusion polypeptide capable of binding Ang-2, said Fc-fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19.
16. The polynucleotide of claim 15 , wherein said Fc in SEQ ID NO:19 is a human IgG1 Fc.
17. The polynucleotide of claim 15 , wherein said Fc in SEQ ID NO:19 has the amino acid sequence set forth in SEQ ID NO:60.
18. An isolated polynucleotide having a nucleic acid sequence as set forth in SEQ ID NO:40.
19. An isolated polynucleotide encoding 2xCon4(N)1K peptibody as set forth in SEO ID NO:19.
20. An expression vector comprising the polynucleotide of claims 1 - 19 .
21. A host cell comprising the expression vector of claim 20 .
22. The host cell of claim 21 , wherein said host cell is a prokaryotic cell.
23. The host cell of claim 22 , wherein said prokaryotic cell is an E. coli.
24. The host cell of claim 21 , wherein said host cell is a eukaryotic cell.