Polypeptides comprising a modified bacteriophage g3p amino acid sequence with reduced immunogenicity
The invention relates to polypeptides that comprise a portion of filamentous bacteriophage gene 3 protein (g3p) sufficient to bind to and/or disaggregate amyloid, e.g., the N1-N2 portion of g3p and mutants and fragments thereof, wherein that g3p amino acid sequence has been modified through amino acid substitution to be substantially less immunogenic than the corresponding wild-type g3p amino acid sequence when used in vivo. The polypeptides of the invention retain their ability bind to and/or disaggregate amyloid. The invention relates furthermore to the use of these variant g3p-polypeptides in the treatment and/or prevention of diseases associated with misfolding or aggregation of amyloid.
1. A polypeptide comprising a variant of a starting amino acid sequence, wherein the starting amino acid sequence is selected from the group consisting of: amino acids 1-217 of SEQ ID NO:1, amino acids 1-217 of SEQ ID NO:3, and amino acids 1-217 of SEQ ID NO:7, wherein:
(a) the variant has from 1 to 9 amino acid substitutions as compared to the starting amino acid sequence, wherein each amino acid substitution is selected from the group of amino acid substitutions set forth below:
Amino Acid present
Amino
in the Starting Amino
Acid #
Acid Sequence
Amino Acid Substitutions
48
G
H, K, R, S, T, D, P
50
E
G, H, K, P, R
51
T
G, H, K, R, P, Q, N, W
53
C
F, H, K, N, Q, R, W, Y
54
Y
G, H, K, R, P
56
T
G, H, K, R, P
135
M
A, D, G, K, N, T, H, R, C, E, P, Q, S
137
Q
D, E
138
N
D, E, G, H, P, Q, S, T
140
R
D, E, H, Q, A, G, M, N, P, S, Y
141
F
D, E
143
N
A, G
173
S
G, P, K, D, H, R, T
174
K
R
175
A
G, H, K, P, R
176
M
G, H, K, N, R, P, Q, W
178
D
G, N, Q, S, T, F, H, K, R, W, Y
179
A
H, K, P, R
181
W
G, H, K, R, P
wherein, when the starting amino acid sequence is amino acids 1-217 of SEQ ID NO:1, any of the 1 to 9 amino acid substitutions is additionally selected from the group consisting of V215S, V215T, V215C, V215D, V215E, V215F, V215H, V215K, V215N, V215P, V215Q, and V215R; and
(b) the variant optionally further comprises one or more of the following modifications as compared to the starting amino acid sequence:
(i) substitution of VVV at amino acids 43-45 with AAA;
(ii) substitution C53W
(iii) deletion of amino acids 96-103;
(iv) substitution of QPP at amino acids 212-214 with AGA;
(v) substitutions W181A, F190A and F194A;
(vi) deletion of amino acid 1; and
(vii) deletion of amino acids 1 and 2;
and wherein the polypeptide binds to and/or disaggregates amyloid and has reduced immunogenicity as compared to a corresponding polypeptide comprising the starting amino acid sequence.
2. The polypeptide of claim 1 , wherein each of the 1 to 9 amino acid substitutions in the starting amino acid sequence is selected from the group of amino acid substitutions set forth below:
Amino Acid present
in the Starting Amino
Amino Acid #
Acid Sequence
Amino Acid Substitutions
48
G
H, K, R, S T
51
T
G, H, K, R, P, Q, N
54
Y
G, H, K, R, P
56
T
G, H, K, R, P
135
M
A, D, G, K, N, T, H, R
140
R
D, E, H, Q, A, G
141
F
D, E
143
N
A, G
173
S
G, P, K
174
K
R
176
M
G, H, K, N, R
178
D
G, N, Q, S, T
181
W
G, H, K, R.
3. The polypeptide of claim 1 , wherein the variant amino acid sequence has 2 to 9 amino acid substitutions relative to the starting amino acid sequence, and wherein at least one substitution is present in at least two of: epitope 1, comprising amino acids 48-56 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7; epitope 2, comprising amino acids 135-143 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7; and epitope 3, comprising amino acids 173-181 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7.
4. The polypeptide of claim 3 , wherein the modified amino acid sequence has only two amino acid substitutions, and wherein the substitutions are selected from the group of two amino acid substitutions set forth below:
Y54K and M135K
Y54K and M135T
Y54K and R140Q
Y54R and M135K
Y54R and M135T
Y54R and R140Q
T56H and M135K
T56H and M135T
T56H and R140Q
T56K and M135K
T56K and M135T
T56K and R140Q
Y54K and D178N
Y54K and W181H
Y54K and W181R
Y54K and K174R
Y54R and D178N
Y54R and W181H
Y54R and W181R
Y54R and K174R
T56H and D178N
T56H and W181H
T56H and W181R
T56H and K174R
T56K and D178N
T56K and W181H
T56K and W181R
T56K and K174R
M135K and D178N
M135K and W181H
M135K and W181R
M135K and K174R
M135T and D178N
M135T and W181H
M135T and W181R
M135T and K174R
R140Q and D178N
R140Q and W181H
R140Q and W181R
R140Q and K174R.
5. The polypeptide of claim 1 , wherein the variant amino acid sequence has 3 to 9 amino acid substitutions relative to the starting amino acid sequence, and wherein at least one substitution is in each of epitope 1, comprising amino acids 48-56 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7; epitope 2, comprising amino acids 135-143 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7; and epitope 3, comprising amino acids 173-181 of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7.
6. The polypeptide of claim 4 , wherein the variant amino acid sequence has only three amino acid substitutions compared to the starting amino acid sequence, and wherein the substitutions are selected from any of the following sets of amino acid substitutions: T56H, M135K and D178N; T56K, M135K and D178N; T56K, M135T and D178N; T56H, M135K and W181R; T56H, M135T and W181R; Y54K, M135T and K174R; Y54R, M135K and K174R; Y54R, M135T and K174R; T56H, M135K and K174R; and T56H, M135T and K174R.
7. The polypeptide of claim 1 , consisting essentially of a human or humanized immunoglobulin Fc polypeptide sequence fused either via a peptide linker or directly to the C-terminus of the variant amino acid sequence.
8. The polypeptide of claim 7 , wherein the immunoglobulin Fc polypeptide sequence is the Fc portion of a human IgG.
9. The polypeptide of claim 8 , wherein the amino acid sequence of the peptide linker and Fc portion of human IgG is selected from amino acids 218-488 of SEQ ID NO:1, amino acids 218-486 of SEQ ID NO:3, and amino acids 218-488 of SEQ ID NO:7.
10. A polypeptide variant comprising a modified starting sequence, wherein the starting sequence is selected from SEQ ID NO:3 or SEQ ID NO:7 and the modification is two or three amino acid substitutions, the polypeptide being selected from any one of the polypeptides set forth below:
Polypeptide
Starting
Epitope 1
Epitope 2
Epitope 3
No.
Sequence
Substitution
Substitution
Substitution
63
SEQ ID NO: 3
Y54K
M135K
None
64
SEQ ID NO: 3
Y54K
M135T
None
65
SEQ ID NO: 3
Y54K
R140Q
None
66
SEQ ID NO: 3
Y54R
M135K
None
67
SEQ ID NO: 3
Y54R
M135T
None
68
SEQ ID NO: 3
Y54R
R140Q
None
69
SEQ ID NO: 3
T56H
M135K
None
70
SEQ ID NO: 3
T56H
M135T
None
71
SEQ ID NO: 3
T56H
R140Q
None
72
SEQ ID NO: 3
T56K
M135K
None
73
SEQ ID NO: 3
T56K
M135T
None
74
SEQ ID NO: 3
T56K
R140Q
None
75
SEQ ID NO: 3
Y54K
None
D178N
76
SEQ ID NO: 3
Y54K
None
W181H
77
SEQ ID NO: 3
Y54K
None
W181R
78
SEQ ID NO: 3
Y54K
None
K174R
79
SEQ ID NO: 3
Y54R
None
D178N
80
SEQ ID NO: 3
Y54R
None
W181H
81
SEQ ID NO: 3
Y54R
None
W181R
82
SEQ ID NO: 3
Y54R
None
K174R
83
SEQ ID NO: 3
T56H
None
D178N
84
SEQ ID NO: 3
T56H
None
W181H
85
SEQ ID NO: 3
T56H
None
W181R
86
SEQ ID NO: 3
T56H
None
K174R
87
SEQ ID NO: 3
T56K
None
D178N
88
SEQ ID NO: 3
T56K
None
W181H
89
SEQ ID NO: 3
T56K
None
W181R
90
SEQ ID NO: 3
T56K
None
K174R
91
SEQ ID NO: 3
None
M135K
D178N
92
SEQ ID NO: 3
None
M135K
W181H
93
SEQ ID NO: 3
None
M135K
W181R
94
SEQ ID NO: 3
None
M135K
K174R
95
SEQ ID NO: 3
None
M135T
D178N
96
SEQ ID NO: 3
None
M135T
W181H
97
SEQ ID NO: 3
None
M135T
W181R
98
SEQ ID NO: 3
None
M135T
K174R
99
SEQ ID NO: 3
None
R140Q
D178N
100
SEQ ID NO: 3
None
R140Q
W181H
101
SEQ ID NO: 3
None
R140Q
W181R
102
SEQ ID NO: 3
None
R140Q
K174R
110
SEQ ID NO: 7
T56H
M135K
D178N
111
SEQ ID NO: 7
T56H
M135K
W181H
112
SEQ ID NO: 7
T56H
M135K
W181R
113
SEQ ID NO: 7
T56H
M135K
K174R
114
SEQ ID NO: 7
T56H
M135T
D178N
115
SEQ ID NO: 7
T56H
M135T
W181H
116
SEQ ID NO: 7
T56H
M135T
W181R
117
SEQ ID NO: 7
T56H
M135T
K174R
118
SEQ ID NO: 7
T56K
M135K
D178N
119
SEQ ID NO: 7
T56K
M135K
W181H
120
SEQ ID NO: 7
T56K
M135K
W181R
121
SEQ ID NO: 7
T56K
M135K
K174R
122
SEQ ID NO: 7
T56K
M135T
D178N
123
SEQ ID NO: 7
T56K
M135T
W181H
124
SEQ ID NO: 7
T56K
M135T
W181R
125
SEQ ID NO: 7
T56K
M135T
K174R
126
SEQ ID NO: 7
Y54K
M135K
K174R
127
SEQ ID NO: 7
Y54K
M135T
K174R
128
SEQ ID NO: 7
Y54R
M135K
K174R
129
SEQ ID NO: 7
Y54R
M135T
K174R
130
SEQ ID NO: 7
T56H
None
D178N
131
SEQ ID NO: 7
T56H
None
W181H
132
SEQ ID NO: 7
T56H
None
W181R
133
SEQ ID NO: 7
T56H
None
K174R
134
SEQ ID NO: 7
T56K
None
D178N
135
SEQ ID NO: 7
T56K
None
W181H
136
SEQ ID NO: 7
T56K
None
W181R
137
SEQ ID NO: 7
T56K
None
K174R.
11. The polypeptide of claim 10 , selected from any one of Polypeptide Nos. 110, 112, 113, 116, 117, 118, 122, 127, 128 or 129.
12. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
13. The polypeptide of claim 9 , wherein the starting amino acid sequence is amino acids 1-217 of SEQ ID NO:1, and wherein any of the 1 to 9 amino acid substitutions is additionally selected from the group of amino acid substitutions set forth below:
Amino Acid
Amino
present in the Starting
Acid #
Amino Acid Sequence
Amino Acid Substitutions
218
G
C, E, N, P, Q, S, T, A, H, W
220
G
E, D, F, W, M, Y
221
S
D, E, G
223
G
D, P, E, K, N, R, T.
14. The polypeptide of claim 9 , wherein the amino acid sequence of the peptide linker and Fc portion of human IgG is selected from amino acids 218-488 of SEQ ID NO:3 with amino acid substitution G220E, an amino acid deletion at 258, and an amino acid deletion at 259.