Recognition molecules for the treatment and detection of tumors
The invention relates to recognition molecules directed towards tumors, and it also relates to pharmaceutical compositions comprising such recognition molecules, methods for the production of such recognition molecules, and to the use of such recognition molecules in the diagnosis and therapy of tumor diseases.
1. A recombinant recognition molecule which comprises a variable heavy chain comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and which comprises a variable light chain of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 and which specifically binds to a glycosylated MUC1 tumor epitope.
2. A recombinant recognition molecule comprising the amino acid sequences set forth in (a)-(f), wherein the variable heavy chain
(a) comprises SEQ ID NO. 1 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 1 comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20;
(b) comprises SEQ ID NO. 3or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 3 comprises SEQ ID NO: 21;
(c) comprises SEQ ID NO. 5;
wherein the variable light chain
(d) comprises SEQ ID NO. 7 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 7 comprises SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26;
(e) comprises SEQ ID NO. 9; and
(f) comprises SEQ ID NO. 11 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 11 comprises SEQ ID NO: 30;
and wherein the recognition molecule specifically binds to a glycosylated MUC1 tumor epitope.
3. The recognition molecule according to claim 1 further comprising one or more antibody framework sequences which separate, enclose and/or flank said amino acid sequences.
4. The recognition molecule according to claim 3 wherein the antibody framework sequence comprises
a) FRH1, FRH2, FRH3 and FRH4 (SEQ ID NO: 82) comprising the following amino acid sequences, the amino acid position corresponding to the numbering according to Kabat:
for FRH1 in position
1
E
(SEQ ID NO: 84)
2
V
3
K
4
L
5
V
6
E
7
S
8
G
9
G
10
G
11
L
12
V
13
Q
14
P
15
G
16
G
17
S
18
M
19
K
20
L
21
S
22
C
23
A or V
24
A, V, S or T
25
S
26
G
27
Y, F, S or D
28
T
29
F, L or I
30
S
for FRH2 in position
36
W
(SEQ ID NO: 85)
37
V
38
R
39
Q
40
S
41
P
42
E
43
K
44
G
45
L
46
E
47
W
48
V
49
A
for FRH3 in position
66
R
(SEQ ID NO: 86)
67
F
68
T
69
I
70
S
71
R
72
D
73
D or V
74
S
75
K
76
S
77
S
78
V
79
Y or S
80
L
81
Q
82
M
82a
N
82b
N
82c
L
83
R
84
A or V
85
E
86
D
87
T
88
G
89
I
90
Y
91
Y
92
C
93
T
94
R, G, N, K or S
for FRH4 in position
103
W
(SEQ ID NO: 87)
104
G
105
Q
106
G
107
T
108
T
109
L
110
T
111
V
112
S
113
S or A
and
b) FRL1, FRL2, FRL3 and FRL4 (SEQ ID NO: 83) comprising the following amino acid sequences, the amino acid position corresponding to the numbering according to Kabat:
for FRL1 in position
1
D
(SEQ ID NO: 88)
2
I, V or L
3
V
4
M or L
5
T
6
Q
7
T or A
8
P or A
9
L or F
10
S
11
L or N
12
P
13
V
14
S or T
15
L
16
G
17
D or T
18
Q or S
19
A
20
S
21
I
22
S
23
C
for FRL2 in position
35
W
(SEQ ID NO: 89)
36
Y
37
L
38
Q
39
K
40
P
41
G
42
Q or L
43
S
44
P
45
K or Q
46
L
47
L
48
I or V
49
Y
for FRL3 in position
57
G
(SEQ ID NO: 90)
58
V
59
P
60
D
61
R
62
F
63
S
64
G or S
65
S
66
G
67
S
68
G
69
T
70
D
71
F
72
T
73
L
74
K or R
75
I
76
S
77
R
78
V
79
E
80
A
81
E
82
D
83
L or V
84
G
85
V
86
Y
87
Y
88
C
for FRL4 in position
98
F
(SEQ ID NO: 91)
99
G
100
G or D
101
G
102
T
103
K
104
L
105
E
106
I or L
106a
K
107
R
108
A.
5. The recognition molecule according to claim 1 , which comprises SEQ ID NO:32 and SEQ ID NO:34.
6. The recognition molecule according to claim 1 , which comprises
(i) at least one sequence set forth in SEQ ID NOs 36 to 47,
(ii) SEQ ID NO: 60 and SEQ ID NO: 62,
(iii) SEQ ID NO: 64 and SEQ ID NO: 66, or
(iv) SEQ ID NO:66 and SEQ ID NO: 68.
7. A composition comprising
(i) at least one recognition molecule according to claim 1 ; and/or
(ii) at least one construct comprising the recognition molecule of claim 1 which is fused, chemically coupled, or covalently or non-covalently associated with
(i) an immunoglobulin domain of various species,
(ii) an enzyme molecule,
(iii) an interaction domain,
(iv) a domain for stabilization,
(v) a signal sequence,
(vi) a fluorescent dye,
(vii) a toxin,
(viii) a catalytic antibody,
(ix) an antibody molecule or a fragment with different specificity,
(x) a cytolytic component,
(xi) an immunomodulator,
(xii) an immunoeffector,
(xiii) an MHC class I or class II antigen,
(xiv) a chelating agent for radioactive labeling,
(xv) a radioisotope,
(xvi) a liposome,
(xvii) a transmembrane domain,
(xviii) a virus or
(xix) a cell;
and/or
(iii) at least one nucleic acid molecule which encodes the recognition molecule of claim 1 ; together with a pharmaceutically tolerable carrier and/or adjuvant.
8. A method for diagnosing, reducing, treating, following-up and/or after-caring tumor diseases and/or metastases in a subject in need thereof comprising administering to said subject a recognition molecule according to claim 1 .
9. An in vitro method for the diagnosis of a tumor comprising detecting a glycosylated MUC1 tumor epitope with at least one recognition molecule according to claim 1 .
10. A recombinant recognition molecule which comprises a variable heavy chain comprising an amino acid sequence which contains the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, and which comprises a variable light chain comprising SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and which specifically binds to a glycosylated MUC1 tumor epitope.
11. The recognition molecule according to claim 10 which comprises SEQ ID NO: 33 and SEQ ID NO: 35.
12. The recognition molecule according to claim 5 which comprises a single-chain antibody fragment, a multibody, a Fab fragment, a fusion protein of an antibody fragment with a peptide or a protein or an immunoglobulin molecule of the IgG, IgM, IgA, IgE, IgD isotype or a subclass thereof.
13. A construct comprising the recognition molecule of claim 12 which is fused, chemically coupled, covalently or non-covalently associated with
(i) an immunoglobulin domain of various species,
(ii) an enzyme molecule,
(iii) an interaction domain,
(iv) a domain for stabilization,
(v) a signal sequence,
(vi) a fluorescent dye,
(vii) a toxin,
(viii) a catalytic antibody,
(ix) an antibody molecule or a fragment with different specificity,
(x) a cytolytic component,
(xi) an immunomodulator,
(xii) an immunoeffector,
(xiii) an MHC class I or class II antigen,
(xiv) a chelating agent for radioactive labeling,
(xv) a radioisotope,
(xvi) a liposome,
(xvii) a transmembrane domain,
(xviii) a virus or
(xix) a cell.
14. A method for the production of the recognition molecule according to claim 1 , comprising:
(i) incorporating one or more nucleic acid molecules encoding the amino acid sequence of at least one recognition molecule according to claim 1 in a virus or in a host cell;
(ii) culturing the host cells or viruses under suitable conditions; and
(iii) obtaining the recognition molecule from the effector cell bearing the recognition molecule or the virus, wherein said recognition molecule specifically binds to the glycosylated MUC 1 tumor epitope.
15. The method according to claim 8 , wherein the recognition molecule comprises an immunoglobulin IgG molecule or a fragment thereof.
16. The method according to claim 8 , wherein the recognition molecules comprise a multibody.
17. A recombinant recognition molecule comprising the amino acid sequences set forth in (a)-(f), wherein the variable chain
(a) comprises SEQ ID NO. 2 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 2 comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16;
(b) comprises SEQ ID NO. 4 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 4 comprises SEQ ID NO: 22 or SEQ ID NO: 23; and
(c) comprises SEQ ID NO. 6;
wherein the variable chain
(d) comprises SEQ ID NO. 8 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 8 comprises SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29;
(e) comprises SEQ ID NO. 10; and
(f) comprises SEQ ID NO. 12 or an equivalent canonical structure variant thereof wherein the equivalent canonical structure variant of SEQ ID NO: 12 comprises SEQ ID NO: 31;
and wherein the recognition molecule specifically binds to a glycosylated MUC1 tumor epitope.
18. The recognition molecule according to claim 10 further comprising one or more antibody framework sequences which separate, enclose and/or flank said amino acid sequences.
19. The recognition molecule according to claim 18 , wherein the antibody framework sequence comprises
a) FRH1, FRH2, FRH3 and FRH4 (SEQ ID NO: 82) comprising the following amino acid sequences, the amino acid position corresponding to the numbering according to Kabat:
for FRH1 in position (SEQ ID NO: 84)
1
E
2
V
3
K
4
L
5
V
6
E
7
S
8
G
9
G
10
G
11
L
12
V
13
Q
14
P
15
G
16
G
17
S
18
M
19
K
20
L
21
S
22
C
23
A or V
24
A, V, S or T
25
S
26
G
27
Y, F, S or D
28
T
29
F, L or I
30
S
for FRH2 in position(SEQ ID NO: 85)
36
W
37
V
38
R
39
Q
40
S
41
P
42
E
43
K
44
G
45
L
46
E
47
W
48
V
49
A
for FRH3 in position (SEQ ID NO: 86)
66
R
67
F
68
T
69
I
70
S
71
R
72
D
73
D or V
74
S
75
K
76
S
77
S
78
V
79
Y or S
80
L
81
Q
82
M
82a
N
82b
N
82c
L
83
R
84
A or V
85
E
86
D
87
T
88
G
89
I
90
Y
91
Y
92
C
93
T
94
R, G, N, K or S
for FRH4 in position (SEQ ID NO: 87)
103
W
104
G
105
Q
106
G
107
T
108
T
109
L
110
T
111
V
112
S
113
S or A
and
b) FRL1, FRL2, FRL3 and FRL4 (SEQ ID NO: 83) comprising the following amino acid sequences, the amino acid position corresponding to the numbering according to Kabat:
for FRL1 in position (SEQ ID NO: 88)
1
D
2
I, V or L
3
V
4
M or L
5
T
6
Q
7
T or A
8
P or A
9
L or F
10
S
11
L or N
12
P
13
V
14
S or T
15
L
16
G
17
D or T
18
Q or S
19
A
20
S
21
I
22
S
23
C
for FRL2 in position (SEQ ID NO: 89)
35
W
36
Y
37
L
38
Q
39
K
40
P
41
G
42
Q or L
43
S
44
P
45
K or Q
46
L
47
L
48
I or V
49
Y
for FRL3 in position (SEQ ID NO: 90)
57
G
58
V
59
P
60
D
61
R
62
F
63
S
64
G or S
65
S
66
G
67
S
68
G
69
T
70
D
71
F
72
T
73
L
74
K or R
75
I
76
S
77
R
78
V
79
E
80
A
81
E
82
D
83
L or V
84
G
85
V
86
Y
87
Y
88
C
for FRL4 in position (SEQ ID NO: 91)
98
F
99
G
100
G or D
101
G
102
T
103
K
104
L
105
E
106
I or L
106a
K
107
R
108
A.
20. The recognition molecule according to claim 11 , wherein it comprises a single-chain antibody fragment, a multibody, a Fab fragment, a fusion protein of an antibody fragment with peptides or proteins and/or an immunoglobulin molecule of the IgG, IgM, IgA, IgE, IgD isotype or a subclasses thereof.
21. The recognition molecule according to claim 10 , which comprises
(i) at least one sequence set forth in SEQ ID NOs 48 to 59,
(ii) SEQ ID NO:61 and SEQ ID NO:63,
(iii) SEQ ID NO:65 and SEQ ID NO:69, or
(iv) SEQ ID NO:67 and SEQ ID NO:69.
22. A construct comprising a recognition molecule according to claim 20 which is fused, chemically coupled, or covalently or non-covalently associated with
(i) an immunoglobulin domain of various species,
(ii) an enzyme molecule,
(iii) an interaction domain,
(iv) a domain for stabilization,
(v) a signal sequence,
(vi) a fluorescent dye,
(vii) a toxin,
(viii) a catalytic antibody,
(ix) an antibody molecule or a fragment with different specificity,
(x) a cytolytic component,
(xi) an immunomodulator,
(xii) an immunoeffector,
(xiii) an MHC class I or class II antigen,
(xiv) a chelating agent for radioactive labeling,
(xv) a radioisotope,
(xvi) a liposome,
(xvii) a transmembrane domain,
(xviii) a virus or
(xix) a cell.
23. A composition comprising
(i) at least one recognition molecule according to claim 10 ; and/or
(ii) a construct comprising at least one recognition molecule of claim 10 which is fused, chemically coupled, or covalently or non-covalently associated with
(i) an immunoglobulin domain of various species,
(ii) an enzyme molecule,
(iii) an interaction domain,
(iv) a domain for stabilization,
(v) a signal sequence,
(vi) a fluorescent dye,
(vii) a toxin,
(viii) a catalytic antibody,
(ix) an antibody molecule or a fragment with different specificity,
(x) a cytolytic component,
(xi) an immunomodulator,
(xii) an immunoeffector,
(xiii) an MHC class I or class II antigen,
(xiv) a chelating agent for radioactive labeling,
(xv) a radioisotope,
(xvi) a liposome,
(xvii) a transmembrane domain,
(xviii) a virus or
(xix) a cell;
and/or
(iii) at least one nucleic acid molecule which encodes the recognition molecule of claim 10 ; together with a pharmaceutically tolerable carrier and/or adjuvant.
24. A method for the production of recognition molecules according to claim 10 comprising
(i) incorporating one or more nucleic acid molecules encoding the amino acid sequence of at least one recognition molecule according to claim 10 in a virus or in a host cell;
(ii) culturing the host cells or viruses under suitable conditions; and
(iii) obtaining the recognition molecule from the effector cell bearing the recognition molecule or construct, or the virus, which specifically recognize the glycosylated MUC 1 tumor epitope.
25. A method for diagnosing, reducing, treating, following-up and/or after-caring tumor diseases and/or metastases in a subject in need thereof comprising administering to said subject a recognition molecule according to claim 10 .
26. The method according to claim 25 , wherein the recognition molecule comprises an immunoglobulin IgG molecule or a fragment thereof.
27. The method according to claim 25 , wherein the recognition molecule comprises a multibody.
28. An in vitro method for the diagnosis of a tumor comprising detecting a glycosylated MUC1 tumor epitope with at least one recognition molecule according to claim 10 .
29. A method for the production of the construct according to claim 13 comprising
(i) incorporating one or more nucleic acid molecules encoding the amino acid sequence of at least one construct comprising said recognition molecule in a virus or in a host cell;
(ii) culturing the host cells or viruses under suitable conditions; and
(iii) obtaining the construct, the effector cell bearing the recognition molecule or construct, or the virus, which specifically recognize the glycosylated MUC 1 tumor epitope.
30. A method for diagnosing, reducing, treating, following-up and/or after-caring tumor diseases and/or metastases in a subject in need thereof comprising administering to said subject a construct according to claim 13 .
31. An in vitro method for the diagnosis of a tumor comprising detecting a glycosylated MUC1 tumor epitope with at least one construct according to claim 13 .
32. A method for diagnosing, reducing, treating, following-up and/or after-caring tumor diseases and/or metastases in a subject in need thereof comprising administering to said subject a construct according to claim 7 .
33. An in vitro method for the diagnosis of a tumor comprising detecting a glycosylated MUC1 tumor epitope with at least one construct according to claim 7 .
34. The recognition molecule according to claim 1 wherein the glycosylated MUC1 tumor epitope comprises a glycosylated PDTRP (SEQ ID NO: 81) region within a MUC1 tandem repeat sequence and is glycosylated with GalNAc or Gal-GalNAc on the PDTRP (SEQ ID NO: 81) threonine.
35. The recognition molecule according to claim 10 wherein the glycosylated MUC1 tumor epitope comprises a glycosylated PDTRP (SEQ ID NO: 81) region within a MUC1 tandem repeat sequence and is glycosylated with GalNAc or Gal-GalNAc on the PDTRP (SEQ ID NO: 81) threonine.
36. The recognition molecule according to claim 34 wherein the glycosylated MUC1 tumor epitope comprises A[HGVTSAPDT(GalNAcα)RPAPGSTAPPA] n wherein n=1, 3, or 5 (SEQ ID NO: 73).
37. The recognition molecule according to claim 35 wherein the glycosylated MUC1 tumor epitope comprises A[HGVTSAPDT(GalNAcα)RPAPGSTAPPA] n wherein n=1, 3, or 5 (SEQ ID NO: 73).